Browsing by Subject "Hepatitis C, Chronic"
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Item Characterizing B cell Phenotype during chronic HCV infection(2009-06-15) Ayers, Christopher Lee; Karandikar, NitinChronic hepatitis C virus (HCV) infection is characterized by an attenuation of virus-specific T cell responses. The mechanisms leading to T cell attenuation are still not well understood, and likely involve several integrating correlates. We hypothesized that dysfunctions of antigen presenting cells (APC) may contribute to the immunosuppresed phenotype. We also reasoned that direct viral interactions of HCV with immune cells may be responsible for such dysfunction. We employed a strand-specific real time RT-PCR assay and found that virus is frequently associated with B cells (predominantly positive strand was detected). Interestingly, we also found that ex-vivo derived B cells from chronic HCV individuals were better inducers of allogeneic T cell proliferation and this ability correlated with the presence of HCV RNA in those B cells. During such enhanced allostimulation, we also found an increase in the proportion of CD4+CD25+FoxP3+ T cells, which correlated with an increased suppressive capacity thereby demonstrating a paradoxical link between hyperactive B cells and the generation of suppressive T cells. Furthermore, ex-vivo derived chronic HCV B cells had an attenuated response to mitogenic stimulation with associated apoptosis. In an effort to determine direct HCV involvement in immune cell dysfunction, we evaluated the possibility of culture adapted JFH-1 virus to infect PBMC populations. While we found no evidence of viral replication in PBMC, exposure to JFH-1 resulted in vigorous activation of B cells. Interestingly, the B cell activation did not require viable virions, but was dependent upon CD81 availability and the presence of monocytes. We also determined that upon viral exposure, these B cells replicated the hyper-activation of MLR responses found in ex-vivo derived B cells from chronic HCV individuals. In all, our results suggest a novel model wherein HCV-B cell interaction leads to B cell hyper-activation and consequent paradoxical T cell suppression.Item Hepatitis C Virus NS3/4A Protease and the Intracellular Antiviral Response: Mapping Complex Virus-Host Interactions(2009-06-17) Johnson, Cynthia L.; Gale, Michael J.Virus infection triggers an innate immune response characterized by host cell production of interferon (IFN). Intermediates of viral replication, including dsRNA, initiate a signaling cascade that is amplified within the cell and alerts neighboring cells of viral invaders. Recognition of dsRNA intermediates occurs through retinoic acid inducible gene-I (RIG-I). RIG-I elicits an antiviral state by binding to the IFN-beta Promoter Stimulator-1 (IPS-1) adaptor protein, activating the latent downstream transcription factors IRF-3 and NF-kappaB. These transcription factors bind to the promoter region of effector genes including IFN-beta, producing an antiviral amplification loop within and around the infected cell. This response is critical for immunity to infection. Hepatitis C virus (HCV) is a serious global health problem with 170 million people chronically infected. HCV persistence is linked to viral regulation of innate host defenses by the nonstructural 3/4A protein complex (NS3/4A) cleavage of IPS-1. NS3 structural composition includes an amino-terminal serine protease and a carboxy-terminal RNA helicase. A structure-function analysis of NS3/4A truncation and deletion mutations was conducted. Mutants lacking the helicase domain retained the ability to control RIG-I signaling, but this regulation was abrogated by truncation of the protease domain. Furthermore, treatment of HCV-infected cells with a NS3/4A protease inhibitor prevented IPS-1 proteolysis, restored RIG-I signaling, and decreased viral protein levels. These results indicate that the NS3/4A protease domain alone can target IPS-1 on the mitochondrial membrane. Current dogma holds that NS3/4A is located on the endoplasmic reticulum, thus the mechanism of NS3/4A targeting IPS-1, a mitochondrial membrane protein, remains unexplained. We have shown that NS3/4A distributes on mitochondria independently of the previously identified NS4A membrane localization motif, in a manner dependent on the first twenty amino acids of the NS3 protease domain. The functional domains of IPS-1 that direct the immune response have not been elucidated. We conducted a structure-function study of IPS-1 that revealed distinct processes of IRF-3 and NF-kappaB activation. Mutational analyses further identified areas of IPS-1 critical for mitochondrial localization, dimerization, and uncoupling IRF-3 and NF-kappaB signaling. These findings improve our understanding of IPS-1 function in innate immunity to virus infection.Item Peripheral Generation Of Regulatory T Cells In Health And Disease(2007-05-23) Pillai, Vinodh; Karandikar, NitinCD4+CD25+FOXP3+ regulatory T cells (Tregs) form an important arm of the immune system responsible for suppressing untoward immune responses. They play a role in autoimmunity, allergy, asthma, transplantation, tumors and infectious diseases. Tregs are increased in the peripheral blood of chronic hepatitis C virus-infected patients and their depletion in-vitro increases anti HCV responses when measured by a sensitive CFSE-based flow cytometric proliferation assay. The CFSE-based assay, developed and validated by my laboratory, has a greater ability to detect low frequency and low avidity type T cell responses in the chronic HCV patients that are difficult to measure using ex-vivo assays. Using this assay in a cross sectional study, I showed that anti-viral immune responses are attenuated in untreated chronic HCV patients and are increased after anti-viral treatment with Interferon and Ribavirin. Interestingly, increase in anti-viral immune responses after Treg depletion was not seen in patients who were successfully treated with interferon and ribavirin. These results suggest that anti-viral therapy may be acting by modulating anti-viral immune responses. Tregs can be thymically derived (natural Tregs) or peripherally induced (adaptive Tregs). FOXP3 expression and in-vitro suppressive activity are considered unique hallmarks of this dedicated and stable lineage of regulatory cells. Initial evidence indicated that it is a specific marker of natural Tregs. It was thought that FOXP3+ T cells cannot arise in the periphery from na?CD4+CD25- T cells. However, using allostimulation of CFSE stained T cells and polycolor flow cytometry, I showed that virtually all human CD4+CD25-FOXP3- T cells and CD8+CD25-FOXP3- T cells attain a transient FOXP3+CD25+ state during activation. In this state of activation, these cells possess the classic phenotype of Tregs, in that they express similar markers and inhibit in-vitro proliferation of autologous CD4+CD25- T cells. This state is characterized by suppressed IFN-gamma production and robust TNF-alpha and IL-10 production. Interestingly, the great majority of the activated cells eventually downregulate FOXP3 expression, with a concomitant drop in suppressive ability. However some of the FOXP3+ T cells continue to maintain FOXP3 expression suggesting that activation might be a mechanism of producing FOXP3+ Regulatory T cells in the periphery during viral infections like chronic HCV infection. Transient FOXP3 expression in activated T cells might also be a mechanism of fine tuning excessive immune activation. These results show that, in humans, FOXP3 expression and Treg functionality are not exclusive features of a stable or unique lineage of T cells, but may also be a transient state attained by almost all T cells. These results warrant caution in interpreting human studies using FOXP3 and suppressive activity as readouts and suggest that attempts to induce " Tregs" may paradoxically result in induction of effector T cells, unless stability is confirmed.