The major goal of the Hepatic Pathogenesis Section is to conduct translational research on the pathogenesis of acute and chronic liver disease, with a major focus on viral hepatitis and its long-term sequelae, cirrhosis and hepatocellular carcinoma (HCC), which contribute to a very large burden of disease worldwide. 1. Molecular Definition of Hepatitis B Virus-Associated Acute Liver Failure (ALF) To better understand the molecular pathogenesis of HBV-associated ALF, we undertook a comprehensive study in which we investigated simultaneously the role of viral and host factors in ALF. Moreover, the availability of archived liver specimens from acutely HBV-infected chimpanzees gave us the opportunity to compare the findings in ALF with those in classic acute hepatitis B. The major aim of this study was to investigate the nature and the pathogenic role of the enhanced immune response in the pathogenesis of ALF, as well as the genetic and immunologic characteristics of the HBV strains isolated from ALF patients. Gene expression profiling showed a complete separation between ALF and controls for both mRNA and microRNA. Both mRNA and microRNA expressions were significantly associated with five major disease categories, with inflammatory and immunological diseases among the most prominent. In contrast to the prominent B-cell gene signature seen in ALF, classic acute hepatitis B was associated with expression of a large number of T-cell-derived and IFN--regulated genes, which reflect the role exerted by the adaptive T-cell response in this disease. Deep sequencing analysis of HBV obtained from ALF patients showed that the rate of mutation was not evenly distributed over the entire HBV genome with the precore/core region being the most variable. In contrast, no mutations were detected in the core antigen (HBcAg) obtained from two chimpanzees or patients with classic acute hepatitis B, indicating that a high HBV heterogeneity was associated with ALF. Next, we investigated the reactivity of intrahepatic antibodies with both homologous and heterologous HBV antigens derived from ALF patients and chimpanzees. Sequence analysis of intrahepatic IgM and IgG from ALF patients demonstrated that the majority of VH genes coding for HBcAg-specific IgM and IgG lack somatic mutations, indicating that HBcAg is the target of germline human antibodies with KD values in the subnanomolar range, an exceptionally high affinity typically seen with somatically hypermutated antibodies. In contrast, all anti-HBc antibodies derived from the liver of two chimpanzees with acute hepatitis B contained somatic hypermutations, consistent with a T-cell dependent B-cell stimulation. Thus, the massive intrahepatic production of anti-HBc antibodies in germline configuration but with high affinity is unique to ALF, and corroborates the pathogenic role of humoral immunity in this dramatic disease. 2. Determinants of fibrogenesis and disease progression in chronic hepatitis C In collaboration with Dr. Alter, we investigated the association between circulating microRNA (miRNA) levels and disease progression in chronic hepatitis C (CHC). We studied 130 CHC patients prospectively followed over several decades. A comprehensive miRNA profile in plasma showed 323 miRNAs differentially expressed between healthy individuals and CHC patients, but only six that distinguished patients with mild versus severe CHC. Let-7a/7c/7d-5p and miR-122-5p were identified as candidate predictors of disease progression. Cross-sectional analyses at the time of initial liver biopsy showed that reduced levels of let-7a/7c/7d-5p (let-7s) in plasma correlated with advanced hepatic fibrosis and other fibrotic markers, whereas miR-122-5p levels were correlated with inflammatory activity, but not fibrosis. Longitudinal analyses showed that let-7s levels in plasma markedly declined over time in parallel with fibrosis progression, thereby providing the best correlation with hepatic fibrosis in CHC. Pathway analysis suggested that low levels of let-7 may influence hepatic fibrogenesis through activation of TGF- signaling in hepatic stellate cells. 3. Host and viral factors in the pathogenesis of hepatocellular carcinoma (HCC) and search for early biomarkers HCC is the third leading cause of cancer-related death worldwide, and chronic infection with hepatitis viruses accounts for over 80% of HCC cases. Cirrhosis is the single most important risk factor for HCC being present in 80-90% of the cases. Although the major etiologic agents and risk factors for HCC are well defined, the molecular mechanisms of hepatocarcinogenesis remain unclear.
Our aim i s to investigate the pathogenesis of HCC by studying both the host and the virus. Diminished Replication and Viral Compartmentalization of Hepatitis C Virus (HCV) in HCC HCC associated with HCV infection is the fastest-rising cause of cancer-related death in the United States. Analysis of HCV replication and quasispecies distribution within the tumor of patients with HCV-associated HCC can provide insight into the role of HCV in hepatocarcinogenesis and conversely the effect that HCC has on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from HCC patients who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared to surrounding non-tumorous tissues, whereas no differences were observed in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared to control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in the viral population between tumorous and non-tumorous areas whose extent correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. In contrast, compartmentalization was not observed between non-tumorous areas and serum, nor in controls between different areas of the cirrhotic liver or between liver and serum. Our findings indicate that HCV replication within the tumor is restricted and compartmentalized, suggesting segregation of viral variants in malignant hepatocytes. Molecular characterization of HCC associated with hepatitis D virus (HDV) HDV causes the most severe and rapidly progressive form of hepatitis, leading to cirrhosis in about 80% of cases. Although the risk of developing HCC dramatically increases at the cirrhosis stage, there are no data on the molecular mechanisms of HDV-associated hepatocarcinogenesis. Moreover, because of the obligatory link of HDV with HBV, the specific role of HDV in promoting liver cancer remains unknown. We used genomic techniques to investigate host-virus interactions in patients with HDV HCC who underwent liver transplantation by using whole liver tissue and laser capture microdissected hepatocytes. Gene expression profiling showed that HDV-associated HCC is characterized by an enrichment of genes related to DNA repair mechanisms pointing to genetic instability as an important mechanism in HDV hepatocarcinogenesis. By contrast, HBV-associated HCC was characterized by a metabolism switch-off. Analysis of the genes expressed in HDV HCC identified specific pathways related to DNA damage-induced signaling, cyclins, cell cycle regulation and tumor cell proliferation. Deep sequencing revealed deletions in HBV genomes from both the tumor and the nontumorous tissues of patients with HBV/HDV coinfection. Altogether, these data emphasize the different molecular features of HCC associated with different hepatitis viruses, suggesting that each virus may promote carcinogenesis by distinct molecular mechanisms.
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