Chronic viral hepatitis and its long-term sequelae, cirrhosis and hepatocellular carcinoma (HCC) contribute to a very large burden of disease both in the United States and worldwide.  The Hepatic Pathogenesis Section (HPS) has developed an extensive program involving basic and clinical research to study the pathogenesis of acute and chronic liver diseases in humans with a major focus on viral hepatitis.  1.  Molecular mechanisms of pathogenesis of acute liver failure (ALF) and liver regeneration.  ALF is a dramatic clinical syndrome characterized by sudden loss of hepatic function leading to multiorgan failure.  By gene expression profiling we found that HBV-associated ALF, unlike classic acute hepatitis B where liver damage is predominantly T-cell mediated, is characterized by an overwhelming intrahepatic B-cell gene signature associated with a T-cell independent, intrahepatic B-cell response with massive production of antibodies in germline configuration, accompanied by complement deposition.  In collaboration with Dr. Stevens from the LSB, NIAMS, the target epitope recognized by these antibodies has been characterized as a novel conformational site on the capsid of HBV, suggesting that antibodies directed to particular HBcAg epitopes may be involved in the pathogenesis of ALF.  ALF has also provided a model to study the complex process of liver regeneration in humans.  We found that HBV-associated ALF is associated with a strong hepatic stem/progenitor cell (HSPC) gene signature, hepatic stellate cell activation and fibrogenesis, along with an overriding tumorigenesis gene signature, which was previously never reported in liver regeneration.  Both HSPC markers and fibrogenesis positively correlated with the extent of histopathological severity, likely reflecting the wound-healing process.  2.  Determinants of disease progression, fibrogenesis, and viral evolution in chronic hepatitis C. Chronic hepatitis C may follow a mild and stable disease course or progress rapidly to cirrhosis and liver-related death.  The mechanisms underlying the different rates of disease progression are unknown.  Using prospectively collected samples from cases of transfusion-associated hepatitis C, we identified outcome-specific features that predict long-term disease severity.  Slowly progressing disease correlated with an early ALT peak and antibody seroconversion, transient control of viremia, and significant induction of IFN-gamma and MIP-1beta, all indicative of an effective, albeit insufficient, adaptive immune response.  By contrast, rapidly progressive disease correlated with persistent and significant elevations of ALT and the profibrogenic chemokine MCP-1 (CCL2) greater viral diversity and divergence, and a higher rate of synonymous substitution.  These studies led to the identification of profibrogenic and proinflammatory chemokines that predict rapid progression of hepatitis C to cirrhosis, providing a new model of HCV-disease pathogenesis and opening new perspectives for early diagnosis and treatment.  3.  Host and Viral Factors in the Pathogenesis of Hepatocellular Carcinoma (HCC) and search for biomarkers for the early detection of HCC.  Hepatitis viruses (HBV, HCV and HDV) account for about 80% of HCC, and cirrhosis is the single most important risk factor.  Although the major etiologic agents and risk factors for HCC are well defined, the molecular mechanisms of hepatocarcinogenesis remain unclear.  Our aim is to investigate the pathogenesis of HCC by studying the host and the virus.  Due to the complexity of HCC, our initial approach was an extensive mapping of gene expression profiles of the entire liver containing HCC associated with HBV, HCV and HDV in order to identify molecular signatures specific for each hepatitis virus as well as early biomarkers of progression to HCC.  Strikingly, we detected a distinct molecular signature specific for each hepatitis virus, which was confirmed by profiling of laser-microdissected hepatocytes.  This different molecular signature was also reflected by the different major canonical pathways.  Altogether, these data emphasize the different molecular features of HCC associated with different hepatitis viruses, suggesting that each virus may induce distinct disease mechanisms.  A. Limited replication of hepatitis viruses within HCC tissue.  We quantified the levels of HBV, HCV and HDV replication in different areas of the liver within and outside the tumor, in non-HCC cirrhotic livers, and in serum samples from the same patients.  Strikingly, we found a significant reduction in the levels of HCV RNA within the tumor compared to the non-tumoral tissue, whereas no differences were seen in multiple liver specimens obtained from non-HCC cirrhosis.  HDV RNA levels were also lower in the tumor compared to the surrounding sites.  By contrast, the levels of HCV RNA and HDV RNA in serum of HCC patients did not differ from those with non-HCC cirrhosis.  Collectively, our data indicate that HBV, HCV and HDV do not grow well in malignant hepatocytes in vivo, in line with the inability or limited efficiency of hepatitis viruses to grow in hepatoma cell lines in vitro, suggesting that malignant hepatocytes express restriction factors that negatively affect viral replication.  B. HCV quasispecies within the tumor and the surrounding non-tumoral tissue.  To investigate whether the reduced HCV replication in the tumor reflected the presence of viral variants selected by malignant hepatocytes, we performed an extensive HCV quasispecies analysis from the E1/E2 region (inclusive of HVR1) from different liver areas and serum.  Cirrhotic patients showed lack of HCV compartmentalization, whereas a different quasispecies distribution was seen in HCC.  Cases with 1-log drop in HCV RNA had a pattern similar to that seen in cirrhosis, while those with the greatest drop showed a shift in quasispecies distribution, consistent with selection of viral variants by malignant hepatocytes.  C. Identification of microRNA specifically expressed in HCV-associated HCC.  Perturbations of miRNAs have been correlated with various diseases and in particular with cancer.  Although several studies have investigated the association of miRNAs with HCC, the data are not univocal.  We identified 18 miRNAs expressed in HCV-associated HCC, some of which were never previously reported in association with HCC.  The HCC-specific miRNAs are connected through a regulatory network pivoting on p53, PTEN and all-trans retinoic acid signaling pathway, which may be directly involved in the pathogenesis of HCC.  These findings suggest that HCC-specific miRNAs form a molecular network directly involved in the pathogenesis of HCC.  Because of the lower HCV RNA levels found in the tumor, we are also investigating the potential role of HCC-specific miRNAs in modulating HCV replication.  4.  Search for new hepatotropic agents: attempt to transmit a putative infectious agent from a patient with primary biliary cirrhosis (PBC).  PBC is a chronic progressive disease of the liver bile ducts of unknown etiology that primarily affects women and may lead to cirrhosis and liver-related death.  Although it is generally thought to be an autoimmune disease, an infectious agent has not been excluded.  The aim of this study was to attempt to transmit a putative infectious agent from PBC to chimpanzees through intravenous inoculation of serum from an Italian patient with early stage PBC (Stage 1) to two chimpanzees, and a second passage to a third animal.  Serial liver biopsies have revealed progressive liver damage with increasing degree of bile duct damage and ductular reaction and, importantly, fibrosis.  This is a particularly striking finding as liver fibrosis is not commonly seen in chimpanzees, even in those chronically infected with hepatitis viruses for more than 10 years.