The major goal of the Hepatic Pathogenesis Section (HPS) 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 mechanisms of pathogenesis of acute liver failure (ALF). The pathogenesis of HBV-associated ALF is poorly understood. Following our initial observation of an overwhelming B cell gene signature in ALF, with massive intrahepatic accumulation of plasma cells secreting IgG and IgM: i) we studied the biological and genetic characteristics of the HBV strains recovered from serum and liver of 4 well-characterized patients with ALF;ii) we cloned and expressed HBsAg and HBcAg from all 4 patients, which were used to screen the corresponding phage-display Fab libraries (IgG1 and IgM) generated from the liver of each patient to identify the molecular targets of the antibodies produced in the liver;iii) we performed extensive sequence analysis of these antibodies to investigate their variable region usage and somatic mutation rates;and iv) we studied the whole set of human miRNAs to investigate the correlation between mRNA and miRNA expression. Next-generation sequencing showed the HBV pre-core stop mutation in almost 100% of the viral populations both in liver and serum of all 4 patients. HBcAg was the most variable region of the entire genome. Amino acid (AA) changes were scattered throughout the protein, with clusters particularly within the immunodominant B-cell epitope, indicating that HBcAg is under strong immune pressure. By contrast, no AA changes within HBcAg were seen in reported sequences of patients with classic acute hepatitis B. Screening of 8 phage libraries showed that the intrahepatic antibodies in ALF are directed against HBcAg. Sequence analysis demonstrated that both IgG and IgM intrahepatic antibodies, albeit produced from unrelated individuals with ALF, display a restricted variable heavy chain (VH) repertoire and lack somatic mutations, providing evidence that HBcAg is the target of antibodies in germline configuration. An integrated analysis of mRNA and miRNA showed that they were strongly correlated. Strikingly, we found that the majority of ALF-specific miRNAs (74%) target B-lineage-associated genes, whereas only a very small number of miRNA target T-cell genes, in line with the limited T-cell signature detected in ALF. Altogether, our data reveal a dominant B-cell-driven disease signature consistent with a major role of B-cell immunity in the pathogenesis of ALF in contrast to the classic acute hepatitis B where the liver damage is believed to be T-cell mediated. 2. Determinants of disease progression, fibrogenesis, and viral evolution in chronic hepatitis C. We have identified 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 the early diagnosis and treatment. We are now attempting to validate these predictive markers in a large cohort of patients with slow and rapid HCV-disease progression. 3. Host and Viral Factors in the Pathogenesis of HCC and search for biomarkers for the early detection HCC is the third leading cause of cancer-related death worldwide. In most patients, HCC arises in the setting of chronic liver disease, with cirrhosis being present in about 80% of the cases. Chronic infection with HBV and HCV are responsible for over 80% of HCC cases worldwide. 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. HBV-associated HCC We used genomic and molecular techniques to investigate host-virus interactions by studying multiple areas of the same liver from patients with HCC. We compared the gene signature of whole liver tissue (WLT) versus laser capture-microdissected (LCM) hepatocytes and correlated it with intrahepatic expression of HBV. We found a sharp change in gene expression within millimeters of the tumor margin, which correlated with a significant decrease in HBsAg expression. The tumor is characterized by a metabolism switch-off of lipids and fatty acids, glucose, amino acids and drugs, with down-regulation of pathways involved in the activation of nuclear receptors, comprising PGC-1a and FOXO1, two key regulators of the hepatic metabolic functions and HBV transcription, which may contribute to the decreased HBsAg expression that we documented within the tumor. LCM allowed to confirm most of the genes detected in whole liver tissue and to identify a series of unique genes associated with cancer and signaling pathways, including two novel HCC-associated cancer testis antigens, NUF2 and TTK. Our data provide new insights for dissecting HCC pathogenesis, which may lead to the discovery of new biomarkers for the early diagnosis of HCC. HCV-associated HCC Over the past two decades, the incidence of HCC has more than tripled in the United States, and this alarming trend is due primarily, if not exclusively, to HCV infection. Whether HCV promotes HCC indirectly through chronic inflammation and fibrosis, or directly through the expression of viral proteins like other human oncogenic viruses, remain to be established. Serum and up to 17 specimens from individual liver (5 from the tumor and 12 outside the tumor) of 8 patients who underwent liver transplantation or resection, were analyzed for HCV RNA levels and gene expression profiling. Strikingly, HCV RNA levels were significantly reduced (up to 3 logs) in the tumor compared to surrounding non-tumorous areas. This drop was associated with dramatic changes in gene expression. Both innate and adaptive immune response genes were downregulated within the tumor, suggesting that the HCV RNA reduction cannot be explained by immune-mediated mechanisms. Expression of miR122, a key regulator of HCV replication, did not differ between tumor and non-tumorous areas. An extensive analysis of the HCV quasispecies from the E1/E2 region in different liver areas and serum demonstrated the presence of HCV compartmentalization in the tumor compared to non-tumor tissue, but not between non-tumor tissue and serum. By contrast, non-HCC cirrhosis showed neither changes in the levels of HCV RNA among different areas of the liver nor HCV compartmentalization between different areas of the liver, and between liver and serum. These findings are consistent with the limited replication of HCV within the tumor and the selection of viral variants by malignant hepatocytes. 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 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. We attempted 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, as well as a second passage from one inoculated chimpanzee to a third animal. Serial liver biopsies revealed in all animals progressive liver damage with increasing bile duct damage and ductular reaction and, most importantly, fibrosis. We are now attempting to identify the putative agent by direct next-generation sequencing of genetic material from the chimpanzees. We are also performing microarray analysis to identify signs of host response to infection and genes associated with chronic liver damage.
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