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Analysis on the Pathogenesis of Symptomatic Pulmonary Embolism with Human Genomics

BACKGROUND: In the present study, the whole human genome oligo microarray was employed to investigate the gene expression profile in symptomatic pulmonary embolism (PE). METHODS: Twenty patients with PE and 20 age and gender matched patients without PE as controls were enrolled into the present study in the same period. The diagnosis of PE was based on the clinical manifestations and findings on imaging examinations. Acute arterial and/or venous thrombosis was excluded in controls. The whole human genome oligo microarray was employed for detection. Statistical analysis was performed with t test following analysis of very small samples of repeated measurements and Gene Ontology (GO) analysis. RESULTS: Genomic data showed no damage to vascular endothelial cells in PE patients. Genomic data only found increased mRNA expression of a small amount of coagulation factors in PE patients. In the PE group, anticoagulant proteins, Fibrinolytic system and proteins related to platelet functions only played partial roles in the pathogenesis of PE. In addition, the mRNA expressions of a fraction of adhesion molecules were markedly up-regulated. Gene Ontology analysis showed the genes with down-regulated expressions mainly explain the compromised T cell immunity. Symptomatic VTE patients have compromised T cell immunity. CONCLUSION: The damage to vascular endothelial cells is not necessary in the pathogenesis of VTE, and only a fraction of factors involved in the shared coagulation cascade are activated. Genomic results may provide a new clue for clinical diagnosis, treatment and prevention of VTE.

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Diversity and roles of (t)RNA ligases

The discovery of discontiguous tRNA genes triggered studies dissecting the process of tRNA splicing. As a result, we have gained detailed mechanistic knowledge on enzymatic removal of tRNA introns catalyzed by endonuclease and ligase proteins. In addition to the elucidation of tRNA processing, these studies facilitated the discovery of additional functions of RNA ligases such as RNA repair and non-conventional mRNA splicing events. Recently, the identification of a new type of RNA ligases in bacteria, archaea, and humans closed a long-standing gap in the field of tRNA processing. This review summarizes past and recent findings in the field of tRNA splicing with a focus on RNA ligation as it preferentially occurs in archaea and humans. In addition to providing an integrated view of the types and phyletic distribution of RNA ligase proteins known to date, this survey also aims at highlighting known and potential accessory biological functions of RNA ligases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00018-012-0944-2) contains supplementary material, which is available to authorized users.

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ESP Abstracts 2012

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Molecular Imaging Reveals a Progressive Pulmonary Inflammation in Lower Airways in Ferrets Infected with 2009 H1N1 Pandemic Influenza Virus

Molecular imaging has gained attention as a possible approach for the study of the progression of inflammation and disease dynamics. Herein we used [(18)F]-2-deoxy-2-fluoro-D-glucose ([(18)F]-FDG) as a radiotracer for PET imaging coupled with CT (FDG-PET/CT) to gain insight into the spatiotemporal progression of the inflammatory response of ferrets infected with a clinical isolate of a pandemic influenza virus, H1N1 (H1N1pdm). The thoracic regions of mock- and H1N1pdm-infected ferrets were imaged prior to infection and at 1, 2, 3 and 6 days post-infection (DPI). On 1 DPI, FDG-PET/CT imaging revealed areas of consolidation in the right caudal lobe which corresponded with elevated [(18)F]-FDG uptake (maximum standardized uptake values (SUVMax), 4.7–7.0). By days 2 and 3, consolidation (CT) and inflammation ([(18)F]-FDG) appeared in the left caudal lobe. By 6 DPI, CT images showed extensive areas of patchy ground-glass opacities (GGO) and consolidations with the largest lesions having high SUVMax (6.0–7.6). Viral shedding and replication were detected in most nasal, throat and rectal swabs and nasal turbinates and lungs on 1, 2 and 3 DPI, but not on day 7, respectively. In conclusion, molecular imaging of infected ferrets revealed a progressive consolidation on CT with corresponding [(18)F]-FDG uptake. Strong positive correlations were measured between SUVMax and bronchiolitis-related pathologic scoring (Spearman’s ρ = 0.75). Importantly, the extensive areas of patchy GGO and consolidation seen on CT in the ferret model at 6 DPI are similar to that reported for human H1N1pdm infections. In summary, these first molecular imaging studies of lower respiratory infection with H1N1pdm show that FDG-PET can give insight into the spatiotemporal progression of the inflammation in real-time.

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The Dynamics, Causes and Possible Prevention of Hepatitis E Outbreaks

Rapidly spreading infectious diseases are a serious risk to public health. The dynamics and the factors causing outbreaks of these diseases can be better understood using mathematical models, which are fit to data. Here we investigate the dynamics of a Hepatitis E outbreak in the Kitgum region of northern Uganda during 2007 to 2009. First, we use the data to determine that [Image: see text] is approximately 2.25 for the outbreak. Secondly, we use a model to estimate that the critical level of latrine and bore hole coverages needed to eradicate the epidemic is at least [Image: see text] and [Image: see text] respectively. Lastly, we further investigate the relationship between the co-infection factor for malaria and Hepatitis E on the value of [Image: see text] for Hepatitis E. Taken together, these results provide us with a better understanding of the dynamics and possible causes of Hepatitis E outbreaks.

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Human Bocaviruses Are Not Significantly Associated with Gastroenteritis: Results of Retesting Archive DNA from a Case Control Study in the UK

Gastroenteritis is a common illness causing considerable morbidity and mortality worldwide. Despite improvements in detection methods, a significant diagnostic gap still remains. Human bocavirus (HBoV)s, which are associated with respiratory infections, have also frequently been detected in stool samples in cases of gastroenteritis, and a tentative association between HBoVs, and in particular type-2 HBoVs, and gastroenteritis has previously been made. The aim of this study was to determine the role of HBoVs in gastroenteritis, using archived DNA samples from the case-control Infectious Intestinal Disease Study (IID). DNA extracted from stool samples from 2,256 cases and 2,124 controls were tested for the presence of HBoV DNA. All samples were screened in a real time PCR pan-HBoV assay, and positive samples were then tested in genotype 1 to 3-specific assays. HBoV was detected in 7.4% but no significantly different prevalence was observed between cases and controls. In the genotype-specific assays 106 of the 324 HBoV-positive samples were genotyped, with HBoV-1 predominantly found in controls whilst HBoV-2 was more frequently associated with cases of gastroenteritis (p<0.01). A significant proportion of HBoV positives could not be typed using the type specific assays, 67% of the total positives, and this was most likely due to low viral loads being present in the samples. However, the distribution of the untyped HBoV strains was no different between cases and controls. In conclusion, HBoVs, including HBoV-2 do not appear to be a significant cause of gastroenteritis in the UK population.

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Structural Origins for the Loss of Catalytic Activities of Bifunctional Human LTA4H Revealed through Molecular Dynamics Simulations

Human leukotriene A4 hydrolase (hLTA4H), which is the final and rate-limiting enzyme of arachidonic acid pathway, converts the unstable epoxide LTA4 to a proinflammatory lipid mediator LTB4 through its hydrolase function. The LTA4H is a bi-functional enzyme that also exhibits aminopeptidase activity with a preference over arginyl tripeptides. Various mutations including E271Q, R563A, and K565A have completely or partially abolished both the functions of this enzyme. The crystal structures with these mutations have not shown any structural changes to address the loss of functions. Molecular dynamics simulations of LTA4 and tripeptide complex structures with functional mutations were performed to investigate the structural and conformation changes that scripts the observed differences in catalytic functions. The observed protein-ligand hydrogen bonds and distances between the important catalytic components have correlated well with the experimental results. This study also confirms based on the structural observation that E271 is very important for both the functions as it holds the catalytic metal ion at its location for the catalysis and it also acts as N-terminal recognition residue during peptide binding. The comparison of binding modes of substrates revealed the structural changes explaining the importance of R563 and K565 residues and the required alignment of substrate at the active site. The results of this study provide valuable information to be utilized in designing potent hLTA4H inhibitors as anti-inflammatory agents.

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Molecular and Microscopic Analysis of Bacteria and Viruses in Exhaled Breath Collected Using a Simple Impaction and Condensing Method

Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. The exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 µL rolling deionized water droplet. The collected EBC was further analyzed using culturing, DNA stain, Scanning Electron Microscope (SEM), polymerase chain reaction (PCR) and colorimetry (VITEK 2) for bacteria and viruses. Experimental data revealed that bacteria and viruses in EBC can be rapidly collected using the method developed here, with an observed efficiency of 100 µL EBC within 1 min. Culturing, DNA stain, SEM, and qPCR methods all detected high bacterial concentrations up to 7000 CFU/m(3) in exhaled breath, including both viable and dead cells of various types. Sphingomonas paucimobilis and Kocuria variants were found dominant in EBC samples using VITEK 2 system. SEM images revealed that most bacteria in exhaled breath are detected in the size range of 0.5–1.0 µm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. Using qPCR, influenza A H3N2 viruses were also detected in one EBC sample. Different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix.

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Seasonal influenza vaccination knowledge, risk perception, health beliefs and vaccination behaviours of nurses

The relationship between knowledge, risk perceptions, health belief towards seasonal influenza and vaccination and the vaccination behaviours of nurses was explored. Qualified nurses attending continuing professional education courses at a large London university between 18 April and 18 October 2010 were surveyed (522/672; response rate 77·7%). Of these, 82·6% worked in hospitals; 37·0% reported receiving seasonal influenza vaccination in the previous season and 44·9% reported never being vaccinated during the last 5 years. All respondents were categorized using two-step cluster analyses into never, occasionally, and continuously vaccinated groups. Nurses vaccinated the season before had higher scores of knowledge and risk perception compared to the unvaccinated (P<0·001). Nurses never vaccinated had the lowest scores of knowledge and risk perception compared to other groups (P<0·001). Nurses' seasonal influenza vaccination behaviours are complex. Knowledge and risk perception predict uptake of vaccination in nurses.

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IFN-γ Signaling to Astrocytes Protects from Autoimmune Mediated Neurological Disability

Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS). Cells resident within the central nervous system (CNS) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB) integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors.

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Influence of Mabs on PrP(Sc) Formation Using In Vitro and Cell-Free Systems

PrP(Sc) is believed to serve as a template for the conversion of PrP(C) to the abnormal isoform. This process requires contact between the two proteins and implies that there may be critical contact sites that are important for conversion. We hypothesized that antibodies binding to either PrP(c)or PrP(Sc) would hinder or prevent the formation of the PrP(C)–PrP(Sc) complex and thus slow down or prevent the conversion process. Two systems were used to analyze the effect of different antibodies on PrP(Sc) formation: (i) neuroblastoma cells persistently infected with the 22L mouse-adapted scrapie stain, and (ii) protein misfolding cyclic amplification (PMCA), which uses PrP(Sc) as a template or seed, and a series of incubations and sonications, to convert PrP(C) to PrP(Sc). The two systems yielded similar results, in most cases, and demonstrate that PrP-specific monoclonal antibodies (Mabs) vary in their ability to inhibit the PrP(C)–PrP(Sc) conversion process. Based on the numerous and varied Mabs analyzed, the inhibitory effect does not appear to be epitope specific, related to PrP(C) conformation, or to cell membrane localization, but is influenced by the targeted PrP region (amino vs carboxy).

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Predictors and outcome of patients with acute respiratory distress syndrome caused by miliary tuberculosis: a retrospective study in Chongqing, China

BACKGROUND: Miliary tuberculosis (TB) is an uncommon cause of acute respiratory distress syndrome (ARDS) with a high mortality. The aim of the present study was to evaluate the clinical characteristics, predictors and outcome of patients with ARDS caused by miliary TB. METHODS: A retrospective study was conducted among patients with a diagnosis of ARDS with miliary TB in four hospitals from 2006 to 2010. Medical records and laboratory examinations of these patients were taken during the first 24 h of admission. RESULTS: Eighty-five patients with miliary TB developed ARDS, 45 of whom survived (52.9%). The median age was 36.6 ± 12.5 years with 38 males (44.7%). Diabetes mellitus (DM) was the most common underlying disease (18.8%).ICU mortality was 47.1%. The time from admission to anti-tuberculosis therapy was 4.5 ± 2.0 days. Mean duration of mechanical ventilation was 8.5 ± 3.0 days in all patients. Duration of time to diagnosis, time from diagnosis to mechanical ventilation, and time to anti-tuberculosis therapy were significantly shorter in survivors than those in non-survivors. Diabetes mellitus (OR 5.431, 95%CI 1.471-20.049; P = 0.005), ALT (70-100U/L, OR 10.029, 95%CI 2.764-36.389; P = 0.001), AST (>94U/L,OR 8.034, 95%CI 2.200-29.341; P = 0.002), D-dimer (>1.6mg/L, OR 3.167, 95%CI 0.896-11.187; P = 0.042), hemoglobin (<90g/L, OR 14.824, 95%CI 3.713-59.179; P = 0.001), albumin (<25g/L, OR 15.896, 95%CI 3.975-63.566; P = 0.001) were independent predictors of ARDS development in the setting of miliary TB. CONCLUSIONS: Accurate diagnosis, early initiation of anti-tuberculosis therapy and mechanical ventilation are important for the outcome of patients with ARDS caused by miliary TB. DM, ALT, AST, D-dimer, hemoglobin, and albumin are independent predictors of ARDS development in patients with miliary TB.

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Symptomatic Venous Thromboembolism Is a Disease Related to Infection and Immune Dysfunction

The characteristics of human genomics and cellular immune function between clinically symptomatic venous thromboembolism (VTE) and controls were systematically compared to explore the immunologic pathogenesis of VTE. Microarray assay showed the mRNA expressions of genes related to non-specific cellarer immune and cytokines were significantly down-regulated. Abnormal expressions of CD3+, CD4+, CD8+, NK marker CD16+56+, CD19 and aberrant CD4+/CD8+ ratio were detected in 54 among 56 patients. In PE patients, microarray assay revealed the imbalance in the expressions of genes related to the immune system. The expressions of genes related to non-specific immune cells and cytokines were markedly up-regulated and those associated with cellular immune were dramatically down-regulated. In VTE patients, cytological examination indicated the functions of NK cells were significantly compromised, and the antigen recognition and killing function of T cells markedly decreased. The consistence between genomic and cytological examination suggests the symptomatic VTE is closely associated with the infection and immune dysfunction.

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Cedar Virus: A Novel Henipavirus Isolated from Australian Bats

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.

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Host Modulators of H1N1 Cytopathogenicity

Influenza A virus infects 5–20% of the population annually, resulting in ∼35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here we employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human bronchial epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to an expanding body of studies directed at decoding vulnerabilities in the command and control networks specified by influenza virulence factors.

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Association of Fcγ Receptor IIB Polymorphism with Cryptococcal Meningitis in HIV-Uninfected Chinese Patients

BACKGROUND: As important regulators of the immune system, the human Fcγ receptors (FcγRs) have been demonstrated to play important roles in the pathogenesis of various infectious diseases. The aim of the present study was to identify the association between FCGR polymorphisms and cryptococcal meningitis. METHODOLOGY/PRINCIPAL FINDINGS: In this case control genetic association study, we genotyped four functional polymorphisms in low-affinity FcγRs, including FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA1/NA2, and FCGR2B 232I/T, in 117 patients with cryptococcal meningitis and 190 healthy controls by multiplex SNaPshot technology. Among the 117 patients with cryptococcal meningitis, 59 had predisposing factors. In patients with cryptococcal meningitis, the FCGR2B 232I/I genotype was over-presented (OR = 1.652, 95% CI [1.02–2.67]; P = 0.039) and the FCGR2B 232I/T genotype was under-presented (OR = 0.542, 95% CI [0.33–0.90]; P = 0.016) in comparison with control group. In cryptococcal meningitis patients without predisposing factors, FCGR2B 232I/I genotype was also more frequently detected (OR = 1.958, 95% CI [1.05–3.66]; P = 0.033), and the FCGR2B 232I/T genotype was also less frequently detected (OR = 0.467, 95% CI [0.24–0.91]; P = 0.023) than in controls. No significant difference was found among FCGR2A 131H/R, FCGR3A 158F/V, and FCGR3B NA1/NA2 genotype frequencies between patients and controls. CONCLUSION/SIGNIFICANCE: We found for the first time associations between cryptococcal meningitis and FCGR2B 232I/T genotypes, which suggested that FcγRIIB might play an important role in the central nervous system infection by Cryptococcus in HIV-uninfected individuals.

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Infectious Disease Transmission during Organ and Tissue Transplantation

Infectious disease transmission through organ and tissue transplantation has been associated with severe complications in recipients. Determination of donor-derived infectious risk associated with organ and tissue transplantation is challenging and limited by availability and performance characteristics of current donor epidemiologic screening (e.g., questionnaire) and laboratory testing tools. Common methods and standards for evaluating potential donors of organs and tissues are needed to facilitate effective data collection for assessing the risk for infectious disease transmission. Research programs can use advanced microbiological technologies to define infectious risks posed by pathogens that are known to be transplant transmissible and provide insights into transmission potential of emerging infectious diseases for which transmission characteristics are unknown. Key research needs are explored. Stakeholder collaboration for surveillance and research infrastructure is required to enhance transplant safety.

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Non-HIV Pneumocystis pneumonia: do conventional community-acquired pneumonia guidelines under estimate its severity?

BACKGROUND: Non-HIV Pneumocystis pneumonia (PCP) can occur in immunosuppressed patients having malignancy or on immunosuppressive agents. To classify severity, the A-DROP scale proposed by the Japanese Respiratory Society (JRS), the CURB-65 score of the British Respiratory Society (BTS) and the Pneumonia Severity Index (PSI) of the Infectious Diseases Society of America (IDSA) are widely used in patients with community-acquired pneumonia (CAP) in Japan. To evaluate how correctly these conventional prognostic guidelines for CAP reflect the severity of non-HIV PCP, we retrospectively analyzed 21 patients with non-HIV PCP. METHODS: A total of 21 patients were diagnosed by conventional staining and polymerase chain reaction (PCR) for respiratory samples with chest x-ray and computed tomography (CT) findings. We compared the severity of 21 patients with PCP classified by A-DROP, CURB-65, and PSI. Also, patients’ characteristics, clinical pictures, laboratory results at first visit or admission and intervals from diagnosis to start of specific-PCP therapy were evaluated in both survivor and non-survivor groups. RESULTS: Based on A-DROP, 18 patients were classified as mild or moderate; respiratory failure developed in 15 of these 18 (83.3%), and 7/15 (46.7%) died. Based on CURB-65, 19 patients were classified as mild or moderate; respiratory failure developed in 16/19 (84.2%), and 8 of the 16 (50%) died. In contrast, PSI classified 14 as severe or extremely severe; all of the 14 (100%) developed respiratory failure and 8/14 (57.1%) died. There were no significant differences in laboratory results in these groups. The time between the initial visit and diagnosis, and the time between the initial visit and starting of specific-PCP therapy were statistically shorter in the survivor group than in the non-survivor group. CONCLUSIONS: Conventional prognostic guidelines for CAP could underestimate the severity of non-HIV PCP, resulting in a therapeutic delay resulting in high mortality. The most important factor to improve the mortality of non-HIV PCP is early diagnosis and starting of specific-PCP therapy as soon as possible.

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Proteomic Investigation of Falciparum and Vivax Malaria for Identification of Surrogate Protein Markers

This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM) (n = 20), vivax malaria (VM) (n = 17) and healthy controls (HC) (n = 20) were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC). Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05) serum proteins were identified in FM and VM respectively, and almost half (46.2%) of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC) were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC) curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates the potential of this analytical approach for the detection of malaria as well as other human diseases.

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Novel Method for Isolation of Murine Clara Cell Secretory Protein-Expressing Cells with Traces of Stemness

Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+) cells. Moreover, CCSP(+) cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(−)expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP(+) cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

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Development of Real-Time PCR Array for Simultaneous Detection of Eight Human Blood-Borne Viral Pathogens

BACKGROUND: Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. FINDINGS: We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100–1,000 geq/ml of plasma of HIV-1 subtypes (A – G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction. CONCLUSIONS: The viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development.

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TcdC Does Not Significantly Repress Toxin Expression in Clostridium difficile 630ΔErm

In the past decade, Clostridium difficile has emerged as an important gut pathogen. Symptoms of C. difficile infection range from mild diarrhea to pseudomembranous colitis, sometimes resulting in colectomy or death. The main virulence factors of C. difficile are toxin A and toxin B. Besides the genes encoding these toxins (tcdA and tcdB), the pathogenicity locus (PaLoc) also contains genes encoding a sigma factor (tcdR) and a putative anti-sigma factor (tcdC). The important role of TcdR as a sigma factor for toxin expression is undisputed, whereas the role of TcdC as an anti-sigma factor, inhibiting toxin expression, is currently the subject of debate. To clarify the role of TcdC in toxin expression, we generated an isogenic ClosTron-based mutant of tcdC in Clostridium difficile strain 630Δ Erm (CT::tcdC) and determined the transcription levels of the PaLoc genes and the expression levels of the toxins in the wild type strain and the tcdC mutant strain. We found only minor differences in transcription levels of the PaLoc genes between the wild type and CT::tcdC strains and total toxin levels did not significantly differ either. These results suggest that in C. difficile 630Δerm TcdC is not a major regulator of toxin expression under the conditions tested.

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Childhood Tuberculosis Presenting with Haemophagocytic Syndrome

Haemophagocytic syndrome is a life threatening complication of systemic infection resulting from an exaggerated immune response to a triggering agent. Prompt recognition and treatment of this disorder can abrogate otherwise high fatality associated with this disorder. A 2 year old girl presented with acute enteritis, developed prolonged fever and organomegaly complicated by multi-organ failure. She fulfilled the diagnostic criteria for haemophagocytic lymphohistiocytosis including bone marrow evidence of haemophagocytosis. In addition she had serological evidence of tubercular infection as well as a positive family history of tuberculosis. She responded rapidly to immunosuppressive therapy and anti-tubercular therapy. Our case illustrates the association of haemophagocytic syndrome with tuberculosis as well as the favourable response obtained with prompt diagnosis and treatment.

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Activation of the Canonical Bone Morphogenetic Protein (BMP) Pathway during Lung Morphogenesis and Adult Lung Tissue Repair

Signaling by Bone Morphogenetic Proteins (BMP) has been implicated in early lung development, adult lung homeostasis and tissue-injury repair. However, the precise mechanism of action and the spatio-temporal pattern of BMP-signaling during these processes remains inadequately described. To address this, we have utilized a transgenic line harboring a BMP-responsive eGFP-reporter allele (BRE-eGFP) to construct the first detailed spatiotemporal map of canonical BMP-pathway activation during lung development, homeostasis and adult-lung injury repair. We demonstrate that during the pseudoglandular stage, when branching morphogenesis progresses in the developing lung, canonical BMP-pathway is active mainly in the vascular network and the sub-epithelial smooth muscle layer of the proximal airways. Activation of the BMP-pathway becomes evident in epithelial compartments only after embryonic day (E) 14.5 primarily in cells negative for epithelial-lineage markers, located in the proximal portion of the airway-tree, clusters adjacent to neuro-epithelial-bodies (NEBs) and in a substantial portion of alveolar epithelial cells. The pathway becomes activated in isolated E12.5 mesenchyme-free distal epithelial buds cultured in Matrigel suggesting that absence of reporter activity in these regions stems from a dynamic cross-talk between endoderm and mesenchyme. Epithelial cells with activated BMP-pathway are enriched in progenitors capable of forming colonies in three-dimensional Matrigel cultures. As lung morphogenesis approaches completion, eGFP-expression declines and in adult lung its expression is barely detectable. However, upon tissue-injury, either with naphthalene or bleomycin, the canonical BMP-pathways is re-activated, in bronchial or alveolar epithelial cells respectively, in a manner reminiscent to early lung development and in tissue areas where reparatory progenitor cells reside. Our studies illustrate the dynamic activation of canonical BMP-pathway during lung development and adult lung tissue-repair and highlight its involvement in two important processes, namely, the early development of the pulmonary vasculature and the management of epithelial progenitor pools both during lung development and repair of adult lung tissue-injury.

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Pulmonary sequelae in a patient recovered from swine flu

The pandemic of swine flu (H1N1) influenza spread to involve the whole world rapidly. Many patients manifested a mild clinical illness but some developed pneumonia and respiratory failure. High mortality was observed in patients with severe disease. Among survivors, studies are limited. Ground-glass opacities on a high-resolution computerized tomography scan and reduced diffusion capacity were noted after 3 months in a study. But long-term complications in patients with swine flu pneumonia have not been studied well. We are presenting an unusual case of swine flu pneumonia who developed interstitial lung disease after recovery.

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Dengue Virus Serotype 2 Blocks Extracellular Signal-Regulated Kinase and Nuclear Factor-κB Activation to Downregulate Cytokine Production

BACKGROUND: Dengue virus (DENV) infection is the most common mosquito-borne viral disease threatening human health around the world. Type I interferon (IFN) and cytokine production are crucial in the innate immune system. We previously reported that DENV serotype 2 (DENV-2) induced low levels of interferon regulatory factor 3 and NF-κB activation, thus leading to reduced production of IFN-β in the early phase of infection. Here, we determined whether DENV infection not only hampers type I IFN activation but also cytokine production triggered by Toll-like receptor (TLR) signaling. METHODOLOGY/PRINCIPAL FINDINGS: We used quantitative RT-PCR and found that only low levels of IFN-β and inflammatory cytokines such as interleukin 10 (IL-10), IL-12 and tumor necrosis factor α (TNFα) mRNA were detected in DENV-2–infected bone-marrow–derived dendritic cells. Furthermore, DENV-2 infection repressed cytokine production triggered by TLR signaling. To elucidate the molecular mechanisms underlying this suppression event, we measured NF-κB activation by p65 nuclear translocation and luciferase reporter assay and found that NF-κB activation triggered by TLR ligands was blocked by DENV-2 infection. As well, extracellular signal-regulated kinase (ERK) activity was suppressed by DENV-2 infection. CONCLUSIONS/SIGNIFICANCE: To downregulate the host innate immunity, DENV-2 by itself is a weak inducer of type I IFN and cytokines, furthermore DENV-2 can also block the TLR-triggered ERK–NF-κB activation and cytokine production.

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High Influenza A Virus Infection Rates in Mallards Bred for Hunting in the Camargue, South of France

During the last decade, the role of wildlife in emerging pathogen transmission to domestic animals has often been pointed out. Conversely, far less attention has been paid to pathogen transmission from domestic animals to wildlife. Here, we focus on the case of game restocking, which implies the release of millions of animals worldwide each year. We conducted a 2-year study in the Camargue (Southern France) to investigate the influence of hand-reared Mallard releases on avian influenza virus dynamics in surrounding wildlife. We sampled Mallards (cloacal swabs) from several game duck facilities in 2009 and 2010 before their release. A very high (99%) infection rate caused by an H10N7 strain was detected in the game bird facility we sampled in 2009. We did not detect this strain in shot ducks we sampled, neither during the 2008/2009 nor the 2009/2010 hunting seasons. In 2010 infection rates ranged from 0 to 24% in hand-reared ducks. The 2009 H10N7 strain was fully sequenced. It results from multiple reassortment events between Eurasian low pathogenic strains. Interestingly, H10N7 strains had previously caused human infections in Egypt and Australia. The H10 and N7 segments we sequenced were clearly distinct from the Australian ones but they belonged to the same large cluster as the Egyptian ones. We did not observe any mutation linked to increased virulence, transmission to mammals, or antiviral resistance in the H10N7 strain we identified. Our results indicate that the potential role of hand-reared Mallards in influenza virus epizootics must be taken into account given the likely risk of viral exchange between game bird facilities and wild habitats, owing to duck rearing conditions. Measures implemented to limit transmission from wildlife to domestic animals as well as measures to control transmission from domestic animals to wild ones need to be equally reinforced.

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Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia

Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis.

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Personal, Occupational, and Public Health Perspectives on Dealing with the First Case of Influenza A (H1N1) in the United Arab Emirates

New epidemics of infectious diseases often involve health care workers. In this short communication we present a case report of a health care professional who became the first case of influenza H1N1 virus to be notified in the United Arab Emirates. There are several issues related to workplace considerations and general public health, including preventive measures, the need for isolation of the patient, dealing with contacts, return to work, and communication with the workforce.

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Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma

BACKGROUND: Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4). METHODOLOGY/PRINCIPAL FINDINGS: In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or “minibody”) of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4(+) tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G(+) FcγRIIIa(CD16A)(+) murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4(+) tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A(+) CD56(+) NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells. CONCLUSIONS/SIGNIFICANCE: Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A(+) immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL.

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The VNTR Polymorphism of the DC-SIGNR Gene and Susceptibility to HIV-1 Infection: A Meta-Analysis

BACKGROUND: Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin related (DC-SIGNR) can bind to the human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein and is thus important for the host-pathogen interaction in HIV-1 infection. Studies of the association between the variable number tandem repeat (VNTR) polymorphism of the DC-SIGNR gene and HIV-1 susceptibility have produced controversial results. METHODS AND FINDINGS: We conducted a meta-analysis of the data contained in the literature to clarify these findings. In total, 10 studies consisting of 2683 HIV-1 patients and 3263 controls (2130 healthy controls and 1133 HIV-1 exposed but seronegative (HESN) controls) were included. Odds ratios (ORs) with 95% confidence intervals (95% CIs) were assessed in the main analyses. Further stratified analyses by ethnicity and sample size were performed. By dividing the controls into two groups, healthy controls and HIV-1 exposed but seronegative (HESN) controls, we explored different genetic models to detect any association between the VNTR polymorphism and predisposition to HIV-1 infection. The results showed that the 5-repeat allele carriers (OR = 0.84, 95% CI = 0.73–0.96) and the 5/5 homozygous (OR = 0.68, 95% CI = 0.50–0.93) had significantly reduced risk when using the HIV-1 exposed but seronegative (HESN) as controls. The stratified analyses by ethnicity and sample size confirmed these findings. However, a low to moderate degree of heterogeneity was also found across studies. CONCLUSIONS: Our findings demonstrate that the VNTR polymorphism of the DC-SIGNR gene is associated with a moderate effect on host susceptibility to HIV-1 infection. Similar to the 32-bp deletion in the chemokine receptor-5 gene (CCR5Δ32), the DC-SIGNR VNTR 5-repeat allele might have a role in resistance to HIV infection, particularly in Asian populations.

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Toll-like receptors are critical for clearance of Brucella and play different roles in development of adaptive immunity following aerosol challenge in mice

Brucella spp. cause undulant fever in humans and brucellosis in variety of other animals. Both innate and adaptive immunity have been shown to be important in controlling Brucella infection. Toll-like receptors (TLRs) represent a group of pattern recognition receptors (PRRs) that play critical roles in the host innate immune response, as well as development of adaptive immunity. In the current report, we investigated the role of TLR signaling in the clearance of Brucella and development of adaptive immunity in TLR2(−/−), TLR4(−/−), or MyD88(−/−) mice following aerosol exposure to B. melitensis 16 M. Consistent with previous reports, MyD88 is required for efficient clearance of Brucella from all three organs (lung, spleen, and liver). The results reveal Th2-skewed immune responses in TLR2(−/−) mice late in infection and support a TLR2 requirement for efficient clearance of Brucella from the lungs, but not from the spleen or liver. Similarly, TLR4 is required for efficient clearance of Brucella from the lung, but exhibits a minor contribution to clearance from the spleen and no demonstrable contribution to clearance from the liver. Lymphocyte proliferation assays suggest that the TLRs are not involved in the development of cell-mediated memory response to Brucella antigen. Antibody detection reveals that TLR2 and 4 are required to generate early antigen-specific IgG, but not during the late stages of infection. TLR2 and 4 are only transiently required for IgM production and not at all for IgA production. In contrast, MyD88 is essential for antigen specific IgG production late in infection, but is not required for IgM generation over the course of infection. Surprisingly, despite the prominent role for MyD88 in clearance from all tissues, MyD88-knockout mice express significantly higher levels of serum IgA. These results confirm the important role of MyD88 in controlling infection in the spleen while providing evidence of a prominent contribution to protection in other tissues. In addition, although TLR4 and TLR2 contribute little to control of spleen infection, a significant contribution to clearance of lung infection is described.

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Diagnostic Devices for Isothermal Nucleic Acid Amplification

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

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Clinical characteristics of pediatric hospitalizations associated with 2009 pandemic influenza A (H1N1) in Northern Bavaria, Germany

BACKGROUND: The 2009 pandemic influenza A (H1N1) (PIA) virus infected large parts of the pediatric population with a wide clinical spectrum and an initially unknown complication rate. The aims of our study were to define clinical characteristics and outcome of pandemic influenza A (H1N1) 2009-associated hospitalizations (PIAH) in children <18 years of age. All hospitalized cases of children <18 years of age with laboratory-confirmed pandemic influenza A (H1N1) 2009 in the region of Wuerzburg (Northern Bavaria, Germany) between July 2009 and March 2010 were identified. For these children a medical chart review was performed to determine their clinical characteristics and complications. RESULTS: Between July 2009 and March 2010, 94 PIAH (62% males) occurred in children <18 years of age, with a median age of 7 years (IQR: 3–12 years). Underlying diseases and predisposing factors were documented in 40 (43%) children; obesity (n = 12, 30%), asthma (n = 10, 25%) and neurologic disorders (n = 8, 20%) were most frequently reported. Sixteen (17%) children received oxygen supplementation; three (3%) children required mechanical ventilation. Six (6%) children were admitted to an intensive care unit, four of them with underlying chronic diseases. CONCLUSIONS: Most PIAH demonstrated a benign course of disease. However, six children (6%) needed treatment at an intensive care unit for severe complications.

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Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment

Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.

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Clinical characteristics and outcome of Penicillium marneffei infection among HIV-infected patients in northern Vietnam

OBJECTIVE: This study reports the clinical characteristics and outcome of HIV-associated Penicilliummarneffei infection in northern Vietnam. METHODS: We conducted a retrospective chart review of all patients with laboratory confirmed Penicilliummarneffei infection admitted to the National Hospital for Tropical Diseases in Hanoi, Vietnam, between July 2006 and September 2009. RESULTS: 127 patients with P. marneffei infection were identified. All were HIV-infected; median CD4+ T-cell count was 24 cells/μl (IQR:12-48); 76% were men. Common clinical features were fever (92.9%), skin lesions (82.6%), hepatomegaly (61.4%), lymphadenopathy (40.2%), weight loss (59.1%) and cough (49.6%). Concurrent opportunistic infections were present in 22.0%; half of those had tuberculosis. Initial treatment regimens were: itraconazole or ketoconazole capsule (77.2%), amphotericin B (20.5%), and fluconazole (1.6%). In-hospital mortality was 12.6% and showed no significant difference in patients treated with itraconazole (or ketoconazole) and amphotericin B (p = 0.43). Dyspnea, ascites, and increased LDH level were independent predictors of mortality. No seasonality was observed. CONCLUSION: The clinical features, treatments and outcomes of HIV-associated P. marneffei infection in northern Vietnam are similar to those reported in other endemic regions. Dyspnea was an important predictor of mortality. More patients were treated with itraconazole than amphotericin B and no significant difference in treatment outcome was observed. It would be of clinical value to compare the efficacy of oral itraconazole and amphotericin B in a clinical trial.

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Prospective application of clinician-performed lung ultrasonography during the 2009 H1N1 influenza A pandemic: distinguishing viral from bacterial pneumonia

BACKGROUND: Emergency department visits quadrupled with the initial onset and surge during the 2009 H1N1 influenza pandemic in New York City from April to June 2009. This time period was unique in that >90% of the circulating virus was surveyed to be the novel 2009 H1N1 influenza A according to the New York City Department of Health. We describe our experience using lung ultrasound in a case series of patients with respiratory symptoms requiring chest X-ray during the initial onset and surge of the 2009 H1N1 influenza pandemic. METHODS: We describe a case series of patients from a prospective observational cohort study of lung ultrasound, enrolling patients requiring chest X-ray for suspected pneumonia that coincided with the onset and surge of the 2009 H1N1 influenza pandemic. RESULTS: Twenty pandemic 2009 H1N1 influenza patients requiring chest X-ray were enrolled during this time period. Median age was 6.7 years. Lung ultrasound via modified Bedside Lung Ultrasound in Emergency protocol assisted in the identification of viral pneumonia (n = 15; 75%), viral pneumonia with superimposed bacterial pneumonia (n = 7; 35%), isolated bacterial pneumonia only (n = 1; 5%), and no findings of viral or bacterial pneumonia (n = 4; 20%) in this cohort of patients. Based on 54 observations, interobserver agreement for distinguishing viral from bacterial pneumonia using lung ultrasound was ĸ = 0.82 (0.63 to 0.99). CONCLUSIONS: Lung ultrasound may be used to distinguish viral from bacterial pneumonia. Lung ultrasound may be useful during epidemics or pandemics of acute respiratory illnesses for rapid point-of-care triage and management of patients.

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Serum Levels of Gelatinase Associated Lipocalin as Indicator of the Inflammatory Status in Coronary Artery Disease

Background. Atherosclerosis is a chronic inflammatory disease and the acute clinical manifestations represent acute on chronic inflammation. Neutrophil gelatinase-associated lipocalin (NGAL) is found in the granules of human neutrophils, with many diverse functions. The aim of this study was to evaluate the hypothesis that levels NGAL in blood may reflect the inflammatory process in various stages of coronary artery disease. Methods. We studied 140 patients, with SA 40, UA 35, NSTEMI 40, and STEMI 25, and 20 healthy controls. Serum NGAL was measured upon admission and before coronary angiography. Results. Significant differences were observed in median serum-NGAL(ng/mL) between patients with SA (79.23 (IQR, 37.50–100.32)), when compared with UA (108.00 (68.34–177.59)), NSTEMI (166.49 (109.24–247.20)), and STEMI (178.63 (111.18–305.92)) patients and controls (50.31 (44.30–69.78)) with significant incremental value from SA to STEMI. We observed a positive and significant correlation between serum-NGAL and hs-CRP (spearman coefficient rho = 0.685, P < 0.0001) as well as with neutrophil counts (r = 0.511, P < 0.0001). Conclusions. In patients with coronary artery disease serum levels of NGAL increase and reflect the degree of inflammatory process. In patients with acute coronary syndromes, serum levels of NGAL have high negative predictive value and reflecting the inflammatory status could show the severity of coronary clinical syndrome.

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Activity based protein profiling to detect serine hydrolase alterations in virus infected cells

Activity-based protein profiling (ABPP) is a newly emerging technique that uses active site-directed probes to monitor the functional status of enzymes. Serine hydrolases are one of the largest families of enzymes in mammals. More than 200 serine hydrolases have been identified, but little is known about their specific roles. Serine hydrolases are involved in a variety of physiological functions, including digestion, immune response, blood coagulation, and reproduction. ABPP has been used recently to investigate host–virus interactions and to understand the molecular pathogenesis of virus infections. Monitoring the altered serine hydrolases during viral infection gives insight into the catalytic activity of these enzymes that will help to identify novel targets for diagnostic and therapeutic application. This review presents the usefulness of ABPP in detecting and analyzing functional annotation of host cell serine hydrolases as a result of host–virus interaction.

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Murphy's law—if anything can go wrong, it will: Problems in phage electron microscopy

The quality of bacteriophage electron microscopy appears to be on a downward course since the 1980s. This coincides with the introduction of digital electron microscopes and a general lowering of standards, possibly due to the disappearance of several world-class electron microscopists The most important problem seems to be poor contrast. Positive staining is frequently not recognized as an undesirable artifact. Phage parts, bacterial debris, and aberrant or damaged phage particles may be misdiagnosed as bacterial viruses. Digital electron microscopes often seem to be operated without magnification control because this is difficult and inconvenient. In summary, most phage electron microscopy problems may be attributed to human failure. Journals are a last-ditch defense and have a heavy responsibility in selecting competent reviewers and rejecting, or not, unsatisfactory articles.

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Herbal Products: Benefits, Limits, and Applications in Chronic Liver Disease

Complementary and alternative medicine soughts and encompasses a wide range of approaches; its use begun in ancient China at the time of Xia dynasty and in India during the Vedic period, but thanks to its long-lasting curative effect, easy availability, natural way of healing, and poor side-effects it is gaining importance throughout the world in clinical practice. We conducted a review describing the effects and the limits of using herbal products in chronic liver disease, focusing our attention on those most known, such as quercetin or curcumin. We tried to describe their pharmacokinetics, biological properties, and their beneficial effects (as antioxidant role) in metabolic, alcoholic, and viral hepatitis (considering that oxidative stress is the common pathway of chronic liver diseases of different etiology). The main limit of applicability of CAM comes from the lacking of randomized, placebo-controlled clinical trials giving a real proof of efficacy of those products, so that anecdotal success and personal experience are frequently the driving force for acceptance of CAM in the population.

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Identification of pneumonia and influenza deaths using the death certificate pipeline

BACKGROUND: Death records are a rich source of data, which can be used to assist with public surveillance and/or decision support. However, to use this type of data for such purposes it has to be transformed into a coded format to make it computable. Because the cause of death in the certificates is reported as free text, encoding the data is currently the single largest barrier of using death certificates for surveillance. Therefore, the purpose of this study was to demonstrate the feasibility of using a pipeline, composed of a detection rule and a natural language processor, for the real time encoding of death certificates using the identification of pneumonia and influenza cases as an example and demonstrating that its accuracy is comparable to existing methods. RESULTS: A Death Certificates Pipeline (DCP) was developed to automatically code death certificates and identify pneumonia and influenza cases. The pipeline used MetaMap to code death certificates from the Utah Department of Health for the year 2008. The output of MetaMap was then accessed by detection rules which flagged pneumonia and influenza cases based on the Centers of Disease and Control and Prevention (CDC) case definition. The output from the DCP was compared with the current method used by the CDC and with a keyword search. Recall, precision, positive predictive value and F-measure with respect to the CDC method were calculated for the two other methods considered here. The two different techniques compared here with the CDC method showed the following recall/ precision results: DCP: 0.998/0.98 and keyword searching: 0.96/0.96. The F-measure were 0.99 and 0.96 respectively (DCP and keyword searching). Both the keyword and the DCP can run in interactive form with modest computer resources, but DCP showed superior performance. CONCLUSION: The pipeline proposed here for coding death certificates and the detection of cases is feasible and can be extended to other conditions. This method provides an alternative that allows for coding free-text death certificates in real time that may increase its utilization not only in the public health domain but also for biomedical researchers and developers. TRIAL REGISTRATION: This study did not involved any clinical trials.

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Functional Analysis of Rift Valley Fever Virus NSs Encoding a Partial Truncation

Rift Valley fever virus (RVFV), belongs to genus Phlebovirus of the family Bunyaviridae, causes high rates of abortion and fetal malformation in infected ruminants as well as causing neurological disorders, blindness, or lethal hemorrhagic fever in humans. RVFV is classified as a category A priority pathogen and a select agent in the U.S., and currently there are no therapeutics available for RVF patients. NSs protein, a major virulence factor of RVFV, inhibits host transcription including interferon (IFN)-β mRNA synthesis and promotes degradation of dsRNA-dependent protein kinase (PKR). NSs self-associates at the C-terminus 17 aa., while NSs at aa.210–230 binds to Sin3A-associated protein (SAP30) to inhibit the activation of IFN-β promoter. Thus, we hypothesize that NSs function(s) can be abolished by truncation of specific domains, and co-expression of nonfunctional NSs with intact NSs will result in the attenuation of NSs function by dominant-negative effect. Unexpectedly, we found that RVFV NSs truncated at aa. 6–30, 31–55, 56–80, 81–105, 106–130, 131–155, 156–180, 181–205, 206–230, 231–248 or 249–265 lack functions of IFN–β mRNA synthesis inhibition and degradation of PKR. Truncated NSs were less stable in infected cells, while nuclear localization was inhibited in NSs lacking either of aa.81–105, 106–130, 131–155, 156–180, 181–205, 206–230 or 231–248. Furthermore, none of truncated NSs had exhibited significant dominant-negative functions for NSs-mediated IFN-β suppression or PKR degradation upon co-expression in cells infected with RVFV. We also found that any of truncated NSs except for intact NSs does not interact with RVFV NSs even in the presence of intact C-terminus self-association domain. Our results suggest that conformational integrity of NSs is important for the stability, cellular localization and biological functions of RVFV NSs, and the co-expression of truncated NSs does not exhibit dominant-negative phenotype.

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Predictive and Reactive Distribution of Vaccines and Antivirals during Cross-Regional Pandemic Outbreaks

As recently pointed out by the Institute of Medicine, the existing pandemic mitigation models lack the dynamic decision support capability. We develop a large-scale simulation-driven optimization model for generating dynamic predictive distribution of vaccines and antivirals over a network of regional pandemic outbreaks. The model incorporates measures of morbidity, mortality, and social distancing, translated into the cost of lost productivity and medical expenses. The performance of the strategy is compared to that of the reactive myopic policy, using a sample outbreak in Fla, USA, with an affected population of over four millions. The comparison is implemented at different levels of vaccine and antiviral availability and administration capacity. Sensitivity analysis is performed to assess the impact of variability of some critical factors on policy performance. The model is intended to support public health policy making for effective distribution of limited mitigation resources.

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A Novel Vaccine Using Nanoparticle Platform to Present Immunogenic M2e against Avian Influenza Infection

Using peptide nanoparticle technology, we have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P < 0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI.

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Factors Associated with Increased Risk Perception of Pandemic Influenza in Australia

The aim of this study was to assess factors associated with increased risk perception of pandemic influenza in Australia. The sample consisted of 2081 Australian adults aged 16 years and older who completed a short three item pandemic influenza question module which was incorporated into the NSW Health Adult Population Health Survey during the first quarter of 2007. After adjusting for covariates, multivariate analysis indicated that those living in rural regions were significantly more likely to perceive a high risk that a pandemic influenza would occur, while those with poor self-rated health perceived both a high likelihood of pandemic and high concern that self/family would be directly affected were such an event to occur. Those who spoke a language other than English at home and those on low incomes and younger people (16–24 years) were significantly more likely to have changed the way they lived their lives due to the possibility of pandemic influenza, compared to those who spoke only English at home, middle-high income earners, and older age groups, respectively. This data provides an Australian population baseline against which the risk perceptions of demographic subgroups regarding the current, and potential future pandemics, can be compared and monitored.

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Blow Flies Were One of the Possible Candidates for Transmission of Highly Pathogenic H5N1 Avian Influenza Virus during the 2004 Outbreaks in Japan

The 2003-2004 H5N1 highly pathogenic avian influenza (HPAI) outbreaks in Japan were the first such outbreaks in 79 years in Japan. Epidemic outbreaks have been occurring in Southeast Asia, with the most recent in 2010. Knowledge of the transmission route responsible for the HPAI outbreaks in these countries remains elusive. Our studies strongly suggested that field and laboratory studies focusing on mechanical transmission by blow flies should be considered to control H5N1 avian influenza outbreaks, in particular in epidemic areas, where there are high densities of different fly species throughout the year. In this paper, we review these field and laboratory entomological studies and discuss the possibility of blow flies transmitting H5N1 viruses.

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Examining the knowledge, attitudes and practices of domestic and international university students towards seasonal and pandemic influenza

BACKGROUND: Prior to the availability of the specific pandemic vaccine, strategies to mitigate the impact of the disease typically involved antiviral treatment and “non-pharmaceutical” community interventions. However, compliance with these strategies is linked to risk perceptions, perceived severity and perceived effectiveness of the strategies. In 2010, we undertook a study to examine the knowledge, attitudes, risk perceptions, practices and barriers towards influenza and infection control strategies amongst domestic and international university students. METHODS: A study using qualitative methods that incorporated 20 semi-structured interviews was undertaken with domestic and international undergraduate and postgraduate university students based at one university in Sydney, Australia. Participants were invited to discuss their perceptions of influenza (seasonal vs. pandemic) in terms of perceived severity and impact, and attitudes towards infection control measures including hand-washing and the use of social distancing, isolation or cough etiquette. RESULTS: While participants were generally knowledgeable about influenza transmission, they were unable to accurately define what ‘pandemic influenza’ meant. While avian flu or SARS were mistaken as examples of past pandemics, almost all participants were able to associate the recent “swine flu” situation as an example of a pandemic event. Not surprisingly, it was uncommon for participants to identify university students as being at risk of catching pandemic influenza. Amongst those interviewed, it was felt that ‘students’ were capable of fighting off any illness. The participant’s nominated hand washing as the most feasible and acceptable compared with social distancing and mask use. CONCLUSIONS: Given the high levels of interaction that occurs in a university setting, it is really important that students are informed about disease transmission and about risk of infection. It may be necessary to emphasize that pandemic influenza could pose a real threat to them, that it is important to protect oneself from infection and that infection control measures can be effective.

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Use of functional gene arrays for elucidating in situ biodegradation

Microarrays have revolutionized the study of microbiology by providing a high-throughput method for examining thousands of genes with a single test and overcome the limitations of many culture-independent approaches. Functional gene arrays (FGA) probe a wide range of genes involved in a variety of functions of interest to microbial ecology (e.g., carbon degradation, N fixation, metal resistance) from many different microorganisms, cultured and uncultured. The most comprehensive FGA to date is the GeoChip array, which targets tens of thousands of genes involved in the geochemical cycling of carbon, nitrogen, phosphorus, and sulfur, metal resistance and reduction, energy processing, antibiotic resistance and contaminant degradation as well as phylogenetic information (gyrB). Since the development of GeoChips, many studies have been performed using this FGA and have shown it to be a powerful tool for rapid, sensitive, and specific examination of microbial communities in a high-throughput manner. As such, the GeoChip is well-suited for linking geochemical processes with microbial community function and structure. This technology has been used successfully to examine microbial communities before, during, and after in situ bioremediation at a variety of contaminated sites. These studies have expanded our understanding of biodegradation and bioremediation processes and the associated microorganisms and environmental conditions responsible. This review provides an overview of FGA development with a focus on the GeoChip and highlights specific GeoChip studies involving in situ bioremediation.

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Functional Studies of ssDNA Binding Ability of MarR Family Protein TcaR from Staphylococcus epidermidis

The negative transcription regulator of the ica locus, TcaR, regulates proteins involved in the biosynthesis of poly-N-acetylglucosamine (PNAG). Absence of TcaR increases PNAG production and promotes biofilm formation in Staphylococci. Previously, the 3D structure of TcaR in its apo form and its complex structure with several antibiotics have been analyzed. However, the detailed mechanism of multiple antibiotic resistance regulator (MarR) family proteins such as TcaR is unclear and only restricted on the binding ability of double-strand DNA (dsDNA). Here we show by electrophoretic mobility shift assay (EMSA), electron microscopy (EM), circular dichroism (CD), and Biacore analysis that TcaR can interact strongly with single-stranded DNA (ssDNA), thereby identifying a new role in MarR family proteins. Moreover, we show that TcaR preferentially binds 33-mer ssDNA over double-stranded DNA and inhibits viral ssDNA replication. In contrast, such ssDNA binding properties were not observed for other MarR family protein and TetR family protein, suggesting that the results from our studies are not an artifact due to simple charge interactions between TcaR and ssDNA. Overall, these results suggest a novel role for TcaR in regulation of DNA replication. We anticipate that the results of this work will extend our understanding of MarR family protein and broaden the development of new therapeutic strategies for Staphylococci.

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Marine Organism Cell Biology and Regulatory Sequence Discoveryin Comparative Functional Genomics

The use of bioinformatics to integrate phenotypic and genomic data from mammalian models is well established as a means of understanding human biology and disease. Beyond direct biomedical applications of these approaches in predicting structure–function relationships between coding sequences and protein activities, comparative studies also promote understanding of molecular evolution and the relationship between genomic sequence and morphological and physiological specialization. Recently recognized is the potential of comparative studies to identify functionally significant regulatory regions and to generate experimentally testable hypotheses that contribute to understanding mechanisms that regulate gene expression, including transcriptional activity, alternative splicing and transcript stability. Functional tests of hypotheses generated by computational approaches require experimentally tractable in vitro systems, including cell cultures. Comparative sequence analysis strategies that use genomic sequences from a variety of evolutionarily diverse organisms are critical for identifying conserved regulatory motifs in the 5′-upstream, 3′-downstream and introns of genes. Genomic sequences and gene orthologues in the first aquatic vertebrate and protovertebrate organisms to be fully sequenced (Fugu rubripes, Ciona intestinalis, Tetraodon nigroviridis, Danio rerio) as well as in the elasmobranchs, spiny dogfish shark (Squalus acanthias) and little skate (Raja erinacea), and marine invertebrate models such as the sea urchin (Strongylocentrotus purpuratus) are valuable in the prediction of putative genomic regulatory regions. Cell cultures have been derived for these and other model species. Data and tools resulting from these kinds of studies will contribute to understanding transcriptional regulation of biomedically important genes and provide new avenues for medical therapeutics and disease prevention.

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Regulated multicistronic expression technology for mammalian metabolic engineering

Contemporary basic research is rapidly revealing increasingly complex molecular regulatory networks which are often interconnected via key signal integrators. These connections among regulatory and catalytic networks often frustrate bioengineers as promising metabolic engineering strategies are bypassed by compensatory metabolic responses or cause unexpected, undesired outcomes such as apoptosis, product protein degradation or inappropriate post- translational modification. Therefore, for metabolic engineering to achieve greater success in mammalian cell culture processes and to become important for future applications such as gene therapy and tissue engineering, this technology must be enhanced to allow simultaneous, in cases conditional, reshaping of metabolic pathways to access difficult-to-attain cell states. Recent advances in this new territory of multigene metabolic engineering are intimately linked to the development of multicistronic expression technology which allows the simultaneous, and in some cases, regulated expression of several genes in mammalian cells. Here we review recent achievements in multicistronic expression technology in view of multigene metabolic engineering.

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Production of high titre disabled infectious single cycle (DISC) HSV from a microcarrier culture

Disabled Infectious Single Cycle (DISC) HSV-2 has been cultured in the complimentary cell line CR2 to provide high titre bulk material suitable for the purification of the virus as a live viral vaccine. CR2 cells are cultured on the microcarrier Cytodex-1 at 5 g l-1 in small scale (1 l) and larger scale (15 l) reactors. The cells are infected at an MOI of 0.01 pfu cell-1 and the culture harvested 60–72 h later. The infected cells are removed from the microcarriers by the addition of a hypotonic saline and the virus released by low-pressure disruption techniques. Virus titres achieved are compared to the standard roller bottle process. The resulting material is the starting point for the purification of the DISC-HSV virus.

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Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.

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Influenza A: From highly pathogenic H5N1 to pandemic 2009 H1N1. Epidemiology and clinical features

The last decade has seen the emergence of two new influenza A subtypes and they have become a cause of concern for the global community. These are the highly pathogenic H5N1 influenza A virus (H5N1) and the Pandemic 2009 influenza H1N1 virus. Since 2003 the H5N1 virus has caused widespread disease and death in poultry, mainly in south East Asia and Africa. In humans the number of cases infected with this virus is few but the mortality has been about 60%. Most patients have presented with severe pneumonia and acute respiratory distress syndrome. The second influenza virus, the pandemic H1N1 2009, emerged in Mexico in March this year. This virus acquired the ability for sustained human to human spread and within a few months spread throughout the world and infected over 4 lakh individuals. The symptoms of infection with this virus are similar to seasonal influenza but it currently affecting younger individuals more often. Fortunately the mortality has been low. Both these new influenza viruses are currently circulating and have different clinical and epidemiological characteristics.

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Lessons learned from the 1918–1919 influenza pandemic

The 1918 influenza pandemic was one of the most virulent strains of influenza in history. Phylogenic evidence of the novel H1N1 strain of influenza discovered in Mexico last spring (2009) links it to the 1918 influenza strain. With information gained from analyzing viral genetics, public health records and advances in medical science we can confront the 2009 H1N1 influenza on a global scale. The paper analyses the causes and characteristics of a pandemic, and major issues in controlling the spread of the disease. Wide public vaccination and open communication between government and health sciences professionals will be an essential and vital component in managing the 2009 H1N1 pandemic and any future pandemics.

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Differential Effects of IL-12 on Tregs and Non-Treg T Cells: Roles of IFN-γ, IL-2 and IL-2R

Complex interactions between effector T cells and Foxp3(+) regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4(+)Foxp3(−) T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression.

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Genetic Variation in the TNF Gene Is Associated with Susceptibility to Severe Sepsis, but Not with Mortality

BACKGROUND: Tumor necrosis factor (TNF) and TNF receptor superfamily (TNFR)-mediated immune response play an essential role in the pathogenesis of severe sepsis. Studies examining associations of TNF and lymphotoxin-α (LTA) single nucleotide polymorphisms (SNPs) with severe sepsis have produced conflicting results. The objective of this study was to investigate whether genetic variation in TNF, LTA, TNFRSF1A and TNFRSF1B was associated with susceptibility to or death from severe sepsis in Chinese Han population. METHODOLOGY/PRINCIPAL FINDINGS: Ten SNPs in TNF, LTA, TNFRSF1A and TNFRSF1B were genotyped in samples of patients with severe sepsis (n = 432), sepsis (n = 384) and healthy controls (n = 624). Our results showed that rs1800629, a SNP in the promoter region of TNF, was significantly associated with risk for severe sepsis. The minor allele frequency of rs1800629 was significantly higher in severe sepsis patients than that in both healthy controls (P(adj) = 0.00046, odds ratio (OR)(adj) = 1.92) and sepsis patients (P(adj) = 0.002, OR(adj) = 1.56). Further, we investigated the correlation between rs1800629 genotypes and TNF-α concentrations in peripheral blood mononuclear cells (PBMCs) of healthy volunteers exposed to lipopolysaccharides (LPS) ex vivo, and the association between rs1800629 and TNF-α serum levels in severe sepsis patients. After exposure to LPS, the TNF-α concentration in culture supernatants of PBMCs was significantly higher in the subjects with AA+AG genotypes than that with GG genotype (P = 0.007). Moreover, in patients with severe sepsis, individuals with AA+AG genotypes had significantly higher TNF-α serum concentrations than those with GG genotype (P(adj) = 0.02). However, there were no significant associations between SNPs in the four candidate genes and 30 day mortality for patients with severe sepsis. CONCLUSIONS/SIGNIFICANCE: Our findings suggested that the functional TNF gene SNP rs1800629 was strongly associated with susceptibility to severe sepsis, but not with lethality in Chinese Han population.

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Identification, Characterization and Application of a G-Quadruplex Structured DNA Aptamer against Cancer Biomarker Protein Anterior Gradient Homolog 2

BACKGROUND: Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with K(d) values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2. CONCLUSION/SIGNIFICANCE: In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.

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Flt3L Combined with Rapamycin Promotes Cardiac Allograft Tolerance by Inducing Regulatory Dendritic Cells and Allograft Autophagy in Mice

The induction of immune tolerance is still a formidable challenge in organ transplantation. Dendritic cells (DCs) play an important role in orchestrating immune responses by either mediating protective immune responses or inducing antigen specific tolerance. Previous studies demonstrated that the fms-like tyrosine kinase 3 receptor (Flt3) and its ligand (Flt3L) play an essential role in the regulation of DC commitment and development. Here, we report a synergic effect between Flt3L and low-dose rapamycin (Rapa) in the protection of allograft rejction. It was found that Flt3L combined with Rapa significantly prolonged murine cardiac allograft survival time as compared with that of untreated recipients or recipients treated with Rapa or Flt3L alone. Mechanistic studies revealed that Flt3L combined with low-dose of Rapa induced the generation of tolerogenic DCs along with the production of CD25(+) Foxp3(+) regulatory T cells and IL-10 secretion. We also observed enhanced autophagy in the cardiac allograft, which could be another asset contributing to the enhanced allograft survival. All together, these data suggest that Flt3L combined with low-dose of Rapa could be an effective therapeutic approach to induce tolerance in clinical setting of transplantation.

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Renalase's Expression and Distribution in Renal Tissue and Cells

To study renalase's expression and distribution in renal tissues and cells, renalase coded DNA vaccine was constructed, and anti-renalase monoclonal antibodies were produced using DNA immunization and hybridoma technique, followed by further investigation with immunological testing and western blotting to detect the expression and distribution of renalase among the renal tissue and cells. Anti-renalase monoclonal antibodies were successfully prepared by using DNA immunization technique. Further studies with anti-renalase monoclonal antibody showed that renalase expressed in glomeruli, tubule, mesangial cells, podocytes, renal tubule epithelial cells and its cells supernatant. Renalase is wildly expressed in kidney, including glomeruli, tubule, mesangial cells, podocytes and tubule epithelial cells, and may be secreted by tubule epithelial cells primarily.

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Virus contaminations of cell cultures – A biotechnological view

In contrast to contamination by microbes and mycoplasma, which can be relatively easily detected, viral contamination present a serious threat because of the difficulty in detecting some viruses and the lack of effective methods of treating infected cell cultures. While some viruses are capable of causing morphological changes to infected cells (e.g. cytopathic effect)which are detectable by microscopy some viral contaminations result in the integration of the viral genome as provirus, this causes no visual evidence, by means of modification of the cellular morphology. Virus production from such cell lines, are potentially dangerous for other cell cultures (in research labs)by cross contaminations, or for operators and patients (in the case of the production of injectable biologicals) because of potential infection. The only way to keep cell cultures for research, development, and the biotech industry virus-free is the prevention of such contaminations. Cell cultures can become contaminated by the following means: firstly, they may already be contaminated as primary cultures (because the source of the cells was already infected), secondly, they were contaminated due to the use of contaminated raw materials, or thirdly, they were contaminated via an animal passage. This overview describes the problems and risks associated with viral contaminations in animal cell culture, describes the origins of these contaminations as well as the most important virsuses associated with viral contaminations in cell culture. In addition, ways to prevent viral contaminations as well as measures undertaken to avoid and assess risks for viral contaminations as performed in the biotech industry are briefly described.

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DENV Inhibits Type I IFN Production in Infected Cells by Cleaving Human STING

Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.

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Acute Reactogenicity after Intramuscular Immunization with Recombinant Vesicular Stomatitis Virus Is Linked to Production of IL-1β

Vaccines based on live viruses are attractive because they are immunogenic, cost-effective, and can be delivered by multiple routes. However, live virus vaccines also cause reactogenic side effects such as fever, myalgia, and injection site pain that have reduced their acceptance in the clinic. Several recent studies have linked vaccine-induced reactogenic side effects to production of the pro-inflammatory cytokine interleukin-1β (IL-1β) in humans. Our objective was therefore to determine whether IL-1β contributed to pathology after immunization with recombinant vesicular stomatitis virus (rVSV) vaccine vectors, and if so, to identify strategies by which IL-1β mediated pathology might be reduced without compromising immunogenicity. We found that an rVSV vaccine induced local and systemic production of IL-1β in vivo, and that accumulation of IL-1β correlated with acute pathology after rVSV immunization. rVSV-induced pathology was reduced in mice deficient in the IL-1 receptor Type I, but the IL-1R−/− mice were fully protected from lethal rechallenge with a high dose of VSV. This result demonstrated that IL-1 contributed to reactogenicity of the rVSV, but was dispensable for induction of protective immunity. The amount of IL-1β detected in mice deficient in either caspase-1 or the inflammasome adaptor molecule ASC after rVSV immunization was not significantly different than that produced by wild type animals, and caspase-1−/− and ASC−/− mice were only partially protected from rVSV-induced pathology. Those data support the idea that some of the IL-1β expressed in vivo in response to VSV may be activated by a caspase-1 and ASC-independent mechanism. Together these results suggest that rVSV vectors engineered to suppress the induction of IL-1β, or signaling through the IL-1R would be less reactogenic in vivo, but would retain their immunogenicity and protective capacity. Such rVSV would be highly desirable as either vaccine vectors or oncolytic therapies, and would likely be better tolerated in human vaccinees.

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Competition between Influenza A Virus Genome Segments

Influenza A virus (IAV) contains a segmented negative-strand RNA genome. How IAV balances the replication and transcription of its multiple genome segments is not understood. We developed a dual competition assay based on the co-transfection of firefly or Gaussia luciferase-encoding genome segments together with plasmids encoding IAV polymerase subunits and nucleoprotein. At limiting amounts of polymerase subunits, expression of the firefly luciferase segment was negatively affected by the presence of its Gaussia luciferase counterpart, indicative of competition between reporter genome segments. This competition could be relieved by increasing or decreasing the relative amounts of firefly or Gaussia reporter segment, respectively. The balance between the luciferase expression levels was also affected by the identity of the untranslated regions (UTRs) as well as segment length. In general it appeared that genome segments displaying inherent higher expression levels were more efficient competitors of another segment. When natural genome segments were tested for their ability to suppress reporter gene expression, shorter genome segments generally reduced firefly luciferase expression to a larger extent, with the M and NS segments having the largest effect. The balance between different reporter segments was most dramatically affected by the introduction of UTR panhandle-stabilizing mutations. Furthermore, only reporter genome segments carrying these mutations were able to efficiently compete with the natural genome segments in infected cells. Our data indicate that IAV genome segments compete for available polymerases. Competition is affected by segment length, coding region, and UTRs. This competition is probably most apparent early during infection, when limiting amounts of polymerases are present, and may contribute to the regulation of segment-specific replication and transcription.

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Oral health in China – trends and challenges

For a long time, oral disease is one of the major problems of the public health for its high prevalence and incidence throughout the world, which is especially true for low-income populations. Since China's economic reform in 1978, great changes have taken place in China. These changes have significant impact on and have been reflected in oral disease trends in China. This paper provides an overview and assessment of the oral health status in China. It focuses on changes in the nation's demographic profile, in the marketplace, the oral disease status and trends. The paper also suggests some possible measures and strategies for bettering oral health in future China.

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National intensive care unit bed capacity and ICU patient characteristics in a low income country

BACKGROUND: Primary health care delivery in the developing world faces many challenges. Priority setting favours HIV, TB and malaria interventions. Little is known about the challenges faced in this setting with regard to critical care medicine. The aim of this study was to analyse and categorise the diagnosis and outcomes of 1,774 patients admitted to a hospital intensive care unit (ICU) in a low-income country over a 7-year period. We also assessed the country’s ICU bed capacity and described the challenges faced in dealing with critically ill patients in this setting. FINDINGS: A retrospective audit was conducted in a general ICU in a university hospital in Uganda. Demographic data, admission diagnosis, and ICU length of stay were recorded for the 1,774 patients who presented to the ICU in the period January 2003 to December 2009. Their mean age was 35.5 years. Males accounted for 56.5% of the study population; 92.8% were indigenous, and 42.9% were referrals from upcountry units. The average mortality rate over the study period was 40.1% (n = 715). The highest mortality rate (44%) was recorded in 2004 and the lowest (33.2%) in 2005. Children accounted for 11.6% of admissions (40.1% mortality). Sepsis, ARDS, traumatic brain injuries and HIV related conditions were the most frequent admission diagnoses. A telephonic survey determined that there are 33 adult ICU beds in the whole country. CONCLUSIONS: Mortality was 40.1%, with sepsis, head injury, acute lung injury and HIV/AIDS the most common admission diagnoses. The country has a very low ICU bed capacity. Prioritising infectious diseases poses a challenge to ensuring that critical care is an essential part of the health care package in Uganda.

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Influenza Virus Infection in Nonhuman Primates

To determine whether nonhuman primates are infected with influenza viruses in nature, we conducted serologic and swab studies among macaques from several parts of the world. Our detection of influenza virus and antibodies to influenza virus raises questions about the role of nonhuman primates in the ecology of influenza.

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Label-Free Electrochemical Diagnosis of Viral Antigens with Genetically Engineered Fusion Protein

We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg.

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Development of a Plastic-Based Microfluidic Immunosensor Chip for Detection of H1N1 Influenza

Lab-on-a-chip can provide convenient and accurate diagnosis tools. In this paper, a plastic-based microfluidic immunosensor chip for the diagnosis of swine flu (H1N1) was developed by immobilizing hemagglutinin antigen on a gold surface using a genetically engineered polypeptide. A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique. For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip. This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device.

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Hepatitis C VLPs Delivered to Dendritic Cells by a TLR2 Targeting Lipopeptide Results in Enhanced Antibody and Cell-Mediated Responses

Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. In this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the TLR2 agonist Pam(2)Cys (E(8)Pam(2)Cys) to enhance the immunogenicity of VLPs containing the HCV structural proteins (core, E1 and E2) of genotype 1a. While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum). Dramatically improved VLP and E2-specific antibody responses were observed in VLP+E(8)Pam(2)Cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted VLPs was required to match the antibody titres obtained with a single dose of VLPs formulated with this lipopeptide. This result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of VLP+E(8)Pam(2)Cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of VLPs by Huh7 cells. Moreover, vaccination of HLA-A2 transgenic mice with this formulation also induced better VLP-specific IFN-γ-mediated responses compared to non-adjuvanted VLPs but comparable levels to that achieved when coadministered with complete freund’s adjuvant. These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV.

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Viral metagenomic analysis of bushpigs (Potamochoerus larvatus) in Uganda identifies novel variants of Porcine parvovirus 4 and Torque teno sus virus 1 and 2

BACKGROUND: As a result of rapidly growing human populations, intensification of livestock production and increasing exploitation of wildlife habitats for animal agriculture, the interface between wildlife, livestock and humans is expanding, with potential impacts on both domestic animal and human health. Wild animals serve as reservoirs for many viruses, which may occasionally result in novel infections of domestic animals and/or the human population. Given this background, we used metagenomics to investigate the presence of viral pathogens in sera collected from bushpigs (Potamochoerus larvatus), a nocturnal species of wild Suid known to move between national parks and farmland, in Uganda. RESULTS: Application of 454 pyrosequencing demonstrated the presence of Torque teno sus virus (TTSuV), porcine parvovirus 4 (PPV4), porcine endogenous retrovirus (PERV), a GB Hepatitis C–like virus, and a Sclerotinia hypovirulence-associated-like virus in sera from the bushpigs. PCR assays for each specific virus combined with Sanger sequencing revealed two TTSuV-1 variants, one TTSuV-2 variant as well as PPV4 in the serum samples and thereby confirming the findings from the 454 sequencing. CONCLUSIONS: Using a viral metagenomic approach we have made an initial analysis of viruses present in bushpig sera and demonstrated for the first time the presence of PPV4 in a wild African Suid. In addition we identified novel variants of TTSuV-1 and 2 in bushpigs.

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A Three-Dimensional Comparison of Tick-Borne Flavivirus Infection in Mammalian and Tick Cell Lines

Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60–100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection.

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SEED Servers: High-Performance Access to the SEED Genomes, Annotations, and Metabolic Models

The remarkable advance in sequencing technology and the rising interest in medical and environmental microbiology, biotechnology, and synthetic biology resulted in a deluge of published microbial genomes. Yet, genome annotation, comparison, and modeling remain a major bottleneck to the translation of sequence information into biological knowledge, hence computational analysis tools are continuously being developed for rapid genome annotation and interpretation. Among the earliest, most comprehensive resources for prokaryotic genome analysis, the SEED project, initiated in 2003 as an integration of genomic data and analysis tools, now contains >5,000 complete genomes, a constantly updated set of curated annotations embodied in a large and growing collection of encoded subsystems, a derived set of protein families, and hundreds of genome-scale metabolic models. Until recently, however, maintaining current copies of the SEED code and data at remote locations has been a pressing issue. To allow high-performance remote access to the SEED database, we developed the SEED Servers (http://www.theseed.org/servers): four network-based servers intended to expose the data in the underlying relational database, support basic annotation services, offer programmatic access to the capabilities of the RAST annotation server, and provide access to a growing collection of metabolic models that support flux balance analysis. The SEED servers offer open access to regularly updated data, the ability to annotate prokaryotic genomes, the ability to create metabolic reconstructions and detailed models of metabolism, and access to hundreds of existing metabolic models. This work offers and supports a framework upon which other groups can build independent research efforts. Large integrations of genomic data represent one of the major intellectual resources driving research in biology, and programmatic access to the SEED data will provide significant utility to a broad collection of potential users.

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ECR 2012 Book of Abstracts - A - Postergraduate Educational Programme

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Discovery and Targeted LC-MS/MS of Purified Polerovirus Reveals Differences in the Virus-Host Interactome Associated with Altered Aphid Transmission

Circulative transmission of viruses in the Luteoviridae, such as cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus, plant, and aphid proteins. Natural selection has favored these viruses to be retained in the phloem to facilitate acquisition and transmission by aphids. We show that treatment of infected oat tissue homogenate with sodium sulfite reduces transmission of the purified virus by aphids. Transmission electron microscopy data indicated no gross change in virion morphology due to treatments. However, treated virions were not acquired by aphids through the hindgut epithelial cells and were not transmitted when injected directly into the hemocoel. Analysis of virus preparations using nanoflow liquid chromatography coupled to tandem mass spectrometry revealed a number of host plant proteins co-purifying with viruses, some of which were lost following sodium sulfite treatment. Using targeted mass spectrometry, we show data suggesting that several of the virus-associated host plant proteins accumulated to higher levels in aphids that were fed on CYDV-infected plants compared to healthy plants. We propose two hypotheses to explain these observations, and these are not mutually exclusive: (a) that sodium sulfite treatment disrupts critical virion-host protein interactions required for aphid transmission, or (b) that host infection with CYDV modulates phloem protein expression in a way that is favorable for virus uptake by aphids. Importantly, the genes coding for the plant proteins associated with virus may be examined as targets in breeding cereal crops for new modes of virus resistance that disrupt phloem-virus or aphid-virus interactions.

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Mortality, Severe Acute Respiratory Infection, and Influenza-Like Illness Associated with Influenza A(H1N1)pdm09 in Argentina, 2009

INTRODUCTION: While there is much information about the burden of influenza A(H1N1)pdm09 in North America, little data exist on its burden in South America. METHODS: During April to December 2009, we actively searched for persons with severe acute respiratory infection and influenza-like illness (ILI) in three sentinel cities. A proportion of case-patients provided swabs for influenza testing. We estimated the number of case-patients that would have tested positive for influenza by multiplying the number of untested case-patients by the proportion who tested positive. We estimated rates by dividing the estimated number of case-patients by the census population after adjusting for the proportion of case-patients with missing illness onset information and ILI case-patients who visited physicians multiple times for one illness event. RESULTS: We estimated that the influenza A(H1N1)pdm09 mortality rate per 100,000 person-years (py) ranged from 1.5 among persons aged 5–44 years to 5.6 among persons aged ≥65 years. A(H1N1)pdm09 hospitalization rates per 100,000 py ranged between 26.9 among children aged <5 years to 41.8 among persons aged ≥65 years. Influenza A(H1N1)pdm09 ILI rates per 100 py ranged between 1.6 among children aged <5 to 17.1 among persons aged 45–64 years. While 9 (53%) of 17 influenza A(H1N1)pdm09 decedents with available data had obesity and 7 (17%) of 40 had diabetes, less than 4% of surviving influenza A(H1N1)pdm09 case-patients had these pre-existing conditions (p≤0.001). CONCLUSION: Influenza A(H1N1)pdm09 caused a similar burden of disease in Argentina as in other countries. Such disease burden suggests the potential value of timely influenza vaccinations.

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C. difficile 630Δerm Spo0A Regulates Sporulation, but Does Not Contribute to Toxin Production, by Direct High-Affinity Binding to Target DNA

Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely.

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Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. DOI: http://dx.doi.org/10.7554/eLife.00049.001

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The Evolutionary Pattern of Glycosylation Sites in Influenza Virus (H5N1) Hemagglutinin and Neuraminidase

Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of influenza viruses play crucial roles in transfaunation, membrane fusion and the release of progeny virions. To explore the distribution of N-glycosylation sites (glycosites) in these two glycoproteins, we collected and aligned the amino acid sequences of all the HA and NA subtypes. Two glycosites were located at HA0 cleavage sites and fusion peptides and were strikingly conserved in all HA subtypes, while the remaining glycosites were unique to their subtypes. Two to four conserved glycosites were found in the stalk domain of NA, but these are affected by the deletion of specific stalk domain sequences. Another highly conserved glycosite appeared at the top center of tetrameric global domain, while the others glycosites were distributed around the global domain. Here we present a detailed investigation of the distribution and the evolutionary pattern of the glycosites in the envelope glycoproteins of IVs, and further focus on the H5N1 virus and conclude that the glycosites in H5N1 have become more complicated in HA and less influential in NA in the last five years.

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Niclosamide Is a Proton Carrier and Targets Acidic Endosomes with Broad Antiviral Effects

Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+)-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

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A Single Native Ganglioside GM(1)-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway

Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM(1) (GM(1)) molecules, and the toxin-GM(1) complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM(1) by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476–1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573–586, 2012), suggesting that multivalent binding to GM(1) is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM(1) binding sites from zero (nonbinding) to five (wild type). We found that a single GM(1) binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM(1) receptors is not essential for toxicity of CT.

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A Single Residue Substitution in the Receptor-Binding Domain of H5N1 Hemagglutinin Is Critical for Packaging into Pseudotyped Lentiviral Particles

BACKGROUND: Serological studies for influenza infection and vaccine response often involve microneutralization and hemagglutination inhibition assays to evaluate neutralizing antibodies against human and avian influenza viruses, including H5N1. We have previously characterized lentiviral particles pseudotyped with H5-HA (H5pp) and validated an H5pp-based assay as a safe alternative for high-throughput serological studies in BSL-2 facilities. Here we show that H5-HAs from different clades do not always give rise to efficient production of H5pp and the underlying mechanisms are addressed. METHODOLOGY/FINDINGS: We have carried out mutational analysis to delineate the molecular determinants responsible for efficient packaging of HA from A/Cambodia/40808/2005 (H5Cam) and A/Anhui/1/2005 (H5Anh) into H5pp. Our results demonstrate that a single A134V mutation in the 130-loop of the receptor binding domain is sufficient to render H5Anh the ability to generate H5Anh-pp efficiently, whereas the reverse V134A mutation greatly hampers production of H5Cam-pp. Although protein expression in total cell lysates is similar for H5Anh and H5Cam, cell surface expression of H5Cam is detected at a significantly higher level than that of H5Anh. We further demonstrate by several independent lines of evidence that the behaviour of H5Anh can be explained by a stronger binding to sialic acid receptors implicating residue 134. CONCLUSIONS: We have identified a single A134V mutation as the molecular determinant in H5-HA for efficient incorporation into H5pp envelope and delineated the underlying mechanism. The reduced binding to sialic acid receptors as a result of the A134V mutation not only exerts a critical influence in pseudotyping efficiency of H5-HA, but has also an impact at the whole virus level. Because A134V substitution has been reported as a naturally occurring mutation in human host, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 viruses.

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Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation

BACKGROUND: Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. OBJECTIVES: We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC) barrier integrity and enhanced cardiopulmonary dysfunction. METHODS: Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER) in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm). Biochemical assessment of ROS generation and Ca(2+) mobilization were also measured. RESULTS: PM exposure induced tight junction protein Zona occludens-1 (ZO-1) relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin). N-acetyl-cysteine (NAC, 5 mM) reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca(2+)-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. CONCLUSIONS: These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.

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The expression of nicotinic receptor alpha7 during cochlear development

Nicotinic acetylcholine receptor alpha7 expression was examined in the developing and adult auditory system using mice that were modified through homologous recombination to coexpress either GFP (alpha7GFP) or Cre (alpha7Cre), respectively. The expression of alpha7GFP is first detected at embryonic (E) day E13.5 in cells of the spiral prominence. By E14.5, sensory regions including the putative outer hair cells and Deiters' cells express alpha7GFP as do solitary efferent fibers. This pattern diminishes after E16.5 in a basal to apex progression, as Hensen's cells and cells of the spiral ligament acquire alpha7GFP expression. At birth and thereafter alpha7GFP also identifies a subset of spiral ganglion cells whose processes terminate on inner hair cells. Efferent fibers identified by peripherin or calcitonin gene-related protein do not coexpress alpha7GFP. In addition to cochlear structures, there is strong expression of alpha7GFP by cells of the central auditory pathways including the ventral posterior cochlear nucleus, lateral lemniscus, central inferior colliculus, and the medial geniculate nucleus. Our findings suggest that alpha7 expression by both neuronal and non-neuronal cells has the potential to impact multiple auditory functions through mechanisms that are not traditionally attributed to this receptor.

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Identification of serum proteomic biomarkers for early porcine reproductive and respiratory syndrome (PRRS) infection

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases worldwide. Despite its relevance, serum biomarkers associated with early-onset viral infection, when clinical signs are not detectable and the disease is characterized by a weak anti-viral response and persistent infection, have not yet been identified. Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) is a reproducible, accurate, and simple method for the identification of biomarker proteins related to disease in serum. This work describes the SELDI-TOF MS analyses of sera of 60 PRRSV-positive and 60 PRRSV-negative, as measured by PCR, asymptomatic Large White piglets at weaning. Sera with comparable and low content of hemoglobin (< 4.52 μg/mL) were fractionated in 6 different fractions by anion-exchange chromatography and protein profiles in the mass range 1–200 kDa were obtained with the CM10, IMAC30, and H50 surfaces. RESULTS: A total of 200 significant peaks (p < 0.05) were identified in the initial discovery phase of the study and 47 of them were confirmed in the validation phase. The majority of peaks (42) were up-regulated in PRRSV-positive piglets, while 5 were down-regulated. A panel of 14 discriminatory peaks identified in fraction 1 (pH = 9), on the surface CM10, and acquired at low focus mass provided a serum protein profile diagnostic pattern that enabled to discriminate between PRRSV-positive and -negative piglets with a sensitivity and specificity of 77% and 73%, respectively. CONCLUSIONS: SELDI-TOF MS profiling of sera from PRRSV-positive and PRRSV-negative asymptomatic piglets provided a proteomic signature with large scale diagnostic potential for early identification of PRRSV infection in weaning piglets. Furthermore, SELDI-TOF protein markers represent a refined phenotype of PRRSV infection that might be useful for whole genome association studies.

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Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20–57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.

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Toxicology, biodistribution and shedding profile of a recombinant measles vaccine vector expressing HIV-1 antigens, in cynomolgus macaques

As a new human immunodeficiency virus type 1 (HIV-1) vaccine approach, the live-attenuated measles virus (MV) Schwarz vaccine strain was genetically engineered to express the F4 antigen (MV1-F4). F4 is a fusion protein comprising HIV-1 antigens p17 and p24, reverse transcriptase and Nef. This study assessed the toxicity, biodistribution and shedding profiles of MV1-F4. Cynomolgus macaques were intramuscularly immunized one or three times with the highest dose of MV1-F4 intended for clinical use, the reference (Schwarz) measles vaccine or saline, and monitored clinically for 11 or 85 days. Toxicological parameters included local and systemic clinical signs, organ weights, haematology, clinical and gross pathology and histopathology. Both vaccines were well tolerated, with no morbidity, clinical signs or gross pathological findings observed. Mean spleen weights were increased after three doses of either vaccine, which corresponded with increased numbers and/or sizes of germinal centers. This was likely a result of the immune response to the vaccines. Either vaccine virus replicated preferentially in secondary lymphoid organs and to a lesser extent in epithelium-rich tissues (e.g., intestine, urinary bladder and trachea) and the liver. At the expected peak of viremia, viral RNA was detected in some biological fluid samples from few animals immunized with either vaccine, but none of these samples contained infectious virus. In conclusion, no shedding of infectious viral particles was identified in cynomolgus monkeys after injection of MV1-F4 or Schwarz measles vaccines. Furthermore, no toxic effect in relation to the MV vaccination was found with these vaccines in this study.

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Forty-Five Years of Marburg Virus Research

In 1967, the first reported filovirus hemorrhagic fever outbreak took place in Germany and the former Yugoslavia. The causative agent that was identified during this outbreak, Marburg virus, is one of the most deadly human pathogens. This article provides a comprehensive overview of our current knowledge about Marburg virus disease ranging from ecology to pathogenesis and molecular biology.

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The Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Is Modified by SUMO in a DNA-Dependent Manner

During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of evidence suggest that UL44 might have additional roles during virus life cycle. To shed light on this, we searched for cellular partners of UL44 by yeast two-hybrid screenings. Intriguingly, we discovered the interaction of UL44 with Ubc9, an enzyme involved in the covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to cellular and viral proteins. We found that UL44 can be extensively sumoylated not only in a cell-free system and in transfected cells, but also in HCMV-infected cells, in which about 50% of the protein resulted to be modified at late times post-infection, when viral genome replication is accomplished. Mass spectrometry studies revealed that UL44 possesses multiple SUMO target sites, located throughout the protein. Remarkably, we observed that binding of UL44 to DNA greatly stimulates its sumoylation both in vitro and in vivo. In addition, we showed that overexpression of SUMO alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both virus production and DNA replication, arguing for an important role for sumoylation in HCMV life cycle and UL44 function(s). These data report for the first time the sumoylation of a viral processivity factor and show that there is a functional interplay between the HCMV UL44 protein and the cellular sumoylation system.

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Fas-deficient mice have impaired alveolar neutrophil recruitment and decreased expression of anti-KC autoantibody:KC complexes in a model of acute lung injury

BACKGROUND: Exposure to mechanical ventilation enhances lung injury in response to various stimuli, such as bacterial endotoxin (LPS). The Fas/FasL system is a receptor ligand system that has dual pro-apoptotic and pro-inflammatory functions and has been implicated in the pathogenesis of lung injury. In this study we test the hypothesis that a functioning Fas/FasL system is required for the development of lung injury in mechanically ventilated mice. METHODS: C57BL/6 (B6) and Fas-deficient lpr mice were exposed to either intra-tracheal PBS followed by spontaneous breathing or intra-tracheal LPS followed by four hours mechanical ventilation with tidal volumes of 10 mL/kg, respiratory rate of 150 breaths per minute, inspired oxygen 0.21 and positive end expiratory pressure (PEEP) of 3 cm of water. RESULTS: Compared with the B6 mice, the lpr mice showed attenuation of the neutrophilic response as measured by decreased numbers of BAL neutrophils and lung myeloperoxidase activity. Interestingly, the B6 and lpr mice had similar concentrations of pro-inflammatory cytokines, including CXCL1 (KC), and similar measurements of permeability and apoptosis. However, the B6 mice showed greater deposition of anti-KC:KC immune complexes in the lungs, as compared with the lpr mice. CONCLUSIONS: We conclude that a functioning Fas/FasL system is required for full neutrophilic response to LPS in mechanically ventilated mice.

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Evolutionary Dynamics of the Interferon-Induced Transmembrane Gene Family in Vertebrates

Vertebrate interferon-induced transmembrane (IFITM) genes have been demonstrated to have extensive and diverse functions, playing important roles in the evolution of vertebrates. Despite observance of their functionality, the evolutionary dynamics of this gene family are complex and currently unknown. Here, we performed detailed evolutionary analyses to unravel the evolutionary history of the vertebrate IFITM family. A total of 174 IFITM orthologous genes and 112 pseudogenes were identified from 27 vertebrate genome sequences. The vertebrate IFITM family can be divided into immunity-related IFITM (IR-IFITM), IFITM5 and IFITM10 sub-families in phylogeny, implying origins from three different progenitors. In general, vertebrate IFITM genes are located in two loci, one containing the IFITM10 gene, and the other locus containing IFITM5 and various numbers of IR-IFITM genes. Conservation of evolutionary synteny was observed in these IFITM genes. Significant functional divergence was detected among the three IFITM sub-families. No gene duplication or positive selection was found in IFITM5 sub-family, implying the functional conservation of IFITM5 in vertebrate evolution, which is involved in bone formation. No IFITM5 locus was identified in the marmoset genome, suggesting a potential association with the tiny size of this monkey. The IFITM10 sub-family was divided into two groups: aquatic and terrestrial types. Functional divergence was detected between the two groups, and five IFITM10-like genes from frog were dispersed into the two groups. Both gene duplication and positive selection were observed in aquatic vertebrate IFITM10-like genes, indicating that IFITM10 might be associated with the adaptation to aquatic environments. A large number of lineage- and species-specific gene duplications were observed in IR-IFITM sub-family and positive selection was detected in IR-IFITM of primates and rodents. Because primates have experienced a long history of viral infection, such rapid expansion and positive selection suggests that the evolution of primate IR-IFITM genes is associated with broad-spectrum antiviral activity.

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Filovirus Research in Gabon and Equatorial Africa: The Experience of a Research Center in the Heart of Africa

Health research programs targeting the population of Gabon and Equatorial Africa at the International Center for Medical Research in Franceville (CIRMF), Gabon, have evolved during the years since its inception in 1979 in accordance with emerging diseases. Since the reemergence of Ebola virus in Central Africa, the CIRMF “Emerging Viral Disease Unit” developed diagnostic tools and epidemiologic strategies and transfers of such technology to support the response of the National Public Health System and the World Health Organization to epidemics of Ebola virus disease. The Unit carries out a unique investigation program on the natural history of the filoviruses, emergence of epidemics, and Ebola virus pathogenesis. In addition, academic training is provided at all levels to regional and international students covering emerging conditions (host factors, molecular biology, genetics) that favor the spread of viral diseases.

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Reproductive Number and Serial Interval of the First Wave of Influenza A(H1N1)pdm09 Virus in South Africa

BACKGROUND/OBJECTIVE: Describing transmissibility parameters of past pandemics from diverse geographic sites remains critical to planning responses to future outbreaks. We characterize the transmissibility of influenza A(H1N1)pdm09 (hereafter pH1N1) in South Africa during 2009 by estimating the serial interval (SI), the initial effective reproductive number (initial R(t)) and the temporal variation of R(t). METHODS: We make use of data from a central registry of all pH1N1 laboratory-confirmed cases detected throughout South Africa. Whenever date of symptom onset is missing, we estimate it from the date of specimen collection using a multiple imputation approach repeated 100 times for each missing value. We apply a likelihood-based method (method 1) for simultaneous estimation of initial R(t) and the SI; estimate initial R(t) from SI distributions established from prior field studies (method 2); and the Wallinga and Teunis method (method 3) to model the temporal variation of R(t). RESULTS: 12,360 confirmed pH1N1 cases were reported in the central registry. During the period of exponential growth of the epidemic (June 21 to August 3, 2009), we simultaneously estimate a mean R(t) of 1.47 (95% CI: 1.30–1.72) and mean SI of 2.78 days (95% CI: 1.80–3.75) (method 1). Field studies found a mean SI of 2.3 days between primary cases and laboratory-confirmed secondary cases, and 2.7 days when considering both suspected and confirmed secondary cases. Incorporating the SI estimate from field studies using laboratory-confirmed cases, we found an initial R(t) of 1.43 (95% CI: 1.38–1.49) (method 2). The mean R(t) peaked at 2.91 (95% CI: 0.85–2.91) on June 21, as the epidemic commenced, and R(t)>1 was sustained until August 22 (method 3). CONCLUSIONS: Transmissibility characteristics of pH1N1 in South Africa are similar to estimates reported by countries outside of Africa. Estimations using the likelihood-based method are in agreement with field findings.

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Measuring healthcare preparedness: an all-hazards approach

In a paper appearing in this issue, Adini, et al. describe a struggle familiar to many emergency planners—the challenge of planning for all scenarios. The authors contend that all-hazards, or capabilities-based planning, in which a set of core capabilities applicable to numerous types of events is developed, is a more efficient way to achieve general health care system emergency preparedness than scenario-based planning. Essentially, the core of what is necessary to plan for and respond to one kind of disaster (e.g. a biologic event) is also necessary for planning and responding to other types of disasters, allowing for improvements in planning and maximizing efficiencies. While Adini, et al. have advanced the science of health care emergency preparedness through their consideration of 490 measures to assess preparedness, a shorter set of validated preparedness measures would support the dual goals of accountability and improved outcomes and could provide the basis for determining which actions in the name of preparedness really matter.

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Presentation of hemophagocytic lymphohistiocytosis due to a novel MUNC 13–4 mutation masked by partial therapeutic immunosuppression

Hemophagocytic lymphohistiocytosis is a potentially fatal disease characterized by excessive macrophage and lymphocyte activity. Patients can be affected following immune activation after an oncologic, autoimmune or infectious trigger. An associated gene mutation may be found which impairs cytolytic lymphocyte function. We describe a pediatric case of hemophagocytic lymphohistiocytosis with a novel mutation of MUNC 13–4 whose diagnosis was confounded by concurrent immunosuppression. Clinical reassessment for hemophagocytic lymphohistiocytosis is necessary in persistently febrile patients with laboratory derangements in the setting of immunosuppressive agent exposure.

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Relative cost and outcomes in the intensive care unit of acute lung injury (ALI) due to pandemic influenza compared with other etiologies: a single-center study

BACKGROUND: Critical illness due to 2009 H1N1 influenza has been characterized by respiratory complications, including acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), and associated with high mortality. We studied the severity, outcomes, and hospital charges of patients with ALI/ARDS secondary to pandemic influenza A infection compared with ALI and ARDS from other etiologies. METHODS: A retrospective review was conducted that included patients admitted to the Cleveland Clinic MICU with ALI/ARDS and confirmed influenza A infection, and all patients admitted with ALI/ARDS from any other etiology from September 2009 to March 2010. An itemized list of individual hospital charges was obtained for each patient from the hospital billing office and organized by billing code into a database. Continuous data that were normally distributed are presented as the mean ± SD and were analyzed by the Student’s t test. The chi-square and Fisher exact tests were used to evaluate differences in proportions between patient subgroups. Data that were not normally distributed were compared with the Wilcoxon rank-sum test. RESULTS: Forty-five patients were studied: 23 in the H1N1 group and 22 in the noninfluenza group. Mean ± SD age was similar (44 ± 13 and 51 ± 17 years, respectively, p = 0.15). H1N1 patients had lower APACHE III scores (66 ± 20 vs. 89 ± 32, p = 0.015) and had higher Pplat and PEEP on days 1, 3, and 14. Hospital and ICU length of stay and duration of mechanical ventilation were comparable. SOFA scores over the first 2 weeks in the ICU indicate more severe organ failure in the noninfluenza group (p = 0.017). Hospital mortality was significantly higher in the noninfluenza group (77 vs. 39%, p = 0.016). The noninfluenza group tended to have higher overall charges, including significantly higher cost of blood products in the ICU. CONCLUSIONS: ALI/ARDS secondary to pandemic influenza infection is associated with more severe respiratory compromise but has lower overall acuity and better survival rates than ALI/ARDS due to other causes. Higher absolute charges in the noninfluenza group are likely due to underlying comorbid medical conditions.

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Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

BACKGROUND: The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. METHODS AND RESULTS: This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched (213)SVQYHPL(219) of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that (213)SVQYHPL(219) was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. CONCLUSIONS AND SIGNIFICANCE: We identified (213)SVQYHPL(219) as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.

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Lower Respiratory Tract Infection in a Renal Transplant Recipient: Do not Forget Metapneumovirus

Human metapneumovirus (hMPV) is emerging as a cause of a severe respiratory tract infection in immunocompromised patients. hMPV pneumonia has only been seldom reported in nonpulmonary solid organ transplanted patients, such as renal transplant recipients. We report here a case of a 39-year-old patient presenting with fever, cough, and interstitial opacities on CT scan diagnosed as a nonsevere hMPV pneumonia 11 years after a renal transplantation. Infection resolved spontaneously. Differential diagnosis with Pneumocystis pneumonia was discussed. We review the medical literature and discuss clinical presentation and detection methods that can be proposed in solid organ transplant recipients.

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What was the primary mode of smallpox transmission? Implications for biodefense

The mode of infection transmission has profound implications for effective containment by public health interventions. The mode of smallpox transmission was never conclusively established. Although, “respiratory droplet” transmission was generally regarded as the primary mode of transmission, the relative importance of large ballistic droplets and fine particle aerosols that remain suspended in air for more than a few seconds was never resolved. This review examines evidence from the history of variolation, data on mucosal infection collected in the last decades of smallpox transmission, aerosol measurements, animal models, reports of smallpox lung among healthcare workers, and the epidemiology of smallpox regarding the potential importance of fine particle aerosol mediated transmission. I introduce briefly the term anisotropic infection to describe the behavior of Variola major in which route of infection appears to have altered the severity of disease.