Considerable progress has been made in the control and treatment of acute and chronic viral hepatitis, but the success is still limited and further progress will depend on a more thorough knowledge of the molecular mechanisms of pathogenesis. In the absence of reliable in vitro cell culture systems and small animal models, the chimpanzee model has been an invaluable resource for the discovery of the etiologic agents and for understanding the diseases caused by the hepatitis viruses. However, chimpanzees are not a suitable model for fulminant hepatitis and liver fibrogenesis. My long-term clinical experience in a geographic area (Sardinia, Italy) with a high endemicity for infection with hepatitis viruses has given me a unique opportunity to select and store clinical samples from well-characterized patients with acute and chronic liver diseases. This collection of specimens serves as the basis for the translational research that I have initiated at the NIH since last year, when I joined the LID as a senior investigator. Our main research strategy is to combine the clinical information, inclusive of a detailed clinical history, as well as biochemical, virologic and histopathologic data, with the molecular analysis using sophisticated methods that have become available in the post-genomic era.? ? 1.Molecular mechanisms of pathogenesis of acute liver failure (ALF). ? Few conditions in medicine are more dramatic and challenging than ALF, a clinical syndrome characterized by a sudden loss of hepatic function leading to multiorgan failure in a person without preexisting liver disease. Viral hepatitis, especially caused by HBV and HDV, and drug-induced liver injury account for most cases of ALF. In collaboration with Dr. Alter, we provided the first evidence that HCV, albeit rarely, may cause ALF and we defined the early molecular events in this dramatic clinical syndrome. Although the pathogenic mechanisms of virally-induced ALF are presently unknown, in HCV the extent of liver damage correlated with the magnitude of viral replication and with an unusually homogeneous viral population, suggesting that in FH there is a trend to preserve the unique fitness of a particular viral variant, supporting the hypothesis that viral factors are important in the pathogenesis of fulminant hepatitis C. By contrast, in fulminant hepatitis B, HBV replication is barely detectable or undetectable concomitant with an early anti-HBsAg seroconversion, suggesting an enhanced immune response to viral antigens as a possible mechanism leading to massive liver necrosis. ? ? The advent of gene array technologies has provided a powerful tool to study the pathogenesis of complex diseases in which thousands of genes are jointly regulated. Until now, however, studies of gene-expression profiling of ALF have been lacking. Access to well-preserved explanted liver tissue samples from two well-defined cases of acute HBV-associated hepatic failure who underwent liver transplantation gave us a unique opportunity to perform a systematic study of gene expression profiling by means of microarray.? ? Microarray data of 16 liver samples, including 8 derived from two patients with HBV-related ALF (4 samples per liver) and 8 derived from normal liver donors, were analyzed using BRB-ArrayTools. Using a multivariate permutation test with a false discovery rate lower than 1 per cent, a total of 1368 genes were differentially expressed between the two classes. Among them, 709 were upregulated and 609 downregulated in ALF. Genes associated with the immune response were the most represented among upregulated genes. In particular, the most striking finding was an overriding signature for B cells. Among the 50 most up-regulated genes (fold changes ranging from 108 to 17), 28 were characteristic of the B-lymphoid lineage, with 21 belonging to the immunoglobulin heavy or light chain families. The latter included a similar representation of ? and ? light chains, indicating a polyclonal B-cell response. Unexpectedly, only 5 transcripts indicative of a T-cell signature were differentially expressed in ALF, with a single transcript appearing among the 50 most upregulated genes. Immunohistochemical analysis confirmed that the prominent B-cell signature observed by gene-expression profiling was reflected by a massive infiltration of the liver parenchima by cells of the B-lymphoid lineage, primarily plasma cells predominantly within the lobules that express IgM and IgG. In parallel with the striking increase in gene expression described above, a dramatic downregulation of multiple genes was detected in ALF livers. Specifically, the profile of downregulated genes in ALF was indicative of massive hepatocellular damage with a consequent shut-off of hepatic synthesis and the metabolic pathways that are regulated by the liver.? ? Because we had observed a massive intrahepatic production of immunoglobulins and a dramatic reduction in serum complement activity (CH50 assay), we investigated the deposition of complement factors in the liver tissue by immunohistochemistry. Strikingly, in both cases we documented a diffuse, granular to globular staining for C1q, C3 and C4d, clearly documenting the deposition of complement in the liver tissue of patients with ALF. The overall picture emerging from combined observations by gene expression profiling and immunohistochemical analysis provide a new possible model for the pathogenesis of ALF associated with HBV infection in humans. The presence of a prominent plasma-cell infiltrate within the liver tissue, with extensive intrahepatic expression of immunoglobulins and complement deposition, strongly suggest a possible role of complement-mediated cytotoxicity in the pathogenesis of the massive liver necrosis that leads to ALF. Thus, our data are compatible with the hypothesis that the intrahepatic production of immunoglobulins, possibly directed against virus-specific or virus-induced antigens, may lead to an extensive formation of antigen-antibody immunocomplexes on the surface of both hepatocytes and other cells, resulting in widespread complement-mediated hepatocyte destruction. ? ? 2. Molecular mechanisms of pathogenesis of chronic liver diseases? Chronic viral hepatitis and its long-term sequelae, cirrhosis and HCC, represent a major public health problem worldwide. The rate of progression to chronicity as well as the rate of progression to cirrhosis varies significantly among the three viruses that cause chronic hepatitis. The mechanisms underlying this variable clinical outcome, as well as the different speed of fibrosis progression, are poorly understood. Equally interesting is the clinical observation that cirrhosis, once established, may be a stable disease for decades in some patients, whereas in others it may be rapidly progressive leading to liver-related death for hepatic decompensation or, eventually, HCC. We have performed gene expression profiling in 104 liver specimens obtained from patients who underwent OLT for end-stage liver disease of different etiology, including alcoholic, HCV infection, HCV plus alcohol, HBV infection, HDV infection, and primary biliary cirrhosis. For the identification of differentially expressed genes between cirrhosis of different etiology, data from each patient were processed by a multivariate permutation test to control the false discovery rate. The highest number of differentially expressed genes was found between alcoholic and HCV cirrhosis, and between alcoholic and HDV cirrhosis. Remarkably, the presence of HCV seems to exert a dominant effect on gene expression profiles. As previously documented in the liver of experimentally infected chimpanzees during the early stages of HCV infection, a signature of IFN-inducible genes was still present in the liver of patients with end-stage HCV liver disease.
