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Economic analysis of pandemic influenza mitigation strategies for five pandemic severity categories

BACKGROUND: The threat of emergence of a human-to-human transmissible strain of highly pathogenic influenza A(H5N1) is very real, and is reinforced by recent results showing that genetically modified A(H5N1) may be readily transmitted between ferrets. Public health authorities are hesitant in introducing social distancing interventions due to societal disruption and productivity losses. This study estimates the effectiveness and total cost (from a societal perspective, with a lifespan time horizon) of a comprehensive range of social distancing and antiviral drug strategies, under a range of pandemic severity categories. METHODS: An economic analysis was conducted using a simulation model of a community of ~30,000 in Australia. Data from the 2009 pandemic was used to derive relationships between the Case Fatality Rate (CFR) and hospitalization rates for each of five pandemic severity categories, with CFR ranging from 0.1% to 2.5%. RESULTS: For a pandemic with basic reproduction number R(0) = 1.8, adopting no interventions resulted in total costs ranging from $441 per person for a pandemic at category 1 (CFR 0.1%) to $8,550 per person at category 5 (CFR 2.5%). For severe pandemics of category 3 (CFR 0.75%) and greater, a strategy combining antiviral treatment and prophylaxis, extended school closure and community contact reduction resulted in the lowest total cost of any strategy, costing $1,584 per person at category 5. This strategy was highly effective, reducing the attack rate to 5%. With low severity pandemics costs are dominated by productivity losses due to illness and social distancing interventions, whereas higher severity pandemic costs are dominated by healthcare costs and costs arising from productivity losses due to death. CONCLUSIONS: For pandemics in high severity categories the strategies with the lowest total cost to society involve rigorous, sustained social distancing, which are considered unacceptable for low severity pandemics due to societal disruption and cost.

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Infiltration of Proinflammatory M1 Macrophages into the Outer Retina Precedes Damage in a Mouse Model of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of blindness in the developed world. Oxidative stress and inflammation are implicated in AMD, but precise mechanisms remain poorly defined. Carboxyethylpyrrole (CEP) is an AMD-associated lipid peroxidation product. We previously demonstrated that mice immunized with CEP-modified albumin developed AMD-like degenerative changes in the outer retina. Here, we examined the kinetics of lesion development in immunized mice and the presence of macrophages within the interphotoreceptor matrix (IPM), between the retinal pigment epithelium and photoreceptor outer segments. We observed a significant and time-dependent increase in the number of macrophages in immunized mice relative to young age-matched controls prior to overt pathology. These changes were more pronounced in BALB/c mice than in C57BL/6 mice. Importantly, IPM-infiltrating macrophages were polarized toward the M1 phenotype but only in immunized mice. Moreover, when Ccr2-deficient mice were immunized, macrophages were not present in the IPM and no retinal lesions were observed, suggesting a deleterious role for these cells in our model. This work provides mechanistic evidence linking immune responses against oxidative damage with the presence of proinflammatory macrophages at sites of future AMD and experimentally demonstrates that manipulating immunity may be a target for modulating the development of AMD.

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A novel submicron emulsion system loaded with vincristine–oleic acid ion-pair complex with improved anticancer effect: in vitro and in vivo studies

BACKGROUND: Vincristine (VCR), which is a widely used antineoplastic drug, was integrated with a submicron-emulsion drug-delivery system to enhance the anticancer effect. METHODS: After the formation of a VCR-oleic acid ion-pair complex (VCR-OA), the VCR-OA-loaded submicron emulsion (VCR-OA-SME), prepared by classical high-pressure homogenization, was characterized and its in vitro anticancer effects were evaluated. RESULTS: The submicron-emulsion formulation exhibited a homogeneous round shape. The mean particle size, zeta potential, and encapsulation efficiency were 157.6 ± 12.6 nm, −26.5 ± 5.0 mV and 78.64% ± 3.44%, respectively. An in vitro release study of the VCR-OA-SME revealed that 12.4% of the VCR was released within the first 2 hours (initial burst-release phase) and the rest of the drug was detected in the subsequent sustained-release phase. Compared with VCR solution, the pharmacokinetic study of VCR-OA-SME showed relatively longer mean residence time (mean residence time [0–∞] increased from 187.19 to 227.56 minutes), higher maximum concentration (from 252.13 ng/mL to 533.34 ng/mL), and greater area under the curve (area under the curve [0–∞] from 11,417.77 μg/L/minute to 17,164.34 μg/L/minute. Moreover, the VCR-OA-SME exhibited higher cytotoxicity (P < 0.05) on tumor cells by inducing cell arrest in the G2/M phase or even apoptosis (P < 0.05). CONCLUSION: The VCR-OA-SME formulation in our study displayed great potential for an anticancer effect for VCR.

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Molecular Profiling of Multiple Human Cancers Defines an Inflammatory Cancer-Associated Molecular Pattern and Uncovers KPNA2 as a Uniform Poor Prognostic Cancer Marker

BACKGROUND: Immune evasion is one of the recognized hallmarks of cancer. Inflammatory responses to cancer can also contribute directly to oncogenesis. Since the immune system is hardwired to protect the host, there is a possibility that cancers, regardless of their histological origins, endow themselves with a common and shared inflammatory cancer-associated molecular pattern (iCAMP) to promote oncoinflammation. However, the definition of iCAMP has not been conceptually and experimentally investigated. METHODS AND FINDINGS: Genome-wide cDNA expression data was analyzed for 221 normal and 324 cancer specimens from 7 cancer types: breast, prostate, lung, colon, gastric, oral and pancreatic. A total of 96 inflammatory genes with consistent dysregulation were identified, including 44 up-regulated and 52 down-regulated genes. Protein expression was confirmed by immunohistochemistry for some of these genes. The iCAMP contains proteins whose roles in cancer have been implicated and others which are yet to be appreciated. The clinical significance of many iCAMP genes was confirmed in multiple independent cohorts of colon and ovarian cancer patients. In both cases, better prognosis correlated strongly with high CXCL13 and low level of GREM1, LOX, TNFAIP6, CD36, and EDNRA. An “Inflammatory Gene Integrated Score” was further developed from the combination of 18 iCAMP genes in ovarian cancer, which predicted overall survival. Noticeably, as a selective nuclear import protein whose immuno-regulatory function just begins to emerge, karyopherin alpha 2 (KPNA2) is uniformly up-regulated across cancer types. For the first time, the cancer-specific up-regulation of KPNA2 and its clinical significance were verified by tissue microarray analysis in colon and head-neck cancers. CONCLUSION: This work defines an inflammatory signature shared by seven epithelial cancer types and KPNA2 as a consistently up-regulated protein in cancer. Identification of iCAMP may not only serve as a novel biomarker for prognostication and individualized treatment of cancer, but also have significant biological implications.

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Interaction of Bordetella bronchiseptica and Its Lipopolysaccharide with In Vitro Culture of Respiratory Nasal Epithelium

The nasal septa of fetal rabbits at 26 days of gestation were harvested by cesarean section of the does while under anesthesia and then exposed to Bordetella bronchiseptica or its lipopolysaccharide (LPS) for periods of 2 and 4 hours. A total of 240 explants were used. The tissues were examined using the Hematoxylin & Eosin technique. Then, semithin sections (0.5 μm) were stained with toluidine blue and examined with indirect immunoperoxidase (IPI) and lectin histochemistry. The most frequent and statistically significant findings were as follows: (1) cell death and increased goblet cell activity when exposed to bacteria and (2) cell death, cytoplasmic vacuolation and infiltration of polymorphonuclear leukocytes when exposed to LPS. The lesions induced by the bacterium were more severe than with LPS alone, except for the cytoplasmic vacuolation in epithelial cells. IPI stained the ciliated border of the epithelium with the bacterium more intensely, while LPS lectin histochemistry preferentially labeled the cytoplasm of goblet cell. These data indicate that B. bronchiseptica and its LPS may have an affinity for specific glycoproteins that would act as adhesion receptors in both locations.

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Knowledge of, attitudes toward, and preventive practices relating to cholera and oral cholera vaccine among urban high-risk groups: findings of a cross-sectional study in Dhaka, Bangladesh

BACKGROUND: In endemic countries such as Bangladesh, consequences of cholera place an enormous financial and social burden on patients and their families. Cholera vaccines not only provide health benefits to susceptible populations but also have effects on the earning capabilities and financial stability of the family. Community-based research and evaluations are necessary to understand perceptions about and practices of the community relating to cholera and oral cholera vaccines. This may help identify the ways in which such vaccines may be successfully introduced, and other preventive measures can be implemented. The present study assessed the knowledge of, attitudes toward, and preventive practices relating to cholera and oral cholera vaccine among an urban population residing in a high cholera-prone setting in Dhaka, Bangladesh. METHODS: This cross-sectional study was conducted in an area of high cholera prevalence in 15 randomly-selected clusters in Mirpur, Dhaka city. A study team collected data through a survey and in-depth interviews during December 2010–February 2011. RESULTS: Of 2,830 families included in the final analysis, 23% could recognize cholera as acute watery diarrhea and 16% had ever heard of oral cholera vaccine. About 54% of the respondents had poor knowledge about cholera-related issues while 97% had a positive attitude toward cholera and oral cholera vaccine. One-third showed poor practice relating to the prevention of cholera. The findings showed a significant (p < 0.05) association between the respondents’ knowledge and sex, education, occupation, monthly overall household expenditure, attitudes and practice. In the adjusted model, male sex, having a lower monthly overall household expenditure, and having a less positive attitude toward cholera were the significant predictors to having poor knowledge. CONCLUSIONS: The findings suggest the strengthening of health education activities to improve knowledge on cholera, its prevention and treatment and information on cholera vaccination among high-risk populations. The data also underscore the potential of mass cholera vaccination to prevent and control cholera.

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Saikosaponin-d Enhances the Anticancer Potency of TNF-α via Overcoming Its Undesirable Response of Activating NF-Kappa B Signalling in Cancer Cells

Tumor necrosis factor-alpha (TNF-α) was reported as anticancer therapy due to its cytotoxic effect against an array of tumor cells. However, its undesirable responses of TNF-α on activating NF-κB signaling and pro-metastatic property limit its clinical application in treating cancers. Therefore, sensitizing agents capable of overcoming this undesirable effect must be valuable for facilitating the usage of TNF-α-mediated apoptosis therapy for cancer patients. Previously, saikosaponin-d (Ssd), a triterpene saponin derived from the medicinal plant, Bupleurum falcatum L. (Umbelliferae), showed to exhibit a variety of pharmacological activities such as antiinflammation, antibacteria, antivirus and anticancer. Recently, we found that Ssd could inhibit the activated T lymphocytes via suppression of NF-κB, NF-AT and AP-1 signaling. Here, we showed that Ssd significantly potentiated TNF-α-mediated cell death in HeLa and HepG2 cancer cells via suppression of TNF-α-induced NF-κB activation and its target genes expression involving cancer cell proliferation, invasion, angiogenesis and survival. Also, Ssd revealed a significant potency of abolishing TNF-α-induced cancer cell invasion and angiogenesis in HUVECs while inducing apoptosis via enhancing the loss of mitochondrial membrane potential in HeLa cells. Collectively, these findings indicate that Ssd has a significant potential to be developed as a combined adjuvant remedy with TNF-α for cancer patients.

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Pandemic influenza in Papua New Guinea: a modelling study comparison with pandemic spread in a developed country

OBJECTIVES: The possible occurrence of a highly pathogenic influenza strain is of concern to health authorities worldwide. It is known that during past influenza pandemics developing countries have experienced considerably higher death rates compared with developed countries. Furthermore, many developing countries lack appropriate pandemic preparedness plans. Mathematical modelling studies to guide the development of such plans are largely focused on predicting pandemic influenza spread in developed nations. However, intervention strategies shown by modelling studies to be highly effective for developed countries give limited guidance as to the impact which an influenza pandemic may have on low-income countries given different demographics and resource constraints. To address this, an individual-based model of a Papua New Guinean (PNG) community was created and used to simulate the spread of a novel influenza strain. The results were compared with those obtained from a comparable Australian model. DESIGN: A modelling study. SETTING: The towns of Madang in PNG (population ∼35 000) and Albany (population ∼30 000) in Australia. OUTCOME MEASURES: Daily and cumulative illness attack rates in both models following introduction of a novel influenza strain into a naive population, for an unmitigated scenario and two social distancing intervention scenarios. RESULTS: The unmitigated scenario indicated an approximately 50% higher attack rate in PNG compared with the Australian model. The two social distancing-based interventions strategies were 60–70% less effective in a PNG setting compared with an Australian setting. CONCLUSIONS: This study provides further evidence that an influenza pandemic occurring in a low-income country such as PNG may have a greater impact than one occurring in a developed country, and that PNG-feasible interventions may be substantially less effective. The larger average household size in PNG, the larger proportion of the population under 18 and greater community-wide contact all contribute to this feature.

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Protection of chickens against infectious bronchitis virus with a multivalent DNA vaccine and boosting with an inactivated vaccine

The protective efficacy of DNA plasmids encoding avian infectious bronchitis virus (IBV) S1, N, or M protein was investigated in chickens. Chickens were inoculated monovalently (with plasmid pVAX1-16S1, pVAX1-16M, or pVAX1-16N alone) or multivalently (combination of the three different plasmids, pVAX1-16S1/M/N). A prime-boost immunization protocol against IBV was developed. Chickens were immunized with the multivalent DNA vaccine twice and then boosted with an inactivated vaccine once. Antibody titers of the chickens immunized with pVAX1-16S1/M/N were much higher than those of the monovalent groups (p < 0.01). A protective rate up to 90% was observed in the pVAX1-16S1/M/N group. The serum antibody titers in the prime-boost birds were significantly higher than those of the multivalent DNA vaccine group (p < 0.01) but not significantly different compared to the inactivated vaccine group at 49 days of age. Additionally, the prime-boost group also showed the highest level of IBV-specific cellular proliferation compared to the monovalent groups (p < 0.01) but no significant difference was found compared to the multivalent DNA vaccine group, and the prime-boost group completely protected from followed viral challenge.

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Advanced Querying Features for Disease Surveillance Systems

Most automated disease surveillance systems notify users of increases in the prevalence of reports in syndrome categories and allow users to view patient level data related to those increases. Occasionally, a more dynamic level of control is required to properly detect an emerging disease in a community. Dynamic querying features are invaluable when using existing surveillance systems to investigate outbreaks of newly emergent diseases or to identify cases of reportable diseases within data being captured for surveillance. The objective of the Advance Querying Tool (AQT) is to build a more flexible query interface for most web-based disease surveillance systems. This interface allows users to define and build their query as if they were writing a logical expression for a mathematical computation. The AQT allows users to develop, investigate, save, and share complex case definitions. It provides a flexible interface that accommodates both advanced and novice users, checks the validity of the expression as it is built, and marks errors for users.

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Monoclonal antibody against CXCL-10/IP-10 ameliorates influenza A (H1N1) virus induced acute lung injury

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This Zoonotic World

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Alginic Acid-Coated Chitosan Nanoparticles Loaded with Legumain DNA Vaccine: Effect against Breast Cancer in Mice

Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a legumain DNA vaccine. First, we tested its characteristic in acidic environments in vitro. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer’s patches. Full-length legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated Salmonella typhimurium and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated Salmonella typhimurium as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl(2), which influenced the translocation of legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3(+)/CD8(+)/CD25(+)) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.

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Multicomponent Therapeutics of Berberine Alkaloids

Although berberine alkaloids (BAs) are reported to be with broad-spectrum antibacterial and antiviral activities, the interactions among BAs have not been elucidated. In the present study, methicillin-resistant Staphylococcus aureus (MRSA) was chosen as a model organism, and modified broth microdilution was applied for the determination of the fluorescence absorption values to calculate the anti-MRSA activity of BAs. We have initiated four steps to seek the optimal combination of BAs that are (1) determining the anti-MRSA activity of single BA, (2) investigating the two-component combination to clarify the interactions among BAs by checkerboard assay, (3) investigating the multicomponent combination to determine the optimal ratio by quadratic rotation-orthogonal combination design, and (4) in vivo and in vitro validation of the optimal combination. The results showed that the interactions among BAs are related to their concentrations. The synergetic combinations included “berberine and epiberberine,” “jatrorrhizine and palmatine” and “jatrorrhizine and coptisine”; the antagonistic combinations included “coptisine and epiberberine”. The optimal combination was berberine : coptisine : jatrorrhizine : palmatine : epiberberine = 0.702 : 0.863 : 1 : 0.491 : 0.526, and the potency of the optimal combination on cyclophosphamide-immunocompromised mouse model was better than the natural combinations of herbs containing BAs.

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Using GPS Technology to Quantify Human Mobility, Dynamic Contacts and Infectious Disease Dynamics in a Resource-Poor Urban Environment

Empiric quantification of human mobility patterns is paramount for better urban planning, understanding social network structure and responding to infectious disease threats, especially in light of rapid growth in urbanization and globalization. This need is of particular relevance for developing countries, since they host the majority of the global urban population and are disproportionally affected by the burden of disease. We used Global Positioning System (GPS) data-loggers to track the fine-scale (within city) mobility patterns of 582 residents from two neighborhoods from the city of Iquitos, Peru. We used ∼2.3 million GPS data-points to quantify age-specific mobility parameters and dynamic co-location networks among all tracked individuals. Geographic space significantly affected human mobility, giving rise to highly local mobility kernels. Most (∼80%) movements occurred within 1 km of an individual’s home. Potential hourly contacts among individuals were highly irregular and temporally unstructured. Only up to 38% of the tracked participants showed a regular and predictable mobility routine, a sharp contrast to the situation in the developed world. As a case study, we quantified the impact of spatially and temporally unstructured routines on the dynamics of transmission of an influenza-like pathogen within an Iquitos neighborhood. Temporally unstructured daily routines (e.g., not dominated by a single location, such as a workplace, where an individual repeatedly spent significant amount of time) increased an epidemic’s final size and effective reproduction number by 20% in comparison to scenarios modeling temporally structured contacts. Our findings provide a mechanistic description of the basic rules that shape human mobility within a resource-poor urban center, and contribute to the understanding of the role of fine-scale patterns of individual movement and co-location in infectious disease dynamics. More generally, this study emphasizes the need for careful consideration of human social interactions when designing infectious disease mitigation strategies, particularly within resource-poor urban environments.

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Clinical Characteristics and Outcomes in Hospitalized Patients with Respiratory Viral Co-Infection during the 2009 H1N1 Influenza Pandemic

BACKGROUND: The clinical consequences of co-infection with two or more respiratory viruses are poorly understood. We sought to determine if co-infection with pandemic 2009–2010 influenza A H1N1 (pH1N1) and another respiratory virus was associated with worse clinical outcomes. METHODS: A retrospective cohort study was performed of all hospitalized patients with a positive respiratory viral panel (RVP) for two or more viruses within 72 hours of admission at our institution from October 2009 to December 2009. We compared patients infected with one respiratory virus to those with respiratory viral co-infection. RESULTS: We identified 617 inpatients with a positive RVP sample with a single virus and 49 inpatients with a positive RVP sample for two viruses (i.e. co-infection). Co-infected patients were significantly younger, more often had fever/chills, tachypnea, and they more often demonstrated interstitial opacities suggestive of viral pneumonia on the presenting chest radiograph (OR 7.5, 95% CI 3.4–16.5). The likelihood of death, length of stay, and requirement for intensive care unit level of care were similar in both groups, but patients with any respiratory virus co-infection were more likely to experience complications, particularly treatment for a secondary bacterial pneumonia (OR 6.8, 95% CI 3.3–14.2). Patients co-infected with pH1N1 and another respiratory virus were more likely to present with chest radiograph changes suggestive of a viral pneumonia, compared to mono-infection with pH1N1 (OR 16.9, 95% CI 4.5–62.7). By logistic regression using mono-infection with non-PH1N1 viruses as the reference group, co-infection with pH1N1 was the strongest independent predictor of treatment for a secondary bacterial pneumonia (OR 17.8, 95% CI 6.7–47.1). CONCLUSION: Patients with viral co-infection, particularly with pH1N1, were more likely to have chest radiograph features compatible with a viral pneumonia and complications during their hospital course, particularly treatment for secondary bacterial pneumonia. Despite this, co-infection was not associated with ICU admission.

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Find novel dual-agonist drugs for treating type 2 diabetes by means of cheminformatics

The high prevalence of type 2 diabetes mellitus in the world as well as the increasing reports about the adverse side effects of the existing diabetes treatment drugs have made developing new and effective drugs against the disease a very high priority. In this study, we report ten novel compounds found by targeting peroxisome proliferator-activated receptors (PPARs) using virtual screening and core hopping approaches. PPARs have drawn increasing attention for developing novel drugs to treat diabetes due to their unique functions in regulating glucose, lipid, and cholesterol metabolism. The reported compounds are featured with dual functions, and hence belong to the category of dual agonists. Compared with the single PPAR agonists, the dual PPAR agonists, formed by combining the lipid benefit of PPARα agonists (such as fibrates) and the glycemic advantages of the PPARγ agonists (such as thiazolidinediones), are much more powerful in treating diabetes because they can enhance metabolic effects while minimizing the side effects. This was observed in the studies on molecular dynamics simulations, as well as on absorption, distribution, metabolism, and excretion, that these novel dual agonists not only possessed the same function as ragaglitazar (an investigational drug developed by Novo Nordisk for treating type 2 diabetes) did in activating PPARα and PPARγ, but they also had more favorable conformation for binding to the two receptors. Moreover, the residues involved in forming the binding pockets of PPARα and PPARγ among the top ten compounds are explicitly presented, and this will be very useful for the in-depth conduction of mutagenesis experiments. It is anticipated that the ten compounds may become potential drug candidates, or at the very least, the findings reported here may stimulate new strategies or provide useful insights for designing new and more powerful dual-agonist drugs for treating type 2 diabetes.

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Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na(+)/H(+) exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.

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The Impact of Model Building on the Transmission Dynamics under Vaccination: Observable (Symptom-Based) versus Unobservable (Contagiousness-Dependent) Approaches

BACKGROUND: The way we formulate a mathematical model of an infectious disease to capture symptomatic and asymptomatic transmission can greatly influence the likely effectiveness of vaccination in the presence of vaccine effect for preventing clinical illness. The present study aims to assess the impact of model building strategy on the epidemic threshold under vaccination. METHODOLOGY/PRINCIPAL FINDINGS: We consider two different types of mathematical models, one based on observable variables including symptom onset and recovery from clinical illness (hereafter, the “observable model”) and the other based on unobservable information of infection event and infectiousness (the “unobservable model”). By imposing a number of modifying assumptions to the observable model, we let it mimic the unobservable model, identifying that the two models are fully consistent only when the incubation period is identical to the latent period and when there is no pre-symptomatic transmission. We also computed the reproduction numbers with and without vaccination, demonstrating that the data generating process of vaccine-induced reduction in symptomatic illness is consistent with the observable model only and examining how the effective reproduction number is differently calculated by two models. CONCLUSIONS: To explicitly incorporate the vaccine effect in reducing the risk of symptomatic illness into the model, it is fruitful to employ a model that directly accounts for disease progression. More modeling studies based on observable epidemiological information are called for.

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Widespread Divergence of the CEACAM/PSG Genes in Vertebrates and Humans Suggests Sensitivity to Selection

In mammals, carcinoembryonic antigen cell adhesion molecules (CEACAMs) and pregnancy-specific glycoproteins (PSGs) play important roles in the regulation of pathogen transmission, tumorigenesis, insulin signaling turnover, and fetal–maternal interactions. However, how these genes evolved and to what extent they diverged in humans remain to be investigated specifically. Based on syntenic mapping of chordate genomes, we reveal that diverging homologs with a prototypic CEACAM architecture–including an extracellular domain with immunoglobulin variable and constant domain-like regions, and an intracellular domain containing ITAM motif–are present from cartilaginous fish to humans, but are absent in sea lamprey, cephalochordate or urochordate. Interestingly, the CEACAM/PSG gene inventory underwent radical divergence in various vertebrate lineages: from zero in avian species to dozens in therian mammals. In addition, analyses of genetic variations in human populations showed the presence of various types of copy number variations (CNVs) at the CEACAM/PSG locus. These copy number polymorphisms have 3–80% frequency in select populations, and encompass single to more than six PSG genes. Furthermore, we found that CEACAM/PSG genes contain a significantly higher density of nonsynonymous single nucleotide polymorphism (SNP) compared to the chromosome average, and many CEACAM/PSG SNPs exhibit high population differentiation. Taken together, our study suggested that CEACAM/PSG genes have had a more dynamic evolutionary history in vertebrates than previously thought. Given that CEACAM/PSGs play important roles in maternal–fetal interaction and pathogen recognition, these data have laid the groundwork for future analysis of adaptive CEACAM/PSG genotype-phenotypic relationships in normal and complicated pregnancies as well as other etiologies.

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Regulation of the Epithelial Adhesion Molecule CEACAM1 Is Important for Palate Formation

Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at various developmental stages before, during, and after palate fusion using GeneChip® microarrays. Ceacam1 was one of the highly up-regulated genes during palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was present in prefusion palatal epithelium and was degraded during fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1 (−/−)) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1 (−/−) mice. TGFβ3 expression, apoptosis, and cell proliferation in palatal epithelium were not affected in the palate of Ceacam1(−/−)mice. However, CEACAM1 expression was retained in the remaining MEE of TGFβ-deficient mice. These results suggest that CEACAM1 has roles in the initiation of palatal fusion via epithelial cell adhesion.

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Identification of Hepatotropic Viruses from Plasma Using Deep Sequencing: A Next Generation Diagnostic Tool

We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.

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Trends in North American Newspaper Reporting of Brain Injury in Ice Hockey

The frequency and potential long-term effects of sport-related traumatic brain injuries (TBI) make it a major public health concern. The culture within contact sports, such as ice hockey, encourages aggression that puts youth at risk of TBI such as concussion. Newspaper reports play an important role in conveying and shaping the culture around health-related behaviors. We qualitatively studied reports about sport-related TBI in four major North American newspapers over the last quarter-century. We used the grounded-theory approach to identify major themes and then did a content analysis to compare the frequency of key themes between 1998–2000 and 2009–2011. The major themes were: perceptions of brain injury, aggression, equipment, rules and regulations, and youth hockey. Across the full study period, newspaper articles from Canada and America portrayed violence and aggression that leads to TBI both as integral to hockey and as an unavoidable risk associated with playing the game. They also condemned violence in ice hockey, criticized the administrative response to TBI, and recognized the significance of TBI. In Canada, aggression was reported more often recently and there was a distinctive shift in portraying protective equipment as a solution to TBI in earlier years to a potential contributing factor to TBI later in the study period. American newspapers gave a greater attention to ‘perception of risks’ and the role of protective equipment, and discussed TBI in a broader context in the recent time period. Newspapers from both countries showed similar recent trends in regards to a need for rule changes to curb youth sport-related TBI. This study provides a rich description of the reporting around TBI in contact sport. Understanding this reporting is important for evaluating whether the dangers of sport-related TBI are being appropriately communicated by the media.

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Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.

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The Development and Application of the Two Real-Time RT-PCR Assays to Detect the Pathogen of HFMD

Large-scale Hand, Foot, and Mouth Disease (HFMD) outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs). Among them, human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1–2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID(50)/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells). The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11) by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.

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Complete Genome Sequence of a Recombinant Porcine Epidemic Diarrhea Virus Strain from Eastern China

A field porcine epidemic diarrhea virus (PEDV) strain, JS2008, was isolated from stool samples of a piglet with acute diarrhea on a vaccinated farm in eastern China. We sequenced and analyzed the complete genome of strain JS2008, which will help increase our understanding of the molecular characteristics of the epidemic PEDV in China.

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In memory of Patrick Manson, founding father of tropical medicine and the discovery of vector-borne infections

Patrick Manson, a clinician-scientist serving in China (1866–1889), discovered that many tropical infectious diseases require a vector peculiar to warm climate for person to person transmission. He demonstrated the nocturnal periodicity of microfilariae in the blood of patients with elephantiasis. These microfilariae undergo metamorphosis when ingested by the mosquito acting as the vector for the completion of their life cycle. Furthermore, he demonstrated the linkage between the lung fluke and endemic haemoptysis by finding operculated eggs in patients' sputa. He predicted that the miracidium from hatched eggs uses crustaceans, such as fresh-water snails found at tropical conditions, as the intermediate hosts in the life cycle of many trematodes. His vector hypothesis leads to vector control which is now the cornerstone for the World Health Organization's programme for the elimination/control of lymphatic filariasis, dracunculiasis and malaria. Before leaving China, he established the Alice Memorial Hospital, the Hong Kong College of Medicine for Chinese (the forerunner of the University of Hong Kong), and the Hong Kong Medical Society for medical service and education. He also incepted the Hong Kong Dairy Farm for supplying hygienic milk affordable by pregnant women, children and patients.

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Marked Variability in the Extent of Protein Disorder within and between Viral Families

Intrinsically disordered regions in eukaryotic proteomes contain key signaling and regulatory modules and mediate interactions with many proteins. Many viral proteomes encode disordered proteins and modulate host factors through the use of short linear motifs (SLiMs) embedded within disordered regions. However, the degree of viral protein disorder across different viruses is not well understood, so we set out to establish the constraints acting on viruses, in terms of their use of disordered protein regions. We surveyed predicted disorder across 2,278 available viral genomes in 41 families, and correlated the extent of disorder with genome size and other factors. Protein disorder varies strikingly between viral families (from 2.9% to 23.1% of residues), and also within families. However, this substantial variation did not follow the established trend among their hosts, with increasing disorder seen across eubacterial, archaebacterial, protists, and multicellular eukaryotes. For example, among large mammalian viruses, poxviruses and herpesviruses showed markedly differing disorder (5.6% and 17.9%, respectively). Viral families with smaller genome sizes have more disorder within each of five main viral types (ssDNA, dsDNA, ssRNA+, dsRNA, retroviruses), except for negative single-stranded RNA viruses, where disorder increased with genome size. However, surveying over all viruses, which compares tiny and enormous viruses over a much bigger range of genome sizes, there is no strong association of genome size with protein disorder. We conclude that there is extensive variation in the disorder content of viral proteomes. While a proportion of this may relate to base composition, to extent of gene overlap, and to genome size within viral types, there remain important additional family and virus-specific effects. Differing disorder strategies are likely to impact on how different viruses modulate host factors, and on how rapidly viruses can evolve novel instances of SLiMs subverting host functions, such as innate and acquired immunity.

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Citrus tristeza virus: Evolution of Complex and Varied Genotypic Groups

Amongst the Closteroviridae, Citrus tristeza virus (CTV) is almost unique in possessing a number of distinct and characterized strains, isolates of which produce a wide range of phenotype combinations among its different hosts. There is little understanding to connect genotypes to phenotypes, and to complicate matters more, these genotypes are found throughout the world as members of mixed populations within a single host plant. There is essentially no understanding of how combinations of genotypes affect symptom expression and disease severity. We know little about the evolution of the genotypes that have been characterized to date, little about the biological role of their diversity and particularly, about the effects of recombination. Additionally, genotype grouping has not been standardized. In this study we utilized an extensive array of CTV genomic information to classify the major genotypes, and to determine the major evolutionary processes that led to their formation and subsequent retention. Our analyses suggest that three major processes act on these genotypes: (1) ancestral diversification of the major CTV lineages, followed by (2) conservation and co-evolution of the major functional domains within, though not between CTV genotypes, and (3) extensive recombination between lineages that have given rise to new genotypes that have subsequently been retained within the global population. The effects of genotype diversity and host-interaction are discussed, as is a proposal for standardizing the classification of existing and novel CTV genotypes.

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Oral presentation in dengue hemorrhagic fever: A rare entity

One of the major health hazards which is prevalent and dangerous is the dengue fever which causes the death of many people. This may be associated with a variety of mucocutaneous manifestations which may be of help in early diagnosis. Many biochemical assays and hematological investigations may aid in the further diagnosis and treatment of the fatal disease. Oral lesions are rare to occur and if present, are often mistaken for platelet abnormality. This case report highlights the importance of oral lesions and it is the first of its kind to be reported as dengue hemorrhagic fever.

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9G DNAChip Technology: Self-Assembled Monolayer (SAM) of ssDNA for Ultra-Sensitive Detection of Biomarkers

A 9G DNAChip obtained by allowing the formation of a self-assembled monolayer (SAM) of oligonucleotides appended with nine consecutive guanines on the chip surface has been applied in the detection of biomarkers. Using a 9G DNAChip, biomarker in the concentration range of 4 pg/mL to 40 fg/mL can be easily differentiated in the buffer matrix. Moreover, it is the first time that a biomarker with a concentration of 40 fg/mL has been detected in a mixture of proteins without use of any signal amplification technique.

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A Study of the Mechanism of the Chaperone-like Function of an scFv of Human Creatine Kinase by Computer Simulation

A new application of antibodies is to use them as macromolecular chaperones. Protein antigens usually have multiple epitopes, thus, there may be a plurality of antibodies binding to one antigen. However, not all antibodies that bind to one antigen could act as a chaperone. Experiments show that some screened anti-human creatine kinase single chain antibodies (scFV) could assist in the folding and stabilizing of the enzyme, while others could not. We built the model of the single chain antibody (scFv-A4) that increased the stability of human creatine kinase (HCK) by the homology modeling method. Epitopes of human creatine kinase were predicted by computer and then the binding of scFv-A4 and HCK was modeled with computer. The calculation results were further combined with the peptide array membrane experiment results to obtain reliable models for the scFv-A4-HCK complex. Based on the above study we gave an explanation about how scFv-A4 could act as a macromolecular chaperone assisting the folding of HCK. This study provides an approach for predicting antigen-antibody binding mode and also a useful theoretical guidance for the study of antibodies' chaperone-like function.

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HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

Upon recognition of viral components by pattern recognition receptors, such as the toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like helicases, cells are activated to produce type I interferon (IFN) and proinflammatory cytokines. These pathways are tightly regulated by the host to prevent an inappropriate cellular response, but viruses can modulate these pathways to proliferate and spread. In this study, we revealed a novel mechanism in which hepatitis C virus (HCV) evades the immune surveillance system to proliferate by activating microRNA-21 (miR-21). We demonstrated that HCV infection upregulates miR-21, which in turn suppresses HCV-triggered type I IFN production, thus promoting HCV replication. Furthermore, we demonstrated that miR-21 targets two important factors in the TLR signaling pathway, myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase 1 (IRAK1), which are involved in HCV-induced type I IFN production. HCV-mediated activation of miR-21 expression requires viral proteins and several signaling components. Moreover, we identified a transcription factor, activating protein-1 (AP-1), which is partly responsible for miR-21 induction in response to HCV infection through PKCε/JNK/c-Jun and PKCα/ERK/c-Fos cascades. Taken together, our results indicate that miR-21 is upregulated during HCV infection and negatively regulates IFN-α signaling through MyD88 and IRAK1 and may be a potential therapeutic target for antiviral intervention.

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Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5′ triphosphate (5′ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5′pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5′pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5′pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5′pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5′pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.

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Immunosenescence in vertebrates and invertebrates

There is an established consensus that it is primarily the adaptive arm of immunity, and the T cell subset in particular, that is most susceptible to the deleterious changes with age known as “immunosenescence”. Can we garner any clues as to why this might be by considering comparative immunology and the evolutionary emergence of adaptive and innate immunity? The immune system is assumed to have evolved to protect the organism against pathogens, but the way in which this is accomplished is different in the innate-vs-adaptive arms, and it is unclear why the latter is necessary. Are there special characteristics of adaptive immunity which might make the system more susceptible to age-associated dysfunction? Given recent accumulating findings that actually there are age-associated changes to innate immunity and that these are broadly similar in vertebrates and invertebrates, we suggest here that it is the special property of memory in the adaptive immune system which results in the accumulation of cells with a restricted receptor repertoire, dependent on the immunological history of the individual’s exposures to pathogens over the lifetime, and which is commonly taken as a hallmark of “immunosenescence”. However, we further hypothesize that this immunological remodelling per se does not necessarily convey a disadvantage to the individual (ie. is not necessarily “senescence” if it is not deleterious). Indeed, under certain circumstances, or potentially even as a rule, this adaptation to the individual host environment may confer an actual survival advantage.

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ACE2-Ang-(1-7)-Mas Axis in Brain: A Potential Target for Prevention and Treatment of Ischemic Stroke

The renin-angiotensin system (RAS) in brain is a crucial regulator for physiological homeostasis and diseases of cerebrovascular system, such as ischemic stroke. Overactivation of brain Angiotensin-converting enzyme (ACE) - Angiotensin II (Ang II) - Angiotensin II type 1 receptor (AT(1)R) axis was found to be involved in the progress of hypertension, atherosclerosis and thrombogenesis, which increased the susceptibility to ischemic stroke. Besides, brain Ang II levels have been revealed to be increased in ischemic tissues after stroke, and contribute to neural damage through elevating oxidative stress levels and inducing inflammatory response in the ischemic hemisphere via AT(1)R. In recent years, new components of RAS have been discovered, including ACE2, Angiotensin-(1–7) [Ang-(1-7)] and Mas, which constitute ACE2-Ang-(1-7)-Mas axis. ACE2 converts Ang II to Ang-(1-7), and Ang-(1-7) binds with its receptor Mas, exerting benefical effects in cerebrovascular disease. Through interacting with nitric oxide and bradykinin, Ang-(1-7) could attenuate the development of hypertension and the pathologic progress of atherosclerosis. Besides, its antithrombotic activity also prevents thrombogenic events, which may contribute to reduce the risk of ischemic stroke. In addition, after ischemia insult, ACE2-Ang-(1-7)-Mas has been shown to reduce the cerebral infarct size and improve neurological deficits through its antioxidative and anti-inflammatory effects. Taken together, activation of the ACE2-Ang-(1-7)-Mas axis may become a novel therapeutic target in prevention and treatment of ischemia stroke, which deserves further investigations.

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Baicalein Reduces Airway Injury in Allergen and IL-13 Induced Airway Inflammation

BACKGROUND: Baicalein, a bioflavone present in the dry roots of Scutellaria baicalensis Georgi, is known to reduce eotaxin production in human fibroblasts. However, there are no reports of its anti-asthma activity or its effect on airway injury. METHODOLOGY/PRINCIPAL FINDINGS: In a standard experimental asthma model, male Balb/c mice that were sensitized with ovalbumin (OVA), treated with baicalein (10 mg/kg, ip) or a vehicle control, either during (preventive use) or after OVA challenge (therapeutic use). In an alternate model, baicalein was administered to male Balb/c mice which were given either IL-4 or IL-13 intranasally. Features of asthma were determined by estimating airway hyperresponsiveness (AHR), histopathological changes and biochemical assays of key inflammatory molecules. Airway injury was determined with apoptotic assays, transmission electron microscopy and assessing key mitochondrial functions. Baicalein treatment reduced AHR and inflammation in both experimental models. TGF-β(1), sub-epithelial fibrosis and goblet cell metaplasia, were also reduced. Furthermore, baicalein treatment significantly reduced 12/15-LOX activity, features of mitochondrial dysfunctions, and apoptosis of bronchial epithelia. CONCLUSION/SIGNIFICANCE: Our findings demonstrate that baicalein can attenuate important features of asthma, possibly through the reduction of airway injury and restoration of mitochondrial function.

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Rosiglitazone Treatment of Type 2 Diabetic db/db Mice Attenuates Urinary Albumin and Angiotensin Converting Enzyme 2 Excretion

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication.

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Awareness and knowledge of disease surveillance and notification by health-care workers and availability of facility records in Anambra state, Nigeria

BACKGROUND: Disease surveillance and notification (DSN) is part of the Health Management Information System (HMIS) which comprises databases, personnel, and materials that are organized to collect data which are utilized for informed decision making. The knowledge about DSN is very important for the reporting of notifiable diseases. OBJECTIVE: The aim of this study is to examine the awareness and knowledge of health-care workers about DSN, and availability of facility records in Anambra State, Nigeria. MATERIALS AND METHODS: The study was a descriptive cross-sectional one in which relevant data were collected from health-care workers selected by a multistage sampling technique. Qualitative information was also elicited by key informant interviews, whereas an observational checklist, preceded by a desk review was used to examine the availability of facility records. RESULTS: Although 89.8% of the health-care workers were aware of the DSN system, only 33.3, 31.1, and 33.7% of them knew the specific uses of forms IDSR 001, IDSR 002, and IDSR 003 (IDSR: Integrated Diseases Surveillance and Response), respectively. Knowledge of use of the various forms at the facility and local government area (LGA) levels were generally low, although the observational checklist revealed that IDSR 001 and IDSR 002 forms were predominantly found in primary health-care facilities. HMIS forms were less likely to be available in secondary health-care facilities (χ(2)=7.67, P=0.005). CONCLUSIONS: Regular training and retraining of concerned health-care workers on DSN at the LGA level is recommended. This should run concurrently with adequate and regular provision of IDSR forms, copies of the standard case definitions, and other necessary logistics to the health-care facilities by the local and state governments.

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Risk factors for severe acute lower respiratory infections in children – a systematic review and meta-analysis

AIM: To identify the risk factors in children under five years of age for severe acute lower respiratory infections (ALRI), which are the leading cause of child mortality. METHODS: We performed a systematic review of published literature available in the public domain. We conducted a quality assessment of all eligible studies according to GRADE criteria and performed a meta-analysis to report the odds ratios for all risk factors identified in these studies. RESULTS: We identified 36 studies that investigated 19 risk factors for severe ALRI. Of these, 7 risk factors were significantly associated with severe ALRI in a consistent manner across studies, with the following meta-analysis estimates of odds ratios (with 95% confidence intervals): low birth weight 3.18 (1.02-9.90), lack of exclusive breastfeeding 2.34 (1.42-3.88), crowding – more than 7 persons per household 1.96 (1.53-2.52), exposure to indoor air pollution 1.57 (1.06-2.31), incomplete immunization 1.83 (1.32-2.52), undernutrition – weight-for-age less than 2 standard deviations 4.47 (2.10-9.49), and HIV infection 4.15 (2.57-9.74). CONCLUSION: This study highlights the role of the above seven risk factors in the development of severe pneumonia in under-five children. In addition, it emphasizes the need for further studies investigating other potential risk factors. Since these risk factors are potentially preventable, health policies targeted at reducing their prevalence provide a basis for decreasing the burden of childhood pneumonia.

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Inference of R (0) and Transmission Heterogeneity from the Size Distribution of Stuttering Chains

For many infectious disease processes such as emerging zoonoses and vaccine-preventable diseases, [Image: see text] and infections occur as self-limited stuttering transmission chains. A mechanistic understanding of transmission is essential for characterizing the risk of emerging diseases and monitoring spatio-temporal dynamics. Thus methods for inferring [Image: see text] and the degree of heterogeneity in transmission from stuttering chain data have important applications in disease surveillance and management. Previous researchers have used chain size distributions to infer [Image: see text], but estimation of the degree of individual-level variation in infectiousness (as quantified by the dispersion parameter, [Image: see text]) has typically required contact tracing data. Utilizing branching process theory along with a negative binomial offspring distribution, we demonstrate how maximum likelihood estimation can be applied to chain size data to infer both [Image: see text] and the dispersion parameter that characterizes heterogeneity. While the maximum likelihood value for [Image: see text] is a simple function of the average chain size, the associated confidence intervals are dependent on the inferred degree of transmission heterogeneity. As demonstrated for monkeypox data from the Democratic Republic of Congo, this impacts when a statistically significant change in [Image: see text] is detectable. In addition, by allowing for superspreading events, inference of [Image: see text] shifts the threshold above which a transmission chain should be considered anomalously large for a given value of [Image: see text] (thus reducing the probability of false alarms about pathogen adaptation). Our analysis of monkeypox also clarifies the various ways that imperfect observation can impact inference of transmission parameters, and highlights the need to quantitatively evaluate whether observation is likely to significantly bias results.

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A study of non-prescription usage of antibiotics in the upper respiratory tract infections in the urban population

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Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome

The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (−) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb(2+) ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5′–3′ terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3′ terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5′ DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via ‘flipping’ mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3′ DB and its complementary region PK1 indicated that communication between 5′–3′ terminal regions strongly depends on structure and sequence composition of the 5′ cyclization region.

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Identifying Early Inflammatory Changes in Monocyte-Derived Macrophages from a Population with IQ-Discrepant Episodic Memory

BACKGROUND: Cells of the innate immune system including monocytes and macrophages are the first line of defence against infections and are critical regulators of the inflammatory response. These cells express toll-like receptors (TLRs), innate immune receptors which govern tailored inflammatory gene expression patterns. Monocytes, which produce pro-inflammatory mediators, are readily recruited to the central nervous system (CNS) in neurodegenerative diseases. METHODS: This study explored the expression of receptors (CD11b, TLR2 and TLR4) on circulating monocyte-derived macrophages (MDMs) and peripheral blood mononuclear cells (PBMCs) isolated from healthy elderly adults who we classified as either IQ memory-consistent (high-performing, HP) or IQ memory-discrepant (low-performing, LP). RESULTS: The expression of CD11b, TLR4 and TLR2 was increased in MDMs from the LP group when compared to HP cohort. MDMs from both groups responded robustly to treatment with the TLR4 activator, lipopolysaccharide (LPS), in terms of cytokine production. Significantly, MDMs from the LP group displayed hypersensitivity to LPS exposure. INTERPRETATION: Overall these findings define differential receptor expression and cytokine profiles that occur in MDMs derived from a cohort of IQ memory-discrepant individuals. These changes are indicative of inflammation and may be involved in the prodromal processes leading to the development of neurodegenerative disease.

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The Transient Nature of Bunyamwera Orthobunyavirus NSs Protein Expression: Effects of Increased Stability of NSs Protein on Virus Replication

The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein.

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Dynamics of translation by single ribosomes through mRNA secondary structures

During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary (2°) structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we employ single molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem loop or pseudoknot mRNA 2° structures. Downstream stem-loops containing 100% G-C base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit (E) site. Downstream stem-loops or pseudoknots containing both G-C and A-U pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat surprisingly, unfolding of mRNA 2° structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.

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H1N1 influenza A virus Related Pneumonia and Respiratory Failure

Influenza viruses are common respiratory pathogens in humans and can cause serious infection that leads to the development of pneumonia. In this study, the clinical and laboratory features of 36 patients from Turkey who are hospitalized in intensive care unit due to pandemic influenza A (H1N1) associated pneumonia and respiratory failure were retrospectively evaluated. The most common symptoms were cough and fever. Consolidation (36.1 %) and interstitial changes (30.6 %) were the most frequently identified findings on chest radiographs at the time of admission. Six of the patients (16.7 %) died. Mortality occurred in 3 of 13 patients (23.1 %) with underlying disease, whilst it occurred in only 3 of 23 patients (13 %) who were previously healthy. Mortality was found to be significantly associated only with an elevated lactate dehydrogenase level. A significant relationship was determined only between the presence of lymphopenia and acute respiratory distress syndrome and the need for intensive care treatment. The average time elapsed from the onset of the symptoms until admission was 8.67 ± 2.87 days for the patients died, and 6.0 ± 3.8 days for the patients survived. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13337-012-0122-z) contains supplementary material, which is available to authorized users.

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Low serum total nitrite and nitrate levels in severe leptospirosis

BACKGROUND: The relationship between inducible nitric oxide synthatase activity and disease severity in leptospirosis is unclear. Nitric oxide is converted to nitrites and nitrates, thus nitrite and nitrate levels (NO(x)) in serum are considered surrogate markers for nitric oxide. NO(x) are excreted through the kidneys, and elimination is diminished in renal impairment. We assessed the correlation of NO(x) with disease severity in patients with leptospirosis, compared with healthy controls and non-leptospirosis fever patients. METHODS: All patients admitted over a two-month period to the National Hospital, Colombo, Sri Lanka with a clinical picture suggestive of leptospirosis were included. Leptospirosis was confirmed by the microscopic agglutination test (titre≥400). Severe leptospirosis was defined by the presence of two or more of the following criteria: jaundice (bilirubin> 51.3 μmol/l), oliguria (urine output < 400 ml/day), serum creatinine> 133 μmol/l or blood urea > 25.5 mmol/l, or the presence of organ dysfunction. Non-leptospirosis fever patients and healthy volunteers were used as control groups. NO(x) levels were measured using a modified Griess reaction. RESULTS: Forty patients were confirmed as having leptospirosis and 26 of them had severe disease. NO(x) levels were significantly higher in confirmed leptospirosis patients compared to healthy controls, MAT equivocal patients and non-leptospirosis fever patients (p<0.001). NO(x) concentrations were also significantly higher in patients with severe compared to mild leptospirosis (p<0.001). Once NO(x) levels were corrected for renal function, by using the ratio NO(x)/creatinine, NO(x) levels were actually significantly lower in patients with severe disease compared to other patients, and values were similar to those of healthy controls. CONCLUSIONS: We postulate that high NOx levels may be protective against severe leptospirosis, and that finding low NOx levels (when corrected for renal function) in patients with leptospirosis may predict the development of severe disease and organ dysfunction.

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Incidence and Mortality Rates of Disasters and Mass Casualty Incidents in Korea: A Population-Based Cross-Sectional Study, 2000-2009

The objective of study was to evaluate the incidence and mortality rates of disasters and mass casualty incidents (MCIs) over the past 10 yr in the administrative system of Korea administrative system and to examine their relationship with population characteristics. This was a population-based cross-sectional study. We calculated the nationwide incidence, as well as the crude mortality and injury incidence rates, of disasters and MCIs. The data were collected from the administrative database of the National Emergency Management Agency (NEMA) and from provincial fire departments from January 2000 to December 2009. A total of 47,169 events were collected from the NEMA administrative database. Of these events, 115 and 3,079 cases were defined as disasters and MCIs that occurred in Korea, respectively. The incidence of technical disasters/MCIs was approximately 12.7 times greater than that of natural disasters/MCIs. Over the past 10 yr, the crude mortality rates for disasters and MCIs were 2.36 deaths per 100,000 persons and 6.78 deaths per 100,000 persons, respectively. The crude injury incidence rates for disasters and MCIs were 25.47 injuries per 100,000 persons and 152 injuries per 100,000 persons, respectively. The incidence and mortality of disasters/MCIs in Korea seem to be low compared to that of trend around the world.

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Carriage of Mycoplasma pneumoniae in the Upper Respiratory Tract of Symptomatic and Asymptomatic Children: An Observational Study

BACKGROUND: Mycoplasma pneumoniae is thought to be a common cause of respiratory tract infections (RTIs) in children. The diagnosis of M. pneumoniae RTIs currently relies on serological methods and/or the detection of bacterial DNA in the upper respiratory tract (URT). It is conceivable, however, that these diagnostic methods also yield positive results if M. pneumoniae is carried asymptomatically in the URT. Positive results from these tests may therefore not always be indicative of a symptomatic infection. The existence of asymptomatic carriage of M. pneumoniae has not been established. We hypothesized that asymptomatic carriage in children exists and investigated whether colonization and symptomatic infection could be differentiated by current diagnostic methods. METHODS AND FINDINGS: This study was conducted at the Erasmus MC–Sophia Children's Hospital and the after-hours General Practitioners Cooperative in Rotterdam, The Netherlands. Asymptomatic children (n = 405) and children with RTI symptoms (n = 321) aged 3 mo to 16 y were enrolled in a cross-sectional study from July 1, 2008, to November 30, 2011. Clinical data, pharyngeal and nasopharyngeal specimens, and serum samples were collected. The primary objective was to differentiate between colonization and symptomatic infection with M. pneumoniae by current diagnostic methods, especially real-time PCR. M. pneumoniae DNA was detected in 21.2% (95% CI 17.2%–25.2%) of the asymptomatic children and in 16.2% (95% CI 12.2%–20.2%) of the symptomatic children (p = 0.11). Neither serology nor quantitative PCR nor culture differentiated asymptomatic carriage from infection. A total of 202 children were tested for the presence of other bacterial and viral pathogens. Two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Finally, longitudinal sampling showed persistence of M. pneumoniae in the URT for up to 4 mo. Fifteen of the 21 asymptomatic children with M. pneumoniae and 19 of the 22 symptomatic children with M. pneumoniae in this longitudinal follow-up tested negative after 1 mo. CONCLUSIONS: Although our study has limitations, such as a single study site and limited sample size, our data indicate that the presence of M. pneumoniae in the URT is common in asymptomatic children. The current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection. Please see later in the article for the Editors' Summary

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Abstracts from the 36th Annual Meeting of the Society of General Internal Medicine

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Recombinant Vaccines against T. gondii: Comparison between Homologous and Heterologous Vaccination Protocols Using Two Viral Vectors Expressing SAG1

The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1) of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector) is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination). Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1), to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

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The Secret Life of Viral Entry Glycoproteins: Moonlighting in Immune Evasion

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Abortive Lytic Reactivation of KSHV in CBF1/CSL Deficient Human B Cell Lines

Since Kaposi's sarcoma associated herpesvirus (KSHV) establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade.

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Reovirus Activates a Caspase-Independent Cell Death Pathway

Virus-induced apoptosis is thought to be the primary mechanism of cell death following reovirus infection. Induction of cell death following reovirus infection is initiated by the incoming viral capsid proteins during cell entry and occurs via NF-κB-dependent activation of classical apoptotic pathways. Prototype reovirus strain T3D displays a higher cell-killing potential than strain T1L. To investigate how signaling pathways initiated by T3D and T1L differ, we methodically analyzed cell death pathways activated by these two viruses in L929 cells. We found that T3D activates NF-κB, initiator caspases, and effector caspases to a significantly greater extent than T1L. Surprisingly, blockade of NF-κB or caspases did not affect T3D-induced cell death. Cell death following T3D infection resulted in a reduction in cellular ATP levels and was sensitive to inhibition of the kinase activity of receptor interacting protein 1 (RIP1). Furthermore, membranes of T3D-infected cells were compromised. Based on the dispensability of caspases, a requirement for RIP1 kinase function, and the physiological status of infected cells, we conclude that reovirus can also induce an alternate, necrotic form of cell death described as necroptosis. We also found that induction of necroptosis requires synthesis of viral RNA or proteins, a step distinct from that necessary for the induction of apoptosis. Thus, our studies reveal that two different events in the reovirus replication cycle can injure host cells by distinct mechanisms.

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Editorial Commentary: Pandemic H5N1: Receding Risk or Coming Catastrophe?

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Unified Theory of Bacterial Sialometabolism: How and Why Bacteria Metabolize Host Sialic Acids

Sialic acids are structurally diverse nine-carbon ketosugars found mostly in humans and other animals as the terminal units on carbohydrate chains linked to proteins or lipids. The sialic acids function in cell-cell and cell-molecule interactions necessary for organismic development and homeostasis. They not only pose a barrier to microorganisms inhabiting or invading an animal mucosal surface, but also present a source of potential carbon, nitrogen, and cell wall metabolites necessary for bacterial colonization, persistence, growth, and, occasionally, disease. The explosion of microbial genomic sequencing projects reveals remarkable diversity in bacterial sialic acid metabolic potential. How bacteria exploit host sialic acids includes a surprisingly complex array of metabolic and regulatory capabilities that is just now entering a mature research stage. This paper attempts to describe the variety of bacterial sialometabolic systems by focusing on recent advances at the molecular and host-microbe-interaction levels. The hope is that this focus will provide a framework for further research that holds promise for better understanding of the metabolic interplay between bacterial growth and the host environment. An ability to modify or block this interplay has already yielded important new insights into potentially new therapeutic approaches for modifying or blocking bacterial colonization or infection.

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A Lattice Model for Influenza Spreading

We construct a stochastic SIR model for influenza spreading on a D-dimensional lattice, which represents the dynamic contact network of individuals. An age distributed population is placed on the lattice and moves on it. The displacement from a site to a nearest neighbor empty site, allows individuals to change the number and identities of their contacts. The dynamics on the lattice is governed by an attractive interaction between individuals belonging to the same age-class. The parameters, which regulate the pattern dynamics, are fixed fitting the data on the age-dependent daily contact numbers, furnished by the Polymod survey. A simple SIR transmission model with a nearest neighbors interaction and some very basic adaptive mobility restrictions complete the model. The model is validated against the age-distributed Italian epidemiological data for the influenza A(H1N1) during the [Image: see text] season, with sensible predictions for the epidemiological parameters. For an appropriate topology of the lattice, we find that, whenever the accordance between the contact patterns of the model and the Polymod data is satisfactory, there is a good agreement between the numerical and the experimental epidemiological data. This result shows how rich is the information encoded in the average contact patterns of individuals, with respect to the analysis of the epidemic spreading of an infectious disease.

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Proteomic analysis identifies highly antigenic proteins on exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages

Exosomes are small 30–100 nm membrane vesicles released from hematopoietic and non-hematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present on exosomes inducing these host responses were not defined. The present study used LC-MS/MS to identify 41 mycobacterial proteins present on exosomes released from M. tuberculosis-infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFP) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP-treated J774 cells, the majority of which were also present on exosomes isolated from M. tuberculosis-infected cells. The exosomes from CFP-treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naïve T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine.

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Induction of Interferon-Stimulated Genes on the IL-4 Response Axis by Epstein-Barr Virus Infected Human B Cells; Relevance to Cellular Transformation

Epstein-Barr virus (EBV) is an oncogenic virus that is associated with the pathogenesis of several human lymphoid malignancies, including Hodgkin's lymphoma. Infection of normal resting B cells with EBV results in activation to lymphoblasts that are phenotypically similar to those generated by physiological stimulation with CD40L plus IL-4. One important difference is that infection leads to the establishment of permanently growing lymphoblastoid cell lines, whereas CD40L/IL-4 blasts have finite proliferation lifespans. To identify early events which might later determine why EBV infected blasts go on to establish transformed cell lines, we performed global transcriptome analyses on resting B cells and on EBV and CD40L/IL-4 blasts after 7 days culture. As anticipated there was considerable overlap in the transcriptomes of the two types of lymphoblasts when compared to the original resting B cells, reflecting common changes associated with lymphocyte activation and proliferation. Of interest to us was a subset of 255 genes that were differentially expressed between EBV and CD40L/IL-4 blasts. Genes which were more highly expressed in EBV blasts were substantially and significantly enriched for a set of interferon-stimulated genes which on further in silico analyses were found to be repressed by IL-4 in other cell contexts and to be up-regulated in micro-dissected malignant cells from Hodgkin's lymphoma biopsies when compared to their normal germinal center cell counterparts. We hypothesized that EBV and IL-4 were targeting and discordantly regulating a common set of genes. This was supported experimentally in our B cell model where IL-4 stimulation partially reversed transcriptional changes which follow EBV infection and it impaired the efficiency of EBV-induced B cell transformation. Taken together, these data suggest that the discordant regulation of interferon and IL-4 pathway genes by EBV that occurs early following infection of B cells has relevance to the development or maintenance of an EBV-associated malignancy.

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Dual Host-Virus Arms Races Shape an Essential Housekeeping Protein

Transferrin Receptor (TfR1) is the cell-surface receptor that regulates iron uptake into cells, a process that is fundamental to life. However, TfR1 also facilitates the cellular entry of multiple mammalian viruses. We use evolutionary and functional analyses of TfR1 in the rodent clade, where two families of viruses bind this receptor, to mechanistically dissect how essential housekeeping genes like TFR1 successfully balance the opposing selective pressures exerted by host and virus. We find that while the sequence of rodent TfR1 is generally conserved, a small set of TfR1 residue positions has evolved rapidly over the speciation of rodents. Remarkably, all of these residues correspond to the two virus binding surfaces of TfR1. We show that naturally occurring mutations at these positions block virus entry while simultaneously preserving iron-uptake functionalities, both in rodent and human TfR1. Thus, by constantly replacing the amino acids encoded at just a few residue positions, TFR1 divorces adaptation to ever-changing viruses from preservation of key cellular functions. These dynamics have driven genetic divergence at the TFR1 locus that now enforces species-specific barriers to virus transmission, limiting both the cross-species and zoonotic transmission of these viruses.

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ECR 2013 Book of Abstracts - B - Scientific Sessions

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ECR 2013 Book of Abstracts - A - Postgraduate Educational Programme

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Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium’s pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is desirable to simplify the identification of structural epitopes, as this would extend the spectrum of novel epitopes to be detected.

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Monitoring Influenza Epidemics in China with Search Query from Baidu

Several approaches have been proposed for near real-time detection and prediction of the spread of influenza. These include search query data for influenza-related terms, which has been explored as a tool for augmenting traditional surveillance methods. In this paper, we present a method that uses Internet search query data from Baidu to model and monitor influenza activity in China. The objectives of the study are to present a comprehensive technique for: (i) keyword selection, (ii) keyword filtering, (iii) index composition and (iv) modeling and detection of influenza activity in China. Sequential time-series for the selected composite keyword index is significantly correlated with Chinese influenza case data. In addition, one-month ahead prediction of influenza cases for the first eight months of 2012 has a mean absolute percent error less than 11%. To our knowledge, this is the first study on the use of search query data from Baidu in conjunction with this approach for estimation of influenza activity in China.

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Double-Stranded RNA Attenuates the Barrier Function of Human Pulmonary Artery Endothelial Cells

Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca(2+)) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca(2+) signaling in hPAECs by inhibiting the sarco-endoplasmic Ca(2+)-ATPase (SERCA) which is involved in the regulation of the intracellular Ca(2+) homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes.

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A Novel Lactococcal Vaccine Expressing a Peptide from the M2 Antigen of H5N2 Highly Pathogenic Avian Influenza A Virus Prolongs Survival of Vaccinated Chickens

A cost-effective and efficacious influenza vaccine for use in commercial poultry farms would help protect against avian influenza outbreaks. Current influenza vaccines for poultry are expensive and subtype specific, and therefore there is an urgent need to develop a universal avian influenza vaccine. We have constructed a live bacterial vaccine against avian influenza by expressing a conserved peptide from the ectodomain of M2 antigen (M2e) on the surface of Lactococcus lactis (LL). Chickens were vaccinated intranasally with the lactococcal vaccine (LL-M2e) or subcutaneously with keyhole-limpet-hemocyanin conjugated M2e (KLH-M2e). Vaccinated and nonvaccinated birds were challenged with high pathogenic avian influenza virus A subtype H5N2. Birds vaccinated with LL-M2e or KLH-M2e had median survival times of 5.5 and 6.0 days, respectively, which were significantly longer than non-vaccinated birds (3.5 days). Birds vaccinated subcutaneously with KLH-M2e had a lower mean viral burden than either of the other two groups. However, there was a significant correlation between the time of survival and M2e-specific serum IgG. The results of these trials show that birds in both vaccinated groups had significantly (P < 0.05) higher median survival times than non-vaccinated birds and that this protection could be due to M2e-specific serum IgG.

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Nanorobot Hardware Architecture for Medical Defense

This work presents a new approach with details on the integrated platform and hardware architecture for nanorobots application in epidemic control, which should enable real time in vivo prognosis of biohazard infection. The recent developments in the field of nanoelectronics, with transducers progressively shrinking down to smaller sizes through nanotechnology and carbon nanotubes, are expected to result in innovative biomedical instrumentation possibilities, with new therapies and efficient diagnosis methodologies. The use of integrated systems, smart biosensors, and programmable nanodevices are advancing nanoelectronics, enabling the progressive research and development of molecular machines. It should provide high precision pervasive biomedical monitoring with real time data transmission. The use of nanobioelectronics as embedded systems is the natural pathway towards manufacturing methodology to achieve nanorobot applications out of laboratories sooner as possible. To demonstrate the practical application of medical nanorobotics, a 3D simulation based on clinical data addresses how to integrate communication with nanorobots using RFID, mobile phones, and satellites, applied to long distance ubiquitous surveillance and health monitoring for troops in conflict zones. Therefore, the current model can also be used to prevent and save a population against the case of some targeted epidemic disease.

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Self-Oligomerization Is Essential for Enhanced Immunological Activities of Soluble Recombinant Calreticulin

We have recently reported that calreticulin (CRT), a luminal resident protein, can be found in the sera of patients with rheumatoid arthritis and also that recombinant CRT (rCRT) exhibits extraordinarily strong immunological activities. We herein further demonstrate that rCRT fragments 18–412 (rCRT/18-412), rCRT/39-272, rCRT/120-308 and rCRT/120-250 can self-oligomerize in solution and are 50–100 fold more potent than native CRT (nCRT, isolated from mouse livers) in activating macrophages in vitro. We narrowed down the active site of CRT to residues 150–230, the activity of which also depends on dimerization. By contrast, rCRT/18-197 is almost completely inactive. When rCRT/18-412 is fractionated into oligomers and monomers by gel filtration, the oligomers maintain most of their immunological activities in terms of activating macrophages in vitro and inducing specific antibodies in vivo, while the monomers were much less active by comparison. Additionally, rCRT/18-412 oligomers are much better than monomers in binding to, and uptake by, macrophages. Inhibition of macrophage endocytosis partially blocks the stimulatory effect of rCRT/18-412. We conclude that the immunologically active site of CRT maps between residues 198–230 and that soluble CRT could acquire potent immuno-pathological activities in microenvironments favoring its oligomerization.

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Epitope Mapping of M36, a Human Antibody Domain with Potent and Broad HIV-1 Inhibitory Activity

M36 is the first member of a novel class of potent HIV-1 entry inhibitors based on human engineered antibody domains (eAds). It exhibits broad inhibitory activity suggesting that its CD4-induced epitope is highly conserved. Here, we describe fine mapping of its epitope by using several approaches. First, a panel of mimotopes was affinity-selected from a random peptide library and potential m36-binding residues were computationally predicted. Second, homology modeling of m36 and molecular docking of m36 onto gp120 revealed potentially important residues in gp120-m36 interactions. Third, the predicted contact residues were verified by site-directed mutagenesis. Taken together, m36 epitope comprising three discontinuous sites including six key gp120 residues (Site C1: Thr123 and Pro124; Site C3: Glu370 and Ile371; Site C4: Met426 and Trp427) were identified. In the 3D structure of gp120, the sites C1 and C4 are located in the bridging sheet and the site C3 is within the β15-α3 excursion, which play essential roles for the receptor- and coreceptor-binding and are major targets of neutralizing antibodies. Based on these results we propose a precise localization of the m36 epitope and suggest a mechanism of its broad inhibitory activity which could help in the development of novel HIV-1 therapeutics based on eAds.

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Stenotrophomonas, Mycobacterium, and Streptomyces in home dust and air: associations with moldiness and other home/family characteristics

Respiratory illnesses have been linked to children's exposures to water‐damaged homes. Therefore, understanding the microbiome in water‐damaged homes is critical to preventing these illnesses. Few studies have quantified bacterial contamination, especially specific species, in water‐damaged homes. We collected air and dust samples in twenty‐one low‐mold homes and twenty‐one high‐mold homes. The concentrations of three bacteria/genera, Stenotrophomonas maltophilia, Streptomyces sp., and Mycobacterium sp., were measured in air and dust samples using quantitative PCR (QPCR). The concentrations of the bacteria measured in the air samples were not associated with any specific home characteristic based on multiple regression models. However, higher concentrations of S. maltophilia in the dust samples were associated with water damage, that is, with higher floor surface moisture and higher concentrations of moisture‐related mold species. The concentrations of Streptomyces and Mycobacterium sp. had similar patterns and may be partially determined by human and animal occupants and outdoor sources of these bacteria.

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Microarray analysis of MicroRNA expression in peripheral blood mononuclear cells of critically ill patients with influenza A (H1N1)

BACKGROUND: With concerns about the disastrous health and economic consequences caused by the influenza pandemic, comprehensively understanding the global host response to influenza virus infection is urgent. The role of microRNA (miRNA) has recently been highlighted in pathogen-host interactions. However, the precise role of miRNAs in the pathogenesis of influenza virus infection in humans, especially in critically ill patients is still unclear. METHODS: We identified cellular miRNAs involved in the host response to influenza virus infection by performing comprehensive miRNA profiling in peripheral blood mononuclear cells (PBMCs) from critically ill patients with swine-origin influenza pandemic H1N1 (2009) virus infection via miRNA microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays. Receiver operator characteristic (ROC) curve analysis was conducted and area under the ROC curve (AUC) was calculated to evaluate the diagnostic accuracy of severe H1N1 influenza virus infection. Furthermore, an integrative network of miRNA-mediated host-influenza virus protein interactions was constructed by integrating the predicted and validated miRNA-gene interaction data with influenza virus and host-protein-protein interaction information using Cytoscape software. Moreover, several hub genes in the network were selected and validated by qRT-PCR. RESULTS: Forty-one significantly differentially expressed miRNAs were found by miRNA microarray; nine were selected and validated by qRT-PCR. QRT-PCR assay and ROC curve analyses revealed that miR-31, miR-29a and miR-148a all had significant potential diagnostic value for critically ill patients infected with H1N1 influenza virus, which yielded AUC of 0.9510, 0.8951 and 0.8811, respectively. We subsequently constructed an integrative network of miRNA-mediated host-influenza virus protein interactions, wherein we found that miRNAs are involved in regulating important pathways, such as mitogen-activated protein kinase signaling pathway, epidermal growth factor receptor signaling pathway, and Toll-like receptor signaling pathway, during influenza virus infection. Some of differentially expressed miRNAs via in silico analysis targeted mRNAs of several key genes in these pathways. The mRNA expression level of tumor protein T53 and transforming growth factor beta receptor 1 were found significantly reduced in critically ill patients, whereas the expression of Janus kinase 2, caspase 3 apoptosis-related cysteine peptidase, interleukin 10, and myxovirus resistance 1 were extremely increased in critically ill patients. CONCLUSIONS: Our data suggest that the dysregulation of miRNAs in the PBMCs of H1N1 critically ill patients can regulate a number of key genes in the major signaling pathways associated with influenza virus infection. These differentially expressed miRNAs could be potential therapeutic targets or biomarkers for severe influenza virus infection.

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A cost effective real-time PCR for the detection of adenovirus from viral swabs

Compared to traditional testing strategies, nucleic acid amplification tests such as real-time PCR offer many advantages for the detection of human adenoviruses. However, commercial assays are expensive and cost prohibitive for many clinical laboratories. To overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an “in-house” real-time PCR. In 196 swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral DNA obtained from a commercial nucleic acid extraction. In addition, the in-house real-time PCR outperformed traditional testing strategies using virus culture, with sensitivities of 100% and 69.2%, respectively. Overall, the combination of homogenization and heat treatment with a sensitive in-house real-time PCR provides accurate results at a cost comparable to viral culture.

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Introducing the Outbreak Threshold in Epidemiology

When a pathogen is rare in a host population, there is a chance that it will die out because of stochastic effects instead of causing a major epidemic. Yet no criteria exist to determine when the pathogen increases to a risky level, from which it has a large chance of dying out, to when a major outbreak is almost certain. We introduce such an outbreak threshold (T(0)), and find that for large and homogeneous host populations, in which the pathogen has a reproductive ratio R(0), on the order of 1/Log(R(0)) infected individuals are needed to prevent stochastic fade-out during the early stages of an epidemic. We also show how this threshold scales with higher heterogeneity and R(0) in the host population. These results have implications for controlling emerging and re-emerging pathogens.

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Identification of canine parvovirus with the Q370R point mutation in the VP2 gene from a giant panda (Ailuropoda melanoleuca)

BACKGROUND: In this study, we sequenced and phylogenetic analyses of the VP2 genes from twelve canine parvovirus (CPV) strains obtained from eleven domestic dogs and a giant panda (Ailuropoda melanoleuca) in China. A novel canine parvovirus (CPV) was detected from the giant panda in China. RESULTS: Nucleotide and phylogenetic analysis of the capsid protein VP2 gene classified the CPV as a new CPV-2a type. Substitution of Gln for Arg at the conserved 370 residue in CPV presents an unusual variation in the new CPV-2a amino acid sequence of the giant panda and is further evidence for the continuing evolution of the virus. CONCLUSIONS: These findings extend the knowledge on CPV molecular epidemiology of particular relevance to wild carnivores.

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Acute demyelinating encephalomyelitis: Clinical characteristics and outcome

BACKGROUND: ADEM, although relatively uncommon, is probably under-recognized. OBJECTIVES: To spotlight the clinical profile and therapeutic outcome of children with ADEM. MATERIALS AND METHODS: This is a prospective study of patients with ADEM who were admitted to the Pediatric Departments in Aladan and Alfarawanya Hospitals in Kuwait, from January 2009 to January 2011. Clinical, microbiological and radiological data were analyzed. RESULTS: Of 48 patients presented with acute neurological symptoms and signs, 21 patients fulfilled criteria for ADEM. 80.95% of cases were presenting in winter and spring, 57% of patients had a history of upper respiratory tract illness. The commonest presentations were motor deficits, convulsions and altered consciousness. CSF virology studies showed herpes simplex virus (HSV) and Epstein-Barr virus (EBV) (3 patients) whereas nasal and nasopharyngeal swab showed evidence of influenza H1N1 virus (1 patient). Brain MRI was performed in all patients and revealed multiple hyperintense supratentorial brain lesions on T2/FLAIR images. 85.7% of patients had cortical and/or subcortical white matter lesions which were bilateral and asymmetric in location and size. CONCLUSION: ADEM although rare must be considered in children with acute onset of neurological signs and symptoms and must be distinguished from any acute neurological insult.

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Novel compounds for the treatment of Duchenne muscular dystrophy: emerging therapeutic agents

The identification of dystrophin and the causative role of mutations in this gene in Duchenne and Becker muscular dystrophies (D/BMD) was expected to lead to timely development of effective therapies. Despite over 20 years of research, corticosteroids remain the only available pharmacological treatment for DMD, although significant benefits and extended life have resulted from advances in the clinical care and management of DMD individuals. Effective treatment of DMD will require dystrophin restitution in skeletal, cardiac, and smooth muscles and nonmuscle tissues; however, modulation of muscle loss and regeneration has the potential to play an important role in altering the natural history of DMD, particularly in combination with other treatments. Emerging biological, molecular, and small molecule therapeutics are showing promise in ameliorating this devastating disease, and it is anticipated that regulatory environments will need to display some flexibility in order to accommodate the new treatment paradigms.

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Correlation between Dengue-Specific Neutralizing Antibodies and Serum Avidity in Primary and Secondary Dengue Virus 3 Natural Infections in Humans

Although heterotypic secondary infection with dengue virus (DENV) is associated with severe disease, the majority of secondary infections are mild or asymptomatic. The mechanisms of antibody-mediated protection are poorly understood. In 2010, 108 DENV3-positive cases were enrolled in a pediatric hospital-based study in Managua, Nicaragua, with 61 primary and 47 secondary infections. We analyzed DENV-specific neutralization titers (NT(50)), IgM and IgG avidity, and antibody titer in serum samples collected during acute and convalescent phases and 3, 6, and 18 months post-infection. NT(50) titers peaked at convalescence and decreased thereafter. IgG avidity to DENV3 significantly increased between convalescent and 3-month time-points in primary DENV infections and between the acute and convalescent phase in secondary DENV infections. While avidity to DENV2, a likely previous infecting serotype, was initially higher than avidity to DENV3 in secondary DENV infections, the opposite relation was observed 3–18 months post-infection. We found significant correlations between IgM avidity and NT(50) in acute primary cases and between IgG avidity and NT(50) in secondary DENV infections. In summary, our findings indicate that IgM antibodies likely play a role in early control of DENV infections. IgG serum avidity to DENV, analyzed for the first time in longitudinal samples, switches from targeting mainly cross-reactive serotype(s) to the current infecting serotype over time. Finally, serum avidity correlates with neutralization capacity.

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Long-term quality of life in patients with acute respiratory distress syndrome requiring extracorporeal membrane oxygenation for refractory hypoxaemia

INTRODUCTION: The purpose of the study was to assess the long term outcome and quality of life of patients with acute respiratory distress syndrome (ARDS) receiving extracorporeal membrane oxygenation (ECMO) for refractory hypoxemia. METHODS: A retrospective observational study with prospective health related quality of life (HRQoL) assessment was conducted in ARDS patients who had ECMO as a rescue therapy for reversible refractory hypoxemia from January 2009 until April 2011 in a tertiary Australian centre. Survival and long-term quality of life assessment, using the Short-Form 36 (SF-36) and the EuroQol health related quality of life questionnaire (EQ5D) were assessed and compared to international data from other research groups. RESULTS: Twenty-one patients (mean age 36.3 years) with ARDS receiving ECMO for refractory hypoxemia were studied. Eighteen (86%) patients were retrieved from external intensive care units (ICUs) by a dedicated ECMO retrieval team. Eleven (55%) had H1N1 influenza A-associated pneumonitis. Eighteen (86%) patients survived to hospital discharge. Of the 18 survivors, ten (56%) were discharged to other hospitals and 8 (44%) were discharged directly home. Sequelae and health related quality of life were evaluated for 15 of the 18 (71%) long-term survivors (assessment at median 8 months). Mean SF-36 scores were significantly lower across all domains compared to age and sex matched Australian norms. Mean SF-36 scores were lower (minimum important difference at least 5 points) than previously described ARDS survivors in the domains of general health, mental health, vitality and social function. One patient had long-term disability as a result of ICU acquired weakness. Only 26% of survivors had returned to previous work levels at the time of follow-up. CONCLUSIONS: This ARDS cohort had a high survival rate (86%) after use of ECMO support for reversible refractory hypoxemia. Long term survivors had similar physical health but decreased mental health, general health, vitality and social function compared to other ARDS survivors and an unexpectedly poor return to work.

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Different Immunity Elicited by Recombinant H5N1 Hemagglutinin Proteins Containing Pauci-Mannose, High-Mannose, or Complex Type N-Glycans

Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA), a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vaccine development. In this study, we conducted a systematic study of the immune response against influenza virus infection following immunization with recombinant HA proteins expressed in insect (Sf9) cells, insect cells that contain exogenous genes for elaborating N-linked glycans (Mimic) and mammalian cells (CHO). While the antibody titers are higher with the insect cell derived HA proteins, the neutralization and HA inhibition titers are much higher with the mammalian cell produced HA proteins. Recombinant HA proteins containing tri- or tetra-antennary complex, terminally sialylated and asialyated-galactose type N-glycans induced better protective immunity in mice to lethal challenge. The results are highly relevant to issues that should be considered in the production of fragment vaccines.

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Clinical characteristics and antimicrobial susceptibilities of viridans streptococcal bacteremia during febrile neutropenia in patients with hematologic malignancies: a comparison between adults and children

BACKGROUND: This study was performed to compare the clinical characteristics and antibiotic susceptibilities of viridans streptococcal bacteremia (VSB) between febrile neutropenic adults and children with hematologic malignancies. METHODS: The consecutive medical records of neutropenic patients with hematologic malignancies who were admitted to the Catholic Blood and Marrow Transplantation Center between April 2009 and July 2012, and who were subsequently diagnosed with VSB were reviewed retrospectively. A comparison was made between the clinical and laboratory characteristics of adults and pediatric patients and also between patients with cefepime susceptible or not susceptible VSB. RESULTS: A total of 202 episodes (141 in adults, 61 in children) of VSB were identified. Among them, 26 (12.9%) cases had severe complications including four (2.0%) cases of death attributable to VSB. For antibacterial prophylaxis, most adults received ciprofloxacin (97.1%), but children more frequently received trimethoprim/sulfamethoxazole (86.9%). Oral mucositis (p = 0.005) and abdominal pain (p = 0.001) were found more frequently in adults, and cough was found more frequently in children (p = 0.004). The occurrence rates of severe complications and death attributable to VSB were not significantly different between adults and children. Susceptibility rate to cefepime was significantly higher in adults than children (85.7% vs. 66.1%, p = 0.002). However, in multivariate analysis, cefepime susceptibility had no impact on clinical outcome. CONCLUSIONS: There was no significant difference in clinical outcome between adults and children with VSB despite a difference in cefepime susceptibility. Hence, different antibiotic treatment strategies may not be necessary.

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Sumoylation at the Host-Pathogen Interface

Many viral proteins have been shown to be sumoylated with corresponding regulatory effects on their protein function, indicating that this host cell modification process is widely exploited by viral pathogens to control viral activity. In addition to using sumoylation to regulate their own proteins, several viral pathogens have been shown to modulate overall host sumoylation levels. Given the large number of cellular targets for SUMO addition and the breadth of critical cellular processes that are regulated via sumoylation, viral modulation of overall sumoylation presumably alters the cellular environment to ensure that it is favorable for viral reproduction and/or persistence. Like some viruses, certain bacterial plant pathogens also target the sumoylation system, usually decreasing sumoylation to disrupt host anti-pathogen responses. The recent demonstration that Listeria monocytogenes also disrupts host sumoylation, and that this is required for efficient infection, extends the plant pathogen observations to a human pathogen and suggests that pathogen modulation of host sumoylation may be more widespread than previously appreciated. This review will focus on recent aspects of how pathogens modulate the host sumoylation system and how this benefits the pathogen.

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Evidence for Novel Hepaciviruses in Rodents

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.

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Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5)AIDITRK(11), (72)RDELNVL(78), (251)KSKHNRREGY(260), (269)DENGIVLD(276), and (341)DETTLVRS(348). Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

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MEK/ERK Dependent Activation of STAT1 Mediates Dasatinib-Induced Differentiation of Acute Myeloid Leukemia

Dasatinib (BMS-354825) is a FDA-approved multitargeted kinase inhibitor of BCR/ABL and Src kinases. It is now used in the treatment of chronic myelogenous leukemia (CML) with resistance or intolerance to prior therapies, including imatinib. Here we report a novel effect of dasatinib on inducing the differentiation of acute myeloid leukemia (AML) cells through MEK/ERK-dependent activation of signal transducer and activator of transcription 1 (STAT1). We found that dasatinib could induce the differentiation of AML cells as demonstrated by the expression of differentiation marker CD11b, G0/G1 phase arrest and decreased ratio of nucleus to cytoplasm. Of note, dasatinib induced robust phosphorylation of STAT1 both at Tyr701 and Ser727 as well as the redistribution of STAT1 from the cytoplasm to the nucleus, thus leading to the transcription of STAT1-targeted genes. Knocking down STAT1 expression by shRNA significantly attenuated dasatinib-induced differentiation, indicating an important role of STAT1 in myeloid maturation. We further found that dasatinib-induced activation of STAT1 was regulated by the MEK/ERK kinases. The phosporylation of MEK and ERK occurred rapidly upon dasatinib treatment and increased progressively as differentiation was induced. MEK inhibitors PD98059 and U0216 not only inhibited the phosphorylation of STAT1, but also abrogated dasatinib-induced myeloid differentiation, suggesting that MEK/ERK dependent phosphorylation of STAT1 might be indispensable for the differentiating effect of dasatinib in AML cells. Taken together, our study suggests that STAT1 is an important mediator in dasatinib-induced differentiation of AML cells, whose activation requires the activation of MEK/ERK cascades.

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A Literature Review and Survey of Childhood Pneumonia Etiology Studies: 2000–2010

The Pneumonia Etiology Research for Child Health (PERCH) project is the largest multicountry etiology study of childhood pneumonia since the Board on Science and Technology in International Development studies of the 1980s. However, it is not the only recent or ongoing pneumonia etiology study, and even with seven sites, it cannot capture all epidemiologic settings in the developing world. Funding providers, researchers and policymakers rely on the best available evidence to strategically plan programs, new research directions and interventions. We aimed to describe the current landscape of recent pneumonia etiology studies in children under 5 years of age in the developed and developing world, as ascertained by a literature review of relevant studies with data since the year 2000 and a survey of researchers in the field of childhood pneumonia. We collected information on the study population, study design, case definitions, laboratory samples and methods and identified pathogens. A literature review identified 88 studies with child pneumonia etiology results. As of June 2010, our survey of researchers identified an additional 65 ongoing and recently completed child pneumonia etiology studies. This demonstrates the broad existing context into which the PERCH study must be placed. However, the landscape analysis also reveals a multiplicity of case definitions, levels of clinician involvement, facility types, specimen collection, and laboratory techniques. It reinforces the need for the standardization of methods and analyses for present and future pneumonia etiology studies in order to optimize their cumulative potential to accurately describe the microbial causes of childhood pneumonia.

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Herpes zoster encephalitis presenting as multiple cerebral hemorrhages – a rare presentation: a case report

INTRODUCTION: An infection by herpes zoster virus is a common and important cause of encephalitis. Herpes zoster virus encephalitis if not treated promptly can result in significant morbidity and mortality. The diagnosis of herpes zoster virus encephalitis is based on clinical history, examination, neuroradiological imaging (magnetic resonance imaging and/or computed tomography scan), cerebrospinal fluid analysis and identification of the pathogen in cerebrospinal fluid by polymerase chain reaction amplification and/or anti-herpes zoster virus immunoglobulin G antibody in cerebrospinal fluid. Although ischemic intracerebral infarcts in patients with herpes zoster virus encephalitis or vasculopathy are reported in the literature, multiple intracerebral hemorrhages as a complication of herpes zoster virus encephalitis in an immunocompetent individual are extremely rare. CASE PRESENTATION: A 40-year-old Indian man presented with an acute history of four episodes of seizures, fever, headache, drowsiness, focal neurological deficits and vesicular eruptions over the abdomen in a typical dermatomal distribution. His head computed tomography scan revealed multiple cerebral hemorrhages. Investigations (positive ratio between the cerebrospinal fluid/serum quotients for anti-herpes zoster virus immunoglobulin G and total immunoglobulin G antibodies) established its infective origin due to herpes zoster virus. He developed bilateral pneumonia during the hospital course. He had an excellent recovery following a 2 weeks’ course of intravenous acyclovir. CONCLUSION: Herpes zoster virus encephalitis or vasculopathy is a rare cause of multiple intracerebral hemorrhages and must be considered in the differential diagnosis of patients presenting with an acute history of fever, altered consciousness, and focal neurologic deficits with history of a typical herpetic rash. Its prompt recognition and treatment could alter the course of illness.

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Neutrophils Turn Plasma Proteins into Weapons against HIV-1

As a consequence of innate immune activation granulocytes and macrophages produce hypochlorite/hypochlorous acid (HOCl) via secretion of myeloperoxidase (MPO) to the outside of the cells, where HOCl immediately reacts with proteins. Most proteins that become altered by this system do not belong to the invading microorganism but to the host. While there is no doubt that the myeloperoxidase system is capable of directly inactivating HIV-1, we hypothesized that it may have an additional indirect mode of action. We show in this article that HOCl is able to chemically alter proteins and thus turn them into Idea-Ps (Idea-P = immune defence-altered protein), potent amyloid-like and SH-groups capturing antiviral weapons against HIV-1. HOCl-altered plasma proteins (Idea-PP) have the capacity to bind efficiently and with high affinity to the HIV-1 envelope protein gp120, and to its receptor CD4 as well as to the protein disulfide isomerase (PDI). Idea-PP was able to inhibit viral infection and replication in a cell culture system as shown by reduced number of infected cells and of syncytia, resulting in reduction of viral capsid protein p24 in the culture supernatant. The unmodified plasma protein fraction had no effect. HOCl-altered isolated proteins antithrombin III and human serum albumin, taken as representative examples of the whole pool of plasma proteins, were both able to exert the same activity of binding to gp120 and inhibition of viral proliferation. These data offer an opportunity to improve the understanding of the intricacies of host-pathogen interactions and allow the generation of the following hypothetical scheme: natural immune defense mechanisms generate by posttranslational modification of plasma proteins a potent virucidal weapon that immobilizes the virus as well as inhibits viral fusion and thus entry into the host cells. Furthermore simulation of this mechanism in vitro might provide an interesting new therapeutic approach against microorganisms.

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Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8(+) T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8(+) T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8(+) T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8(+) T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.

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Towards Systematic Discovery of Signaling Networks in Budding Yeast Filamentous Growth Stress Response Using Interventional Phosphorylation Data

Reversible phosphorylation is one of the major mechanisms of signal transduction, and signaling networks are critical regulators of cell growth and development. However, few of these networks have been delineated completely. Towards this end, quantitative phosphoproteomics is emerging as a useful tool enabling large-scale determination of relative phosphorylation levels. However, phosphoproteomics differs from classical proteomics by a more extensive sampling limitation due to the limited number of detectable sites per protein. Here, we propose a comprehensive quantitative analysis pipeline customized for phosphoproteome data from interventional experiments for identifying key proteins in specific pathways, discovering the protein-protein interactions and inferring the signaling network. We also made an effort to partially compensate for the missing value problem, a chronic issue for proteomics studies. The dataset used for this study was generated using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) technique with interventional experiments (kinase-dead mutations). The major components of the pipeline include phosphopeptide meta-analysis, correlation network analysis and causal relationship discovery. We have successfully applied our pipeline to interventional experiments identifying phosphorylation events underlying the transition to a filamentous growth form in Saccharomyces cerevisiae. We identified 5 high-confidence proteins from meta-analysis, and 19 hub proteins from correlation analysis (Pbi2p and Hsp42p were identified by both analyses). All these proteins are involved in stress responses. Nine of them have direct or indirect evidence of involvement in filamentous growth. In addition, we tested four of our predicted proteins, Nth1p, Pbi2p, Pdr12p and Rcn2p, by interventional phenotypic experiments and all of them present differential invasive growth, providing prospective validation of our approach. This comprehensive pipeline presents a systematic way for discovering signaling networks using interventional phosphoproteome data and can suggest candidate proteins for further investigation. We anticipate the methodology to be applicable as well to other interventional studies via different experimental platforms.

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A Simulation Optimization Approach to Epidemic Forecasting

Reliable forecasts of influenza can aid in the control of both seasonal and pandemic outbreaks. We introduce a simulation optimization (SIMOP) approach for forecasting the influenza epidemic curve. This study represents the final step of a project aimed at using a combination of simulation, classification, statistical and optimization techniques to forecast the epidemic curve and infer underlying model parameters during an influenza outbreak. The SIMOP procedure combines an individual-based model and the Nelder-Mead simplex optimization method. The method is used to forecast epidemics simulated over synthetic social networks representing Montgomery County in Virginia, Miami, Seattle and surrounding metropolitan regions. The results are presented for the first four weeks. Depending on the synthetic network, the peak time could be predicted within a 95% CI as early as seven weeks before the actual peak. The peak infected and total infected were also accurately forecasted for Montgomery County in Virginia within the forecasting period. Forecasting of the epidemic curve for both seasonal and pandemic influenza outbreaks is a complex problem, however this is a preliminary step and the results suggest that more can be achieved in this area.

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Identification of Recombinant Human Rhinovirus A and C in Circulating Strains from Upper and Lower Respiratory Infections

Human rhinoviruses (HRVs), in the Enterovirus genus within the family Picornaviridae, are a highly prevalent cause of acute respiratory infection (ARI). Enteroviruses are genetically highly variable, and recombination between serotypes is known to be a major contribution to their diversity. Recently it was reported that recombination events in HRVs cause the diversity of HRV-C. This study analyzed parts of the viral genes spanning the 5′ non- coding region (NCR) through to the viral protein (VP) encoding sequences of 105 HRV field isolates from 51 outpatient cases of Acute Respiratory Infectious Network (ARINET) and 54 inpatient cases of severe lower respiratory infection (SLRI) surveillance, in order to identify recombination in field samples. When analyzing parts of the 5′NCR and VP4/VP2 encoding sequences, we found intra- and interspecies recombinants in field strains of HRV-A and -C. Nineteen cases of recombination events (18.1%) were found among 105 field strains. For HRV-A, there were five cases (4.8%) of intraspecies recombination events and three cases (2.8%) of interspecies recombination events. For HRV-C, there were four cases (3.8%) of intraspecies recombination events and seven cases (6.7%) of interspecies recombination events. Recombination events were significantly more frequently observed in the ARINET samples (18 cases) than in the SLRI samples (1 case; P< 0.0001). The recombination breakpoints were located in nucleotides (nt) 472–554, which comprise stem-loop 5 in the internal ribosomal entry site (IRES), based on the HRV-B 35 sequence (accession no. FJ445187). Our findings regarding genomic recombination in circulating HRV-A and -C strains suggest that recombination might play a role in HRV fitness and could be a possible determinant of disease severity caused by various HRV infections in patients with ARI.

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Esoteric infections and anaesthesiologist: Need for self protection

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TRAF molecules in cell signaling and in human diseases

The tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of intracellular proteins were originally identified as signaling adaptors that bind directly to the cytoplasmic regions of receptors of the TNF-R superfamily. The past decade has witnessed rapid expansion of receptor families identified to employ TRAFs for signaling. These include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), T cell receptor, IL-1 receptor family, IL-17 receptors, IFN receptors and TGFβ receptors. In addition to their role as adaptor proteins, most TRAFs also act as E3 ubiquitin ligases to activate downstream signaling events. TRAF-dependent signaling pathways typically lead to the activation of nuclear factor-κBs (NF-κBs), mitogen-activated protein kinases (MAPKs), or interferon-regulatory factors (IRFs). Compelling evidence obtained from germ-line and cell-specific TRAF-deficient mice demonstrates that each TRAF plays indispensable and non-redundant physiological roles, regulating innate and adaptive immunity, embryonic development, tissue homeostasis, stress response, and bone metabolism. Notably, mounting evidence implicates TRAFs in the pathogenesis of human diseases such as cancers and autoimmune diseases, which has sparked new appreciation and interest in TRAF research. This review presents an overview of the current knowledge of TRAFs, with an emphasis on recent findings concerning TRAF molecules in signaling and in human diseases.

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Plasma in the PICU: why and when should we transfuse?

Whereas red blood cell transfusions have been used since the 19th century, plasma has only been available since 1941. It was originally mainly used as volume replacement, mostly during World War II and the Korean War. Over the years, its indication has shifted to correct coagulation factors deficiencies or to prevent bleeding. Currently, it remains a frequent treatment in the intensive care unit, both for critically ill adults and children. However, observational studies have shown that plasma transfusion fail to correct mildly abnormal coagulation tests. Furthermore, recent epidemiological studies have shown that plasma transfusions are associated with an increased morbidity and mortality in critically ill patients. Therefore, plasma, as any other treatment, has to be used when the benefits outweigh the risks. Based on observational data, most experts suggest limiting its use either to massively bleeding patients or bleeding patients who have documented abnormal coagulation tests, and refraining for transfusing plasma to nonbleeding patients whatever their coagulation tests. In this paper, we will review current evidence on plasma transfusions and discuss its indications.

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Hepatic Deficiency of COP9 Signalosome Subunit 8 Induces Ubiquitin-Proteasome System Impairment and Bim-Mediated Apoptosis in Murine Livers

The COP9 signalosome (CSN), an evolutionally highly conserved protein complex composed of 8 unique subunits (CSN1 through CSN8) in higher eukaryotes, is purported to modulate protein degradation mediated by the ubiquitin-proteasome system (UPS) but this has not been demonstrated in a critical mitotic parenchymal organ of vertebrates. Hepatocyte-specific knockout of the Cops8 gene (HS-Csn8KO) was shown to cause massive hepatocyte apoptosis and liver malfunction but the underlying mechanism remains unclear. Here, we report that Csn8/CSN exerts profound impacts on hepatic UPS function and is critical to the stability of the pro-apoptotic protein Bim. Significant decreases in CIS (cytokine-inducible Src homology 2 domain-containing protein), a Bim receptor of a cullin2-based ubiquitin ligase, were found to co-exist with a marked increase of Bim proteins. Csn8 deficiency also significantly decreased 19S proteasome subunit Rpt5 and markedly increased high molecular weight neddylated and ubiquitinated proteins. The use of a surrogate UPS substrate further reveals severe impairment of UPS-mediated proteolysis in HS-Csn8KO livers. Inclusion body-like materials were accumulated in Csn8 deficient hepatocytes. In addition to Bim, massive hepatocyte apoptosis in HS-Csn8KO livers is also associated with elevated expression of other members of the Bcl2 family, including pro-apoptotic Bax as well as anti-apoptotic Bcl2 and Bcl-XL. Increased interaction between Bcl2 and Bim, but not between Bcl2 and Bax, was detected. Hence, it is concluded that hepatic CSN8 deficiency impairs the UPS in the liver and the resultant Bim upregulation likely plays an important role in triggering hepatocyte apoptosis via sequestering Bcl2 away from Bax.

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Enterococcus faecalis FK-23 affects alveolar-capillary permeability to attenuate leukocyte influx in lung after influenza virus infection

Infection with influenza A virus, one of the most common life-threatening viruses, causes the accumulation of inflammatory cells in the lung, which is directly correlated with influenza-associated morbidity and mortality. In this study, we investigated the potential of lysozyme-treated Enterococcus faecalis FK-23 (LFK) to prevent influenza in influenza virus-infected mice. C57BL/6N mice were orally administered LFK and intranasally infected with influenza virus A/Puerto Rico/8/34 (H1N1) at lethal doses. After infection with influenza A virus, the survival rate of the LFK-administered mice was significantly higher than that of saline-administered mice. Staining of lung sections with hematoxylin-eosin, and cell counts of lung and bronchoalveolar lavage fluid showed that oral administration of LFK suppressed the excessive infiltration of leukocytes into the lung after viral infection. Extravasation assay revealed that the arrest was mediated by modulation of pulmonary alveolar-capillary permeability. Expression levels of genes involved in matrix degradation, which are correlated with vascular permeability, were downregulated in LFK-administered mice. These findings suggest that stabilizing the integrity of the alveolar-capillary barrier by the administration of LFK improves survival rate. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/2193-1801-2-269) contains supplementary material, which is available to authorized users.

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Human H5N1 influenza infections in Cambodia 2005–2011: case series and cost-of-illness

BACKGROUND: Southeast Asia has been identified as a potential epicentre of emerging diseases with pandemic capacity, including highly pathogenic influenza. Cambodia in particular has the potential for high rates of avoidable deaths from pandemic influenza due to large gaps in health system resources. This study seeks to better understand the course and cost-of-illness for cases of highly pathogenic avian influenza in Cambodia. METHODS: We studied the 18 laboratory-confirmed cases of avian influenza subtype H5N1 identified in Cambodia between January 2005 and August 2011. Medical records for all patients were reviewed to extract information on patient characteristics, travel to hospital, time to admission, diagnostic testing, treatment and disease outcomes. Further data related to costs was collected through interviews with key informants at district and provincial hospitals, the Ministry of Health and non-governmental organisations. An ingredient-based approach was used to estimate the total economic cost for each study patient. Costing was conducted from a societal perspective and included both financial and opportunity costs to the patient or carer. Sensitivity analysis was undertaken to evaluate potential change or variation in the cost-of-illness. RESULTS: Of the 18 patients studied, 11 (61%) were under the age of 18 years. The majority of patients (16, 89%) died, eight (44%) within 24 hours of hospital admission. There was an average delay of seven days between symptom onset and hospitalisation with patients travelling an average of 148 kilometres (8-476 km) to the admitting hospital. Five patients were treated with oseltamivir of whom two received the recommended dose. For the 16 patients who received all their treatment in Cambodia the average per patient cost of H5N1 influenza illness was US$300 of which 85.0% comprised direct medical provider costs, including diagnostic testing (41.2%), pharmaceuticals (28.4%), hospitalisation (10.4%), oxygen (4.4%) and outpatient consultations (0.6%). Patient or family costs were US$45 per patient (15.0%) of total economic cost. CONCLUSION: Cases of avian influenza in Cambodia were characterised by delays in hospitalisation, deficiencies in some aspects of treatment and a high fatality rate. The costs associated with medical care, particularly diagnostic testing and pharmaceutical therapy, were major contributors to the relatively high cost-of-illness.

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Intranasally Administered Antigen 85B Gene Vaccine in Non-Replicating Human Parainfluenza Type 2 Virus Vector Ameliorates Mouse Atopic Dermatitis

Atopic dermatitis (AD) is a refractory and recurrent inflammatory skin disease. Various factors including heredity, environmental agent, innate and acquired immunity, and skin barrier function participate in the pathogenesis of AD. T -helper (Th) 2-dominant immunological milieu has been suggested in the acute phase of AD. Antigen 85B (Ag85B) is a 30-kDa secretory protein well conserved in Mycobacterium species. Ag85B has strong Th1-type cytokine inducing activity, and is expected to ameliorate Th2 condition in allergic disease. To perform Ag85B function in vivo, effective and less invasive vaccination method is required. Recently, we have established a novel functional virus vector; recombinant human parainfluenza type 2 virus vector (rhPIV2): highly expressive, replication-deficient, and very low-pathogenic vector. In this study, we investigated the efficacy of rhPIV2 engineered to express Ag85B (rhPIV2/Ag85B) in a mouse AD model induced by repeated oxazolone (OX) challenge. Ear swelling, dermal cell infiltrations and serum IgE level were significantly suppressed in the rhPIV2/Ag85B treated mouse group accompanied with elevated IFN-γ and IL-10 mRNA expressions, and suppressed IL-4, TNF-α and MIP-2 mRNA expressions. The treated mice showed no clinical symptom of croup or systemic adverse reactions. The respiratory tract epithelium captured rhPIV2 effectively without remarkable cytotoxic effects. These results suggested that rhPIV2/Ag85B might be a potent therapeutic tool to control allergic disorders.

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Chronic psychosocial stress: does it modulate immunity to the influenza vaccine in Hong Kong Chinese elderly caregivers?

Previous studies evaluated the effects of psychosocial stress on influenza vaccine responses. However, there were methodological limitations. This study aims to determine whether chronic stress is associated with poorer influenza-specific immune responses to influenza vaccines in Hong Kong Chinese elderly people. This is a prospective study with a 12-week follow-up. Subjects were recruited from government general out-patient clinics, non-government organizations, and public housing estates in Hong Kong. Participants include 55 caregivers of spouses with chronic conditions that impaired their activities of daily living and 61 age- and sex-matched non-caregivers. A single-dose trivalent influenza vaccine was given to all subjects by intramuscular ingestion. Blood samples were collected before vaccination, at 6 weeks, and at 12 weeks after vaccination. Influenza vaccine strain-specific antibody titers were measured by the hemagglutination inhibition method. Lymphocyte subsets were analyzed for ratios and absolute counts, and cytokine concentration were measured by flow cytometry. Validated scales were used to assess psychological (depressive symptoms, perceived stress, and caregiver strain), social (multidimensional social support scale), and lifestyle factors (physical exercise, cigarette smoking, and alcohol consumption) at baseline prior to vaccination. Demographic and socioeconomic variables were also collected. Albumin levels were measured as an indicator for nutritional status in subjects. Caregivers had statistically significant (p < 0.05) lower cell-mediated immune responses to influenza vaccination at 12 weeks when compared with those of the controls. No differences in humoral immune response to vaccination were observed between caregivers and controls. Hong Kong Chinese elderly who experience chronic stress have a significantly lower cell-mediated immune response to influenza vaccination when compared with non-caregivers.