Chronic viral hepatitis and its long-term sequelae, cirrhosis and hepatocellular carcinoma (HCC), represent a major global health problem. Considerable progress has been made in the control and treatment of chronic viral hepatitis, but further progress will depend on a more thorough knowledge of the molecular mechanisms of pathogenesis. The Hepatic Pathogenesis Section (HPS) has developed a comprehensive research program to study the pathogenesis of acute and chronic liver diseases in humans. 1. Molecular mechanisms of pathogenesis of acute liver failure (ALF) and liver regeneration.
Our aim was to establish a molecular definition of liver regeneration associated with ALF, and to correlate the molecular analysis with the severity of liver necrosis. Gene expression profiling of ALF cases that underwent liver transplantation, and individual specimens from liver donors documented a distinct gene signature for ALF. Unsupervised multidimensional scaling and hierarchical clustering identified two clusters of ALF that segregated according to histopathological severity: massive hepatic necrosis (MHN) and submassive hepatic necrosis (SHN). We found that ALF is characterized by a strong hepatic stem/progenitor cell (HSPC) gene signature, along with ductular reaction, both of which were more prominent in MHN. Strikingly, ALF was associated with a strong tumorigenesis gene signature. MHN had the greatest upregulation of stem cell genes, whereas the most upregulated genes in SHN were related to cellular growth and proliferation. Our data provide evidence that the intensity of the gene signature in HBV-associated ALF is directly correlated with the histopathological severity. HSPC activation and fibrogenesis positively correlated with the extent of liver necrosis. Moreover, we detected a strong tumorigenesis gene signature in ALF, emphasizing the close relationship between liver regeneration and liver cancer. 2. Molecular mechanisms of pathogenesis of chronic liver diseases and predictive factors of disease progression. Our project was aimed at investigating the mechanisms responsible for the different rates of disease progression in chronic hepatitis C. The clinical course of chronic hepatitis C are highly variable with stable or slowly progressive disease in about 70% of chronically infected patients, and progressive liver disease culminating in cirrhosis and possibly HCC in 30%. Cirrhosis usually develops in 20-40 years, but in some patients severe fibrosis can develop within 5 to 10 years, and is not possible to predict which individuals will rapidly evolve to severe fibrosis and liver failure. Using serial serum samples prospectively collected from 6 patients with transfusion-associated hepatitis C (followed for up to 30 years), we identified outcome-specific features that predict long-term disease severity. Slowly evolving disease correlated with an early ALT peak and antibody seroconversion, transient control of viremia and significant induction of IFN-gamma and MIP-1b, 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, greater viral diversity and divergence, and a higher rate of synonymous substitutions. This study suggests the long-term course of chronic hepatitis C is determined early in infection and disease severity is predicted by the evolutionary dynamics of HCV and the level of MCP-1, a chemokine that appears critical to the induction of progressive fibrogenesis and, ultimately, the ominous complications of cirrhosis. 3. Pathogenesis of 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. However, the molecular mechanisms of hepatocarcinogenesis are still unclear. Most of the studies of HCC have been limited to the host, whereas data on the role of hepatitis B, C and D viruses in hepatocarcinogenesis are very limited. Moreover, in many studies HCC cases are not divided according to the different etiologies and are often analyzed as a single group regardless of the hepatitis virus involved. Our major goal is to investigate the molecular mechanisms of hepatocarcinogenesis by studying simultaneously both the host and the oncogenic role of the hepatitis viruses. We investigated by microarray the molecular heterogeneity within and outside the tumor by mapping the entire liver containing HCC (up to 21 specimens for each patient). We have so far analyzed by microarray 462 liver specimens from patients with HCC of different etiology. Selected samples from the tumor and the periphery were also analyzed by laser capture microdissection. Remarkably, we found a high proportion of differentially expressed genes that were confirmed using both whole and microdissected tissue, confirming the robustness of our data. Interestingly, each tumor showed a distinct molecular signature with pathways specific for each tumor. In parallel with gene expression profiling, our unique collection of samples has allowed us to quantify by real time PCR the levels of HBV, HCV and HDV replication. Strikingly, we found a considerable difference in the levels of viral replication within and outside the tumor. By contrast, no differences in viral load were seen in multiple liver specimens obtained from different areas of livers with non-HCC cirrhosis. 4. Identification of microRNA specifically expressed in HCV-associated HCC. Although several studies have investigated the association of miRNAs with HCC, the data are not univocal. A reason for these discrepancies may be the fact that most studies used as a control the non-tumorous tissue surrounding the HCC lesion, which is almost invariably affected by cirrhosis or chronic hepatitis, as well as, possibly, other pathological conditions such as HBV or HCV infection. In this study, we investigated miRNAs differentially expressed in HCV-related HCC by comparing the tumorous tissues to a wide range of liver specimens, including healthy livers obtained from liver donors and patients who underwent liver resection for angioma in addition to tissues from various acute and chronic liver diseases, including HCV-related cirrhosis not associated with HCC, HCV-related cirrhosis associated with HCC, and HBV-associated ALF. Our study examined the whole set of 2,226 human miRNAs. By applying stringent statistical methods, our data identified 18 miRNAs exclusively expressed in HCV-associated HCC, connected through a regulatory network pivoting on p53, PTEN and all-trans retinoic acid. Thus, findings suggest that HCC-exclusive miRNAs form a molecular network directly involved in the pathogenesis of HCC. 5. Search for new hepatotropic agents. Primary biliary cirrhosis (PBC) is generally thought to be an autoimmune disease though an infectious agent has not been excluded. We have identified a Sardinian patient with early PBC (stage 1).
The aim of this study was to attempt to transmit a putative infectious agent from PBC to chimpanzees. The animal inoculated with serum from the patient with PBC has been followed for more than 3 years, and serial liver biopsies have revealed bile duct damage and increasing degrees of ductular reaction along with fibrosis detection. Based on these promising results, we inoculated the same serum into a second animal, and we have attempted a second passage from the first inoculated animal. Both animals showed similar changes as the first inoculated animal. Microarray analysis is in progress to identify specific genes related to innate immunity or mitochondria or other cellular genes. Finally, we are planning to attempt to identify the putative agent by molecular techniques such as high-throughput sequencing.

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Chen, Jian; Zaidi, Sobia; Rao, Shuyun et al. (2018) Analysis of Genomes and Transcriptomes of Hepatocellular Carcinomas Identifies Mutations and Gene Expression Changes in the Transforming Growth Factor-? Pathway. Gastroenterology 154:195-210
Diaz, Giacomo; Engle, Ronald E; Tice, Ashley et al. (2018) Molecular Signature and Mechanisms of Hepatitis D Virus-Associated Hepatocellular Carcinoma. Mol Cancer Res 16:1406-1419
Sekhar, Vandana; Pollicino, Teresa; Diaz, Giacomo et al. (2018) Infection with hepatitis C virus depends on TACSTD2, a regulator of claudin-1 and occludin highly downregulated in hepatocellular carcinoma. PLoS Pathog 14:e1006916
Matsuura, Kentaro; De Giorgi, Valeria; Schechterly, Cathy et al. (2016) Circulating let-7 levels in plasma and extracellular vesicles correlate with hepatic fibrosis progression in chronic hepatitis C. Hepatology 64:732-45
Raghwani, Jayna; Rose, Rebecca; Sheridan, Isabelle et al. (2016) Exceptional Heterogeneity in Viral Evolutionary Dynamics Characterises Chronic Hepatitis C Virus Infection. PLoS Pathog 12:e1005894
Zhang, Quan; Matsuura, Kentaro; Kleiner, David E et al. (2016) Analysis of long noncoding RNA expression in hepatocellular carcinoma of different viral etiology. J Transl Med 14:328
Harouaka, Djamila; Engle, Ronald E; Wollenberg, Kurt et al. (2016) Diminished viral replication and compartmentalization of hepatitis C virus in hepatocellular carcinoma tissue. Proc Natl Acad Sci U S A 113:1375-80
Zhu, Ruixin; Baker, Susan S; Moylan, Cynthia A et al. (2016) Systematic transcriptome analysis reveals elevated expression of alcohol-metabolizing genes in NAFLD livers. J Pathol 238:531-42
Diaz, Giacomo; Zamboni, Fausto; Tice, Ashley et al. (2015) Integrated ordination of miRNA and mRNA expression profiles. BMC Genomics 16:767
Bukh, Jens; Engle, Ronald E; Faulk, Kristina et al. (2015) Immunoglobulin with High-Titer In Vitro Cross-Neutralizing Hepatitis C Virus Antibodies Passively Protects Chimpanzees from Homologous, but Not Heterologous, Challenge. J Virol 89:9128-32

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