Although chronic cough is a common problem in clinical practice, data on the prevalence and characteristics of cough in the general population are scarce. Our aim was to determine the prevalence of chronic cough that is not associated with diagnosed respiratory conditions and examine the impact on health status and psychological health, in a representative adult population cohortNorth West Adelaide Health Study  is a representative population adult cohort. Clinical assessment included spirometry, anthropometry and skin tests. Questionnaires assessed demographics, lifestyle risk factors, quality of life, mental health and respiratory symptoms, doctor diagnosed conditions and medication use.Of the 3355 people without identified lung disease at baseline, 18.2% reported chronic cough. In multiple logistic regression models, at follow-up, dry chronic cough without sputum production was significantly more common in males , current smokers , obesity , use of ACE inhibitors , severe mental health disturbance  and older age . Among non-smokers only, all cough was significantly more common in men, those with severe mental health disturbance and obesity.Chronic cough is a major cause of morbidity. Attention to cough is indicated in patients with obesity, psychological symptoms or smokers. Inquiring about cough in those with mental health problems may identify reversible morbidity. Cough is the commonest symptom seen in primary care -3, and cChronic cough is associated with adverse effects on health-related quality of life -17. SuccOur aim was to determine the prevalence of chronic cough in a representative adult population cohort, particularly cough that is not associated with diagnosed respiratory conditions, and examine the impact on health status and psychological health.The North West Adelaide Health Study (NWAHS) is a representative biomedical longitudinal population cohort study of people aged eighteen years or older, randomly selected from the electronic white pages telephone directory and living in the north western suburbs of Adelaide, South Australia . NWAHS initially recruited between 2000 and 2002 with follow-up in 2004-05. The methods of the North West Adelaide Health Study  and the At stage 1, 4060 adults underwent biomedical examination, representing 69% of those who completed the telephone interview. Overall, at Stage 2 follow-up  survey data was obtained on 88% (n = 3574) and clinic data on 79% (n = 3206) of the Stage 1 NWAHS population using the same methodology and questions. One hundred subjects were deceased, 226 persons were unable to be contacted, and 160 refused further participation in the study.2: Underweight: ≤ 18.49; Normal: 18.5-24.9; Overweight: 25.0-29.9; Obesity: ≥ 30.0. Medication use was identified when participants were also asked to bring all current medicines (including complementary medicines) into the clinic at their appointment.Telephone interviews investigated self-reported health status (including asthma and COPD), smoking status and demographic variables. A self-completed questionnaire comprised items on demographic information, risk factors , quality of life, mental health and respiratory symptoms. Smoking was categorized into self-reported current, former or never smoker. Clinic assessment by trained technicians included spirometry according to American Thoracic Society criteria , skin pr1 in the absence of a doctor diagnosis of asthma [1/FVC ratio less than 0.70) [Asthma was defined as current self-reported physician-diagnosed asthma or demonstration of a significant bronchodilator response (SBR) of at least 12% of baseline FEVf asthma ,27. Partan 0.70)  were idean 0.70) ,30. ThisHealth-related quality of life was assessed using the Medical Outcomes Study Short Form 36 Health Survey (SF-36) Physical Health Component Summary (PCS) and Mental Health Component Summary (MCS) scores . The PCSSPSS Version 15.0, SPSS Inc, Chicago, IL). Multivariable logistic regression analyses were conducted to assess the association of GHQ disturbance with chronic cough  after adjustment for sex, age, smoking, BMI and ACE inhibitor use. The models were also adjusted for reflux medication use as a proxy for GERD. An additional model assessing the association of all cough and GHQ disturbance was conducted in the population of never/former smokers.Data were weighted to the 1999 Estimated Residential Population for South Australia  and CensApproval for the NWAHS study was obtained from institutional ethics committees of the North West Adelaide Health Service, and all subjects gave written informed consent.The socio-demographic characteristics and health status of the study subjects at Stage 1 baseline have been described in detail previously ,23. Of tThe prevalence of chronic cough at baseline within various demographic and clinical groups is shown in Table In multiple regression analysis Table , chronicParticipants reporting cough at any time were significantly more likely to have psychological disturbance on the GHQ-12 and report significantly lower quality of life compared to those without cough at any time Table . CompareIn a representative population sample we have shown that chronic, dry cough is common among people without known respiratory disease, with a prevalence of nearly 9% among adults. Cough productive of sputum occurs in around a further 4% of those without known lung disease. People with chronic cough report significant impairments in quality of life and psychological health, compared to those without cough. Across the population, chronic cough was significantly associated with obesity and severe depression, and was more common in men and in people aged over 60 years. Although cough was more common in people who currently smoke, when only non-smokers were analyzed, the significant associations seen with depression, obesity, men and age persisted. The prevalence of cough was not significantly different between former smokers and those who had never smoked.The frequency of chronic cough independent of other lung disease, with its strong associations with impaired mental health, particularly depression, and significantly reduced quality of life, indicates cough is a major contributor to morbidity in the community. The reduction in quality of life in general physical health is similar to that previously reported in Australian populations for asthma , diabeteOur data demonstrates that careful attention should be given to assessment and management of psychological morbidity in the large number of patients with chronic cough in the community, as well as those seen in referral centers. This may be especially the case in people in whom coughing persists in the absence of an identifiable cause and despite extended trials of empirical therapy . ChronicWe found obesity to be significantly associated with dry cough and cough in never/ex-smokers. Janson et al have reported cough was significantly associated with obesity. However, the study population of 20-48 year olds included people with asthma and other respiratory diseases . As obesComparison with previous studies examining the prevalence and associations of chronic cough are difficult due to differences in sampling and other methodological questions. We used a validated symptom score of chronic lung disease to identify cough frequency over the previous 3 months. The use of this tool differs from prior studies and makes direct comparison of prevalence between studies difficult. Studies using selected age groups have either excluded adults aged > 50  or > 60 Similar to other reports we found chronic cough is associated with adverse effects on health-related quality of life -17 and pThe direction of causality regarding cough and psychological problems is difficult to determine. We found that in terms of disturbance on the GHQ-28 that the group with cough a follow-up only was not significantly different from those with cough at both time points, suggesting there may be little effect of chronicity over our follow-up period of 4 to 5 years. However, we do not know if people had cough for all the follow-up period or recurrent cough only in the 3 months prior to each clinic assessment. Although those people with cough at baseline but who were no longer coughing had significantly higher physical health quality of life and were less likely to report disturbance on the GHQ. This can be interpreted as indicating chronic cough has both immediate and longer-term consequences for psychological health that may stem from the significant impact on general health experienced with cough. Alternatively, this may suggest chronic cough is more likely to be seen in those with underlying anxiety or depression, and this may influence an individual's awareness of symptoms. However, anxiety about underlying serious illness has been identified as a concern for most patients with chronic cough . McGarveContrary to anecdotal observations, and consequent to the representativeness of our sample we found cough to be more common in men and in people aged over 60 years, two groups where there is evidence to suggest there is a tendency to under-report symptoms to clinicians . Older pOur study is limited by a lack of specific information regarding some of the common causes of chronic cough, such as upper airways syndrome or gastro-esophageal reflux disease ,5. HowevIn summary, chronic cough is a common problem that is significantly associated with reductions in physical and mental health. Investigation and management of chronic cough is therefore an important medical need. Patients with a history of smoking, obesity, allergy, or use of ACE inhibitors should be questioned regarding cough and active clinical care pursued. Careful attention to symptoms of psychological disturbance, including somatic symptoms, and their management may help identify depression and reduce the burden of this problem. Conversely, specifically inquiring about cough in patients with mental health problems may identify reversible physical and psychological morbidity in this group.SA, DW and AT have no competing interests to declare. RA and RR have received honoraria and speakers fees from Glaxo-Smith Kline over the last 5 years.RA took main responsibility for conceiving the analysis and for drafting of the manuscript. SA undertook the analysis and contributed to revision of the manuscript. DW, AT and RR contributed to the conception and conduct of the study, to analytical approaches and methods, and to revising of the manuscript. All authors have read and approved the final version of the manuscript.


The down-regulation of the cough reflex in patients with aspiration pneumonia can involve both cortical facilitatory pathways for cough and medullary reflex pathways. In order to study the possible involvement of the supramedullary system in the down-regulation of cough reflex, we evaluated the urge-to-cough in patients with aspiration pneumonia.2) and five or more coughs (C5). The urge-to-cough scores at the concentration of C2 and C5, and at the concentration of two times dilution of C2 (C2/2) and C5 (C5/2) were estimated for each subject.Cough reflex sensitivity and the urge-to-cough to inhaled citric acid were evaluated in patients with at least a history of aspiration pneumonia and age-matched healthy elderly people. The cough reflex sensitivities were defined as the lowest concentration of citric acid that elicited two or more coughs (C2 and C5 in the control subjects were significantly greater than those for patients with aspiration pneumonia. There were no significant differences in the urge-to-cough at C2 and C5 between control subjects and patients with aspiration pneumonia. However, the urge-to-cough scores at both C2/2 and C5/2 in patients with aspiration pneumonia were significantly lower than those in control subjects. The number of coughs at C5/2 was significantly greater in the control subjects than those in the patients with aspiration pneumonia whereas the number of coughs at C2/2 did not show a significant difference between the control subjects and the patients with aspiration pneumonia.Both CThe study suggests the involvement of supramedullary dysfunction in the etiology of aspiration pneumonia in the elderly. Therefore, restoration of the cough motivation system could be a new strategy to prevent aspiration pneumonia in the elderly. Morbidity and mortality from aspiration pneumonia continues to be a major health problem in the elderly. A marked depression of cough reflex sensitivity is reported in elderly patients with aspiration pneumonia who show cerebral atrophy and lacunar infarction in the brain . The risCough is usually referred to as a reflex defense mechanism mediated at the brainstem level, where sensory information arising from airway sensory receptors in response to an appropriate stimulus is processed by the medullary respiratory network to produce the motor pattern of cough. However, there is accumulating evidence indicating that human cough is under voluntary control and that higher centers such as the cerebral cortex or subcortical regions have an important role in both initiating and inhibiting reflexive cough ,5. AlthoCough is typically preceded by an awareness of an irritating stimulus and is perceived as a need to cough, termed the urge-to-cough . In a caCough reflex sensitivity and the urge-to-cough to inhaled citric acid were evaluated in patients with at least one history of aspiration pneumonia and age-matched healthy elderly people.Patients were prospectively and consecutively recruited from those referred and admitted to the Geriatric Unit, Tohoku University Hospital for treatment of pneumonia from May 2007 to April 2008. Pneumonia was diagnosed by the presence of pulmonary infiltration on chest radiograph and computed tomography (CT) and according to systemic inflammation as determined according to white blood cell (WBC) count and C-reactive protein (CRP). The criteria for pneumonia were established according to the pneumonia guidelines of the Japan Respiratory Society . In the During the entry period, 41 patients with pneumonia without an apparent past- and present-history of stroke were admitted to our 20 bed geriatric unit, and 34 patients (83%) were diagnosed as aspiration pneumonia. We performed simple chest X-ray in all of them. Among 34 patients, we performed chest CT scan in 30 patients. All 34 patients showed characteristic images of infiltrates compatible with aspiration pneumonia in the posterior segment of any of the lobes and/or lower lobe by simple chest X-ray and/or CT scan. Of 34 patients, 2 patients died and 3 patients eternally tracheostomized. Of 29 recovered patients, due to the difficulty of urge-to-cough estimation, we excluded patients with dementia using the mini-Mental State Examination (MMSE). Of 29 patients who recovered from aspiration pneumonia, 18 subjects with a MMSE score < 24 were excluded. Three patients with apparent paralysis were excluded. Finally, 8 patients (3 men) with aspiration pneumonia (70–88 years old) were enrolled for this study. From 6 patients among 8, we obtained brain images with non-contrast CT scan. The CT scan revealed that 2 patients had infarct in the deep region of middle cerebral artery territory, 2 patients in the superficial region  of middle cerebral artery territory, and 1 patient in both the deep and superficial region of middle cerebral artery territory. One patient had infarct in the superficial region of the posterior cerebral artery territory. The diameters of all infarcts were within 1 cm.Eleven age and sex-matched healthy elderly people (72–84 years old) as control subjects were recruited from the community by advertisement. None of the subjects were demented (MMSE scores > 23). All control subjects were never-smokers, and had no previous history of pneumonia and other respiratory diseases. None of the patients or controls were taking medication which might affect cough sensitivity such as antitussives, narcotics, or ACE inhibitors. A CT scan was obtained from only one control subject.Cough reflex and urge-to-cough was examined more than 3 months after negative conversion of C reactive protein after pneumonia had responded to antibiotics treatment . At the time of evaluation, the subjects were in a stable state until at least 3 months before. Simple standard instructions were given to each subject.2) and the lowest concentration of citric acid that elicited five or more coughs (C5).We evaluated the cough reflex sensitivities using citric acid because we had previously used this method to observe depressed cough in the elderly ,3. CoughImmediately after the completion of each nebulizer application, the subject made an estimate of the urge-to-cough. The modified Borg scale was used to allow subjects to estimate the urge-to-cough . The scaU test or the chi-square test were used to compare patients with controls. A p value of < 0.05 was considered significant.The study protocol was approved by the local ethics committee and informed consent was obtained from all subjects. Data are expressed as mean (SD) except where specified otherwise. The Mann-Whitney All 19 subjects completed the experiments without any difficulty or side effects. Among the 8 patients with aspiration pneumonia, 3 patients had a history of recurrent pneumonia (2–3 episodes). All subjects were leading an independent life. The characteristics of subjects are summarized in Table 2, in patients with aspiration pneumonia (1.5 ± 0.6 g/l) was significantly higher than those of control . The urge-to-cough scores at the concentration of C2 and at the concentration of two times dilution of C2 (C2/2) were estimated for each subject. There were no significant differences in the urge-to-cough at C2 between control subjects (3.0 ± 1.8 points) and patients with aspiration pneumonia (3.3 ± 3.0 points)  were significantly lower than those in control subjects (1.2 ± 0.8 points)  than in patients with aspiration pneumonia (0.0 ± 0.0 times). At C2/2, only one control subject coughed among all subjects.As shown in Figure ) Figure . However) Figure . There w5, in patients with aspiration pneumonia (1.6 ± 0.5 g/l) was significantly higher than those of control . The urge-to-cough scores at the concentration of C5 and at the concentration of two times dilution of C5 (C5/2) were estimated for each subject. There were no significant differences in the urge-to-cough at C5 between control subjects (7.5 ± 1.8 points) and patients with aspiration pneumonia (5.3 ± 3.4 points)  were significantly lower than those in control subjects (3.0 ± 1.9 points)  than in patients with aspiration pneumonia . Actually, 6 patients (75.0%) with aspiration pneumonia did not cough at all at C5/2 whereas 2 control subjects (18.2%) did not.As shown in Figure ) Figure . However) Figure . The num2 and C5 are same value in 1 subject in control group and 5 subjects in the patients with aspiration pneumonia.In the present study, CThis study shows, for the first time to our knowledge, that the urge-to-cough is significantly attenuated in elderly patients with aspiration pneumonia. It has been suggested that the aspiration pneumonia is, at least in part, a consequence of cough reflex impairment. Sekizawa and coworkers demonstrated a marked depression of the cough reflex in elderly patients with aspiration pneumonia . Nakajoh2 and C5, this might be due to too small sample number in this preliminary study. However, as the urge-to-cough precedes the actual cough [2 was not significant between groups. Moreover, the actual cough has possibility to affect the urge-to-cough. In the study, all patients with aspiration pneumonia did not cough at C2/2, and 6 of 8 did not at C5/2. If the actual cough has ameliorating effect on the depressed urge-to-cough in the patients with aspiration pneumonia, the urge-to-cough scores at C2 and C5 became not different between groups. Well-designed and larger sample studies are warranted to clarify this.Although we did not observe significant difference in the urge-to-cough at Cal cough , the dif2 and C5. C5 is considered as a clinically superior value based on better reproducibility compared to C2 [2/2 in order to avoid the effect of actual cough on the result [5/2 whereas there is no significant difference in the number of cough between controls and patients with aspiration pneumonia at C2/2. Therefore, the urge-to-cough at C2/2 may more purely reflect the supramedually involvement of urge-to-cough.In the present study, we estimated the cough reflex sensitivity using Ced to C2 . Howevere result . In the Due to a lack of flow monitoring, we could not accurately distinguish between cough reflex and expiration reflex, both of which are defensive reflexes to remove foreign substances from the airway by producing the expiratory airflow. However, the latency from stimuli to induce expiration reflex was much shorter than that of cough reflex, suggesting that cortical involvement is unlikely in the expiration reflex . TherefoIn stroke patients, an impaired cough capacity is now regarded as one of the main factors accounting for the increased prevalence of aspiration pneumonia -18. The Since it has been proposed that initiation of a reflex cough response requires the urge-to-cough to facilitate it , the depThis study suggests the involvement of supramedullary dysfunction, at least in a part, in the etiology of aspiration pneumonia in the elderly. Therefore, the restoration of the cough motivation system could be a new strategy to prevent aspiration pneumonia in the elderly.2: the lowest concentration of citric acid that elicited five or more coughs; C2/2: The urge-to-cough scores at the concentration of C2 and at the concentration of two times dilution of C2; C5: the lowest concentration of citric acid that elicited five or more coughs; C5/2: Urge-to-cough scores at the concentration of C5 and at the concentration of two times dilution of C5.MMSE: mini-Mental State Examination; CThe authors declare that they have no competing interests.SY, SE and TE participated in the design of the study, collected and analyzed data, and drafted the manuscript. MY, TA, MA and KU participated in the design of the study and collected the data. HA participated in design of the study and helped to draft the manuscript. All the authors read and approved the final manuscript.


The experimental data providing evidence for the above roles of tryptase are summarized in the present review, as well as the effects of tryptase inhibition in animal asthma models. The potential strategies for the development of anti-asthmatic agents based on the inhibition of tryptase are discussed.Recent physiological and pharmacological studies have indicated the potential importance of tryptase, the major protein component in mast cells, in inflammatory diseases . Being released at inflammatory sites after the activation of mast cells, tryptase is capable of causing bronchohyperresponsiveness and infiltration of eosinophils, neutrophils, etc. in animal airways. The mechanisms by which tryptase causes bronchoconstriction involve probably the potentiation of other chemical mediators such as histamine, production of bradykinin via the hydrolysis of kininogen, and cleavage of the bronchodilating peptides VIP  and PHM (peptide histidine-methionine). Tryptase has also been found to be a potent mitogen


Because 24-h esophageal pH monitoring is quite invasive, the diagnosis of gastroesophageal reflux disease (GERD)-associated cough has usually been made based merely on the clinical efficacy of treatment with proton pump inhibitor (PPI).We recently encountered two patients with PPI-responsive chronic non-productive cough for whom switching from bronchodilators and glucocorticosteroids to PPI resulted in improvement of cough. The cough returned nearly to pre-administration level a few weeks after discontinuation of PPI. Though GERD-associated cough was suspected, 24-h esophageal pH monitoring revealed that the cough rarely involved gastric acid reflux. Following re-initiation of PPI, the cough disappeared again.PPI may improve cough unrelated to gastric acid reflux. Gastroesophageal reflux disease (GERD)-associated cough is a well-known type of chronic, non-productive cough . Asthma,In our institutions, chronic cough lasting more than 8 weeks without history of wheezing was assessed as described in Figure We recently encountered two patients with PPI-responsive chronic non-productive cough in whom 24-h esophageal pH monitoring showed cough rarely associated with a reflux episode. These cases show that PPI may improve cough unrelated to an acid reflux episode.The patient was a 60-year-old man who had been suffering from isolated chronic non-productive cough for about 1 year. He discontinued smoking 9 months before the first visit following development of this cough and had never taken an ACE-I. Although he did not complain of heartburn and other symptoms suggestive of GERD, endoscopic assessment of the esophagus revealed reflux esophagitis (Los Angeles classification Grade B). He had had no respiratory infections during the 8-week period preceding the first visit. No abnormal shadows were noted on chest or paranasal sinus X-rays and chest CT scan. Cutoff points in testing of bronchial hyperresponsiveness and cough reflex hypersensitivity were set at below 10000 μg/ml  and 3.9 The patient was a 34-year-old man who had been suffering from isolated chronic non-productive cough since 31 years of age. He had stopped smoking at 32 years of age and had never taken an ACE-I. Although he did not complain of heartburn or other symptoms suggestive of GERD, endoscopic assessment of the esophagus revealed reflux esophagitis (Los Angeles classification Grade M). He had had no respiratory infections within the 8-week period preceding the first visit. No abnormal shadows were noted on chest or paranasal sinus X-rays and chest CT scan. Airway reversibility to inhaled β2 agonist was 13%, and testing of bronchial responsiveness to methacholine and cough reflex sensitivity revealed hyperresponsiveness (1208 μg/ml) without hypersensitivity (31.2 μM). Cell fractionation of bronchoalveolar lavage fluid revealed percentages of macrophages, lymphocytes, neutrophils, and eosinophils of 92%, 5%, 3%, and 0%, respectively. Neither bronchodilator therapy nor anti-inflammatory therapy improved the cough. PPI was given after discontinuation of bronchodilator and anti-inflammatory therapy. The cough was markedly improved 1 week after initiation of PPI (cough level 1), but had returned nearly to pre-administration level by 3 weeks after discontinuation of PPI (cough level 8). On 24-h esophageal pH monitoring performed prior to re-initiation of PPI, some cough and acid reflux were observed, but little cough-related acid reflux was noted  declare that they have no competing interests.All authors have read and approved the final manuscript. KN had primary responsibility for drafting and submitting the manuscript. HA was involved in the patients' clinical assessment and treatment.


Several studies have suggested that elevated serum alanine aminotransferase (ALT) and asparte aminotransferase (AST) may be markers of hepatitis E virus (HEV) infection. Thus, individuals with elevated ALT and AST may have ongoing subclinical infection of HEV. We estimated the prevalence of anti-HEV antibodies and serum ALT and AST levels among persons who work with pigs in Accra, Ghana.Three hundred and fifty- persons who work with pigs provided blood samples for unlinked anonymous testing for the presence of antibodies to HEV, ALT and AST levels. The median age of participants was 32.85 ± 11.38 years (range 15-70 years). HEV seroprevelance was 34.84%. Anti-HEV IgG was detected in 19.26% while anti-HEV IgM was detected in 15.58% of the persons who tested positive. On multivariate analysis, the independent determinants of HEV infection were, being employed on the farm for less than six months , having piped water in the household and/or on the farm  and consumption of alcohol (OR 4.91: 95% CI 2.65-9.10). Levels >3× the expected maximum were found for both ALT and AST among individuals who tested positive for anti-HEV IgG  and anti-HEV IgM .Consistent with similar studies worldwide, the results of our studies revealed a high prevalence of HEV infection, ALT and AST values in pig handlers. Hepatitis E virus (HEV) infection is one of the major cause of human viral disease with clinical and pathological features of acute hepatitis. The infection represents an important public health concern in many developing countries, where it is primarily transmitted through the faecal oral route due to contaminated water and food , and is The first animal strain of HEV was characterised in pigs in the United States of America ,5 and siGrowing evidence suggests that individuals who work in contact with swine such as pig farmers, veterinarians and slaughterhouse workers are at increased risk of acquiring HEV infection -12. We rSeveral studies -17 suggeA cross-sectional study was carried out between the months of January and October 2008 among workers in 6 commercial pig farms in the Greater Accra Region of Ghana. The pig rearing facilities used for the study ranged from small family-run piggeries (~200 pigs) to large-scale pig farming operations (~4000 pigs) where animal housing conditions, sanitation and overall management were generally of a lower standard. All the farms are situated within the communities in high population density areas. Two of the farms are close to each other while the rest of the farms are about 95 km from the other two farms. The study was approved by the Ethical and Protocol Review Committee of the University of Ghana Medical School, Accra, Ghana.Subjects for the study were male workers of the Farms. The study population was of similar socio-economic and cultural backgrounds. In general, participants had been residing in their respective communities for most part of their lives. Farming is the major source of income; most farmers rear pigs, and other domestic animals such as goats, sheep, cows and poultry for their own consumption and for sale to supplement the family incomes. After an explanation of the purpose of the study, all the workers were invited to participate. They were informed that the study was confidential and that information provided by them would not affect their employment status. A total of 353 persons joined in the research. Written and informed consent was obtained from each participant, and the information regarding the protocol and informed consent was presented at the appropriate literacy level. The study was conducted in a confidential manner and random unique study generated numbers were employed to identify the participants.All the 353 consenting participants completed a structured questionnaire assessing socio-demographic characteristics, and risk factor profile for the infection under investigation.Blood (10 ml) samples were collected from each of the consenting participants in plain tubes. Samples were centrifuged and the sera kept frozen at -20°C until analysed. Sera were tested at the Public Health Reference Laboratories, Korle-Bu, Accra, Ghana, for the presence of IgM and IgG antibodies  to HEV, in accordance with the manufacturer's instructions. The results were scored as positive or negative according to the standard procedures recommended by the manufacturer. Positive and negative controls were included in all the ELISA microplates assayed. The International Immuno-Diagnostics assays were chosen for this investigation because our validation exercises (data not shown) had demonstrated these assays to be sensitive and specific than some other commercially available assays. Liver function tests, particularly ALT  and AST  in serum were determined at the Chemical Pathology Department, Korle Bu Teaching Hospital Central Laboratory, Accra, Ghana, using the Thermo Spectronic spectrophotometer .The Statistical Analysis Software  version 9.1 was used to complete all data analyses. For each generally accepted risk factor for HEV infection, the odds ratio (OR) and the 95% confidence interval (95% CI) were calculated to assess associations with sociodemographic and behavioural variables in univariate analysis. A P value of < 0.05 was considered significant. Independent determinants of anti-HEV reactivity were evaluated by calculating the adjusted OR by univariate and multivariate analysis for the risk factor variables found to be significant and to estimate their joint influence.To examine whether persons who work with pigs are at high risk for HEV infection, 353 subjects , who had been handling pigs for more than 3 months in 6 pig farms in Accra, were recruited to participate in the study between January and October, 2008. The serum samples were assayed for the presence of anti-HEV IgG, anti-HEV IgM, ALT and AST. All the subjects were occupationally exposed to the pigs . Of the subjects occupationally exposed to the pigs, 39.94% (141 out of 353) had been working in the same farm setting for less than 6 months whilst 60.06% (212 out of 353) had been working in the same farm setting for more than 6 months.The overall prevalence of anti-HEV was 34.84% (123 out of 353). Of the persons who tested positive, anti-HEV IgG was detected in 19.26% (68 out of 123) while anti-IgM was detected in 15.58% (55 out of 123). There was a significant difference between them, P < 0.05.Table However, the overall prevalence of antibodies to HEV-IgG was highest 35.71%) among persons 51-55 years of age, followed by 23.08% in 46-50 year group, then 21.82% in 21-25 year group  and anti-HEV IgM . Compared to the different age groups, a higher number of pig handlers, 26-30 years of age, who tested positive for either anti-HEV IgM or anti-HEV IgG had ALT  and a 6.97 fold (95% CI 3.19-15.24) higher risk of elevated ALT and AST levels, respectively. Similarly, pig handlers 26-30 years of age who tested positive to antibodies to HEV IgM had a 41.71 fold (95% CI 18.80-92.56) and a 3.73 fold (95% CI 1.79-7.75) higher risk of increased ALT and AST levels, respectively.Potential risk factors for infection were examined, to determine whether there were associations with anti-HEV prevalence.As shown in Table A similar pattern was observed among persons who work with pigs and had water piped into their homes and/or on the farms and clean barns, assist sows at birth, butcher pigs at the farm, and eat pork (undercooked pork). As shown in Table Of the pig handlers who tested positive to antibodies to HEV, alcohol was consumed by 88.61% (109 out of 123). Our analysis indicated association with alcohol consumption. In univariate analysis, persons who work with pigs and consume alcohol, were at an increased risk (OR 4.91: 95% CI 2.65-9.10) of HEV infection. Pig handlers who consume alcohol above the recommended weekly intake (defined as a maximum weekly intake of 21 units) had a 3.48 fold (95% CI 1.96-6.17) higher risk of HEV infection. In a multivariate analysis, HEV infection was independently associated with length of time a person has been working on the farm, having piped water into their homes and/or the farms and consumption of alcohol.This is the first study in Ghana reporting ALT, AST values and risk factors associated with HEV infection in persons who work with pigs, and demonstrates the high prevalence of and the considerable potential for the transmission of HEV infection in pig farms in Ghana. Although there is no report from the Ministry of Health, Accra, Ghana indicating that Ghana is an endemic area for hepatitis E, this study found very high overall prevalence rates (34.84%) of HEV antibody. In addition, the study found significantly high ALT and AST levels >3× the expected maximum among persons occupationally exposed to pigs, suggesting the possibility of subclinical infections in the country. Further studies with a larger number of pig handlers will be necessary to draw a definitive conclusion. Moreover, additional studies need to be done to isolate both the pig and human HEV strains in Ghana and determine the genomic relationship between them if both the human and pig HEV are isolated.The risk of HEV infection correlated with length of time employed, close contact with animals or animal waste, such as cleaning barns or assisting sows at birth, butchering pigs, consumption of alcohol, and having piped water in homes and/or on the farms. The finding of higher HEV antibody prevalence in persons who work with pigs in Ghana is consistent with literature, and is widely attributable to work-related behaviours practised on the farm settings, although transmission of HEV has also been documented among individuals outside the farm setting and persons who are not occupationally exposed to pigs -22. The Another finding of interest reported herein in our study is that anti-HEV prevalence was significantly higher (P < 0.001) among persons who had been working with pigs in the same farm setting for less than 6 months compared to those who had been working in the same farm setting for more than 6 months . The reason(s) for this disparity cannot be discerned from our study. However, it was observed that newly recruited individuals spend more time cleaning barns and assisting sows at birth, and this perhaps may have accounted for the high seroprevalence rate of HEV infection. Further studies need to be done to define the high prevalence of anti-HEV antibodies in such population.Of particular note, pig handlers studied who tested positive for antibodies to IgG anti-HEV or IgM anti-HEV had ALT  levels >3× the expected maximum. A similar pattern was noted in serum AST levels among pig handlers. Levels 3× the expected maximum were found in pig handlers studied who tested positive for antibodies to IgG anti-HEV  or IgM anti-HEV . Interestingly, among the different age groups, pig handlers, 26-30 years of age, had higher levels of ALT and AST (data not shown).Growing evidence suggest that elevated ALT and AST levels are associated with recent acute HEV infection -17,23. SAnother finding of interest reported herein in our study is that anti-HEV prevalence was associated with consumption of alcohol (OR 4.91: 95% CI 2.65-9.10) among persons who had been working with pigs. Although excess alcohol consumption could compromise hepatic function and predispose pig handlers to HEV infection as suggested by our study, consumption of alcohol may not be probably linked to exposure to HEV infection. However, from our studies it is also possible that excess consumption of alcohol may have resulted in lack of self control therefore leading to higher risk behaviour of HEV infection. Further studies need to be conducted to define the link between alcohol consumption and HEV infection among persons who work with pigs.The small sample size and our inability to test for anti-HEV reactivity in pigs, may be the limitations of this study. However, the detection and prevalence of HEV infection coupled with significantly high values of ALT and AST in persons who work with pigs in Ghana may reflect the prevalence of past and recent HEV infections among pig handlers in the country. Further studies need to be done to define clearly the natural history of HEV infection and transmission in Ghana in order to effectively control and prevent HEV zoonosis.The results reported herein have significant implications for veterinarians, public health officials, persons who work with pigs and farm managers, and suggest urgent need for the introduction of policies to prevent the transmission of HEV on the farms and the general population. These policy strategies must include increasing education of persons who work with pigs about the need for HEV testing and prevention in infected pig handlers. The implementation of a HEV infection prevention programme in pig farms in Ghana should be seen as an opportunity to improve the health status of the infected persons who work with pigs and to prevent further transmission of HEV, within and without the farm settings. The argument for HEV testing among persons who work with pigs in Ghana is compelling, because of the precarious sanitary conditions in most urban and rural areas, increased incidence of acute viral hepatitis without a defined aetiology , and the high infant and maternal mortality. Our findings re-emphasize the suggestion that targeting high-risk pig handlers or universal testing in high prevalence areas, which includes Ghana, could identify most pig handlers, pregnant women, and blood donors infected with HEV at a relatively low cost ,23.This is the first study in Ghana reporting a high prevalence of IgG, IgM anti-HEV antibodies and elevated ALT and AST levels in persons who are occupationally exposed to pigs. The high prevalence of HEV infection coupled with the elevated ALT and AST values suggest that HEV infection should be treated as an occupational illness in persons who work with pigs in Ghana and therefore suggest the urgent need for the introduction of some of the range of effective preventive strategies employed in pig farm settings elsewhere.Fully informed consent was obtained from each study subject. When study subjects were younger than 18 years, informed consent was obtained from their parents.The authors declare that they have no competing interests.AAA, YA, JTA, CAG, JAA, ETN conceived of the study, participated in the design and coordination. All the authors read and approved the final manuscript.


Cough reflex can be induced by the pepper extract capsaicin and by low pH in guinea-pig airways. Transient receptor potential vanniloid-1 (TPRV-1) is expressed in the sensory and afferent nerve fibres in airways.We hypothesized that a novel pyridazinylpiperazine analog TPRV-1 inhibitor can effectively reduce cough reflex stimulated by citric acid and capsaicin.-4M) aerosols, in succession separately by 2 hours.Guinea pigs were injected with specific TPRV-1 inhibitor, V112220, a pyridazinylpiperazine analog of N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carbox-amide (BCTC) (3 mg/kg) intra-peritoneally. One hour before cough response assessment. Coughs were recorded using a recorder system that identified cough sound and accompanying expiratory flows, distinct from sneezes. Guinea-pigs exposed to citric acid (0.4 M) and to capsaicin  and capsaicin  compared to vehicle control.A novel pyridazinylpiperazine analog TPRV-1 inhibitor can inhibit the cough reflex, induced by both low pH and capsaicin, suggesting that it could be clinically beneficial in treatment of cough. Capsaicin is a potent tussive agent in most species including humans. It activates a capsaicin receptor, transient receptor potential vanilloid-1 (TRPV-1), which is a polymodal ion channel  and endoSeveral antagonists of TRPV-1 have now been described . CapsazeThe protocols were approved by the Imperial College BioSciences Group and performed under a Project License from the British Home Office, UK, under the Animals (Scientific Procedures) Act 1986.Pathogen free Male Hartley guinea pigs (600 – 700 g) were used for the study. Animals were screened one week before the in vivo cough examination.Materials used in the study including: V112220, a selective TRPV1 antagonist ; vehicle, 20% hydroxypropyl-β cyclodextrin ; Procaterol hydrochloride ; Citric Acid  and Capsaicin .Conscious guinea pigs were pre-screened to assess their cough response to 0.4 M citric acid one week before the cough study with V112220 or diluent. Low responders (number of coughs < 3) and high responders (number of coughs > 20) were excluded from the study. After pre-screening, animals were allocated into 3 different groups, the control group (n = 4) and two treatment groups .Conscious animals were placed in a 4 L plethysmograph which was equipped with an internal microphone and a pressure transducer, and were connected to a Amplifier Interface Unit series pre-amplifier . Aerosols were generated with an ultrasonic nebuliser  which was connected to a Basic Flow Supplier AIR 200 . Airflow was set at 8 L/min. Coughs were detected in three ways: via the microphone, via the pressure transducer and by observing the guinea-pig behaviour which was also captured with an external camera. Data acquisition was performed with the eDacq  acquisition software.-4 M Capsaicin for 10 min and the cough response was assessed.One week following screening, guinea pigs in the treatment groups were injected with either 1 ml of vehicle or 3 mg/kg V112220 intra-peritoneally (i.p.) 1 hour before cough response assessment. Each guinea pig received 0.1 mg/kg procaterol hydrochloride i.p. injection 10 minutes prior to each cough assessment in order to minimise bronchoconstriction. For cough assessment, animals were exposed to 0.4 M citric acid for 10 min and a 10 min cough response was recorded. Two hours following citric acid inhalation, the same animal was exposed to 10Data were recorded as number of coughs per 10 min assessment. Cough numbers of individual animal were compared among pre-screening, following citric acid inhalation and following capsaicin inhalation. Data from the treatment groups were compared with the control group. Mean values were statistically analyzed by one-way analysis of variance (ANOVA) to evaluate significant differences between groups. Values are expressed as means and 95%CI, with p < 0.05 being considered significant.Guinea-pigs (n = 24) were pre-screened regarding their cough response with citric acid. Ten guinea-pigs (8 low and 2 high cough responders) were excluded and subsequently, fourteen guinea-pigs were divided into 3 groups. No significant difference in baseline cough response was noted among the 3 groups of guinea pigs. Figure Figure Our study demonstrated that blockade of the TRPV-1 receptors with a selective inhibitor, V112220, which is a pyridazinylpiperazine derivative, effectively decreased by 70% coughs evoked by citric acid or capsaicin aerosol exposure in the guinea pig. This is in agreement with a previous study using the earlier TRPV-1 antagonist, capsazepine, which inhibited coughs induced by citric acid or capsaicin but not coughs induced by 7% hypertonic saline solution,15.Recently, 4-(2-pyridyl)piperazine-1-carboxamide analogues as potent TRPV-1 antagonists have been developed . N-(4-teTRPV1 is sensitive to vanilloid molecules, including capsaicin. It can be activated by low extracellular pH ,6,20,21,Chronic persistent cough is a clinical problem, since antitussives available to control cough are often not effective . More poSYL carried out the drugs administration and cough measurements, and performed the statistical analysis. AN helped in the design of the study. ASW & PN participated in animal maintenance. F-XB & QTD assisted in cough measurement, and drugs preparation. KFC conceived of the study, and participated in its coordination. All authors read and approved the final manuscript.


Stimulation of phosphoinositide hydrolysis in myocardium from autoimmune myocarditis mice by ThEA and histamine was assayed. Myocardium from autoimmune heart, but not the normal forms, specifically increased phosphoinositide turnover in the presence of histaminergic agonists. This increment was blocked by a specific H1 antagonist mepyramine and to the same extent by the phospholipase C inhibitor NCDC. By using a binding assay H1 histaminergic receptors were detected in autoimmune heart membrane preparations, but this was not observed in normal heart. These data suggest that autoimmune myocardium expressed a functional H1 receptor that could involve a distinctive mechanism operating in the disease.


Hepatitis E virus (HEV) is a zoonotic pathogen of which several species of animal were reported as reservoirs. Swine stands out as the major reservoir for HEV infection in humans, as suggested by the close genetic relationship of swine and human virus. Since 2000, Genotype 4 HEV has become the dominant cause of hepatitis E disease in China. Recent reports showed that genotype 4 HEV is freely transmitted between humans and swine in eastern and southern China. However, the infection status of HEV in human and swine populations in central China is still unclear. This study was conducted in a rural area of central China, where there are many commercial swine farms. A total of 1476 serum and 554 fecal specimens were collected from the general human and swine populations in this area, respectively. The seroepidemiological study was conducted by enzyme-linked immunosorbent assay. Conserved genomic sequences of open reading frame 2 were detected using reverse transcription-PCR. The results indicated that the overall viral burden of the general human subjects was 0.95% (14/1476), while 7.0% (39/554) of the swine excreted HEV in stool. The positive rate of anti-HEV IgG and IgM in the serum samples was 7.9% (117/1476) and 1.6% (24/1476), respectively. Phylogenetic analysis based on the 150 nt partial sequence of the capsid protein gene showed that the 53 swine and human HEV isolates in the current study all belonged to genotype 4, clustering into three major groups. However, the HEV isolates prevalent in the human and swine populations were classified into known distinct subgenotypes, which suggested that no cross-species transmission between swine and humans had taken place in this area. This result was confirmed by cloning and phylogenetic analysis of the complete capsid protein gene sequence of three representative HEV strains in the three major groups. The cross reactivity between anti-HEV IgG from human sera and the two representative strains from swine in central China was confirmed by Dot-blot assay. In conclusion, although all the HEV strains prevalent in central China belonged to genotype 4, there is no evidence of cross-species transmission between human and swine in this area. Hepatitis E virus (HEV), a member of the genus Hepevirus, is a non-enveloped virus with a positive-stranded RNA genome approximately 7.2 kb in length HEV and antibodies to HEV have been found in a wide variety of animals HEV isolates were divided into four distinct genotypes according to sequence and phylogenetic analyses. Genotype 1 was previously believed to only infect humans, but this has been disputed recently by a report of this subtype in a pig from Cambodia P = 0.462). Our questionnaire showed that 176 individuals had a history of frequent travel to other regions, 9.1% (16/176) of them tested positive for IgG, which was not significantly different from those who did not report frequent travel to other regions (7.8% or 101/1300 P = 0.271). Of the 98 individuals who were positive for anti-HEV IgG, only two have ever been admitted to a hospital because of hepatitis E.The 150-nt PCR-amplified products from 39 swine and 14 human HEV isolates were sequenced. The results identified 28 swine and 14 human HEV isolates with distinct nucleotide sequences. The sequences were deposited in GenBank, isolate names were from Ch-MD-hu1 to Ch-MD-hu14 and from Ch-MD-sw1 to Ch-MD-sw28, and the corresponding accession numbers were FJ641988-FJ641993 and FJ641994-FJ642021, respectively. A BLAST search using these 42 sequences showed that these isolates all belonged to genotype 4 HEV, and some of the matching sequences from GenBank are included in From the three major groups, three representative strains, Ch-MD-sw1, Ch-MD-sw23 and Ch-MD-hu1, were selected and their complete ORF2 sequences were determined. Sequence analysis based on the complete ORF2 sequence indicated that the two swine HEV strains, Ch-MD-sw23 and Ch-MD-sw1, shared 91.7% sequence homology with each other, and 89.7% and 89.3% with the human isolate Ch-MD-hu1, respectively. Their putative amino acid sequences shared 98.5–99.4% sequence identity. Phylogenetic analysis was performed based on the three representative isolates in the present study and 17 referenced, and well characterized, genotype 4 HEV isolates Combining these results, it suggests that there are two different subgenotypes of genotype 4 HEV strains prevalent in swine populations in central China, and they are genetically distinct from the strains which are prevalent in general human populations in this area.The reactivity of the two representative swine HEV strains (Ch-MD-sw1 and Ch-MD-sw23) with the 29 serum samples which were positive for anti-HEV IgG was determined by Dot-blot assay. There is gathering evidence that HEV is enzootic and pigs are considered one of the major reservoirs of human infection with this virus. The zoonotic transmission of HEV from pigs to humans has been suggested previously, particularly for cases in non-endemic areas. This hypothesis was mainly based on phylogenetic analysis, which showed that swine and human HEV strains from same geographic regions share a high genomic similarity Based on the phylogenetic analysis of the 150 nt partial ORF2 HEV sequences from both humans and pigs, Zheng et al reported that genotype 4 HEV transmitted freely between swine and humans in eastern China From the sequencing results, some of the 39 swine HEV strains showed non-unique sequences over the 150nt region. Among these were four strains which had identical sequences but came from two different farms. Phylogenetic analysis also indicated that some of swine or human strains were very closely related  that is being shed enterically from humans and animals may lead to large outbreaks of Hepatitis E Over the last few years there have been a flurry of case reports, case series and descriptions of small scale outbreaks relating to hepatitis E from Japan In the present study, our research was conducted in central China, which is an area of plains with few rivers. Most of the local rural residents consume deeply drilled borehole water. Moreover, the residents in this area are not known to have a penchant for eating raw meat. All these factors make the circulation of HEV in swine or humans independent in central China and thus significantly reduce the chance of HEV cross-species transmission from animals to human. Whether there are other factors which hinder human HEV strains from infecting pigs or vice versa will need to be elucidated by further research.In the present study, all the human serum samples were collected from the general population. None of the 24 persons who were positive for anti-HEV IgM showed evident clinical symptoms of hepatitis, and only two of the 98 persons who were only positive for anti-HEV IgG had a history of being diagnosed with hepatitis E. This suggests that HEV infection in central China is commonly sub-clinical. Thus, HEV infection may persist in disease-endemic populations by serial transmission among susceptible persons in the form of subclinical and asymptomatic infection. These subclinical cases, which go unnoticed, may act as one of the reservoirs for persistence of this infection in the population and as a source of sporadic acute hepatitis E. A similar continual transmission of subclinical infection was the main reservoir of poliovirus in areas in which the disease was endemic. Subclinical HEV infection in laboratory infected cynomolgus macaques was observed by Aggarwal, who reported that subclinical HEV infection in some animals was associated with failure of the development of an immune response when compared with animals with clinical HEV infection Most of the evidence supporting animal–human transmission of HEV is from regions where HEV infection is not endemic. In India, an endemic area for HEV, some reports indicate that zoonotic transmission of HEV from pigs to humans does not exist, and the strains prevalent in human populations all belong to genotype 1 HEV while those prevalent in pigs belong to genotype 4 In the present study, although HEV strains prevalent in human and swine groups in central China all belonged to genotype 4, and the complete ORF2 sequence of representative strains shared nucleotide and amino acid identity of more than 89% and 98%, respectively, they clustered separately into genetically distinct clusters. This suggested that the swine and human HEV isolates in central China are distinct from each other, this phenomenon is not attributed to virus genotype but non-viral factors, such as human living habits, living environments, and local climate.Phylogenetic analysis based on the full or partial ORF2 sequences showed that the human or swine isolates in the present study cross clustered with the swine and human isolates from other regions of China and even Japan  and 2, wSo far, genotype 1 and 4 HEV viruses have been found to be prevalent in human populations in China where genotype 4 HEV has become the dominant cause of hepatitis E disease since 2000 In the present study, we used the primers which had been optimized for genotypes 1 and 4 virus RNA detection In the present study, the age group 20–39 years had the highest positive rate of IgM, suggesting that people in this age group have the highest clinical attack rates in central China. This is consistent with the current opinion that the peak incidence in sporadic cases of hepatitis E in endemic regions occurs in 15-35-year-olds HEV has been isolated and fully sequenced. Sequence analysis of the various genotypes of HEV isolates shows significant similarity of the genomes and many cross-reactive epitopes between these different genotypes. It has been shown that antibodies to one strain are cross-reactive with the other strains, indicating cross-protection between strains. The anti-HEV assays used in the current study used a recombinant peptide of a genotype 1 HEV structural protein that occurs naturally as a homodimer In conclusion, the present study investigated the HEV infection status in the general human population and swine groups in central China. The positive rate of anti-HEV IgG and IgM of the studied subjects was 7.9% (117/1476) and 1.6% (24/1476), respectively. HEV RNA was detected in 0.95% (14/1476) of the general human subjects and 7.0% (39/554) of the swine stool samples. Phylogenetic analysis indicated that although all the HEV strains prevalent in central China belonged to genotype 4, the strains prevalent in swine were genetically distinct from those prevalent in the human populations, which suggested that cross-species transmission of HEV between human and swine might not exist in this area.From September in 2007 to September in 2008, a total of 1476 serum specimens were collected from general populations  of rural communities in central China, including Fuyang ( lies on the boundary of Anhui and Henan Province), Huaibei (lies on the boundary of Anhui and Shandong Province ), and Suzhou (lies on the boundary of Anhui and Jiangsu Province). Most of the participants in the present study were farmers who mainly working on crops growing, and agricultural products processing. All participants ≥16 years of age gave written consent after receiving a full explanation of the study, and written consent was also obtained from the parents of the participants <16 years of age. The Ethics Committee of Shanghai Research Center for Biomodel Organism approved the study. A questionnaire was used to record individual's history of ever being admitted to a hospital because of hepatitis E and frequently tripping to other regions. A total of 554 fecal samples were obtained from pigs aged from 4 to 26 weeks old from March to August in 2008. The swine fecal samples were obtained from 11 swine farms in Fuyang, Huaibei, and Suzhou. Fuyang lies about 200 km from Suzhou and Huaibei. Huaibei lies about 100 km from Suzhou. All the swine farms are formalized and have good sanitation status.Anti-HEV IgG and IgM levels were determined using commercial ELISAs . The ELISAs were performed with a recombinant peptide corresponding to aa 394–606 of the major structural protein specified by open reading frame (ORF) 2 of the HEV genome. Testing was performed in accordance with the manufacturer's instructions.5′-CTGTTTAAYCTTGCTGACAC- 3′) and E5 (5′-WGARAGCCAAAGCACATC-3′) for the first round of PCR and primers E2 (5′-GACAGAATTGATTTCGTCG- 3′) and E4 (5′-TGYTGGTTRTCRTAATCCTG-3′) for the second round. PCR cycling conditions for both rounds consisted of 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 53°C, and extension for 40 s at 72°C.Total RNA was extracted from 100 uL of serum sample or feacal suspension, using Trizol . RT-PCR was performed as described elsewhere The complete sequences of ORF2 of the three representative strains of the three major clusters in the phylogenetic analysis were determined using RT-PCR method with the primers designed according to the related sequences available in GenBank.Specific bands for HEV were purified from 1% agarose gel using the QIAquick Gel Extraction kit . The purified PCR products were ligated into pMD18-T Vector . For each product, three positive colonies were selected and sequenced. A consensus sequence of each fragment was saved and used for phylogenetic analysis or assemble of the full ORF2 sequence.www.megasoftware.net/). Prototype HEV strains used as references in the analysis and their GenBank accession numbers and source of origin are marked in the phylogenetic tree.The viral nucleotide or putative amino-acid sequences were aligned using ClustaX1.8 or DNAstar software. Phylogenetic trees were constructed using the neighbor-joining method and evaluated using the interior branch test method with MEGA software  suspensions in PBS, and clarified by centrifugation at 10000 g for 10 min. Then about 30 ul suspension were spotted onto PVDF membrane strips. Blots were blocked by shaking the at 37°C for one hour with TBS-T  containing 5% skim milk powder. Then, blots were washed three times with TBS-T and incubated with each serum diluted 1∶200 in TBS-T (1 h at room temperature with constant shaking). A human serum negative for anti-HEV IgG were included as negative control. After incubation, blots were washed four times with TBS-T, and incubated with a horseradish peroxidaseconjugated goat anti-human IgG  at a 1∶10,000 dilution. After 1 h of incubation at room temperature, blots were washed four times with TBS-T and color development was carried out using a chemiluminescence detection reagent .


Hidradenoma papilliferum is an uncommon, benign, cystic, papillary tumor that occurs almost exclusively in the female anogenital region. Bartholin's abscess is also an anogenital cystic lesion caused by obstruction of Bartholin's duct with an overlying infection. Concomitant presentation of Bartholin's abscess and Hidradenoma papilliferum is unique.A 43-year-old African American woman presented with a painful cystic mass on the left labia majora. A preoperative diagnosis of Bartholin's abscess was made. During excision and draining, an additional tan-brown dermal nodule was removed which demonstrated histological features of Hidradenoma papilliferum.We present what we believe to be the first case of Bartholin's abscess arising in hidradenoma papilliferum and its clinical significance. Hidradenoma papilliferum (HP) is an uncommon, benign, cystic, papillary tumor that occurs primarily in the vulva of Caucasian women, thought to arise from anogenital glands, and exhibits both eccrine and apocrine differentiation, with the latter being more common . BartholA 43-year-old African American woman presented with a painful cystic mass on the left labia majora. Preoperatively, the lesion was diagnosed as Bartholin's abscess. During the excision and drainage, an additional 2.0 × 0.8 × 0.8 cm tan-brown dermal nodule was identified and submitted for histological analysis. Microscopic examination revealed multiple pieces of fibro-necrotic tissues Fig.  associatPrevious literature has described instances of pathology occurring concurrently with Bartholin's abscess or cyst. Literature describes a case of cellular angiofibroma for which the preoperative diagnosis was Bartholin's glandular cyst . AnotherBartholin's glands are bilaterally located at the base of the labia minora and drain through 2- to 2.5-cm-long ducts that empty into the vestibule at the 4 o'clock and 8 o'clock positions . HidradeDue to hidradenoma papilliferum's mixed eccrine and apocrine features, cyst or abscess formation secondary to the disruption of ductal drainage should be considered . HidradeDiagnostically, hidradenoma papilliferum tends to occur exclusively in post-pubescent white women between the ages of 30 and 70, occurring most commonly in the fourth decade ,9. InterThough it has been suggested surgical excision of Bartholin's abscess is unnecessary due to the low risk of Bartholin's gland cancer , our finWhile hidradenoma papilliferum is a benign tumor, malignant transformations to adenocarcinoma or adenosquamous carcinoma have been reported, although rare -16. TherWe concluded the diagnosed Bartholin's abscess was most likely caused by obstruction of the duct of Bartholin's gland by hidradenoma papilliferum. A diagnosis of Bartholin's abscess should not signal a cessation in further investigation for other pathologies. The clinical presentation of hidradenoma papilliferum itself becomes increasingly indistinct when simultaneously occurring with other cystic lesions of the vulva. As noted previously, though rare, hidradenoma papilliferum does have a potential for malignant conversion. Thus, one should remain diligent in finding a cause of a Bartholin's abscess.Written informed consent could not be obtained. The patient was brought into the emergency room and treated as needed. However, consent was not obtained as the patient left the hospital quickly following treatment. The patient was lost to follow-up as she cannot be contacted due to her unique legal situation. Furthermore, no family contact information was made available. We, the authors, do not believe the patient would object to the writing of our report or the information provided. Lastly, the patient's confidentiality has been protected as no identifiers are given in the report except for age and gender.The authors declare that they have no competing interests.DE was involved in the intellectual analysis of the data and contributed to the writing of the manuscript. WS was involved in the intellectual analysis, carrying out of experimental work on the project, and editing. SD was involved in the intellectual analysis and the major contributor to the writing of this case report. All authors read and approved the final manuscript.


Speech language pathology is an effective management intervention for chronic cough that persists despite medical treatment. The mechanism behind the improvement has not been determined but may include active cough suppression, reduced cough sensitivity or increased cough threshold from reduced laryngeal irritation. Objective measures such as cough reflex sensitivity and cough frequency could be used to determine whether the treatment response was due to reduced underlying cough sensitivity or to more deliberate control exerted by individual patients. The number of treatments required to effect a response was also assessed.The aim of this study was to investigate subjective and objective measures of cough before, during and after speech language pathology treatment for refractory chronic cough and the mechanism underlying the improvement.Adults with chronic cough (n = 17) were assessed before, during and after speech language pathology intervention for refractory chronic cough. The primary outcome measures were capsaicin cough reflex sensitivity, automated cough frequency detection and cough-related quality of life.Following treatment there was a significant improvement in cough related quality of life (Median (IQR) at baseline: 13.5 (6.3) vs. post treatment: 16.9 (4.9), p = 0.002), objective cough frequency , and cough reflex sensitivity .This is the first study to show that speech language pathology management is an effective intervention for refractory chronic cough and that the mechanism behind the improvement is due to reduced laryngeal irritation which results in decreased cough sensitivity, decreased urge to cough and an increased cough threshold. Speech language pathology may be a useful and sustained treatment for refractory chronic cough.Australian New Zealand Clinical Trials Register, ACTRN12608000284369. Chronic cough that persists despite medical treatment (termed refractory cough) is a difficult problem frequently associated with increased cough reflex sensitivity -3. ManagThis study sought to investigate capsaicin cough reflex sensitivity and automated cough frequency monitoring in patients with refractory chronic cough undergoing speech language pathology intervention. Cough reflex sensitivity testing and cough frequency monitoring are two objective measures allowing standardized assessment as well as providing an understanding of possible mechanisms of effect. Capsaicin is an extract of hot peppers and is commonly used as a tussive agent in clinical research because it induces cough in a safe, dose-dependent and reproducible manner -10. Our It was hypothesised that speech language pathology intervention for chronic cough would result in decreased cough reflex sensitivity, reduced cough frequency, improvement in clinical outcome and improvement in cough and laryngeal subjective measures. We also sought to determine how many treatment sessions a patient required to show an improvement and if these benefits were maintained post intervention.A previous pilot study compared 2 behavioral approaches (isolated cough suppression techniques and supportive counselling) for refractory chronic cough (CC) to a CC control group and showed that there was no change in cough reflex sensitivity (CRS) measured as C5 after 1 hour of intervention. These were used to establish the current study in the following ways;1) C5 does not respond to isolated behavioural approaches,2) C5 does not change after 1 × 1 hour session of an isolated behavioural approach, and, 3) CRS testing measured as C5 is a highly reproducible test."For detailed description of the participants, procedures, and analysis, see additional file Participant details and results."Adult non-smokers n = 17) with chronic persistent cough that was refractory to medical assessment and treatment ,12 and w7 with chnormal to severe. A clinical research officer then administered several questionnaires, [Participants attended for a maximum of 6 visits  over a period of 14 to18 weeks. At visit 1, there was a voice assessment by a qualified speech language pathologist. This involved a clinical case history, symptom frequency and severity rating , auditornnaires, -20 and cnnaires,  and cougnnaires,  during tThe speech pathology programme for chronic cough has been described previously  and consCapsaicin CRS was performed as previously reported with the addition of a participant urge-to-cough score  where thThe LCM is a digital ambulatory cough monitor and external free-field microphone . This waAll analyses were performed using statistical and data analysis software STATA . Comparisons of log cough sensitivity (measured as C5 and cough threshold) between baseline and each visit was undertaken using a generalised linear mixed model (GLMM) with a random intercept term which takes into account the repeated observations on individuals. Standard errors were estimated using bootstrapping  and resuFigures were produced using GraphPad Prism 4 .Seventeen participants  with a chronic persistent cough participated in the study. The participants had a median (IQR) cough duration of 60 147) months and age of 61 (20) years with normal spirometry [Table 47 monthsParticipants responded to the treatment with a significant improvement in cough-related quality of life , laryngeal dysfunction symptom questionnaire score , cough score, p= 0.04 and total symptoms score, p = 0.002 Table . There wThere was also a significant decrease in cough frequency with the speech language pathology treatment for chronic persistent cough. The cough count at baseline was reduced after treatment: Mean ± SD cough frequency, 72.5 ± 55.8 vs. 25 ± 27.9 coughs/hr, p = 0.009 [Individual cough frequency data (baseline v post treatment) represented in Figure Cough threshold at baseline was Mean ± SD log CT 0.47 ± 0.38 and was significantly altered during treatment: treatment visit 1, cough threshold (T1) log CT 0.72 ± 0.60, p = 0.024, treatment visit 2 (T2) log CT 0.80 ± 0.60, p = 0.025, treatment visit 3 (T3) log CT 0.69 ± 0.23, p = 0.002, until maximum effect had been achieved with no significant change at treatment visit 4 (T4) log CT 0.66 ± 0.65, p = 0.122. After completion of therapy, cough threshold improved significantly: log CT 1.14 ± 0.76, p = 0.001 [Individual cough threshold data (baseline v post treatment) represented in Figure There was a significant decrease in urge-to-cough with the speech language pathology treatment for chronic persistent cough. The urge-to-cough at baseline was reduced after treatment: Median (IQR), 5 (1) vs. 1 (4), p = 0.01 [Individual urge to cough data (baseline v post treatment) represented in Figure This is the first study to objectively assess response to a speech language pathology programme for refractory chronic cough using measures of cough sensitivity and cough frequency. We have shown that patients with refractory chronic cough have significantly decreased cough sensitivity and cough frequency together with an improvement in clinical outcome and cough and laryngeal symptoms following the speech language pathology intervention. Participants had an early symptom response to the speech language pathology program that was further improved upon throughout subsequent treatment sessions. Generally, a patient needed 3 to 4 treatment sessions and the response was maintained after the intervention ceased. it was found that isolated components such as specific cough suppression techniques or counselling were not enough for a patient to achieve a clinical response. In a previous study [The speech language pathology program for refractory CC includes several components, and from a previously conducted pilot study us study , we showus study  on coughWithin the large population of patients with CC, there is a small subgroup that does not respond to usual medical treatment ,28. In tThe study does suggest that the effective speech language pathology programme components reduce cough reflex sensitivity. This effect could occur by improvement in vocal hygiene leading to reduce sensory nerve stimulation, and is supported by the improvements in C5 and urge to cough during the programme. It is also possible that the reduction in cough frequency subsequently reduces cough-related airway trauma, and this explains the delayed improvement in cough threshold.In this study we used an open design with objective measures to assess outcome. Although a nonrandomized design is a limitation, our primary purpose was to treat refractory cough patients and determine their response to a therapy outside normal chronic cough treatment. We achieved this aim by using objective measures and presenting novel data showing that cough frequency and cough reflex hypersensitivity significantly improve after speech language pathology treatment. It is possible that a placebo effect such as cough suppression -33 may hWe did not find a heightened cough reflex sensitivity in CC females compared to CC males (power 90%) and this was consistent with our previous research ,27 (for In conclusion, this is the first study to show that speech language pathology management is an effective intervention for refractory chronic cough and that the mechanism behind the improvement is due to reduced laryngeal irritation which results in decreased cough sensitivity, decreased urge to cough and an increased cough threshold. This is accompanied with an improvement in cough symptoms, associated laryngeal symptoms, and cough quality of life. Speech language pathology may be a useful therapy for refractory chronic cough.CC: Chronic Cough; CRS: Cough Reflex Sensitivity; C5: concentration of capsaicin required to elicit 5 or more coughs within 30 secs after dose administration; LCM: Leicester Cough Monitor; LCQ: Leicester Cough Questionnaire; GLMM: Generalized linear mixed model; LDQ: Laryngeal Dysfunction Questionnaire; PVFM: Paradoxical Vocal Fold Movement; IQR: InterQuartile Range; T1-T4: Treatment No.; GORD: Gastro Oesophageal Reflux Disease; Snot-20: 20-item sino-nasal outcome test; HADS: Hospital Anxiety and Depression Scale.The authors declare that they have no competing interests.NR, AV and PG planned the study. AV, SB recruited the subjects, NR performed the objective cough tests. NR, AV and SB performed questionnaires, collected data and calculated scores. NR analysed the data. AV, SB performed speech pathology treatment. AV participated in the manuscript drafting. PG participated in the data interpretation, manuscript drafting and coordination of the manuscript. All authors read and approved the final manuscript.Participant details, supplemental methods and results.Click here for file


Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections.Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B  For example, the malto-oligosaccharide/maltodextrin–binding component of the Group A streptococcus malto-oligosaccharide ABC transporter has been shown to be directly involved in virulence in a mouse model of oropharynx infection et al. demonstrated that in human saliva the transcript levels of several GAS carbohydrate utilization proteins other than glucose are highly expressed S. pneumoniae (SPN) highlighted that a number of α-glucan–active enzymes seems to be virulence factors in a mouse model of lung infection The use of carbon sources is essential to the ability of bacteria to colonize the host and potentially cause disease in humans. In particular, highly polymerized α-glucan polysaccharides, such as starch and glycogen, are most likely to be found in environmental niches. Indeed, it is known that dietary-derived starches are very abundant in the human colon Streptococcus pneumoniae (SPN) pullulanases, named PulA and SpuA respectively, have been recently described Thermotoga maritime and it shares a high specificity for α-glucans. Of interest, PulA has been described to have multifunctional activities as the capability to hydrolyze pullulan, a linear polysaccharide of maltotriosyl repeating units linked by α- glycosidic linkage S. pyogenes gene expression Because of the complex structures of highly polymerized α-glucans, bacteria require an appropriate combination of enzymes for de-polymerization to oligo- and monosaccharides. Among these enzymes are ascribed pullulanases. Pullulanases have a glycosidic hydrolase activity towards α-glucan polysaccharides and are considered key extracellular components in bacterial metabolism. GAS and streptococcus (GBS) is an extracellular mucosal pathogen causing neonatal meningitis and invasive diseases in non-pregnant adults. GBS colonizes the lower gastrointestinal and genital tracts of healthy adults, as approximately 20–30% of healthy women are colonized rectovaginally with GBS Group B streptococcus (GAS) infections In silico analysis of recently sequenced GBS genomes Streptococcus agalactiae pullulanase (SAP). The sap gene is present in all sequenced GBS strains and the protein is highly conserved, with an amino acid identity ranges between 98 and 100%. SAP repeated CBMs bore ∼20% amino acid sequence identity to family 41 CBM found in a secreted Termotoga maritime pullulanase. CBM41 binds tightly to α-glucan polysaccharide comprising primarily α- glycosidic linkages Streptococcus pneumoniae (SPN)  pullulanases respectively  expression vector. As shown in E. coli. This is in agreement with previous data reported in the literature et al., unpublished observations) indicating the same protein pattern for recombinant PulA. On the basis of N-terminal sequencing analysis of the low MW form of SAP, which revealed the MKVQPNDYVF motif, we predicted a second putative GTG translational start codon within the COH1 sap ORF at position +1036 and a possible Shine-Dalgarno region (5′-AGGAGA-3′) 4 bp upstream of this point. The resulting translation product obtained from this start site yield a smaller protein lacking both CBMs. A mixture of the high and low molecular weight forms of SAP (H+L) was purified by affinity chromatography. Moreover, by anionic exchange chromatography, we were also able to separate SAP (L) from SAP (H+L). Both SAP recombinant preparations were used to demonstrate that a pullulanase enzymatic activity was associated to the protein.The The capacity of recombinant SAP to catalyze the degradation of α-glucan polysaccharides was tested by 3,5-dinitrosalicylic (DNS) acid assay . As shown in Of interest, comparison of SAP(H+L) versus SAP(L) preparations, showed that they were both active on pullulan, starch and amylopectin, while only SAP(H+L) was able to degrade glycogen. This finding suggests that the CBM contributes to the specific interaction with glycogen, in agreement with previous reports In addition, we compared the enzymatic activity of SAP versus GAS pullulanase (PulA) using different carbohydrates as substrates. As shown in 1H-1H 2D NMR scalar chemical shift correlation spectroscopy, which gave results in agreement with the assignments reported in the literature 1H NMR spectrum of pullulan after the addition of SAP(H+L)  Maltotriose+H1β(C) Maltotriose) and H1α1-4 (A+B) Maltotriose is 1∶2, we can conclude that the SAP cleaves α- glycosidic linkages between the units A and C generating maltotriose units In order to confirm the classification of SAP as a type I pullulanase we evaluated the modifications of the structure of pullulan after incubation with SAP by NMR spectroscopy. SAP(H+L)  containsGlycogen molecular size distribution before and after the addition of SAP(H+L) was instead determined by size exclusion chromatography. As reported in sap deletion mutant. As expected, the mutant strain was negative for SAP expression as confirmed by RT-PCR . Since bacterial pullulanases are known to be regulated by specific carbon sources y RT-PCR , WB analy RT-PCR  and FACSy RT-PCR .sap strain for the ability to grow in CM supplemented with different carbohydrates. As expected no growth differences were observed among these strains when glucose or maltose, that are not pullulanase substrates, were added to the CM  or anti-SAP antibodies purified from human sera was unsuccessful (data not shown). We hypothesize that the quantity and quality of anti-SAP antibodies derived from adult healthy volunteers that have been in contact with GBS, is not sufficient for our in vitro assay. Unfortunately, no sera from convalescent patients or with GBS invasive disease are at the moment available in our laboratory.In order to test whether the immunoglobulin-mediated response towards SAP impaired bacterial metabolic activity versus α-glucans, we tested by DNS acid assay the capacity of mouse and rabbit anti-SAP sera to prevent GBS pullulanase activity. We performed dose-dependent experiments incubating SAP-expressing bacteria with different sera dilutions in a range between 0.1–2%. As shown in Since SAP CBM appears to be a very immunogenic domain  and thatin vivo during the establishment of vaginal colonization and confirms the specificity of CBMs for glycogen in silico analysis of GBS available genomes revealed that SAP is the only surface associated protein containing glycosidic domains  and since SAP appears to be the only enzyme expressed by GBS capable to catabolize α-glucans, we suggest that this enzyme may be vital for GBS permanence in environmental niches poor in glucose. We are currently testing this hypothesis, and preliminary data have shown that deletion of the sap gene reduces the capacity of GBS to colonize the vagina in a mouse model of infection .The increasing need of new vaccine-based preventive strategies replacing the antibiotic prophylaxis used for eradicating GBS colonization of the genital tract of pregnant women, has recently led to identification of antigens conferring a broad protection in mice in vivo during GBS infections. However, to our knowledge the functionality of streptococcal pullulanase-induced antibody-mediated immune response has not been yet addressed. The fact that incubation of bacteria with an anti-SAP serum reduces GBS pullulanase activity, led us to hypothesize that in vivo antibody-mediated immune response towards SAP might affect GBS adaptive metabolism resulting in a decreased capacity to survive in the host.The importance of α-glucans metabolizing enzymes in streptococcal adaptation to the host is further highlighted by recent reports revealing they are immunogenic. Indeed, 81% of convalescent-phase sera from patients with invasive GAS infections had antibodies reactive with PulA Emerging theories on human-microbe mutualism suggest that the mechanisms that underlie microbial community structure and host–symbiont relationships should be considered for planning prevention strategies for human health. Indeed, as recently proposed by David A. Relman In conclusion, the evidence reported in this paper may draw up the basis for preventing streptococcal infections by using immunogenic metabolic enzymes as target molecules for vaccine development. The fact that, at least for pathogenic streptococci, such enzymes are well conserved opens new perspectives in the development of strategies preventing infections from multiple species.sap gene from 2603 V/R (TIGR Accession SAG_1216), 515 Ia (SAL_1339), NEM316 (gbs1288), H36B (SAI_1308), CJB111 (SAM_1238), A909 (SAK_1302) and COH1 (SAN_1346) strain as well as SpuA (SP_0268) and PulA (Spy_1972) was performed using ClustalW The alignment of SAP protein encoded by S. agalactiae strains COH1 serotype III was used in this study. Escherichia coli DH5α and DH10BT1 were used for cloning purposes and E. coli BL21 (λDE3) for expression of SAP fusion protein. S. agalactiae was cultivated at 37°C in Todd-Hewitt broth (THB) up to desired OD600. E. coli was grown in Luria-Bertani broth (LB); E. coli clones carrying the plasmids pJRS233 or pET21(b)+ and derivates were grown in the presence erythromycin (400 µg/mL) or ampicillin (100 µg/mL), respectively. The complex medium  was used for GBS growth with defined carbon sources. The sugar concentrations were 1% final. To evaluate growth in CM, GBS was initially grown to log phase (OD600 0.3) in THB. The cells were harvested by centrifugation, washed twice in an equivalent volume of phosphate-buffered saline (PBS) and diluted 1 to 50 in CM. Growth was monitored spectrophotometrically at a wavelength of 600 nm.sap gene from S. agalactiae COH1 serotype III was used as a template. The construct was amplified by PCR using specific primers GBS5F and GBS5R introducing NdeI and XhoI restriction enzyme sites  cells. BL21 (DE3) cells were grown in LB-Amp (100 µg/ml ampicillin) and induced with IPTG at a final concentration of 1 mM for 3 hours. The resulting biomass was suspended in 0.3 M NaCl, 50 mM Na-PO4 buffer, pH 8.0 and cells were lysed by enzymatic digestion. The sample was then loaded onto a His-Trap Ni-Activated Chelating Sepharose FF column  at a flow rate of 5 ml/min. Bound proteins were then eluted from the column by running a gradient from 0 to 50% of 500 mM Imidazole, 0.3 M NaCl, 50 mM Na phosphate buffer, pH 8.0 in 12 CV. The IMAC eluted material was collected in 2.5-ml fractions and those ones containing the SAP-His protein pooled. An anionic exchange chromatography was used to separate the two forms of SAP. The pooled fractions from Ni-IMAC were dialyzed against 30mM TRIS, pH 8.0 and then loaded on to a HiTrap Q HP 5 ml column (GE) to further purify the two forms of recombinant SAP. The purification was achieved by running a gradient from 0 to 50% 1M NaCl in 30mM TRIS, pH 8.0 in 16 CV at 5 ml/min. The collected fractions were analyzed by SDS-PAGE  and pooled according to apparent MW. The final preparation of the protein was obtained in PBS, pH 7.4 after dialysis.The PCR products were cloned into the pET21(b)+ vector and the plasmid transformed in sap gene was deleted in GBS strain COH1, according to the procedure previously described XhoI restriction enzyme cleavage sites were incorporated at the 5′-end of the primer to clone the fragment into the XhoI-digested pJRS233 plasmid. After cloning the in frame deletion fragment in pJRS233, the plasmid pJRS233Δsap was obtained.The sap was then transformed into the COH1 strain by electroporation and transformants were selected after growth at 30°C on agar plates containing 1 µg/ml erythromycin. Transformants were then grown at 37°C with erythromycin selection as previously described sap, resulting in the sap deletion on the chromosome. Dilutions of the serially passaged cultures were plated onto agar plates, and single colonies were tested for erythromycin sensitivity to confirm the excision of pJRS233Δsap. The resulting strain was named COH1Δsap.The plasmid pJRS233Δ600 0.4 in CM plus sugars, washed in PBS and incubated for 1 h at 37°C in 500 µl of Tris-HCl 50 mM (pH6.8) containing protease inhibitors and 400 U/ml of mutanolysin . The bacterial suspension was then pelleted and the supernatants containing peptidoglycan associated proteins used for western blotting analysis of SAP. In order to prepare GBS extracts relative to the secreted protein fraction, supernatant of bacteria cultures grown to OD600 0.4 were collected. Proteins in 1 ml of supernatant were precipitated with 10% of trichloroaceticacid (TCA) for 1 hr at 4°C. Protein were then pelletted, washed with cold acetone and resuspended in Tris-HCl pH 6,8.GBS protein extracts were prepared by growing bacteria up to OD600 0.4. Total GBS RNA was isolated using the Rneasy mini kit (Qiagen) according to manufacturer's instructions, except that bacteria were lysed with 100 µl of lysozyme (30 mg/ml) in Tris-EDTA buffer and 2,000 U of mutanolysin, and the mixture was incubated for 15 min at 37°C. Quantification of the transcripts was completed by reverse transcription and semi-quantitative RT-PCR using ImPromII RT (Promega) following manufacturer's instructions. Briefly 2 µg of sample and 0.5 µg of random hexamers were added to a final volume of 5 µl. Samples were incubated in a thermocycler (Biometra) at 70°C for 5 min followed by a quick chill at 4°C. The mixture was used in a 20-µl  cDNA synthesis reaction mixture comprising 4 µl of Improm-II 5× reaction buffer (Promega), 2.4 µl MgCl2 at 25 mM, 2 µl of dNTP mix (each dNTP at 2.5 mM), 0.25 µl of Rnasin RNase inhibitor (Promega) and 1 µl of Improm-II reverse transcriptase. The reaction was performed at 42°C for 60 min. In the negative controls, the reverse transcriptase was substituted with water. 2 µl of cDNA were then added to the PCR reaction consisting of 1× reaction buffer, 200 µM dNTP's, 0.2 µM primer pair, 1 U PlatinumTaq dna polymerase (Invitrogen). GBS Gyrase A (GyrA) was used as an internal housekeeping control. PCR reactions consisted of a 7-min denaturation step 94°C, followed by a variable number of cycles. PCR products were electrophoresed through 2% agarose gels and images were acquired by laser densitometry (Gel-Doc Imaging System).COH1 was grown in CM medium plus sugars up to OD2O, 99.9% atom D – Aldrich was used) to a uniform concentration. Samples were therefore transferred to 5-mm NMR tubes (Wilmad Glass. Co.). 70 µL of SAP were therefore added to the pullulan and glycogen samples in the NMR tubes. For every sample, two NMR spectra were recorded, the first on the native polysaccharide and the second 1 hour later (incubation at 25°C) after the addition of enzyme. Samples of maltose (Sigma) and maltotriose were also prepared by dissolving 10 mg of powder in 0.7 mL of deuterated PBS buffer at pH 7.2. 1H NMR experiments were recorded at 25°C on Bruker Avance 600 MHz spectrometer and using 5-mm probe (Bruker). For data acquisition and processing XWINNMR software package (Bruker) was used. 1-D proton NMR spectra were recorded using a standard one-pulse experiment. 64 scans were collected and averaged, giving a total acquisition time of ca. 10 min. The transmitter was set at the HDO frequency, collecting 32 k data points over a spectral window of 6,000 Hz. 1H NMR spectra were obtained in quantitative matter using a total recycle time to ensure a full recovery of each signal . Spectra were Fourier Transformed to 32 k data points after applying a 0.2 Hz line broadening function and referenced relative to the HDO resonance at 4.79 ppm.Samples of pullulan (Sigma) and glycogen (Sigma) were prepared by dissolving polysaccaride powder (10 mg) in 0.7 mL of deuterated PBS buffer at pH 7.2 (DSamples of glycogen were prepared by dissolving polysaccaride powder (1 mg) in 0.1 mL of PBS at pH 7.2 to a uniform concentration. Samples were therefore transferred to 1 mL vials (Waters). 10 µL of SAP were therefore added to the glycogen sample. Two chromatograms were recorded, the first on the native polysaccharide and the second 1 hour later (incubation at 25°C) after the addition of enzyme.5 Da was used. Samples were loaded onto the gel filtration column and eluted isocratically in 100 mM sodium phosphate + 100 mM NaCl buffer pH 7.2 at a flow rate of 0.5 ml min−1 for 50 min. The elution was monitored with a Ultimate 3000 Photodiode Array detector (Dionex) coupled with the Ultimate 3000 HPLC system (Dionex). For data acquisition and processing Chromeleon software package (Dionex) was used.A TSK G4000PW (TosoHaas) gel filtration analytical column (7.5 mm×30.0 cm) with a fractionation range of Mw PEG/PEO 2,000–3×10600 0.4 in CM with 1% sugar, fixed with 1% PFA for 20 min at RT and incubated with mouse anti-SAP serum or mouse anti-PBS serum (negative control) in 0.1% BSA plus 20% of Normal Calf Serum (NCS) for 1 h at 4°C. Bacteria were then washed in PBS containing 0.1% BSA and incubated with the phicoerytrin (PE) conjugated secondary antibodies  for 45 min at 4°C. After washing bacteria were resuspended in 200 µl of PBS and analyzed by a FACSscan flow cytometer (Becton Dickinson) by using FlowJo software program.In order to quantify the exposure of SAP on the bacterial surface, GBS was grown up to OD600 0.3  in CM plus 1% sugars. Bacteria were then centrifuged for 10 min at 3000 rpm (RT), washed and resuspended in 1 ml of PBS. Formvar-carbon-coated nickel grids were floated on drops of GBS suspensions for 5 min. The grids were then fixed in 2% PFA for 5 min, and placed in blocking solution  for 30 min. The grids were then floated on drops of primary antiserum against the SAP protein diluted 1∶20 in blocking solution for 30 min at RT, washed with six drops of blocking solution, and floated on secondary antibody conjugated to 10-nm gold particles diluted 1∶20 in 1% BSA for 30 min. The grids were examined using a TEM GEOL 1200EX II transmission electron microscope.GBS strains COH1 were grown at 37°C up to OD600 0.4 and washed in PBS. Bacterial pellet were fixed in 2% PFA for 20 min at RT and spotted on POLYSINE™ slides (Menzel-Glaser). The slides were then blocked with 3% BSA for 1 h and incubated with a mix of rabbit anti-serotype III capsule and mouse anti-SAP antibodies diluted in 1% BSA for 1 h at RT. Bacteria were then stained with goat anti-mouse and anti-rabbit Alexa Fluor conjugated antibodies  (Molecular Probes) for 20 min at RT. Slow Fade reagent kit (Molecular Probes) was then used to mount cover slips. The slides were analysed with a Bio-Rad confocal scanning microscope.In order to visualize SAP on bacterial surface, COH1 was grown in CM plus sugars up to ODPullulanase activity was determined by measuring the enzymatic release of reducing groups from α-glucans by the DNS colorimetric method 600 0.6) in THB and THY, respectively. Bacteria were then re-inoculated in CM containing 1% pullulan and grown to log phase (OD600 0.4) to allow the expression of pullulanases on bacterial surface. The cells were then harvested by centrifugation, washed twice with PBS and resuspended in PBS. Bacteria (∼5×108 CFU) were pre-incubated with sera dilutions at 37°C for 15 min, then pullulan was added  and the incubation prolonged for other 2 hours. Samples were centrifuged and supernatants were used for the determination of reducing sugars by DNS acid assay.GBS and GAS were grown at 37°C up to mid-late exponential phase (OD