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The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30-dependent changes in lymphocyte effector function. Thus, we conclude that receptor–ligand signaling interactions can be selectively regulated by an extracellular redox catalyst. | [
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] |
BACKGROUND: The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15. RESULTS: We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome. CONCLUSIONS: By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice. | [
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] |
The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor™) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine. | [
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To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. | [
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] |
Influenza A viruses are hosted by numerous avian and mammalian species, which have shaped their evolution into distinct lineages worldwide. The viral genome consists of eight RNA segments that are frequently exchanged between different viruses via a process known as genetic reassortment. A complete genotype nomenclature is essential to describe gene segment reassortment. Specialized bioinformatic tools to analyze reassortment are not available, which hampers progress in understanding its role in host range, virulence and transmissibility of influenza viruses. To meet this need, we have developed a nomenclature to name influenza A genotypes and implemented a web server, FluGenome (http://www.flugenome.org/), for the assignment of lineages and genotypes. FluGenome provides functions for the user to interrogate the database in different modalities and get detailed reports on lineages and genotypes. These features make FluGenome unique in its ability to automatically detect genotype differences attributable to reassortment events in influenza A virus evolution. | [
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BACKGROUND: Influenza pandemic preparedness plans are currently developed and refined on national and international levels. Much attention has been given to the administration of antiviral drugs, but contact reduction can also be an effective part of mitigation strategies and has the advantage to be not limited per se. The effectiveness of these interventions depends on various factors which must be explored by sensitivity analyses, based on mathematical models. METHODS: We use the freely available planning tool InfluSim to investigate how pharmaceutical and non-pharmaceutical interventions can mitigate an influenza pandemic. In particular, we examine how intervention schedules, restricted stockpiles and contact reduction (social distancing measures and isolation of cases) determine the course of a pandemic wave and the success of interventions. RESULTS: A timely application of antiviral drugs combined with a quick implementation of contact reduction measures is required to substantially protract the peak of the epidemic and reduce its height. Delays in the initiation of antiviral treatment (e.g. because of parsimonious use of a limited stockpile) result in much more pessimistic outcomes and can even lead to the paradoxical effect that the stockpile is depleted earlier compared to early distribution of antiviral drugs. CONCLUSION: Pharmaceutical and non-pharmaceutical measures should not be used exclusively. The protraction of the pandemic wave is essential to win time while waiting for vaccine development and production. However, it is the height of the peak of an epidemic which can easily overtax general practitioners, hospitals or even whole public health systems, causing bottlenecks in basic and emergency medical care. | [
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Over the past 15 years, DNA vaccines have gone from a scientific curiosity to one of the most dynamic research field and may offer new alternatives for the control of parasitic diseases such as leishmaniasis and Chagas disease. We review here some of the advances and challenges for the development of DNA vaccines against these diseases. Many studies have validated the concept of using DNA vaccines for both protection and therapy against these protozoan parasites in a variety of mouse models. The challenge now is to translate what has been achieved in these models into veterinary or human vaccines of comparable efficacy. Also, genome-mining and new antigen discovery strategies may provide new tools for a more rational search of novel vaccine candidates. | [
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Reproduction numbers, defined as averages of the number of people infected by a typical case, play a central role in tracking infectious disease outbreaks. The aim of this paper is to develop methods for estimating reproduction numbers which are simple enough that they could be applied with limited data or in real time during an outbreak. I present a new estimator for the individual reproduction number, which describes the state of the epidemic at a point in time rather than tracking individuals over time, and discuss some potential benefits. Then, to capture more of the detail that micro-simulations have shown is important in outbreak dynamics, I analyse a model of transmission within and between households, and develop a method to estimate the household reproduction number, defined as the number of households infected by each infected household. This method is validated by numerical simulations of the spread of influenza and measles using historical data, and estimates are obtained for would-be emerging epidemics of these viruses. I argue that the household reproduction number is useful in assessing the impact of measures that target the household for isolation, quarantine, vaccination or prophylactic treatment, and measures such as social distancing and school or workplace closures which limit between-household transmission, all of which play a key role in current thinking on future infectious disease mitigation. | [
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Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-α/β production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-α administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the promotion of adaptive immunity. | [
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We conducted a study in the summer of 2004 at county fairs in the Midwest to investigate the role poultry exhibits have in spreading avian pathogens to humans. A nearly invisible powder (pathogen surrogate) that fluoresces under UV light was surreptitiously sprinkled each day on 1 show bird at each of 2 fairs. A UV light box was used to daily examine the hands of 94 poultry-exhibit participants (blinded regarding UV box results) for up to 4 days during the poultry shows. Enrollment and end-of-study questionnaires collected data on pathogen risk factors. Eight (8.5%) of 94 participants had evidence of fluorescent powder contamination (95% confidence interval 2.76%–14.26%). This contamination and infrequent handwashing practices suggest that county fairs are a possible venue for animal-to-human pathogen transmission. | [
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The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections. | [
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BACKGROUND: In 2005 huge epizooty of H5N1 HPAI occurred in Russia. It had been clear that territory of Russia becoming endemic for H5N1 HPAI. In 2006 several outbreaks have occurred. To develop new vaccines and antiviral therapies, animal models had to be investigated. We choose highly pathogenic strain for these studies. RESULTS: A/duck/Tuva/01/06 belongs to Quinghai-like group viruses. Molecular markers – cleavage site, K627 in PB2 characterize this virus as highly pathogenic. This data was confirmed by direct pathogenic tests: IVPI = 3.0, MLD(50 )= 1,4Log10EID(50). Also molecular analysis showed sensivity of the virus to adamantanes and neuraminidase inhibitors. Serological analysis showed wide cross-reactivity of this virus with sera produced to H5N1 HPAI viruses isolated earlier in South-East Asia. Mean time to death of infected animals was 8,19+/-0,18 days. First time acute delayed hemorrhagic syndrome was observed in mice lethal model. Hypercytokinemia was determined by elevated sera levels of IFN-gamma, IL-6, IL-10. CONCLUSION: Assuming all obtained data we can conclude that basic model parameters were characterized and virus A/duck/Tuva/01/06 can be used to evaluate anti-influenza vaccines and therapeutics. | [
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Predicting RNA secondary structure is often the first step to determining the structure of RNA. Prediction approaches have historically avoided searching for pseudoknots because of the extreme combinatorial and time complexity of the problem. Yet neglecting pseudoknots limits the utility of such approaches. Here, an algorithm utilizing structure mapping and thermodynamics is introduced for RNA pseudoknot prediction that finds the minimum free energy and identifies information about the flexibility of the RNA. The heuristic approach takes advantage of the 5′ to 3′ folding direction of many biological RNA molecules and is consistent with the hierarchical folding hypothesis and the contact order model. Mapping methods are used to build and analyze the folded structure for pseudoknots and to add important 3D structural considerations. The program can predict some well known pseudoknot structures correctly. The results of this study suggest that many functional RNA sequences are optimized for proper folding. They also suggest directions we can proceed in the future to achieve even better results. | [
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] |
Arginine-rich cell-penetrating peptides (CPPs) are promising transporters for intracellular delivery of antisense morpholino oligomers (PMO). Here, we determined the effect of L-arginine, D-arginine and non-α amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24 CPP−PMOs. Insertion of 6-aminohexanoic acid (X) or β-alanine (B) residues into oligoarginine R(8) decreased the cellular uptake but increased the splice-correction activity of the resulting compound, with a greater increase for the sequences containing more X residues. Cellular toxicity was not observed for any of the conjugates up to 10 μM. Up to 60 μM, only the conjugates with ⩾ 5 Xs exhibited time- and concentration-dependent toxicity. Substitution of L-arginine with D-arginine did not increase uptake or splice-correction activity. High concentration of serum significantly decreased the uptake and splice-correction activity of oligoarginine conjugates, but had much less effect on the conjugates containing X or B. In summary, incorporation of X/B into oligoarginine enhanced the antisense activity and serum-binding profile of CPP−PMO. Toxicity of X/B-containing conjugates was affected by the number of Xs, treatment time and concentration. More active, stable and less toxic CPPs can be designed by optimizing the position and number of R, D-R, X and B residues. | [
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We've always wanted to save the world from the scourges of virus infection by developing better drugs and vaccines. But fully understanding the intricacies of virus-host interactions, the first step in achieving this goal, requires the ability to view the process on a grand scale. The advent of high-throughput technologies, such as DNA microarrays and mass spectrometry, provided the first opportunities to obtain such a view. Here we describe our efforts to use these tools to focus on the changes in cellular gene expression and protein abundance that occur in response to virus infection. By examining these changes in a comprehensive manner, we have been able to discover exciting new insights into innate immunity, interferon and cytokine signaling, and the strategies used by viruses to overcome these cellular defenses. Functional genomics may yet save the world from killer viruses. | [
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] |
The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens. | [
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] |
Aberrant expression of DNA polymerase β, a key enzyme involved in base excision repair, leads to genetic instability and carcinogenesis. Pol β expression has been previously shown to be regulated at the level of transcription, but there is also evidence of post-transcriptional regulation, since rat transcripts undergo alternative polyadenylation, and the resulting 3′UTR contain at least one regulatory element. Data presented here indicate that RNA of the short 3′UTR folds to form a strong secondary structure (hairpin). Its regulatory role was established utilizing a luciferase-based reporter system. Further studies led to the identification of a protein factor, which binds to this element—the anti-apoptotic, cytoskeleton-related protein Hax-1. The results of in vitro binding analysis indicate that the formation of the RNA–protein complex is significantly impaired by disruption of the hairpin motif. We demonstrate that Hax-1 binds to Pol β mRNA exclusively in the form of a dimer. Biochemical analysis revealed the presence of Hax-1 in mitochondria, but also in the nuclear matrix, which, along with its transcript-binding properties, suggests that Hax-1 plays a role in post-transcriptional regulation of expression of Pol β. | [
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The 'global public good' (GPG) concept has gained increasing attention, in health as well as development circles. However, it has suffered in finding currency as a general tool for global resource mobilisation, and is at risk of being attached to almost anything promoting development. This overstretches and devalues the validity and usefulness of the concept. This paper first defines GPGs and describes the policy challenge that they pose. Second, it identifies two key areas, health R&D and communicable disease control, in which the GPG concept is clearly relevant and considers the extent to which it has been applied. We point out that that, while there have been many new initiatives, it is not clear that additional resources from non-traditional sources have been forthcoming. Yet achieving this is, in effect, the entire purpose of applying the GPG concept in global health. Moreover, the proliferation of disease-specific programs associated with GPG reasoning has tended to promote vertical interventions at the expense of more general health sector strengthening. Third, we examine two major global health policy initiatives, the Global Fund against AIDS, Tuberculosis and Malaria (GFATM) and the bundling of long-standing international health goals in the form of Millennium Development Goals (MDG), asking how the GPG perspective has contributed to defining objectives and strategies. We conclude that both initiatives are best interpreted in the context of traditional development assistance and, one-world rhetoric aside, have little to do with the challenge posed by GPGs for health. The paper concludes by considering how the GPG concept can be more effectively used to promote global health. | [
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The development of paediatric intensive care has contributed to the improved survival of critically ill children. Physical and psychological sequelae and consequences for quality of life (QoL) in survivors might be significant, as has been determined in adult intensive care unit (ICU) survivors. Awareness of sequelae due to the original illness and its treatment may result in changes in treatment and support during and after the acute phase. To determine the current knowledge on physical and psychological sequelae and the quality of life in survivors of paediatric intensive care, we undertook a computerised comprehensive search of online databases for studies reporting sequelae in survivors of paediatric intensive care. Studies reporting sequelae in paediatric survivors of cardiothoracic surgery and trauma were excluded, as were studies reporting only mortality. All other studies reporting aspects of physical and psychological sequelae were analysed. Twenty-seven studies consisting of 3,444 survivors met the selection criteria. Distinct physical and psychological sequelae in patients have been determined and seemed to interfere with quality of life. Psychological sequelae in parents seem to be common. Small numbers, methodological limitations and quantitative and qualitative heterogeneity hamper the interpretation of data. We conclude that paediatric intensive care survivors and their parents have physical and psychological sequelae affecting quality of life. Further well-designed prospective studies evaluating sequelae of the original illness and its treatment are warranted. | [
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BACKGROUND: With the development of new technology, it has recently become practical to resequence the genome of a bacterium after experimental manipulation. It is critical though to know the accuracy of the technique used, and to establish confidence that all of the mutations were detected. RESULTS: In order to evaluate the accuracy of genome resequencing using the microarray-based Comparative Genome Sequencing service provided by Nimblegen Systems Inc., we resequenced the E. coli strain W3110 Kohara using MG1655 as a reference, both of which have been completely sequenced using traditional sequencing methods. CGS detected 7 of 8 small sequence differences, one large deletion, and 9 of 12 IS element insertions present in W3110, but did not detect a large chromosomal inversion. In addition, we confirmed that CGS also detected 2 SNPs, one deletion and 7 IS element insertions that are not present in the genome sequence, which we attribute to changes that occurred after the creation of the W3110 lambda clone library. The false positive rate for SNPs was one per 244 Kb of genome sequence. CONCLUSION: CGS is an effective way to detect multiple mutations present in one bacterium relative to another, and while highly cost-effective, is prone to certain errors. Mutations occurring in repeated sequences or in sequences with a high degree of secondary structure may go undetected. It is also critical to follow up on regions of interest in which SNPs were not called because they often indicate deletions or IS element insertions. | [
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The authors discuss humoral immune responses to HIV and approaches to designing vaccines that induce viral neutralizing and other potentially protective antibodies. | [
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BACKGROUND: Despite the seriousness of dengue-related disease, with an estimated 50–100 million cases of dengue fever and 250,000–500,000 cases of dengue hemorrhagic fever/dengue shock syndrome each year, a clear understanding of dengue pathogenesis remains elusive. Because of the lack of a disease model in animals and the complex immune interaction in dengue infection, the study of host response and immunopathogenesis is difficult. The development of genomics technology, microarray and high throughput quantitative PCR have allowed researchers to study gene expression changes on a much broader scale. We therefore used this approach to investigate the host response in dengue virus-infected cell lines and in patients developing dengue fever. METHODOLOGY/PRINCIPAL FINDINGS: Using microarray and high throughput quantitative PCR method to monitor the host response to dengue viral replication in cell line infection models and in dengue patient blood samples, we identified differentially expressed genes along three major pathways; NF-κB initiated immune responses, type I interferon (IFN) and the ubiquitin proteasome pathway. Among the most highly upregulated genes were the chemokines IP-10 and I-TAC, both ligands of the CXCR3 receptor. Increased expression of IP-10 and I-TAC in the peripheral blood of ten patients at the early onset of fever was confirmed by ELISA. A highly upregulated gene in the IFN pathway, viperin, was overexpressed in A549 cells resulting in a significant reduction in viral replication. The upregulation of genes in the ubiquitin-proteasome pathway prompted the testing of proteasome inhibitors MG-132 and ALLN, both of which reduced viral replication. CONCLUSION/SIGNIFICANCE: Unbiased gene expression analysis has identified new host genes associated with dengue infection, which we have validated in functional studies. We showed that some parts of the host response can be used as potential biomarkers for the disease while others can be used to control dengue viral replication, thus representing viable targets for drug therapy. | [
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] |
BACKGROUND: Although the impact of pathogens on the evolution of the mammalian immune system is still under debate, proteins, which both regulate immune responses and serve as cellular receptors for pathogens should be at the forefront of pathogen-driven host evolution. The CEA (carcinoembryonic antigen) gene family codes for such proteins and indeed shows tremendous species-specific variation between human and rodents. Since little is known about the CEA gene family in other lineages of placental mammals, we expected to gain new insights into the evolution of the rapidly diverging CEA family by analyzing the CEA family of the dog. RESULTS: Here we describe the complete CEA gene family in the dog. We found that the gene coding for the ITIM-bearing immunoregulatory molecule CEACAM1 gave rise to a recent expansion of the canine CEA gene family by gene duplication, similar to that previously found in humans and mice. However, while the murine and human CEACAMs (carcinoembryonic antigen-related cell adhesion molecules) are predominantly secreted and GPI-anchored, respectively, in the dog, most of the CEACAMs represent ITAM-bearing transmembrane proteins. One of these proteins, CEACAM28, exhibits nearly complete sequence identity with the ligand-binding N domain of CEACAM1, but antagonizing signaling motifs in the cytoplasmic tail. Comparison of nonsynonymous and synonymous substitutions indicates that the CEACAM28 N domain is under the strongest purifying selection of all canine CEACAM1-related CEACAMs. In addition, CEACAM28 shows a similar expression pattern in resting immune cells and tissues as CEACAM1. However, upon activation CEACAM28 mRNA and CEACAM1 mRNA are differentially regulated. CONCLUSION: Thus, CEACAM1 and CEACAM28 are the first paired immune receptors identified within the CEA gene family, which are expressed on T cells and are most likely involved in the fine-tuning of T cell responses. The direction of gene conversion accompanied by purifying selection and expression in immune cells suggests the possibility that CEACAM28 evolved in response to selective pressure imposed by species-specific pathogens. | [
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Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed. | [
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The leaves of Lagerstroemia speciosa (Lythraceae), a Southeast Asian tree more commonly known as banaba, have been traditionally consumed in various forms by Philippinos for treatment of diabetes and kidney related diseases. In the 1990s, the popularity of this herbal medicine began to attract the attention of scientists worldwide. Since then, researchers have conducted numerous in vitro and in vivo studies that consistently confirmed the antidiabetic activity of banaba. Scientists have identified different components of banaba to be responsible for its activity. Using tumor cells as a cell model, corosolic acid was isolated from the methanol extract of banaba and shown to be an active compound. More recently, a different cell model and the focus on the water soluble fraction of the extract led to the discovery of other compounds. The ellagitannin Lagerstroemin was identified as an effective component of the banaba extract responsible for the activity. In a different approach, using 3T3-L1 adipocytes as a cell model and a glucose uptake assay as the functional screening method, Chen et al. showed that the banaba water extract exhibited an insulin-like glucose transport inducing activity. Coupling HPLC fractionation with a glucose uptake assay, gallotannins were identified in the banaba extract as components responsible for the activity, not corosolic acid. Penta-O-galloyl-glucopyranose (PGG) was identified as the most potent gallotannin. A comparison of published data with results obtained for PGG indicates that PGG has a significantly higher glucose transport stimulatory activity than Lagerstroemin. Chen et al. have also shown that PGG exhibits anti-adipogenic properties in addition to stimulating the glucose uptake in adipocytes. The combination of glucose uptake and anti-adipogenesis activity is not found in the current insulin mimetic drugs and may indicate a great therapeutic potential of PGG. | [
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] |
BACKGROUND: With a heightened increase in concern for an influenza pandemic we sought to better understand the 1918 Influenza pandemic, the most devastating epidemic of the previous century. METHODOLOGY/PRINCIPAL FINDINGS: We use data from several communities in Maryland, USA as well as two ships that experienced well-documented outbreaks of influenza in 1918. Using a likelihood-based method and a nonparametric method, we estimate the serial interval and reproductive number throughout the course of each outbreak. This analysis shows the basic reproductive number to be slightly lower in the Maryland communities (between 1.34 and 3.21) than for the enclosed populations on the ships (R(0) = 4.97, SE = 3.31). Additionally the effective reproductive number declined to sub epidemic levels more quickly on the ships (within around 10 days) than in the communities (within 30–40 days). The mean serial interval for the ships was consistent (3.33, SE = 5.96 and 3.81, SE = 3.69), while the serial intervals in the communities varied substantially (between 2.83, SE = 0.53 and 8.28, SE = 951.95). CONCLUSIONS/SIGNIFICANCE: These results illustrate the importance of considering the population dynamics when making statements about the epidemiological parameters of Influenza. The methods that we employ for estimation of the reproductive numbers and the serial interval can be easily replicated in other populations and with other diseases. | [
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INTRODUCTION: Exogenous surfactant is used to treat acute respiratory failure in children, although the benefits and harms in this setting are not clear. The objective of the present systematic review is to assess the effect of exogenous pulmonary surfactant on all-cause mortality in children mechanically ventilated for acute respiratory failure. METHODS: We searched the MEDLINE, EMBASE, CINAHL and Ovid Healthstar databases, the bibliographies of included trials and review articles, conference proceedings and trial registries. We included prospective, randomized, controlled trials of pulmonary surfactant that enrolled intubated and mechanically ventilated children with acute respiratory failure. We excluded trials that exclusively enrolled neonates or patients with asthma. Two reviewers independently rated trials for inclusion, extracted data and assessed the methodologic quality. We quantitatively pooled the results of trials, where suitable, using a random effects model. RESULTS: Six trials randomizing 314 patients were included. Surfactant use reduced mortality (relative risk = 0.7, 95% confidence interval = 0.4 to 0.97, P = 0.04), was associated with increased ventilator-free days (weighted mean difference = 2.5 days, 95% confidence interval = 0.3 to 4.6 days, P = 0.02) and reduced the duration of ventilation (weighted mean difference = 2.3 days, 95% confidence interval = 0.1 to 4.4 days, P = 0.04). CONCLUSION: Surfactant use decreased mortality, was associated with more ventilator-free days and reduced the duration of ventilation. No serious adverse events were reported. | [
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Influenza A viruses have a wide host range for infection, from wild waterfowl to poultry to humans. Recently, the cross-species transmission of avian influenza A, particularly subtype H5N1, has highlighted the importance of the non-human subtypes and their incidence in the human population has increased over the past decade. During cross-species transmission, human disease can range from the asymptomatic to mild conjunctivitis to fulminant pneumonia and death. With these cases, however, the risk for genetic change and development of a novel virus increases, heightening the need for public health and hospital measures. This review discusses the epidemiology, host range, human disease, outcome, treatment, and prevention of cross-transmission of avian influenza A into humans. | [
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Worst case scenarios for pandemic influenza planning in the US involve over 700,000 patients requiring mechanical ventilation. UK planning predicts a 231% occupancy of current level 3 (intensive care unit) bed capacity. Critical care planners need to recognise that mortality is likely to be high and the risk to healthcare workers significant. Contingency planning should, therefore, be multi-faceted, involving a robust health command structure, the facility to expand critical care provision in terms of space, equipment and staff and cohorting of affected patients in the early stages. It should also be recognised that despite this expansion of critical care, demand will exceed supply and a process for triage needs to be developed that is valid, reproducible, transparent and consistent with distributive justice. We advocate the development and validation of physiological scores for use as a triage tool, coupled with candid public discussion of the process. | [
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BACKGROUND: Avian influenza threatens public health worldwide because it is usually associated with severe illness and, consequently, a higher risk of death. During the first months of 2006, Turkey experienced its first human avian influenza epidemic. A total of 21 human cases were identified, 12 of which were confirmed by the National Institute for Medical Research. Nine of the cases, including the four fatal ones, were from the Dogubeyazit-Van region. This study aims to evaluate the efforts at the avian influenza outbreak control in the Van-Dogubeyazit region in 2006 through the experiences of health personnel. METHODS: We conducted in-depth interviews with seventeen key informants who took active roles during the avian influenza outbreak in East Turkey during the first months of 2006. We gathered information about the initial responses, the progress and management of the outbreak control, and the reactions of the health professionals and the public. The findings of the study are reported according to the topics that appeared through thematic analysis of the interview transcripts. RESULTS: Following the first suspected avian influenza cases, a Van Crisis Coordination Committee was formed as the coordinating and decision-making body and played an important role in the appropriate timing of decisions. The health and agriculture services could not be well coordinated owing to the lack of integrated planning in preparation for outbreak and of integrated surveillance programs. Traditional poultry practice together with the low socio-economic status of the people and the lack of health care access in the region seemed to be a major risk for animal to animal and animal to human transmission. The strengths and weaknesses of the present health system – primary health care services, national surveillance and notification systems, human resource and management – affected the inter organizational coordination during the outbreak. Open communication between the government and the public played an important part in overcoming difficulties. CONCLUSION: Although there were problems during the avian influenza outbreak in Turkey, the rapid responses of the central and regional health authorities and the performance of the health workers were the key points in controlling the epidemic. The lessons from this outbreak should provide an opportunity for integrating the preparation plans of the health and agricultural organizations, and for revising the surveillance system and enhancing the role of the primary health care services in controlling epidemic disease. Developing successful strategies based on knowledge and experience may play a valuable role in delaying an avian influenza pandemic. | [
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BACKGROUND: The European Influenza Surveillance Scheme (EISS) has collected clinical and virological data on influenza since 1996 in an increasing number of countries. The EISS dataset was used to characterise important epidemiological features of influenza activity in Europe during eight winters (1999–2007). The following questions were addressed: 1) are the sentinel clinical reports a good measure of influenza activity? 2) how long is a typical influenza season in Europe? 3) is there a west-east and/or south-north course of peak activity ('spread') of influenza in Europe? METHODS: Influenza activity was measured by collecting data from sentinel general practitioners (GPs) and reports by national reference laboratories. The sentinel reports were first evaluated by comparing them to the laboratory reports and were then used to assess the timing and spread of influenza activity across Europe during eight seasons. RESULTS: We found a good match between the clinical sentinel data and laboratory reports of influenza collected by sentinel physicians (overall match of 72% for +/- 1 week difference). We also found a moderate to good match between the clinical sentinel data and laboratory reports of influenza from non-sentinel sources (overall match of 60% for +/- 1 week). There were no statistically significant differences between countries using ILI (influenza-like illness) or ARI (acute respiratory disease) as case definition. When looking at the peak-weeks of clinical activity, the average length of an influenza season in Europe was 15.6 weeks (median 15 weeks; range 12–19 weeks). Plotting the peak weeks of clinical influenza activity reported by sentinel GPs against the longitude or latitude of each country indicated that there was a west-east spread of peak activity (spread) of influenza across Europe in four winters (2001–2002, 2002–2003, 2003–2004 and 2004–2005) and a south-north spread in three winters (2001–2002, 2004–2005 and 2006–2007). CONCLUSION: We found that: 1) the clinical data reported by sentinel physicians is a valid indicator of influenza activity; 2) the length of influenza activity across the whole of Europe was surprisingly long, ranging from 12–19 weeks; 3) in 4 out of the 8 seasons, there was a west-east spread of influenza, in 3 seasons a south-north spread; not associated with type of dominant virus in those seasons. | [
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The burden of infection in industrialized countries has prompted considerable effort to improve the outcomes of patients with sepsis. This has been formalized through the Surviving Sepsis Campaign 'bundles', derived from the recommendations of 11 professional societies, which have promoted global improvement in those practices whose primary goal it is to reduce sepsis-related death. However, difficulties remain in implementing all of the procedures recommended by the experts, despite the apparent pragmatism of those procedures. We summarize the main proposals made by the Surviving Sepsis Campaign and focus on the difficulties associated with making a proper diagnosis and supplying adequate treatment promptly to septic patients. | [
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BACKGROUND: Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS). Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40) vectors for pulmonary gene therapy. METHODS: Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP). SV40 vectors carrying the luciferase reporter gene (SV/luc) were administered intratracheally immediately after sepsis induction. Sham operated (SO) as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C). Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector. RESULTS: Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response. CONCLUSION: In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool. | [
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] |
BACKGROUND: We previously identified unusual variants of Moloney and Friend ecotropic mouse gammaretroviruses that have altered host range and are cytopathic in cells of the wild mouse species Mus dunni. Cytopathicity was attributed to different amino acid substitutions at the same critical env residue involved in receptor interaction: S82F in the Moloney variant Spl574, and S84A in the Friend mouse leukemia virus F-S MLV. Because M. dunni cells carry a variant CAT-1 cell surface virus receptor (dCAT-1), we examined the role of this receptor variant in cytopathicity and host range. RESULTS: We expressed dCAT-1 or mCAT-1 of NIH 3T3 origin in cells that are not normally infectible with ecotropic MLVs and evaluated the transfectants for susceptibility to virus infection and to virus-induced syncytium formation. The dCAT-1 transfectants, but not the mCAT-1 transfectants, were susceptible to virus-induced cytopathicity, and this cytopathic response was accompanied by the accumulation of unintegrated viral DNA. The dCAT-1 transfectants, however, did not also reproduce the relative resistance of M. dunni cells to Moloney MLV, and the mCAT-1 transfectants did not show the relative resistance of NIH 3T3 cells to Spl574. Western analysis, use of glycosylation inhibitors and mutagenesis to remove receptor glycosylation sites identified a possible role for cell-specific glycosylation in the modulation of virus entry. CONCLUSION: Virus entry and virus-induced syncytium formation using the CAT-1 receptor are mediated by a small number of critical amino acid residues in receptor and virus Env. Virus entry is modulated by glycosylation of cellular proteins, and this effect is cell and virus-specific. | [
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] |
HIV-1 uses a programmed -1 ribosomal frameshift to synthesize the precursor of its enzymes, Gag-Pol. The frameshift efficiency that is critical for the virus replication, is controlled by an interaction between the ribosome and a specific structure on the viral mRNA, the frameshift stimulatory signal. The rate of cap-dependent translation initiation is known to be altered by the TAR RNA structure, present at the 5′ and 3′ end of all HIV-1 mRNAs. Depending upon its concentration, TAR activates or inhibits the double-stranded RNA-dependent protein kinase (PKR). We investigated here whether changes in translation initiation caused by TAR affect HIV-1 frameshift efficiency. CD4+ T cells and 293T cells were transfected with a dual-luciferase construct where the firefly luciferase expression depends upon the HIV-1 frameshift. Translation initiation was altered by adding TAR in cis or trans of the reporter mRNA. We show that HIV-1 frameshift efficiency correlates negatively with changes in the rate of translation initiation caused by TAR and mediated by PKR. A model is presented where changes in the rate of initiation affect the probability of frameshifting by altering the distance between elongating ribosomes on the mRNA, which influences the frequency of encounter between these ribosomes and the frameshift stimulatory signal. | [
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BACKGROUND: With international concern over emerging infectious diseases (EID) and bioterrorist attacks, public health is being required to have early outbreak detection systems. A disease surveillance team was organized to establish a hospital emergency department-based syndromic surveillance system (ED-SSS) capable of automatically transmitting patient data electronically from the hospitals responsible for emergency care throughout the country to the Centers for Disease Control in Taiwan (Taiwan-CDC) starting March, 2004. This report describes the challenges and steps involved in developing ED-SSS and the timely information it provides to improve in public health decision-making. METHODS: Between June 2003 and March 2004, after comparing various surveillance systems used around the world and consulting with ED physicians, pediatricians and internal medicine physicians involved in infectious disease control, the Syndromic Surveillance Research Team in Taiwan worked with the Real-time Outbreak and Disease Surveillance (RODS) Laboratory at the University of Pittsburgh to create Taiwan's ED-SSS. The system was evaluated by analyzing daily electronic ED data received in real-time from the 189 hospitals participating in this system between April 1, 2004 and March 31, 2005. RESULTS: Taiwan's ED-SSS identified winter and summer spikes in two syndrome groups: influenza-like illnesses and respiratory syndrome illnesses, while total numbers of ED visits were significantly higher on weekends, national holidays and the days of Chinese lunar new year than weekdays (p < 0.001). It also identified increases in the upper, lower, and total gastrointestinal (GI) syndrome groups starting in November 2004 and two clear spikes in enterovirus-like infections coinciding with the two school semesters. Using ED-SSS for surveillance of influenza-like illnesses and enteroviruses-related infections has improved Taiwan's pandemic flu preparedness and disease control capabilities. CONCLUSION: Taiwan's ED-SSS represents the first nationwide real-time syndromic surveillance system ever established in Asia. The experiences reported herein can encourage other countries to develop their own surveillance systems. The system can be adapted to other cultural and language environments for better global surveillance of infectious diseases and international collaboration. | [
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] |
BACKGROUND: Cytotoxic T cell (CTL) cross-reactivity is believed to play a pivotal role in generating immune responses but the extent and mechanisms of CTL cross-reactivity remain largely unknown. Several studies suggest that CTL clones can recognize highly diverse peptides, some sharing no obvious sequence identity. The emerging realization in the field is that T cell receptors (TcR) recognize multiple distinct ligands. PRINCIPAL FINDINGS: First, we analyzed peptide scans of the HIV epitope SLFNTVATL (SFL9) and found that TCR specificity is position dependent and that biochemically similar amino acid substitutions do not drastically affect recognition. Inspired by this, we developed a general model of TCR peptide recognition using amino acid similarity matrices and found that such a model was able to predict the cross-reactivity of a diverse set of CTL epitopes. With this model, we were able to demonstrate that seemingly distinct T cell epitopes, i.e., ones with low sequence identity, are in fact more biochemically similar than expected. Additionally, an analysis of HIV immunogenicity data with our model showed that CTLs have the tendency to respond mostly to peptides that do not resemble self-antigens. CONCLUSIONS: T cell cross-reactivity can thus, to an extent greater than earlier appreciated, be explained by amino acid similarity. The results presented in this paper will help resolving some of the long-lasting discussions in the field of T cell cross-reactivity. | [
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BACKGROUND: Human rhinoviruses (HRVs) are the most frequently detected pathogens in acute respiratory tract infections (ARTIs) and yet little is known about the prevalence, recurrence, structure and clinical impact of individual members. During 2007, the complete coding sequences of six previously unknown and highly divergent HRV strains were reported. To catalogue the molecular and clinical features distinguishing the divergent HRV strains, we undertook, for the first time, in silico analyses of all available polyprotein sequences and performed retrospective reviews of the medical records of cases in which variants of the prototype strain, HRV-QPM, had been detected. METHODOLOGY/PRINCIPLE FINDINGS: Genomic analyses revealed that the six divergent strains, residing within a clade we previously called HRV A2, had the shortest polyprotein of all picornaviruses investigated. Structure-based amino acid alignments identified conserved motifs shared among members of the genus Rhinovirus as well as substantive deletions and insertions unique to the divergent strains. Deletions mostly affected regions encoding proteins traditionally involved in antigenicity and serving as HRV and HEV receptor footprints. Because the HRV A2 strains cannot yet be cultured, we created homology models of predicted HRV-QPM structural proteins. In silico comparisons confirmed that HRV-QPM was most closely related to the major group HRVs. HRV-QPM was most frequently detected in infants with expiratory wheezing or persistent cough who had been admitted to hospital and required supplemental oxygen. It was the only virus detected in 65% of positive individuals. These observations contributed to an objective clinical impact ranging from mild to severe. CONCLUSIONS: The divergent strains did not meet classification requirements for any existing species of the genus Rhinovirus or Enterovirus. HRV A2 strains should be partitioned into at least one new species, putatively called Human rhinovirus C, populated by members detected with high frequency, from individuals with respiratory symptoms requiring hospital admission. | [
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] |
In spite of protective ventilatory strategies, Acute Lung Injury (ALI) remains associated with high morbidity and mortality. One reason for the lack of therapeutic options might be that ALI is a co-morbid event associated with a diverse family of diseases and, thus, may be the result of distinct pathological processes. Among them, activated neutrophil- (PMN-) induced tissue injury and epithelial cell apoptosis mediated lung damage represent two potentially important candidate pathomechanisms that have been put forward. Several approaches have been undertaken to test these hypotheses, with substantial success in the treatment of experimental forms of ALI. With this in mind, we will summarize these two current hypotheses of ALI briefly, emphasizing the role of apoptosis in regulating PMN and/or lung epithelial cell responses. In addition, the contribution that Fas-mediated inflammation may play as a potential biological link between lung cell apoptosis and PMN recruitment will be considered, as well as the in vivo application of small interfering RNA (siRNA) as a novel approach to the inhibition of ALI and its therapeutic implications. | [
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RNA–RNA recombination is an important pathway in virus evolution and has been described for many viruses. However, the factors driving recombination or promoting the selection of recombinants are still unclear. Here, we show that the small movement protein (2b) was able to promote selection of RNA 1/2–RNA 3 recombinants within a chimeric virus having RNAs 1 and 2 from cucumber mosaic virus, and RNA 3 from the related tomato aspermy virus, along with heterologous 2b genes. The source of the 2b also determined the selection of the acceptor RNA and the crossover site, as well as affecting the rate of selection of the recombinant RNAs. The nature of the RNA 3 also influenced the selection of the recombinant RNAs. A 163-nt tandem repeat in RNA 3 significantly affected the rate of selection of the recombinant RNA, while a single nucleotide within the repeat affected the crossover site. The recombination occurred in a non-random manner, involved no intermediates and probably was generated via a copy-choice mechanism during (+) strand RNA synthesis. | [
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BACKGROUND: The construction of complex spatial simulation models such as those used in network epidemiology, is a daunting task due to the large amount of data involved in their parameterization. Such data, which frequently resides on large geo-referenced databases, has to be processed and assigned to the various components of the model. All this just to construct the model, then it still has to be simulated and analyzed under different epidemiological scenarios. This workflow can only be achieved efficiently by computational tools that can automate most, if not all, these time-consuming tasks. In this paper, we present a simulation software, Epigrass, aimed to help designing and simulating network-epidemic models with any kind of node behavior. RESULTS: A Network epidemiological model representing the spread of a directly transmitted disease through a bus-transportation network connecting mid-size cities in Brazil. Results show that the topological context of the starting point of the epidemic is of great importance from both control and preventive perspectives. CONCLUSION: Epigrass is shown to facilitate greatly the construction, simulation and analysis of complex network models. The output of model results in standard GIS file formats facilitate the post-processing and analysis of results by means of sophisticated GIS software. | [
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Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by intrahepatic bile duct destruction and the production of anti-mitochondrial antibodies (AMA). The absence of an animal model has been a striking impedance in defining the molecular basis of disease. Previous work has suggested that SJL/J mice immunize with the pyruvate dehydrogenase complex (PDC-E2), the major mitochondrial autoantigen of PBC, leads to the development of lymphoid cell infiltration in portal tracts and a model system coined autoimmune cholangitis. We hypothesized that this pathology would be augmented if immunization occurred in the presence of IFN-γ injections. Accordingly, SJL/J mice were immunized with PDC-E2 and, for purpose of control, α-casein. Subgroups of mice were also treated with exogenous IFN-γ. As expected, mice immunized with PDC-E2, with or without IFN-γ, developed high titer AMAs. In contrast, mice immunized with α-casein, develop antinuclear antibodies. More importantly, the livers from mice immunized with PDC-E2 and/or those immunized with α-casein all displayed lymphoid cell infiltration to the portal tracts, irrespective of bile duct size. Indeed, there was no significant difference between the experimental and the control groups by histologic analysis. Thus, autoimmune cholangitis in these mice is antigen non-specific. | [
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BACKGROUND: La Crosse virus (LACV), family Bunyaviridae, was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl with fatal encephalitis in La Crosse, Wisconsin. LACV is a major cause of pediatric encephalitis in North America and infects up to 300,000 persons each year of which 70–130 result in severe disease of the central nervous system (CNS). As an initial step in the establishment of useful animal models to support vaccine development, we examined LACV infectivity, pathogenesis, and immunogenicity in both weanling mice and rhesus monkeys. RESULTS: Following intraperitoneal inoculation of mice, LACV replicated in various organs before reaching the CNS where it replicates to high titer causing death from neurological disease. The peripheral site where LACV replicates to highest titer is the nasal turbinates, and, presumably, LACV can enter the CNS via the olfactory neurons from nasal olfactory epithelium. The mouse infectious dose(50 )and lethal dose(50 )was similar for LACV administered either intranasally or intraperitoneally. LACV was highly infectious for rhesus monkeys and infected 100% of the animals at 10 PFU. However, the infection was asymptomatic, and the monkeys developed a strong neutralizing antibody response. CONCLUSION: In mice, LACV likely gains access to the CNS via the blood stream or via olfactory neurons. The ability to efficiently infect mice intranasally raises the possibility that LACV might use this route to infect its natural hosts. Rhesus monkeys are susceptible to LACV infection and develop strong neutralizing antibody responses after inoculation with as little as 10 PFU. Mice and rhesus monkeys are useful animal models for LACV vaccine immunologic testing although the rhesus monkey model is not optimal. | [
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Measles virus is a negative‐sense, single‐stranded RNA virus within theMononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. Themeasles virus nucleoprotein consists of a structured N‐terminal domain, and of an intrinsically disordered C‐terminal domain, N(TAIL) (aa 401–525), which undergoes induced folding in the presence of the C‐terminal domain (XD, aa 459–507) of the viral phosphoprotein. With in N(TAIL), an α‐helical molecular recognition element (α‐MoRE, aa 488–499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small‐angle X‐ray scattering, we have derived a low‐resolution structural model of the complex between XD and N(TAIL), which shows that most of N(TAIL) remains disordered in the complex despite P‐induced folding within the α‐MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N‐terminal region of N(TAIL), and of a bulky globular region, corresponding to XD and to the C terminus of N(TAIL) (aa 486–525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that N(TAIL) has an additional site (aa 517–525) involved in binding to XD but not in the unstructured‐to‐structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure. | [
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BACKGROUND: Several public health strategic interventions are required for effective prevention and control of avian influenza (AI) and it is necessary to create a communication plan to keep families adequately informed on how to avoid or reduce exposure. This investigation determined the knowledge, attitudes, and behaviors relating to AI among an adult population in Italy. METHODS: From December 2005 to February 2006 a random sample of 1020 adults received a questionnaire about socio-demographic characteristics, knowledge of transmission and prevention about AI, attitudes towards AI, behaviors regarding use of preventive measures and food-handling practices, and sources of information about AI. RESULTS: A response rate of 67% was achieved. Those in higher socioeconomic classes were more likely to identify the modes of transmission and the animals' vehicles for AI. Those older, who knew the modes of transmission and the animals' vehicles for AI, and who still need information, were more likely to know that washing hands soap before and after touching raw poultry meat and using gloves is recommended to avoid spreading of AI through food. The risk of being infected was significantly higher in those from lower socioeconomic classes, if they did not know the definition of AI, if they knew that AI could be transmitted by eating and touching raw eggs and poultry foods, and if they did not need information. Compliance with the hygienic practices during handling of raw poultry meat was more likely in those who perceived to be at higher risk, who knew the hygienic practices, who knew the modes of transmission and the animals' vehicles for AI, and who received information from health professionals and scientific journals. CONCLUSION: Respondents demonstrate no detailed understanding of AI, a greater perceived risk, and a lower compliance with precautions behaviors and health educational strategies are strongly needed. | [
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BACKGROUND: Knowledge about the complete genome constellation of seasonal influenza A viruses from different countries is valuable for monitoring and understanding of the evolution and migration of strains. Few complete genome sequences of influenza A viruses from Europe are publicly available at the present time and there have been few longitudinal genome studies of human influenza A viruses. We have studied the evolution of circulating human H3N2, H1N1 and H1N2 influenza A viruses from 1999 to 2006, we analysed 234 Danish human influenza A viruses and characterised 24 complete genomes. RESULTS: H3N2 was the prevalent strain in Denmark during the study period, but H1N1 dominated the 2000–2001 season. H1N2 viruses were first observed in Denmark in 2002–2003. After years of little genetic change in the H1N1 viruses the 2005–2006 season presented H1N1 of greater variability than before. This indicates that H1N1 viruses are evolving and that H1N1 soon is likely to be the prevalent strain again. Generally, the influenza A haemagglutinin (HA) of H3N2 viruses formed seasonal phylogenetic clusters. Different lineages co-circulating within the same season were also observed. The evolution has been stochastic, influenced by small "jumps" in genetic distance rather than constant drift, especially with the introduction of the Fujian-like viruses in 2002–2003. Also evolutionary stasis-periods were observed which might indicate well fit viruses. The evolution of H3N2 viruses have also been influenced by gene reassortments between lineages from different seasons. None of the influenza genes were influenced by strong positive selection pressure. The antigenic site B in H3N2 HA was the preferred site for genetic change during the study period probably because the site A has been masked by glycosylations. Substitutions at CTL-epitopes in the genes coding for the neuraminidase (NA), polymerase acidic protein (PA), matrix protein 1 (M1), non-structural protein 1 (NS1) and especially the nucleoprotein (NP) were observed. The N-linked glycosylation pattern varied during the study period and the H3N2 isolates from 2004 to 2006 were highly glycosylated with ten predicted sequons in HA, the highest amount of glycosylations observed in this study period. CONCLUSION: The present study is the first to our knowledge to characterise the evolution of complete genomes of influenza A H3N2, H1N1 and H1N2 isolates from Europe over a time period of seven years from 1999 to 2006. More precise knowledge about the circulating strains may have implications for predicting the following season strains and thereby better matching the vaccine composition. | [
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BACKGROUND: AIDS, SARS, and the recent epidemics of the avian-flu have all served to remind us the debate over the limits of the moral duty to care. It is important to first consider the question of whether or not the "duty to treat" might be subject to contextual constraints. The purpose of this study was to investigate the opinions and beliefs held by both physicians and dentists regarding the occupational risks of infectious diseases, and to analyze the argument that the notion of "presumed consent" on the part of professionals may be grounds for supporting the duty to treat. METHODS: For this cross-sectional survey, the study population was selected from among physicians and dentists in Ankara. All of the 373 participants were given a self-administered questionnaire. RESULTS: In total, 79.6% of the participants said that they either had some degree of knowledge about the risks when they chose their profession or that they learned of the risks later during their education and training. Of the participants, 5.2% said that they would not have chosen this profession if they had been informed of the risks. It was found that 57% of the participants believed that there is a standard level of risk, and 52% of the participants stated that certain diseases would exceed the level of acceptable risk unless specific protective measures were implemented. CONCLUSION: If we use the presumed consent argument to establish the duty of the HCW to provide care, we are confronted with problems ranging over the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the near-impossibility of being able to evaluate retrospectively whether every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases. Our findings suggest that another problem can be added to the list: one-fifth of the participants in this study either lacked adequate knowledge of the occupational risks when they chose the medical profession or were not sufficiently informed of these risks during their faculty education and training. Furthermore, in terms of the moral duty to provide care, it seems that most HCWs are more concerned about the availability of protective measures than about whether they had been informed of a particular risk beforehand. For all these reasons, the presumed consent argument is not persuasive enough, and cannot be used to justify the duty to provide care. It is therefore more useful to emphasize justifications other than presumed consent when defining the duty of HCWs to provide care, such as the social contract between society and the medical profession and the fact that HCWs have a greater ability to provide medical aid. | [
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BACKGROUND: Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control. RESULTS: By applying on-line pO(2 )monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles. By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels. CONCLUSION: The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems. | [
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While foreign pathogens and their products have long been known to activate the innate immune system, the recent recognition of a group of endogenous molecules that serve a similar function has provided a framework for understanding the overlap between the inflammatory responses activated by pathogens and injury. These endogenous molecules, termed alarmins, are normal cell constituents that can be released into the extracellular milieu during states of cellular stress or damage and subsequently activate the immune system. One nuclear protein, High mobility group box-1 (HMGB1), has received particular attention as fulfilling the functions of an alarmin by being involved in both infectious and non-infectious inflammatory conditions. Once released, HMGB1 signals through various receptors to activate immune cells involved in the immune process. Although initial studies demonstrated HMGB1 as a late mediator of sepsis, recent findings indicate HMGB1 to have an important role in models of non-infectious inflammation, such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. Furthermore, in contrast to its pro-inflammatory functions, there is evidence that HMGB1 also has restorative effects leading to tissue repair and regeneration. The complex functions of HMGB1 as an archetypical alarmin are outlined here to review our current understanding of a molecule that holds the potential for treatment in many important human conditions. | [
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] |
Previous studies have reported the clinical usefulness of reverse transcription-polymerase chain reaction (RT-PCR) detection of thyroglobulin (TG) mRNA in the peripheral blood of patients with differentiated thyroid carcinoma. To evaluate this usefulness, we measured TG mRNA in the peripheral blood of patients diagnosed with thyroid carcinoma after total thyroidectomy by real-time quantitative RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. Surprisingly, we detected TG mRNA in all samples obtained after total thyroidectomy, including those from 4 medullary carcinomas. Further, there was no statistical difference in expression levels of TG mRNA in the patients with or without metastasis, and no significant correlation was found between serum TG concentrations and the expression levels of TG mRNA. These results give rise to a question regarding the clinical applications of not only RT-PCR detection but also quantitative measurement of TG mRNA in peripheral blood. © 2001 Cancer Research Campaign http://www.bjcancer.com | [
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] |
BACKGROUND: Jena Virus (JV), a bovine Norovirus, causes enteric disease in cattle and represents a potential model for the study of enteric norovirus infection and pathogenesis. The positive sense RNA genome of JV is organised into ORF1 (non-structural proteins), ORF2 (major capsid protein) and ORF3 (minor capsid protein). The lack of a cell culture system for studying JV replication has meant that work to date has relied upon in vitro systems to study non-structural protein synthesis and processing. PRINCIPAL FINDINGS: Only two of the three major ORF1 proteins were identified (p110 and 2C) following in vitro translation of JV RNA, the N-term protein was not detected. The N-term encoding genomic sequence (5′GS) was tested for IRES-like function in a bi-cistronic system and displayed no evidence of IRES-like activity. The site of translation initiation in JV was determined to be at the predicted nucleotide 22. Following the insertion of an epitope within the 5′GS the JV N-term protein was identified in vitro and within RNA transfected cells. CONCLUSIONS: The in vitro transcription/translation system is currently the best system for analysing protein synthesis and processing in JV. Unlike similarly studied human noroviruses JV initially did not appear to express the N-terminal protein, presenting the possibility that the encoding RNA sequence had a regulatory function, most likely involved in translation initiation in an IRES-like manner. This was not the case and, following determination of the site of translation initiation the N-term protein was detected using an epitope tag, both in vitro and in vivo. Although slightly larger than predicted the N-term protein was detected in a processed form in vivo, thus not only demonstrating initial translation of the ORF1 polyprotein but also activity of the viral protease. These findings indicate that the block to noroviral replication in cultured cells lies elsewhere. | [
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] |
BACKGROUND: There are sparse data on whether non-pharmaceutical interventions can reduce the spread of influenza. We implemented a study of the feasibility and efficacy of face masks and hand hygiene to reduce influenza transmission among Hong Kong household members. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cluster randomized controlled trial of households (composed of at least 3 members) where an index subject presented with influenza-like-illness of <48 hours duration. After influenza was confirmed in an index case by the QuickVue Influenza A+B rapid test, the household of the index subject was randomized to 1) control or 2) surgical face masks or 3) hand hygiene. Households were visited within 36 hours, and 3, 6 and 9 days later. Nose and throat swabs were collected from index subjects and all household contacts at each home visit and tested by viral culture. The primary outcome measure was laboratory culture confirmed influenza in a household contact; the secondary outcome was clinically diagnosed influenza (by self-reported symptoms). We randomized 198 households and completed follow up home visits in 128; the index cases in 122 of those households had laboratory-confirmed influenza. There were 21 household contacts with laboratory confirmed influenza corresponding to a secondary attack ratio of 6%. Clinical secondary attack ratios varied from 5% to 18% depending on case definitions. The laboratory-based or clinical secondary attack ratios did not significantly differ across the intervention arms. Adherence to interventions was variable. CONCLUSIONS/SIGNIFICANCE: The secondary attack ratios were lower than anticipated, and lower than reported in other countries, perhaps due to differing patterns of susceptibility, lack of significant antigenic drift in circulating influenza virus strains recently, and/or issues related to the symptomatic recruitment design. Lessons learnt from this pilot have informed changes for the main study in 2008. TRIAL REGISTRATION: ClinicalTrials.gov NCT00425893 HKClinicalTrials.com HKCTR-365 | [
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] |
BACKGROUND: Fast changes in human demographics worldwide, coupled with increased mobility, and modified land uses make the threat of emerging infectious diseases increasingly important. Currently there is worldwide alert for H5N1 avian influenza becoming as transmissible in humans as seasonal influenza, and potentially causing a pandemic of unprecedented proportions. Here we show how epidemiological surveillance data for emerging infectious diseases can be interpreted in real time to assess changes in transmissibility with quantified uncertainty, and to perform running time predictions of new cases and guide logistics allocations. METHODOLOGY/PRINCIPAL FINDINGS: We develop an extension of standard epidemiological models, appropriate for emerging infectious diseases, that describes the probabilistic progression of case numbers due to the concurrent effects of (incipient) human transmission and multiple introductions from a reservoir. The model is cast in terms of surveillance observables and immediately suggests a simple graphical estimation procedure for the effective reproductive number R (mean number of cases generated by an infectious individual) of standard epidemics. For emerging infectious diseases, which typically show large relative case number fluctuations over time, we develop a Bayesian scheme for real time estimation of the probability distribution of the effective reproduction number and show how to use such inferences to formulate significance tests on future epidemiological observations. CONCLUSIONS/SIGNIFICANCE: Violations of these significance tests define statistical anomalies that may signal changes in the epidemiology of emerging diseases and should trigger further field investigation. We apply the methodology to case data from World Health Organization reports to place bounds on the current transmissibility of H5N1 influenza in humans and establish a statistical basis for monitoring its evolution in real time. | [
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] |
DNA microarrays offer the possibility of testing for the presence of thousands of micro-organisms in a single experiment. However, there is a lack of reliable bioinformatics tools for the analysis of such data. We have developed DetectiV, a package for the statistical software R. DetectiV offers powerful yet simple visualization, normalization and significance testing tools. We show that DetectiV performs better than previously published software on a large, publicly available dataset. | [
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BACKGROUND: Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines. METHODOLOGY AND PRINCIPAL FINDINGS: A recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD(50) (50% bovine protective dose) test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD(50) per dose. CONCLUSION: The results suggest that this strategy might be used to develop the new subunit FMDV vaccine. | [
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In networks, nodes may preferentially contact other nodes with similar (assortatively mixed) or dissimilar (disassortatively mixed) numbers of contacts. Different patterns of contact support different epidemic dynamics, potentially affecting the efficacy of control measures such as contact tracing, which aims to identify and isolate nodes with infectious contacts. We used stochastic simulations to investigate the effects of mixing patterns on epidemic dynamics and contact-tracing efficacy. For uncontrolled epidemics, outbreaks occur at lower infection rates for more assortatively mixed networks, with faster initial epidemic growth rate and shorter epidemic duration than for disassortatively mixed networks. Contact tracing performs better for assortative mixing where epidemic size is large and tracing rate low, but it performs better for disassortative mixing at higher contact rates. For assortatively mixed networks, disease spreads first to highly connected nodes, but this is balanced by contact tracing quickly identifying these same nodes. The converse is true for disassortative mixing, where both disease and tracing are less likely to target highly connected nodes. For small epidemics, contact tracing is more effective on disassortative networks due to the greater resilience of assortative networks to link removal. Multi-step contact tracing is more effective than single-step tracing for assortative mixing, but this effect is smaller for disassortatively mixed networks. | [
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BACKGROUND: Although primary health care, and in particular, general practice will be at the frontline in the response to pandemic influenza, there are no frameworks to guide systematic planning for this task or to appraise available plans for their relevance to general practice. We aimed to develop a framework that will facilitate planning for general practice, and used it to appraise pandemic plans from Australia, England, USA, New Zealand and Canada. METHODOLOGY/PRINCIPAL FINDINGS: We adapted the Haddon matrix to develop the framework, populating its cells through a multi-method study that incorporated the peer-reviewed and grey literature, interviews with general practitioners, practice nurses and senior decision-makers, and desktop simulation exercises. We used the framework to analyse 89 publicly-available jurisdictional plans at similar managerial levels in the five countries. The framework identifies four functional domains: clinical care for influenza and other needs, public health responsibilities, the internal environment and the macro-environment of general practice. No plan addressed all four domains. Most plans either ignored or were sketchy about non-influenza clinical needs, and about the contribution of general practice to public health beyond surveillance. Collaborations between general practices were addressed in few plans, and inter-relationships with the broader health system, even less frequently. CONCLUSIONS: This is the first study to provide a framework to guide general practice planning for pandemic influenza. The framework helped identify critical shortcomings in available plans. Engaging general practice effectively in planning is challenging, particularly where governance structures for primary health care are weak. We identify implications for practice and for research. | [
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Organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. Stress response can either repair the damage or activate one of the programmed cell death (PCD) mechanisms, for example apoptosis, and finally end in cell death. One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. This process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rRNA is less susceptible to degradation in respiring cells with functional defence against oxidative stress. In addition, RNA fragmentation is independent of two yeast apoptotic factors, metacaspase Yca1 and apoptosis-inducing factor Aif1, but it relies on the apoptotic chromatin condensation induced by histone H2B modifications. These data describe a novel phenotype for certain stress- and ageing-related PCD pathways in yeast. | [
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Moraxella catarrhalis is a ubiquitous human-specific bacterium commonly associated with upper and lower respiratory tract infections, including otitis media, sinusitis and chronic obstructive pulmonary disease. The bacterium uses an autotransporter protein UspA1 to target an important human cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Using X-ray crystallography, we show that the CEACAM1 receptor-binding region of UspA1 unusually consists of an extended, rod-like left-handed trimeric coiled-coil. Mutagenesis and binding studies of UspA1 and the N-domain of CEACAM1 have been used to delineate the interacting surfaces between ligand and receptor and guide assembly of the complex. However, solution scattering, molecular modelling and electron microscopy analyses all indicate that significant bending of the UspA1 coiled-coil stalk also occurs. This explains how UspA1 can engage CEACAM1 at a site far distant from its head group, permitting closer proximity of the respective cell surfaces during infection. | [
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Viruses adapt to their hosts by evading defense mechanisms and taking over cellular metabolism for their own benefit. Alterations in cell metabolism as well as side-effects of antiviral responses contribute to symptoms development and virulence. Sometimes, a virus may spill over from its usual host species into a novel one, where usually will fail to successfully infect and further transmit to new host. However, in some cases, the virus transmits and persists after fixing beneficial mutations that allow for a better exploitation of the new host. This situation would represent a case for a new emerging virus. Here we report results from an evolution experiment in which a plant virus was allowed to infect and evolve on a naïve host. After 17 serial passages, the viral genome has accumulated only five changes, three of which were non-synonymous. An amino acid substitution in the viral VPg protein was responsible for the appearance of symptoms, whereas one substitution in the viral P3 protein the epistatically contributed to exacerbate severity. DNA microarray analyses show that the evolved and ancestral viruses affect the global patterns of host gene expression in radically different ways. A major difference is that genes involved in stress and pathogen response are not activated upon infection with the evolved virus, suggesting that selection has favored viral strategies to escape from host defenses. | [
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BACKGROUND: Human metapneumovirus (hMPV) infection can cause acute lower respiratory tract illness in infants, the immunocompromised, and the elderly. Currently there are no licensed preventative measures for hMPV infections. Using a variant of hMPV/NL/1/00 that does not require trypsin supplementation for growth in tissue culture, we deleted the M2-2 gene and evaluated the replication of rhMPV/ΔM2-2 virus in vitro and in vivo. RESULTS: In vitro studies showed that the ablation of M2-2 increased the propensity for insertion of U nucleotides in poly-U tracts of the genomic RNA. In addition, viral transcription was up-regulated although the level of genomic RNA remained comparable to rhMPV. Thus, deletion of M2-2 alters the ratio between hMPV genome copies and transcripts. In vivo, rhMPV/ΔM2-2 was attenuated compared to rhMPV in the lungs and nasal turbinates of hamsters. Hamsters immunized with one dose of rhMPV/ΔM2-2 were protected from challenge with 10(6 )PFU of wild type (wt) hMPV/NL/1/00. CONCLUSION: Our results suggest that hMPV M2-2 alters regulation of transcription and influences the fidelity of the polymerase complex during viral genome replication. In the hamster model, rhMPVΔM2-2 is attenuated and protective suggesting that deletion of M2-2 may result in a potential live vaccine candidate. A more thorough knowledge of the hMPV polymerase complex and the role of M2-2 during hMPV replication are being studied as we develop a potential live hMPV vaccine candidate that lacks M2-2 expression. | [
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Diseases of mucosal inflammation represent important causes of morbidity and mortality, and have led to intense research efforts to understand the factors that lead to their development. It is well accepted that a breakdown of the normally impermeant epithelial barrier of the intestine, the lung, and the kidney is associated with the development of inflammatory disease in these organs, yet significant controversy exists as to how this breakdown actually occurs, and how such a breakdown may lead to inflammation. In this regard, much work has focused upon the role of the epithelium as an “innocent bystander,” a target of a leukocyte-mediated inflammatory cascade that leads to its destruction in the mucosal inflammatory process. However, recent evidence from a variety of laboratories indicates that the epithelium is not merely a passive component in the steps that lead to mucosal inflammation, but is a central participant in the process. In addressing this controversy, we and others have determined that epithelial cells express Toll-like receptors (TLRs) of the innate immune system, and that activation of TLRs by endogenous and exogenous ligands may play a central role in determining the balance between a state of “mucosal homeostasis,” as is required for optimal organ function, and “mucosal injury,” leading to mucosal inflammation and barrier breakdown. In particular, activation of TLRs within intestinal epithelial cells leads to the development of cellular injury and impairment in mucosal repair in the pathogenesis of intestinal inflammation, while activation of TLRs in the lung and kidney may participate in the development of pneumonitis and nephritis respectively. Recent work in support of these concepts is extensively reviewed, while essential areas of further study that are required to determine the significance of epithelial TLR signaling during states of health and disease are outlined. | [
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BACKGROUND: Protein ubiquitination and/or degradation by the ubiquitin/proteasome system (UPS) have been recognized as critical mechanisms in the regulation of numerous essential cellular functions. The importance of the UPS in viral pathogenesis has become increasingly apparent. Using murine cardiomyocytes, we have previously demonstrated that the UPS plays a key role in the replication of coxsackievirus B3 (CVB3), an important human pathogen associated with various diseases. To further elucidate the underlying mechanisms, we examined the interplay between the UPS and CVB3, focusing on the role of ubiquitination in viral lifecycle. METHODOLOGY/PRINCIPAL FINDINGS: As assessed by in situ hybridization, Western blot, and plaque assay, we showed that proteasome inhibition decreased CVB3 RNA replication, protein synthesis, and viral titers in HeLa cells. There were no apparent changes in 20S proteasome activities following CVB3 infection. However, we found viral infection led to an accumulation of protein-ubiquitin conjugates, accompanied by a decreased protein expression of free ubiquitin, implicating an important role of ubiquitination in the UPS-mediated viral replication. Using small-interfering RNA, we demonstrated that gene-silencing of ubiquitin significantly reduced viral titers, possibly through downregulation of protein ubiquitination and subsequent alteration of protein function and/or degradation. Inhibition of deubiquitinating enzymes apparently enhances the inhibitory effects of proteasome inhibitors on CVB3 replication. Finally, by immunoprecipitation, we showed that coxsackieviral polymerase 3D was post-translationally modified by ubiquitination and such modification might be a prerequisite for its function in transcriptional regulation of viral genome. CONCLUSION: Coxsackievirus infection promotes protein ubiquitination, contributing to effective viral replication, probably through ubiquitin modification of viral polymerase. | [
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] |
BACKGROUND: Governments are preparing for a potential influenza pandemic. Therefore they need data to assess the possible impact of interventions. Face-masks worn by the general population could be an accessible and affordable intervention, if effective when worn under routine circumstances. METHODOLOGY: We assessed transmission reduction potential provided by personal respirators, surgical masks and home-made masks when worn during a variety of activities by healthy volunteers and a simulated patient. PRINCIPAL FINDINGS: All types of masks reduced aerosol exposure, relatively stable over time, unaffected by duration of wear or type of activity, but with a high degree of individual variation. Personal respirators were more efficient than surgical masks, which were more efficient than home-made masks. Regardless of mask type, children were less well protected. Outward protection (mask wearing by a mechanical head) was less effective than inward protection (mask wearing by healthy volunteers). CONCLUSIONS/SIGNIFICANCE: Any type of general mask use is likely to decrease viral exposure and infection risk on a population level, in spite of imperfect fit and imperfect adherence, personal respirators providing most protection. Masks worn by patients may not offer as great a degree of protection against aerosol transmission. | [
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CD133 (prominin-1), a 5-transmembrane glycoprotein, has recently been considered to be an important marker that represents the subset population of cancer stem-like cells. Herein we report the isolation of CD133-positive cells (LC-CD133(+)) and CD133-negative cells (LC-CD133(−)) from tissue samples of ten patients with non-small cell lung cancer (LC) and five LC cell lines. LC-CD133(+) displayed higher Oct-4 expressions with the ability to self-renew and may represent a reservoir with proliferative potential for generating lung cancer cells. Furthermore, LC-CD133(+), unlike LC-CD133(−), highly co-expressed the multiple drug-resistant marker ABCG2 and showed significant resistance to chemotherapy agents (i.e., cisplatin, etoposide, doxorubicin, and paclitaxel) and radiotherapy. The treatment of Oct-4 siRNA with lentiviral vector can specifically block the capability of LC-CD133(+) to form spheres and can further facilitate LC-CD133(+) to differentiate into LC-CD133(−). In addition, knock-down of Oct-4 expression in LC-CD133(+) can significantly inhibit the abilities of tumor invasion and colony formation, and increase apoptotic activities of caspase 3 and poly (ADP-ribose) polymerase (PARP). Finally, in vitro and in vivo studies further confirm that the treatment effect of chemoradiotherapy for LC-CD133(+) can be improved by the treatment of Oct-4 siRNA. In conclusion, we demonstrated that Oct-4 expression plays a crucial role in maintaining the self-renewing, cancer stem-like, and chemoradioresistant properties of LC-CD133(+). Future research is warranted regarding the up-regulated expression of Oct-4 in LC-CD133(+) and malignant lung cancer. | [
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Germ-free piglets were orally infected with virulent rotavirus to collect jejunal mucosal scrapings at 12 and 18 hours post infection (two piglets per time point). IFN-gamma mRNA expression was stimulated in the mucosa of all four infected piglets, indicating that they all responded to the rotavirus infection. RNA pools prepared from two infected piglets were used to compare whole mucosal gene expression at 12 and 18 hpi to expression in uninfected germ-free piglets (n = 3) using a porcine intestinal cDNA microarray. Microarray analysis identified 13 down-regulated and 17 up-regulated genes. Northern blot analysis of a selected group of genes confirmed the data of the microarray. Genes were functionally clustered in interferon-regulated genes, proliferation/differentiation genes, apoptosis genes, cytoskeleton genes, signal transduction genes, and enterocyte digestive, absorptive, and transport genes. Down-regulation of the transport gene cluster reflected in part the loss of rotavirus-infected enterocytes from the villous tips. Data mining suggested that several genes were regulated in lower- or mid-villus immature enterocytes and goblet cells, probably to support repair of the damaged epithelial cell layer at the villous tips. Furthermore, up-regulation was observed for IFN-γ induced guanylate binding protein 2, a protein that effectively inhibited VSV and EMCV replication in vitro (Arch Virol 150:1213–1220, 2005). This protein may play a role in the small intestine’s innate defense against enteric viruses like rotavirus. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00705-008-0118-6) contains supplementary material, which is available to authorized users. | [
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BACKGROUND: Enteric outbreaks associated with child care centres (CCC) have been well documented internationally and in Canada. The current literature focuses on identifying potential risk factors for introduction and transmission of enteric disease, but does not examine why these risk factors happen, how the risk is understood and managed by the staff of CCCs, or what challenges they experience responding to enteric illness. The purpose of this study was to explore the understanding, knowledge and actions of CCC staff regarding enteric illness and outbreaks, and to identify challenges that staff encounter while managing them. METHODS: Focus groups were conducted with staff of regulated CCCs in Southern Ontario. Five focus groups were held with 40 participants. An open ended style of interviewing was used. Data were analyzed using content analysis. RESULTS: CCC staff play an important role in preventing and managing enteric illness. Staff used in-depth knowledge of the children, the centre and their personal experiences to assist in making decisions related to enteric illness. The decisions and actions may differ from guidance provided by public health officials, particularly when faced with challenges related to time, money, staffing and parents. CONCLUSION: CCC staff relied on experience and judgment in coordination with public health information to assist decision-making in the management of enteric illness and outbreaks. Advice and guidance from public health officials to CCC staff needs to be consistent yet flexible so that it may be adapted in a variety of situations and meet regulatory and public health requirements. | [
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Precise dating of viral subtype divergence enables researchers to correlate divergence with geographic and demographic occurrences. When historical data are absent (that is, the overwhelming majority), viral sequence sampling on a time scale commensurate with the rate of substitution permits the inference of the times of subtype divergence. Currently, researchers use two strategies to approach this task, both requiring strong conditions on the molecular clock assumption of substitution rate. As the underlying structure of the substitution rate process at the time of subtype divergence is not understood and likely highly variable, we present a simple method that estimates rates of substitution, and from there, times of divergence, without use of an assumed molecular clock. We accomplish this by blending estimates of the substitution rate for triplets of dated sequences where each sequence draws from a distinct viral subtype, providing a zeroth-order approximation for the rate between subtypes. As an example, we calculate the time of divergence for three genes among influenza subtypes A-H3N2 and B using subtype C as an outgroup. We show a time of divergence approximately 100 years ago, substantially more recent than previous estimates which range from 250 to 3800 years ago. | [
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Sepsis remains the most common cause of death in intensive care units in the USA, with a current estimate of at least 750,000 cases per year, and 215,000 deaths annually. Despite extensive research still we do not quite understand the cellular and molecular mechanisms that are involved in triggering and propagation of septic injury. Endotoxin (lipopolysaccharide from Gram-negative bacteria, or LPS) has been implicated as a major cause of this syndrome. Inflammatory shock as a consequence of LPS release remains a serious clinical concern. In humans, inflammatory responses to LPS result in the release of cytokines and other cell mediators from monocytes and macrophages, which can cause fever, shock, organ failure and death. A number of different approaches have been investigated to try to treat and/or prevent the septic shock associated with infections caused by Gram-negative bacteria, including blockage of one or more of the cytokines induced by LPS. Recently several novel amphipathic compounds have been developed as direct LPS antagonists at the LPS receptor, TLR4. This review article will outline the current knowledge on the TLR4-LPS synthesis and discuss the signaling, in vitro pre-clinical and in vivo clinical evaluation of TLR4 antagonists and their potential use in sepsis and a variety of diseases such as atherosclerosis as well as hepatic and renal malfunction. | [
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] |
BACKGROUND: Dengue virus pathogenesis is not yet fully understood and the identification of patients at high risk for developing severe disease forms is still a great challenge in dengue patient care. During the present study, we evaluated prospectively the potential of cytokines present in plasma from patients with dengue in stratifying disease severity. METHODS: Seventeen-cytokine multiplex fluorescent microbead immunoassay was used for the simultaneous detection in 59 dengue patients. GLM models using bimodal or Gaussian family were determined in order to associate cytokines with clinical manifestations and laboratory diagnosis. RESULTS: IL-1β, IFN-γ, IL-4, IL-6, IL-13, IL-7 and GM-CSF were significantly increased in patients with severe clinical manifestations (severe dengue) when compared to mild disease forms (mild dengue). In contrast, increased MIP-1β levels were observed in patients with mild dengue. MIP-1β was also associated with CD56+NK cell circulating rates. IL-1β, IL-8, TNF-α and MCP-1 were associated with marked thrombocytopenia. Increased MCP-1 and GM-CSF levels correlated with hypotension. Moreover, MIP-1β and IFN-γ were independently associated with both dengue severity and disease outcome. CONCLUSION: Our data demonstrated that the use of a multiple cytokine assay platform was suitable for identifying distinct cytokine profiles associated with the dengue clinical manifestations and severity. MIP-β is indicated for the first time as a good prognostic marker in contrast to IFN-γ that was associated with disease severity. | [
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] |
On average, more than two new species of human virus are reported every year. We constructed the cumulative species discovery curve for human viruses going back to 1901. We fitted a statistical model to these data; the shape of the curve strongly suggests that the process of virus discovery is far from complete. We generated a 95% credible interval for the pool of as yet undiscovered virus species of 38–562. We extrapolated the curve and generated an estimate of 10–40 new species to be discovered by 2020. Although we cannot predict the level of health threat that these new viruses will present, we conclude that novel virus species must be anticipated in public health planning. More systematic virus discovery programmes, covering both humans and potential animal reservoirs of human viruses, should be considered. | [
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BACKGROUND: Two problems complicate the study of selection in viral genomes: Firstly, the presence of genes in overlapping reading frames implies that selection in one reading frame can bias our estimates of neutral mutation rates in another reading frame. Secondly, the high mutation rates we are likely to encounter complicate the inference of a reliable alignment of genomes. To address these issues, we develop a model that explicitly models selection in overlapping reading frames. We then integrate this model into a statistical alignment framework, enabling us to estimate selection while explicitly dealing with the uncertainty of individual alignments. We show that in this way we obtain un-biased selection parameters for different genomic regions of interest, and can improve in accuracy compared to using a fixed alignment. RESULTS: We run a series of simulation studies to gauge how well we do in selection estimation, especially in comparison to the use of a fixed alignment. We show that the standard practice of using a ClustalW alignment can lead to considerable biases and that estimation accuracy increases substantially when explicitly integrating over the uncertainty in inferred alignments. We even manage to compete favourably for general evolutionary distances with an alignment produced by GenAl. We subsequently run our method on HIV2 and Hepatitis B sequences. CONCLUSION: We propose that marginalizing over all alignments, as opposed to using a fixed one, should be considered in any parametric inference from divergent sequence data for which the alignments are not known with certainty. Moreover, we discover in HIV2 that double coding regions appear to be under less stringent selection than single coding ones. Additionally, there appears to be evidence for differential selection, where one overlapping reading frame is under positive and the other under negative selection. | [
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BACKGROUND: The underlying basis for the seasonality of influenza A viruses is still uncertain. Phylogenetic studies investigated this phenomenon but have lacked sequences from more subtropical and tropical regions, particularly from Southeast Asia. METHODOLOGY/PRINCIPAL FINDINGS: 281 complete hemagglutinin (HA) and neuraminidase (NA) sequences were obtained from influenza A(H3N2) viruses, collected over 10 years (1997–2006) from Hong Kong. These dated sequences were analyzed with influenza A(H3N2) vaccine strain sequences (Syd/5/97, Mos/10/99, Fuj/411/02, Cal/7/04) and 315 other publicly available dated sequences from elsewhere, worldwide. In addition, the NA sequence alignment was inspected for the presence of any naturally occurring, known, neuraminidase inhibitor (NAI) resistance-associated amino acid mutations (R292K and E119V). Before 2001, the Hong Kong HA and NA sequences clustered more closely with the older vaccine sequences (Syd/5/97, Mos/10/99) than did sequences from elsewhere. After 2001, this trend reversed with significant clusters containing HA and NA sequences from different locations, isolated at different times, suggesting that viral migration may account for much of the influenza A(H3N2) seasonality during this 10-year period. However, at least one example from Hong Kong was found suggesting that in some years, influenza A(H3N2) viruses may persist in the same location, perhaps continuing to circulate, sub-clinically, at low levels between seasons, to re-emerge in the influenza season the following year, relatively unchanged. None of these Hong Kong influenza A(H3N2) NA sequences contained any of the known NAI-resistance associated mutations. CONCLUSIONS/SIGNIFICANCE: The seasonality of influenza A(H3N2) may be largely due to global migration, with similar viruses appearing in different countries at different times. However, occasionally, some viruses may remain within a single location and continue to circulate within that population, to re-emerge during the next influenza season, with relatively little genetic change. Naturally occurring NAI resistance mutations were absent or, at least, very rare in this population. | [
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Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. These results together show that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection. In addition, primary macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro and in infected mouse lung tissue. | [
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Genome-wide scans for positively selected genes (PSGs) in mammals have provided insight into the dynamics of genome evolution, the genetic basis of differences between species, and the functions of individual genes. However, previous scans have been limited in power and accuracy owing to small numbers of available genomes. Here we present the most comprehensive examination of mammalian PSGs to date, using the six high-coverage genome assemblies now available for eutherian mammals. The increased phylogenetic depth of this dataset results in substantially improved statistical power, and permits several new lineage- and clade-specific tests to be applied. Of ∼16,500 human genes with high-confidence orthologs in at least two other species, 400 genes showed significant evidence of positive selection (FDR<0.05), according to a standard likelihood ratio test. An additional 144 genes showed evidence of positive selection on particular lineages or clades. As in previous studies, the identified PSGs were enriched for roles in defense/immunity, chemosensory perception, and reproduction, but enrichments were also evident for more specific functions, such as complement-mediated immunity and taste perception. Several pathways were strongly enriched for PSGs, suggesting possible co-evolution of interacting genes. A novel Bayesian analysis of the possible “selection histories” of each gene indicated that most PSGs have switched multiple times between positive selection and nonselection, suggesting that positive selection is often episodic. A detailed analysis of Affymetrix exon array data indicated that PSGs are expressed at significantly lower levels, and in a more tissue-specific manner, than non-PSGs. Genes that are specifically expressed in the spleen, testes, liver, and breast are significantly enriched for PSGs, but no evidence was found for an enrichment for PSGs among brain-specific genes. This study provides additional evidence for widespread positive selection in mammalian evolution and new genome-wide insights into the functional implications of positive selection. | [
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Copy Number Variations (CNVs) of regions of the human genome have been associated with multiple diseases. We present an algorithm which is mathematically sound and computationally efficient to accurately analyze CNV in a DNA sample utilizing a nanofluidic device, known as the digital array. This numerical algorithm is utilized to compute copy number variation and the associated statistical confidence interval and is based on results from probability theory and statistics. We also provide formulas which can be used as close approximations. | [
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BACKGROUND: Since 1999, the expansion of the West Nile virus (WNV) epizooty has led public health authorities to build and operate surveillance systems in North America. These systems are very useful to collect data, but cannot be used to forecast the probable spread of the virus in coming years. Such forecasts, if proven reliable, would permit preventive measures to be put into place at the appropriate level of expected risk and at the appropriate time. It is within this context that the Multi-Agent GeoSimulation approach has been selected to develop a system that simulates the interactions of populations of mosquitoes and birds over space and time in relation to the spread and transmission of WNV. This simulation takes place in a virtual mapping environment representing a large administrative territory (e.g. province, state) and carried out under various climate scenarios in order to simulate the effects of vector control measures such as larviciding at scales of 1/20 000 or smaller. RESULTS: After setting some hypotheses, a conceptual model and system architecture were developed to describe the population dynamics and interactions of mosquitoes (genus Culex) and American crows, which were chosen as the main actors in the simulation. Based on a mathematical compartment model used to simulate the population dynamics, an operational prototype was developed for the Southern part of Quebec (Canada). The system allows users to modify the parameters of the model, to select various climate and larviciding scenarios, to visualize on a digital map the progression (on a weekly or daily basis) of the infection in and around the crows' roosts and to generate graphs showing the evolution of the populations. The basic units for visualisation are municipalities. CONCLUSION: In all likelihood this system might be used to support short term decision-making related to WNV vector control measures, including the use of larvicides, according to climatic scenarios. Once fully calibrated in several real-life contexts, this promising approach opens the door to the study and management of other zoonotic diseases such as Lyme disease. | [
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The emergence of alternative medicines for AIDS in Asia and Africa was discussed at a satellite symposium and the parallel session on alternative and traditional treatments of the AIDSImpact meeting, held in Marseille, in July 2007. These medicines are heterogeneous, both in their presentation and in their geographic and cultural origin. The sessions focused on the role of these medications in selected resource poor settings in Africa and Asia now that access to anti-retroviral therapy is increasing. The aims of the sessions were to (1) identify the actors involved in the diffusion of these alternative medicines for HIV/AIDS, (2) explore uses and forms, and the way these medicines are given legitimacy, (3) reflect on underlying processes of globalisation and cultural differentiation, and (4) define priority questions for future research in this area. This article presents the insights generated at the meeting, illustrated with some findings from the case studies (Uganda, Senegal, Benin, Burkina Faso, China and Indonesia) that were presented. These case studies reveal the wide range of actors who are involved in the marketing and supply of alternative medicines. Regulatory mechanisms are weak. The efficacy claims of alternative medicines often reinforce a biomedical paradigm for HIV/AIDS, and fit with a healthy living ideology promoted by AIDS care programs and support groups. The AIDSImpact session concluded that more interdisciplinary research is needed on the experience of people living with HIV/AIDS with these alternative medicines, and on the ways in which these products interact (or not) with anti-retroviral therapy at pharmacological as well as psychosocial levels. | [
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H5N1 highly pathogenic avian influenza (HPAI) viruses have seriously affected the Asian poultry industry since their recurrence in 2003. The viruses pose a threat of emergence of a global pandemic influenza through point mutation or reassortment leading to a strain that can effectively transmit among humans. In this study, we present phylogenetic evidences for the interlineage reassortment among H5N1 HPAI viruses isolated from humans, cats, and birds in Indonesia, and identify the potential genetic parents of the reassorted genome segments. Parsimony analyses of viral phylogeography suggest that the reassortant viruses may have originated from greater Jakarta and surroundings, and subsequently spread to other regions in the West Java province. In addition, Bayesian methods were used to elucidate the genetic diversity dynamics of the reassortant strain and one of its genetic parents, which revealed a more rapid initial growth of genetic diversity in the reassortant viruses relative to their genetic parent. These results demonstrate that interlineage exchange of genetic information may play a pivotal role in determining viral genetic diversity in a focal population. Moreover, our study also revealed significantly stronger diversifying selection on the M1 and PB2 genes in the lineages preceding and subsequent to the emergence of the reassortant viruses, respectively. We discuss how the corresponding mutations might drive the adaptation and onward transmission of the newly formed reassortant viruses. | [
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A variety of human diseases are associated with gammaherpesviruses, including neoplasms of lymphocytes (e.g. Burkitt's lymphoma) and endothelial cells (e.g. Kaposi's sarcoma). Gammaherpesvirus infections usually result in either a productive lytic infection, characterized by expression of all viral genes and rapid cell lysis, or latent infection, characterized by limited viral gene expression and no cell lysis. Here, we report characterization of endothelial cell infection with murine gammaherpesvirus 68 (γHV68), a virus phylogenetically related and biologically similar to the human gammaherpesviruses. Endothelial cells supported γHV68 replication in vitro, but were unique in that a significant proportion of the cells escaped lysis, proliferated, and remained viable in culture for an extended time. Upon infection, endothelial cells became non-adherent and altered in size, complexity, and cell-surface protein expression. These cells were uniformly infected and expressed the lytic transcription program based on detection of abundant viral gene transcripts, GFP fluorescence from the viral genome, and viral surface protein expression. Additionally, endothelial cells continued to produce new infectious virions as late as 30 days post-infection. The outcome of this long-term infection was promoted by the γHV68 v-cyclin, because in the absence of the v-cyclin, viability was significantly reduced following infection. Importantly, infected primary endothelial cells also demonstrated increased viability relative to infected primary fibroblasts, and this increased viability was dependent on the v-cyclin. Finally, we provide evidence for infection of endothelial cells in vivo in immune-deficient mice. The extended viability and virus production of infected endothelial cells indicated that endothelial cells provided a source of prolonged virus production and identify a cell-type specific adaptation of gammaherpesvirus replication. While infected endothelial cells would likely be cleared in a healthy individual, persistently infected endothelial cells could provide a source of continued virus replication in immune-compromised individuals, a context in which gammaherpesvirus-associated pathology frequently occurs. | [
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Begomoviruses are single-stranded DNA viruses and cause severe diseases in major crop plants worldwide. Based on current genome sequence analyses, we found that synonymous codon usage variations in the protein-coding genes of begomoviruses are mainly influenced by mutation bias. Base composition analysis suggested that the codon usage bias of AV1 and BV1 genes is significant and their expressions are high. Fourteen codons were determined as translational optimal ones according to the comparison of codon usage patterns between highly and lowly expressed genes. Interestingly the codon usages between begomoviruses from the Old and the New Worlds are apparently different, which supports the idea that the bipartite begomoviruses of the New World might originate from bipartite ones of the Old World, whereas the latter evolve from the Old World monopartite begomoviruses. | [
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Acute lung injury in humans is characterized histopathologically by neutrophilic alveolitis, injury of the alveolar epithelium and endothelium, hyaline membrane formation, and microvascular thrombi. Different animal models of experimental lung injury have been used to investigate mechanisms of lung injury. Most are based on reproducing in animals known risk factors for ARDS, such as sepsis, lipid embolism secondary to bone fracture, acid aspiration, ischemia-reperfusion of pulmonary or distal vascular beds, and other clinical risks. However, none of these models fully reproduces the features of human lung injury. The goal of this review is to summarize the strengths and weaknesses of existing models of lung injury. We review the specific features of human ARDS that should be modeled in experimental lung injury and then discuss specific characteristics of animal species that may affect the pulmonary host response to noxious stimuli. We emphasize those models of lung injury that are based on reproducing risk factors for human ARDS in animals and discuss the advantages and disadvantages of each model and the extent to which each model reproduces human ARDS. The present review will help guide investigators in the design and interpretation of animal studies of acute lung injury. | [
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BACKGROUND: Diffuse alveolar haemorrhage (DAH) is a serious pulmonary complication characterised by a high mortality rate and the absence of specific treatment. The intrapulmonary administration of activated recombinant factor VII (rFVIIa) in DAH was recently published in six patients by Heslet et al with an efficient hemostatic effect. We describe two cases of DAH treated with intrapulmonary rFVIIa. METHODS: Two cases of DAH were admitted to the ICU after presenting abrupt desaturation, tachypnea, cough and haemoptysis, requiring orotracheal intubation and mechanical ventilation. The diagnosis was achieved by the bloody return during the bronchoalveolar lavage, during the procedure rFVIIa (50 μg/Kg in 50 ml of isotonic saline) was administered via the bronchoscope. RESULTS: Immediate cessation of bleeding was observed. Prior to intrapulmonary administration of rFVIIa, the FiO(2 )was 1, which was reduced to 0.4 24 hours later. Following the procedure, the haemostatic effect made blood transfusion superfluous. No thrombotic complications associated with administration of the drug were observed. After the intervention both cases progressed fast and was discharged from the ICU with no further episodes of bleeding. CONCLUSION: 1. Local intrabronchial deposition of DAH with rFVIIa has been shown to be effective in controlling life-threatening DAH. 2. In the case described above, no thrombotic complications were observed following the intrapulmonary administration of rFVIIa. | [
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Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack. | [
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BACKGROUND: Studies of wild animals responding to their native parasites are essential if we are to understand how the immune system functions in the natural environment. While immune defence may bring increased survival, this may come at a resource cost to other physiological traits, including reproduction. Here, we tested the hypothesis that wild common shrews (Sorex araneus), which produce large numbers of offspring during the one breeding season of their short life span, forgo investment in immunity and immune system maintenance, as increased longevity is unlikely to bring further opportunities for mating. In particular, we predicted that adult shrews, with shorter expected lifespans, would not respond as effectively as young animals to infection. METHODOLOGY/PRINCIPAL FINDINGS: We examined haemolymphatic tissues from wild-caught common shrews using light and transmission electron microscopy, applied in conjunction with immunohistology. We compared composition and function of these tissues in shrews of different ages, and the extent and type of inflammatory reactions observed in response to natural parasitic infections. All ages seemed able to mount systemic, specific immune responses, but adult shrews showed some signs of lymphatic tissue exhaustion: lymphatic follicles in adults (n = 21) were both smaller than those in sub-adults (n = 18; Wald = 11.1, p<0.05) and exhibited greater levels of depletion (Wald = 13.3, p<0.05). CONCLUSIONS/SIGNIFICANCE: Contrary to our expectations, shrews respond effectively to their natural parasites, and show little indication of immunosenescence as adults. The pancreas of Aselli, a unique lymphoid organ, may aid in providing efficient immune responses through the storage of large numbers of plasma cells. This may allow older animals to react effectively to previously encountered parasites, but infection by novel agents, and eventual depletion of plasma cell reserves, could both still be factors in the near-synchronous mortality of adult shrews observed shortly after breeding. | [
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] |
High affinity antigen-specific T cells play a critical role during protective immune responses. Epitope enhancement can elicit more potent T cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. We used the consensus peptide-binding motif for the Major Histocompatibility Complex molecule H-2K(b) to design a heteroclitic version of the mouse hepatitis virus-specific subdominant S598 determinant. We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2K(b). The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution. Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice. Collectively, our findings provide a basis for the enhanced immunogenicity of an engineered determinant that will serve as a template for guiding the development of heteroclitic T cell determinants with applications in prevention of CTL escape in chronic viral infections as well as in tumor immunity. | [
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] |
BACKGROUND AND AIMS: Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses. METHODS: Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry. RESULTS: HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4(+) T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8(+) and CD4(+) T-cells producing IFN-γ and TNF-α were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8(+) T-cells produced more of the fibrogenic cytokine, TNF-α. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4(+) T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses. CONCLUSION: The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease. | [
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INTRODUCTION: Hemophagocytic syndrome (HFS) is a potentially lethal disorder due to an uncontrolled immune response to a triggering agent. Our objective is to raise the importance of HFS early diagnosis by presenting a representative case. CASE PRESENTATION: A sixteen-year-old girl with Still disease diagnosis developed a progressive multiorgan failure including acute respiratory distress (ARDS), anemia and thrombopenia, elevated liver enzymes, renal failure, coagulopathy with hypofibrinogenemia, and acute phase reactants elevation despite broad-spectrum antibiotics. A bone marrow puncture-biopsy was performed, and hemophagocytosis was found. Prolonged fever, splenomegaly, bicytopenia, hypofibrinogenemia, hyperferritinemia and hypertriglyceridemia confirmed HFS diagnosis. She received intensive care support therapy including mechanical ventilation and specific therapy according to HLH 2004 protocol, with a very good response. CONCLUSION: Our case shows complexity of HFS diagnosis, due to septic shock-like manifestations. Early diagnosis is essential to start appropriate treatment achieving a better outcome. | [
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] |
Copy number variations (CNVs) in the human genome are conventionally detected using high-throughput scanning technologies, such as comparative genomic hybridization and high-density single nucleotide polymorphism (SNP) microarrays, or relatively low-throughput techniques, such as quantitative polymerase chain reaction (PCR). All these approaches are limited in resolution and can at best distinguish a twofold (or 50%) difference in copy number. We have developed a new technology to study copy numbers using a platform known as the digital array, a nanofluidic biochip capable of accurately quantitating genes of interest in DNA samples. We have evaluated the digital array's performance using a model system, to show that this technology is exquisitely sensitive, capable of differentiating as little as a 15% difference in gene copy number (or between 6 and 7 copies of a target gene). We have also analyzed commercial DNA samples for their CYP2D6 copy numbers and confirmed that our results were consistent with those obtained independently using conventional techniques. In a screening experiment with breast cancer and normal DNA samples, the ERBB2 gene was found to be amplified in about 35% of breast cancer samples. The use of the digital array enables accurate measurement of gene copy numbers and is of significant value in CNV studies. | [
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BACKGROUND: Hospital preparedness is critical for the early detection and management of public health emergency (PHE). Understanding the current status of PHE preparedness is the first step in planning to enhance hospitals' capacities for emergency response. The objective of this study is to understand the current status of hospital PHE preparedness in China. METHODS: Four hundred hospitals in four city and provinces of China were surveyed using a standardized questionnaire. Data related to hospital demographic data; PHE preparation; response to PHE in community; stockpiles of drugs and materials; detection and identification of PHE; procedures for medical treatment; laboratory diagnosis and management; staff training; and risk communication were collected and analyzed. RESULTS: Valid responses were received from 318 (79.5%) of the 400 hospitals surveyed. Of the valid responses, 264 (85.2%) hospitals had emergency plans; 93.3% had command centres and personnel for PHE; 22.9% included community organisations during the training for PHE; 97.4% could transport needed medical staff to a PHE; 53.1% had evaluated stockpiles of drugs; 61.5% had evaluated their supply systems; 55.5% had developed surveillance systems; and 74.6% could monitor the abnormity(See in appendix). Physicians in 80.2% of the analyzed hospitals reported up-to-date knowledge of their institution's PHE protocol. Of the 318 respondents, 97.4% followed strict laboratory regulations, however, only about 33.5% had protocols for suspected samples. Furthermore, only 59.0% could isolate and identify salmonella and staphylococcus and less than 5% could isolate and identify human H5N1 avian flu and SARS. Staff training or drill programs were reported in 94.5% of the institutions; 50.3% periodically assessed the efficacy of staff training; 45% had experts to provide psychological counselling; 12.1% had provided training for their medical staff to assess PHE-related stress. All of the above capacities related to the demographic characteristics of hospitals and will be discussed in-depth in this paper. CONCLUSION: Our survey suggested that, at the time of the survey, hospital preparedness for PHE in China was at an early stage of development. Comprehensive measures should be taken to enhance hospital capacity in the prevention and management of PHE. | [
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] |
BACKGROUND: Female endoparasitic ichneumonid wasps inject virus-like particles into their caterpillar hosts to suppress immunity. These particles are classified as ichnovirus virions and resemble ascovirus virions, which are also transmitted by parasitic wasps and attack caterpillars. Ascoviruses replicate DNA and produce virions. Polydnavirus DNA consists of wasp DNA replicated by the wasp from its genome, which also directs particle synthesis. Structural similarities between ascovirus and ichnovirus particles and the biology of their transmission suggest that ichnoviruses evolved from ascoviruses, although molecular evidence for this hypothesis is lacking. RESULTS: Here we show that a family of unique pox-D5 NTPase proteins in the Glypta fumiferanae ichnovirus are related to three Diadromus pulchellus ascovirus proteins encoded by ORFs 90, 91 and 93. A new alignment technique also shows that two proteins from a related ichnovirus are orthologs of other ascovirus virion proteins. CONCLUSION: Our results provide molecular evidence supporting the origin of ichnoviruses from ascoviruses by lateral transfer of ascoviral genes into ichneumonid wasp genomes, perhaps the first example of symbiogenesis between large DNA viruses and eukaryotic organisms. We also discuss the limits of this evidence through complementary studies, which revealed that passive lateral transfer of viral genes among polydnaviral, bacterial, and wasp genomes may have occurred repeatedly through an intimate coupling of both recombination and replication of viral genomes during evolution. The impact of passive lateral transfers on evolutionary relationships between polydnaviruses and viruses with large double-stranded genomes is considered in the context of the theory of symbiogenesis. | [
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Pre-existing immunity to human adenovirus serotype 5 (Ad5) is common in the general population. Bypassing pre-existing immunity could maximize Ad5 vaccine efficacy. Vaccination by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route with Ad5 expressing Ebola Zaire glycoprotein (Ad5-ZGP) fully protected naïve mice against lethal challenge with Ebola. In the presence of pre-existing immunity, only mice vaccinated I.N. survived. The frequency of IFN-γ+ CD8+ T cells was reduced by 80% and by 15% in animals vaccinated by the I.M. and P.O. routes respectively. Neutralizing antibodies could not be detected in serum from either treatment group. Pre-existing immunity did not compromise the frequency of IFN-γ+ CD8+ T cells (3.9±1% naïve vs. 3.6±1% pre-existing immunity, PEI) nor anti-Ebola neutralizing antibody (NAB, 40±10 reciprocal dilution, both groups). The number of INF-γ+ CD8+ cells detected in bronchioalveolar lavage fluid (BAL) after I.N. immunization was not compromised by pre-existing immunity to Ad5 (146±14, naïve vs. 120±16 SFC/million MNCs, PEI). However, pre-existing immunity reduced NAB levels in BAL by ∼25% in this group. To improve the immune response after oral vaccination, the Ad5-based vaccine was PEGylated. Mice given the modified vaccine did not survive challenge and had reduced levels of IFN-γ+ CD8+ T cells 10 days after administration (0.3±0.3% PEG vs. 1.7±0.5% unmodified). PEGylation did increase NAB levels 2-fold. These results provide some insight about the degree of T and B cell mediated immunity necessary for protection against Ebola virus and suggest that modification of the virus capsid can influence the type of immune response elicited by an Ad5-based vaccine. | [
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Acanthamoeba polyphaga mimivirus (APM), a virus of free-living amebae, has reportedly caused human respiratory disease. Using 2 newly developed real-time PCR assays, we screened 496 respiratory specimens from 9 pneumonia-patient populations for APM. This virus was not detected in any specimen, which suggests it is not a common respiratory pathogen. | [
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|
BACKGROUND: In 2007 Australia experienced its first outbreak of highly infectious equine influenza. Government disease control measures were put in place to control, contain, and eradicate the disease; these measures included movement restrictions and quarantining of properties. This study was conducted to assess the psycho-social impacts of this disease, and this paper reports the prevalence of, and factors influencing, psychological distress during this outbreak. METHODS: Data were collected using an online survey, with a link directed to the affected population via a number of industry groups. Psychological distress, as determined by the Kessler 10 Psychological Distress Scale, was the main outcome measure. RESULTS: In total, 2760 people participated in this study. Extremely high levels of non-specific psychological distress were reported by respondents in this study, with 34% reporting high psychological distress (K10 > 22), compared to levels of around 12% in the Australian general population. Analysis, using backward stepwise binary logistic regression analysis, revealed that those living in high risk infection (red) zones (OR = 2.00; 95% CI: 1.57–2.55; p < 0.001) and disease buffer (amber) zones (OR = 1.83; 95% CI: 1.36–2.46; p < 0.001) were at much greater risk of high psychological distress than those living in uninfected (white zones). Although prevalence of high psychological distress was greater in infected EI zones and States, elevated levels of psychological distress were experienced in horse-owners nationally. Statistical analysis indicated that certain groups were more vulnerable to high psychological distress; specifically younger people, and those with lower levels of formal educational qualifications. Respondents whose principal source of income was from horse-related industry were more than twice as likely to have high psychological distress than those whose primary source of income was not linked to horse-related industry (OR = 2.23; 95% CI: 1.82–2.73; p < 0.001). CONCLUSION: Although, methodologically, this study had good internal validity, it has limited generalisability because it was not possible to identify, bound, or sample the target population accurately. However, this study is the first to collect psychological distress data from an affected population during such a disease outbreak and has potential to inform those involved in assessing the potential psychological impacts of human infectious diseases, such as pandemic influenza. | [
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The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNβ gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNβ gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNα/β, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5′ triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-κB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response. | [
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Genomics was essential for identifying the source of the deadly anthrax strain released after the September 11 terrorist attacks in the US. The same research that is needed to combat low-probability bioterror attacks is needed to combat high-probability natural infectious agents. | [
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The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR(−/−) mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease. | [
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] |
BACKGROUND: The hemorrhagic shock and encephalopathy syndrome (HSES) is a devastating disease that affects young children. The outcomes of HSES patients are often fatal or manifesting severe neurological sequelae. We reviewed the markers for an early diagnosis of HSES. METHODS: We examined the clinical, biological and radiological findings of 8 patients (4 months to 9 years old) who met the HSES criteria. RESULTS: Although cerebral edema, disseminated intravascular coagulopathy (DIC), and multiple organ failure were seen in all 8 cases during their clinical courses, brain computed tomography (CT) scans showed normal or only slight edema in 5 patients upon admission. All 8 patients had normal platelet counts, and none were in shock. However, they all had severe metabolic acidosis, which persisted even after 3 hours (median base excess (BE), -7.6 mmol/L). And at 6 hours after admission (BE, -5.7 mmol/L) they required mechanical ventilation. Within 12 hours after admission, fluid resuscitation and vasopressor infusion for hypotension was required. Seven of the patients had elevated liver enzymes and creatine kinase (CK) upon admission. Twenty-four hours after admission, all 8 patients needed vasopressor infusion to maintain blood pressure. CONCLUSION: CT scan, platelet count, hemoglobin level and renal function upon admission are not useful for an early diagnosis of HSES. However, the elevated liver enzymes and CK upon admission, hypotension in the early stage after admission with refractory acid-base disturbance to fluid resuscitation and vasopressor infusion are useful markers for an early HSES diagnosis and helpful to indicate starting intensive neurological treatment. | [
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