The expression of ten tuberculosis candidate genes in lung and lymph nodes of cynomologus macaques vaccinated and experimentally infected with Mycobacterium tuberculosis (Mtb) was quantified. The expression of TNFα, IL10, IL1β, TLR4, IL17, IL6, IL12, and iNOS in the lungs of vaccinated animals was higher than that of non‐vaccinated animals.

ABSTRACTSimian immunodeficiency virus (SIV) infection disseminated into the oropharyngeal tissues of rhesus macaques 6 weeks following intravenous inoculation. Severe local CD4+T-cell depletion coincided with increases in NK cell and proinflammatory biomarkers and the disruption of growth-associated gene transcription, demonstrating the rapid establishment of pathogenesis in the oral mucosa.

Purpose: With the increasing use of immunomodulators and biologics, concerns have been raised regarding the risk of development of lymphoma in IBD patients. EBV has been associated with lymphomas in post-transplant and immunosuppressed patients. The aim of this study is to prospectively determine EBV viral replication in association with disease activity and medications in patients with IBD. Methods: 29 patients with IBD were studied (Table 1). CDAI and IBDQ values were determined using standard questionnaires. DNA was extracted from peripheral blood mononuclear cells (PBMC) of patients with IBD. Cell numbers were calculated using a CCR5 standard curve. EBV loads per million PBMC were determined using a real-time PCR assay. Statistical significance of the data was determined using the Chi square and Student T tests.Table 1: Clinical Characteristics.Results: Our preliminary findings reveal that EBV viral replication is low in 62.2% of patients who are in remission or have mild disease activity as measured by CDAI (Table 2). For those with at least moderate disease activity (CDAI > 220), 3 (60%) patients have intermediate or high EBV viral replication. EBV viral replication is low in 92.9% of patients whose relapses of IBD are not affecting the quality of life (IBDQ > 170). For those with IBDQ < 170, 19 (59.5%) patients have intermediate or high EBV viral replication. The relationship between IBDQ and EBV viral replication appears to be statistically significant (P < 0.01).Table 2: EBV viral replication and disease activity.Conclusion: EBV viral replication may be significant in more than half of the IBD patients with at least moderate disease activity as measured by the CDAI and quality of life impairment produced by relapses of IBD as measured by the IBDQ. The relationship between medications and EBV viral replication in this group has yet to be analyzed. The significance of the association between EBV viral replication and disease activity will need to be determined.

Salmonella typhimurium causes a localized enteric infection in immunocompetent individuals, whereas HIV-infected individuals develop a life-threatening bacteremia. Here we show that simian immunodeficiency virus (SIV) infection results in depletion of T helper type 17 (TH17) cells in the ileal mucosa of rhesus macaques, thereby impairing mucosal barrier functions to S. typhimurium dissemination. In SIV-negative macaques, the gene expression profile induced by S. typhimurium in ligated ileal loops was dominated by TH17 responses, including the expression of interleukin-17 (IL-17) and IL-22. TH17 cells were markedly depleted in SIV-infected rhesus macaques, resulting in blunted TH17 responses to S. typhimurium infection and increased bacterial dissemination. IL-17 receptor–deficient mice showed increased systemic dissemination of S. typhimurium from the gut, suggesting that IL-17 deficiency causes defects in mucosal barrier function. We conclude that SIV infection impairs the IL-17 axis, an arm of the mucosal immune response preventing systemic microbial dissemination from the gastrointestinal tract.

Human immunodeficiency virus (HIV) infection leads to severe CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) that persists despite the initiation of highly active antiretroviral therapy (HAART). It is not known whether restoration of gut mucosal CD4+ T cells and their functions is feasible during therapy and how that relates to immune correlates and viral reservoirs. Intestinal biopsies and peripheral blood samples from HIV-infected patients who were either HAART naive or on long-term HAART were evaluated. Our data demonstrated that gut CD4+ T-cell restoration ranged from modest (<50%) to high (>50%), compared with uninfected controls. Despite persistent CD4+ T-cell proviral burden and residual immune activation in GALT during HAART, effective CD4+ T-cell restoration (>50%) was achieved, which was associated with enhanced Th17 CD4+ T-cell accumulation and polyfunctional anti-HIV cellular responses. Our findings suggest that a threshold of>50% CD4+ T-cell restoration may be sufficient for polyfunctional HIV-specific T cells with implications in the evaluation of vaccines and therapeutics.

The development of susceptibility to secondary pathogenic infections in the oral cavity during HIV infection is likely to result from or coincide with deterioration of the local mucosal immune system.We have utilized the SIV model to investigate the kinetics and magnitude of oral pathogenesis during systemic dissemination of intravenously inoculated SIVmac251.Viral replication was detected in oropharyngeal lymph nodes at 6 weeks post-infection and shown to be coincident with a broad scale loss of growth factor receptor transcription in the oral mucosa, providing multiple avenues for blocking the normal activity of apoptosis inhibitors that function through Bcl2- and p53-dependent pathways.Our findings suggest that the normal balance between cell death and regeneration may be rapidly disrupted in the oral mucosa during the early stages of immunodeficiency virus infection, setting the stage for continuing deterioration of immune function and the development of susceptibility to secondary infections.

ABSTRACT Gut-associated lymphoid tissue (GALT) is an early target for human immunodeficiency virus type 1 (HIV-1) infection and is a site for severe CD4 + T-cell depletion. HIV-associated enteropathy is well-documented in chronic HIV-1 infection. However, the initial host responses to HIV infection in GALT and the early molecular correlates of HIV enteropathogenesis have not been characterized during primary HIV infection. In this study, we provide evidence of viral replication in GALT resident CD4 + T cells and macrophages in primary-stage patients and identify early patterns of host mucosal responses and changes in the molecular microenvironment through gene expression profiling. High levels of viral replication in GALT and marked CD4 + T-cell depletion correlated with decreased expression levels of genes regulating epithelial barrier maintenance and digestive/metabolic functions. These changes coincided with a marked increase in the transcription of immune activation-, inflammation-, and apoptosis-associated genes. Our findings indicate that HIV-induced pathogenesis in GALT emerges at both the molecular and cellular levels prior to seroconversion in primary HIV infection, potentially setting the stage for disease progression by impairing the ability to control viral replication and repair and regenerate intestinal mucosal tissues.

Gut-associated lymphoid tissue (GALT) is an early target of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) and a site for severe CD4+ T-cell depletion. Although antiretroviral therapy (ART) is effective in suppressing HIV replication and restoring CD4+ T cells in peripheral blood, restoration in GALT is delayed. The role of restored CD4+ T-cell help in GALT during ART and its impact on antiviral CD8+ T-cell responses have not been investigated. Using the SIV model, we investigated gut CD4+ T-cell restoration in infected macaques, initiating ART during either the primary stage (1 week postinfection), prior to acute CD4+ cell loss (PSI), or during the chronic stage at 10 weeks postinfection (CSI). ART led to viral suppression in GALT and peripheral blood mononuclear cells of PSI and CSI animals at comparable levels. CSI animals had incomplete CD4+ T-cell restoration in GALT. In PSI animals, ART did not prevent acute CD4+ T-cell loss by 2 weeks postinfection in GALT but supported rapid and complete CD4+ T-cell restoration thereafter. This correlated with an accumulation of central memory CD4+ T cells and better suppression of inflammation. Restoration of CD4+ T cells in GALT correlated with qualitative changes in SIV gag-specific CD8+ T-cell responses, with a dominance of interleukin-2-producing responses in PSI animals, while both CSI macaques and untreated SIV-infected controls were dominated by gamma interferon responses. Thus, central memory CD4+ T-cell levels and qualitative antiviral CD8+ T-cell responses, independent of viral suppression, were the immune correlates of gut mucosal immune restoration during ART.

Since its discovery in 1983, Human Immunodeficiency Virus-1 (HIV) infection has resulted in the death of several million people worldwide. Control of the spread of HIV infections is one of the major concerns of the scientific community today. This retrovirus houses a genome encoding three viral structural proteins: group-specific antigen, polymerase and envelope. The envelope gp120 binds to cell surface receptors on susceptible target cells in the host. Although HIV is known to infect various cell types, cells expressing the CD4 receptor support high levels of viral replication in vivo. Gastrointestinal complications are commonly seen in patients with HIV-1 infections progressing to AIDS. Recent studies have shown that HIV causes massive disruption of the Gut Associated Lymphoid Tissue (GALT) very early in infection. Intestinal dysfunction is often not resolved despite the success of HAART in improving the clinical status of these patients. GALT supports robust viral replication in mucosal memory CD4+ T cells during primary HIV infection, leading to a severe depletion of the CD4+ T cells which persists through all stages of HIV infection in the absence of therapeutic interventions. Functional genomic analysis of host responses to HIV shows that molecular processes regulating inflammation and immune activation are dominantly expressed in GALT of therapy-nave HIV-1 infected patients with a progressive clinical course. Heterosexual transmission is the primary mode for the spread of HIV infection and the virus gains entry through mucosal surfaces and infects susceptible target cells. Since a majority of new infections are acquired through the mucosal route, induction of mucosal immunity is an important factor in vaccine design.

Background: Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients.Objectives: The objective of this study was to develop and validate an affordable one step real-time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients.Results: We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients.Conclusions: This RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India. (C) 2007 Elsevier B.V. All rights reserved.

BACKGROUND:Simian immunodeficiency virus (SIV) infection leads to severe loss of intestinal CD4(+) T cells and, as compared to peripheral blood, restoration of these cells is slow during antiretroviral therapy (ART). Mechanisms for this delay have not been examined in context of which specific CD4(+) memory subsets or lost and fail to regenerate during ART.METHODS:Fifteen rhesus macaques were infected with SIV, five of which received ART (FTC/PMPA) for 30 weeks. Viral loads were measured by real-time PCR. Flow cytometric analysis determined changes in T-cell subsets and their proliferative state.RESULTS:Changes in proliferative CD4(+) memory subsets during infection accelerated their depletion. This reduced the central memory CD4(+) T-cell pool and contributed to slow CD4(+) T-cell restoration during ART.CONCLUSION:There was a lack of restoration of the CD4(+) central memory and effector memory T-cell subsets in gut-associated lymphoid tissue during ART, which may contribute to the altered intestinal T-cell homeostasis in SIV infection.

Purpose: Inflammatory bowel disease (IBD) (Crohn's Disease and Ulcerative Colitis) is characterized by chronic inflammation of the gastrointestinal tract. IBD is often treated with steroids, mesalamines, immunomodulators and biologics. With the increasing use of immunomodulators and biologics, concerns have been raised regarding the risk of development of lymphoma in IBD patients. The incidence of lymphomas in IBD is controversial. Studies from tertiary care centers seem to associate lymphomas with IBD. Epstein-Barr Virus (EBV), a herpes virus, has been associated with lymphomas in post-transplant and immunosuppressed patients. One major barrier to understanding etiology of this increased risk is delineating between disease activity, treatment and the possible role of oncogenic viruses. Our aim is to determine the prevalence of EBV in patients who are currently being followed in our IBD clinic compared to a selected group of controls. Methods: 79 patients with IBD and 25 control subjects were studied. DNA was extracted from peripheral blood mononuclear cells (PBMC) of patients with IBD and from healthy IBD negative controls. Cell numbers were calculated using a CCR5 standard curve. EBV loads per million PBMC were determined using a real-time PCR assay. Results: A higher incidence of EBV DNA was observed in the IBD positive group compared to the IBD negative group. EBV was detected in 49% of IBD patients and in 32% of controls. Results indicate higher average levels of EBV DNA in IBD positive patients (1.29*108 compared to 1.76*103 copies/million PBMC). Conclusion: The prevalence of EBV is higher in patients with IBD than that of controls. EBV viral load averaged 5 logs greater than that of controls. Contributing factors to these findings may include immunosuppressive medications, biologics and the severity of disease activity. Due to our smaller control sample size, the difference in EBV detection was not statistically significant (P= 0.13). The significance of higher prevalence of EBV and higher EBV viral load will need to be determined in a prospective matter.

Hepatitis C virus (HCV) Core protein is implicated in the development of hepatocellular carcinoma (HCC). We utilized a HepG2 human hepatocyte cell line with inducible expression of HCV Core protein (HCV-1b) to investigate the early effects of Core protein on hepatocyte gene expression and to identify molecular processes modulated by the Core protein. A significant change was observed in the expression of 407 genes, which included genes regulating apoptosis, immune response, and cell cycle. Some of these genes were previously known to be tumor markers. The decreased expression of chemo-attractants such as TNFSF10, CCL20, and osteopontin was observed, which suggested that HCV Core expression could lead to suppression of inflammatory response as well as trafficking of macrophages and neutrophils to the site of HCV infection. An increased expression of anti-apoptosis factors including PAK2, API5, BH1, Tax1BP1, DAXX, and TNFAIP3/A20 was observed. Some of these genes were also linked to the regulation of NFKB activation and that the alteration of their expression levels, by HCV Core, might lead to the suppression NFKB activation of inflammatory responses. Our data suggested that Core expression may contribute to the viral persistence by protecting infected hepatocytes from cell death by the suppressing apoptosis and inflammatory reaction to HCV viral infection.

Although the gut-associated lymphoid tissue (GALT) is an important early site for human immunodeficiency virus (HIV) replication and severe CD4+ T-cell depletion, our understanding is limited about the restoration of the gut mucosal immune system during highly active antiretroviral therapy (HAART). We evaluated the kinetics of viral suppression, CD4+ T-cell restoration, gene expression, and HIV-specific CD8+ T-cell responses in longitudinal gastrointestinal biopsy and peripheral blood samples from patients initiating HAART during primary HIV infection (PHI) or chronic HIV infection (CHI) using flow cytometry, real-time PCR, and DNA microarray analysis. Viral suppression was more effective in GALT of PHI patients than CHI patients during HAART. Mucosal CD4+ T-cell restoration was delayed compared to peripheral blood and independent of the time of HAART initiation. Immunophenotypic analysis showed that repopulating mucosal CD4+ T cells were predominantly of a memory phenotype and expressed CD11 alpha, alpha(E)beta 7, CCR5, and CXCR4. Incomplete suppression of viral replication in GALT during HAART correlated with increased HIV-specific CD8+ T-cell responses. DNA microarray analysis revealed that genes involved in inflammation and cell activation were up regulated in patients who did not replenish mucosal CD4+ T cells efficiently, while expression of genes involved in growth and repair was increased in patients with efficient mucosal CD4+ T-cell restoration. Our findings suggest that the discordance in CD4+ T-cell restoration between GALT and peripheral blood during therapy can be attributed to the incomplete viral suppression and increased immune activation and inflammation that may prevent restoration of CD4+ T cells and the gut microenvironment.

Limited information is available on the molecular mechanisms by which long-term HIV-1-infected nonprogressors suppress HIV-1 infection and maintain immune functions. The intestinal mucosal immune system is an early target for HIV-1 infection and severe CD4+ T cell depletion. We evaluated mucosal T lymphocyte subsets, virus-specific cellular responses, gene expression profiles, and viral loads in intestinal mucosal biopsies of long-term nonprogressor (LTNP) patients as compared to chronically HIV-1-infected patients with high viral loads (HVLs) and CD4+ T cell loss, as well as HIV-seronegative healthy individuals. This study aims to identify the mucosal correlates of HIV disease progression and to determine the molecular changes associated with immune and intestinal dysfunction. LTNP patients had undetectable viral loads, normal CD4+ T cell levels, and virus-specific cellular responses in peripheral blood and mucosal compartments. Microarray analysis revealed a significant increase in gene expression regulating immune activation, cell trafficking, and inflammatory response in intestinal mucosa of HVL patients as compared to LTNP patients. Genes associated with cell cycle regulation, lipid metabolism, and epithelial cell barrier and digestive functions were down-regulated in both HVL and LTNP patients. This may adversely influence nutrient adsorption and digestive functions, with the potential to impact the efficacy of antiretroviral therapy. We demonstrate that the maintenance of mucosal T cells, virus-specific responses, and distinct gene expression profiles correlate with clinical outcome in LTNP patients. However, the intestinal mucosal immune system remains an important target of HIV-1 infection in LTNP, and these effects may ultimately contribute toward disease progression.

ABSTRACT Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections lead to rapid depletion of CD4+ T cells from gut-associated lymphoid tissue (GALT). Although the administration of antiretroviral therapy (ART) has been shown to increase CD4+ T-cell levels in the peripheral blood in both SIV and HIV infections, its efficacy in restoring intestinal mucosal CD4+ T cells has not been well investigated. To gain insights into the molecular mechanisms of virally induced disruptions in the mucosal immune system, we have evaluated longitudinal changes in viral burden, T-cell subsets, and mucosal gene expression profiles in SIV-infected rhesus macaques in the absence or presence of ART. Our results demonstrate a dramatic suppression of mucosal viral loads and rapid reconstitution of CD4+ T cells in GALT in animals receiving ART that were not observed in untreated SIV-infected animals. DNA microarray-based gene expression profiling indicated that CD4+ T-cell restoration in GALT was associated with up regulation of growth factors and genes involved in repair and regeneration of the mucosal epithelium. In contrast, untreated SIV-infected animals increased expression of lymphocyte activation and inflammatory response-associated genes and did not up regulate mucosal growth and repair associated transcription. In conclusion, these data indicate that initiating ART in primary SIV infection may lead to the restoration of the mucosal immune system through reduction of inflammation and promotion of epithelial repair in the intestinal mucosa.