Hepatitis E rORF2p stimulated and unstimulated peripheral expression profiling in patients with self-limiting hepatitis E infection

To improve the current knowledge on the involvement of peripheral lymphocytes in hepatitis E virus (HEV) associated pathogenesis, we analyzed alterations in (1) immunophenotypic expressions (by flow cytometry) and (2) gene expression patterns (by TaqMan Low Density Array) of activatory, inhibitory, integrin, homing, ectonucleotidase machinery, costimulatory, inflammatory markers, and T regulatory cells (Treg) associated cytokines on HEV rORF2p stimulated and unstimulated PBMCs of 43 acute HEV patients, 30 recovered individuals, and 43 controls. The phenotypic expressions of key molecules CTLA-4, GITR, CD103, CD25, CD69, IL10 and TGF-1 in the acute patients and TGF-1 in the recovered individuals were significantly elevated on both unstimulated and stimulated PBMCs. Gene expression array data revealed upregulations of CD25, PD1, CD103, CCR4, IL10, and TGF-1 on both unstimulated and HEV rORF2p stimulated PBMCs of acute patients. The observed upregulations of inhibitory, integrin, activatory, and Treg-associated cytokine genes on the PBMCs of acute HEV patients complemented by their frequency data suggest them as the major players in the fine-tuning of immune response in self-limiting hepatitis E infection

Role of Host Immune Response and Viral Load in the Differential Outcome of Pandemic H1N1 (2009) Influenza Virus Infection in Indian Patients

BACKGROUND: An unusually high number of severe pneumonia cases with considerable mortality is being observed with the pandemic H1N1 2009 virus infections globally. In India, all mild as well as critically ill cases were admitted and treated in the government hospitals during the initial phase of the pandemic. The present study was undertaken during this early phase of the pandemic. METHODOLOGY: The role of viral load and host factors in the pathogenesis were assessed by examining 26 mild (MP), 15 critically ill patients (CIP) and 20 healthy controls from Pune, India. Sequential blood and lung aspirate samples were collected from CIP. Viral load and cytokines/chemokine levels were determined from the plasma and lung aspirates of the patients. TLR levels were determined by staining and FACS analysis. Gene profiling was done for both cells in the lung aspirates and PBMCs using TaqMan Low Density arrays. Antibody titres and isotyping was done using HA protein based ELISAs. PRINCIPAL FINDINGS: 13/15 critically ill patients expired. All plasma samples were negative for the virus irrespective of the patient's category. Sequential lung samples from CIP showed lower viral loads questioning association of viral replication with the severity. Anti-rpH1N1-09-HA-IgG titres were significantly higher in critically ill patients and both categories circulated exclusively IgG1 isotype. Critically ill patients exhibited increase in TLR-3, 4, 7 and decrease in TLR-2 expressions. The disease severity correlated with increased plasma levels of IL1RA, IL2, IL6, CCL3, CCL4 and IL10. Majority of the immune-function genes were down-regulated in the PBMCs and up-regulated in the cells from lung aspirates of critically ill patients. No distinct pattern differentiating fatal and surviving patients was observed when sequential samples were examined for various parameters. CONCLUSIONS: Disease severity was associated with pronounced impairment of host immune response

Clinical Findings and Pro-Inflammatory Cytokines in Dengue Patients in Western India: A Facility-Based Study

BACKGROUND: Descriptions of dengue immunopathogenesis have largely relied on data from South-east Asia and America, while India is poorly represented. This study characterizes dengue cases from Pune, Western India, with respect to clinical profile and pro-inflammatory cytokines. METHODOLOGY/PRINCIPAL FINDINGS: In 2005, 372 clinically suspected dengue cases were tested by MAC-ELISA and RT-PCR for dengue virus (DENV) aetiology. The clinical profile was recorded at the hospital. Circulating levels of IFN-gamma, TNF-alpha, IL-6, and IL-8 were assessed by ELISA and secondary infections were defined by IgM to IgG ratio. Statistical analysis was carried out using the SPSS 11.0 version. Of the 372 individuals, 221 were confirmed to be dengue cases. Three serotypes, DENV-1, 2 and 3 were co-circulating and one case of dual infection was identified. Of 221 cases, 159 presented with Dengue fever (DF) and 62 with Dengue hemorrhagic fever (DHF) of which six had severe DHF and one died of shock. There was a strong association of rash, abdominal pain and conjunctival congestion with DHF. Levels of IFN-gamma were higher in DF whereas IL-6 and IL-8 were higher in DHF cases (p<0.05). The mean levels of the three cytokines were higher in secondary compared to primary infections. Levels of IFN-gamma and IL-8 were higher in early samples collected 2-5 days after onset than late samples collected 6-15 days after onset. IFN-gamma showed significant decreasing time trend (p = 0.005) and IL-8 levels showed increasing trend towards significance in DHF cases (interaction p = 0.059). There was a significant association of IL-8 levels with thrombocytopenia and both IFN-gamma and IL-8 were positively associated with alanine transaminase levels. CONCLUSIONS/SIGNIFICANCE: Rash, abdominal pain and conjunctival congestion could be prognostic symptoms for DHF. High levels of IL-6 and IL-8 were shown to associate with DHF. The time trend of IFN-gamma and IL-8 levels had greater significance than absolute values in DHF pathogenesis

Hepatitis E rORF2p Stimulated and Unstimulated Peripheral Expression Profiling in Patients with Self-Limiting Hepatitis E Infection

To improve the current knowledge on the involvement of peripheral lymphocytes in hepatitis E virus (HEV) associated pathogenesis, we analyzed alterations in (1) immunophenotypic expressions (by flow cytometry) and (2) gene expression patterns (by TaqMan Low Density Array) of activatory, inhibitory, integrin, homing, ectonucleotidase machinery, costimulatory, inflammatory markers, and T regulatory cells (Treg) associated cytokines on HEV rORF2p stimulated and unstimulated PBMCs of 43 acute HEV patients, 30 recovered individuals, and 43 controls. The phenotypic expressions of key molecules CTLA-4, GITR, CD103, CD25, CD69, IL10 and TGF-β1 in the acute patients and TGF-β1 in the recovered individuals were significantly elevated on both unstimulated and stimulated PBMCs. Gene expression array data revealed upregulations of CD25, PD1, CD103, CCR4, IL10, and TGF-β1 on both unstimulated and HEV rORF2p stimulated PBMCs of acute patients. The observed upregulations of inhibitory, integrin, activatory, and Treg-associated cytokine genes on the PBMCs of acute HEV patients complemented by their frequency data suggest them as the major players in the fine-tuning of immune response in self-limiting hepatitis E infection

Phenotypic and functional analyses of NK and NKT-like populations during the early stages of chikungunya infection

The aim of this study was to characterize NK (CD56+CD3−) and NKT-like cell (CD56+CD3+) responses early after chikungunya infection. Expression profiling and functional analysis of T/NK/NKT-like cells were performed on samples from 56 acute and 31 convalescent chikungunya patients and 56 control individuals. The percentages of NK cells were high in both patient groups, whereas NKT-like cell percentages were high only in the convalescent group. The percentages of NKp30+CD3-CD56+, NKp30+CD3+CD56+, CD244+CD3-CD56+, and CD244+CD3+CD56+cells were high, whereas the percentages of NKG2D+CD3-CD56+ and NKG2D+CD3+CD56+cells were low in both patient groups. The percentages of NKp44+CD3-CD56+ cells were high in both patient groups, whereas the percentages of NKp44+CD3+CD56+ cells were higher in the acute group than in convalescent and control groups. The percentages of NKp46+CD3-CD56+ cells were high in both patient groups. Higher percentages of perforin+CD3-CD56+ and perforin+CD3+CD56+ cells were observed in acute and convalescent patients, respectively. Higher cytotoxic activity was observed in acute patients than in controls. IFN-γ expression on NK cells of convalescent patients and on NKT-like cells of both patient groups was indicative of the regulatory role of NK and NKT-like cells. Collectively, these data showed that higher expression of activating receptors on NK/NKT-like cells and perforin+ NK cells in acute patients could be responsible for increased cytotoxicity. The observed expression of perforin+ NK cells in the acute phase and IFN-γ+ NKT-like cells in the subsequent convalescent stage showed that NK/NKT-like cells mount an early and efficient response to chikungunya virus. Further study of the molecular mechanisms that limit viral dissemination/establishment of chronic disease will aid in understanding how NK/NKT-like cells control chikungunya infection

Differential inhibitory and activating NK cell receptor levels and NK/NKT-like cell functionality in chronic and recovered stages of chikungunya

<div><p>The role of natural killer (NK; CD3^-CD56⁺)/NKT (CD3⁺CD56⁺)-like cells in chikungunya virus (CHIKV) disease progression/recovery remains unclear. Here, we investigated the expression profiles and function of NK and NKT-like cells from 35 chronic chikungunya patients, 30 recovered individuals, and 69 controls. Percentage of NKT-like cells was low in chronic chikungunya patients. NKp30⁺, CD244⁺, DNAM-1⁺, and NKG2D⁺ NK cell percentages were also lower (MFI and/or percentage), while those of CD94⁺ and NKG2A⁺ NKT-like cells were higher (MFI and/or percentage) in chronic patients than in recovered subjects. IFN-γ and TNF-α expression on NKT-like cells was high in the chronic patients, while only IFN-γ expression on NK cells was high in the recovered individuals. Furthermore, percentage of perforin⁺NK cells was low in the chronic patients. Lower cytotoxic activity was observed in the chronic patients than in the controls. CD107a expression on NK and NKT-like cells post anti-CD94/anti-NKG2A blocking was comparable among the patients and controls. Upregulated inhibitory and downregulated activating NK receptor expressions on NK/NKT-like cells, lower perforin⁺ and CD107a⁺NK cells are likely responsible for inhibiting the NK and NKT-like cell function in the chronic stage of chikungunya. Therefore, deregulation of NKR expression might underlie CHIKV-induced chronicity.</p></div

Cytokine Profiles, CTL Response and T Cell Frequencies in the Peripheral Blood of Acute Patients and Individuals Recovered from Hepatitis E Infection

Background: Hepatitis E is a major public health problem in the developing countries. Pathogenesis of hepatitis E virus (HEV) infection is poorly understood. Methods: This case-control study included 124 Hepatitis E patients (46 acute and 78 recovered), 9 with prior exposure to HEV and 71 anti-HEV negative healthy controls. HEV induced CTL response by Elispot, cytokines/chemokines quantitation by Milliplex assay and peripheral CD4+ &amp; CD8+ T cell frequencies by flow cytometry were assessed. Results: Among the patient categories, HEV specific IFN-c responses as recorded by Elispot were comparable. Comparisons of cytokines/chemokines revealed significantly high levels of IL-1a and sIL-2Ra during acute phase. Circulating peripheral CD4/CD8+ T-cell subsets in acute and recovered individuals were comparable compared to controls, while among patient categories CD8+T cell subset was significantly higher in recovered individuals. Conclusions: Our findings suggest that IL-1a and sIL-2Ra play a role in the pathogenesis of acute Hepatitis E infection. Lack of robust HEV ORF2-specific CTL response in the peripheral blood of HEV infected patients during the acute and recovered phases of the disease may be associated with involvement of innate immune cells/localization of the immune events at the site of infection

Immunogenicity of candidate hepatitis E virus DNA vaccine expressing complete and truncated ORF2 in mice

Hepatitis E virus (HEV) is a major cause of enterically transmitted acute hepatitis of adults in developing nations. Our present studies show that, the complete ORF2 gene (1-660 amino acids, a.a.) coding for capsid protein of HEV as candidate DNA vaccine induced significant specific humoral and cellular immune responses in mice. Gene gun based DNA administration led to higher seroconversion rates and HEV-specific antibody titers as against needle-injection method. The region (458-607 a.a.) within ORF2 protein is reported to harbour the predominant neutralization epitope/s (NE) of HEV. The NE DNA also induced HEV-specific immune responses in mice. NE-based DNA-prime-protein boost approach was observed to be superior to NE DNA based approach. Co-administration of plasmid expressing mouse granulocyte macrophage colony stimulating factor (GM-CSF) induced immune response at similar level as that with ORF2/NE plasmid alone. IgG1 was the predominant isotype irrespective of the approach used. HEV-specific antibodies in seroconverted mice sera could bind/neutralize HEV in an in vitro ELISA-based assay. In conclusion, efficacy of ORF2 and NE based DNA/DNA-prime-protein-boost approaches are worth exploring in monkey model

Characteristics of study population.

<p>Characteristics of study population.</p