Considerable progress has been made in the control and treatment of 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. The chimpanzee model has been fundamental 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. To investigate the pathogenesis of liver diseases in humans, we started a collaboration with the Liver Transplanation Center and the Liver Unit of the University of Cagliari, (Sardinia, Italy), which is an invaluable resource of 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 I joined the LID as a senior investigator in 2007. Our main research strategy is to combine the molecular analysis with the clinical, biochemical, virologic and histopathologic data. 1. Molecular mechanisms of pathogenesis of acute liver failure Few conditions in medicine are more dramatic and challenging than acute liver failure (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 and drug-induced liver injury account for most cases of ALF. Although the pathogenic mechanisms of virally-induced ALF are presently unknown, we demonstrated that in HCV ALF the extent of liver damage correlated with the magnitude of viral replication and with an unusually homogeneous viral population. By contrast, in fulminant hepatitis B, HBV replication is barely detectable or undetectable concomitant with an early and enhanced immune response to several viral antigens. Taking advantage of gene expression analysis and phage-display library, we have provided evidence that in fulminant hepatitis B the liver damage is mediated by B-cell immunity. Liver specimens obtained from two patients with HBV-associated ALF (4 samples per liver) and individual liver specimens from 8 liver donors were processed by microarray. Multivariate permutation analysis identified 1,368 transcripts that were differentially expressed in ALF;709 were up-regulated and 659 down-regulated. ALF was characterized by an overriding B-cell signature comprising genes related to mature B cells and plasma cells with abundant polyclonal expression of immunoglobulin genes. By contrast, there was a limited T-cell signature and up-regulation of several inhibitors of T-cell activation. Immunohistochemical analysis confirmed the prominent B-cell signature showing diffuse liver infiltration by plasma blasts and plasma cells with strong cytoplasmic staining for IgM and IgG, associated with a significant deposition of complement factors. Analysis of 4 phage display Fab libraries of IgG1 and IgM generated from the liver of the 2 patients with ALF identified HBcAg as the major target of the antibodies produced in the liver. In conclusion, the prominent B-cell gene signature with intrahepatic Ig production primarily directed against HBcAg, associated with complement deposition, suggests a pivotal role of the humoral immunity in the pathogenesis of HBV-associated ALF. 2. Molecular mechanisms of pathogenesis of chronic liver diseases The proportion of patients who develop cirrhosis is 30-40% among patients with chronic hepatitis B and C, and up to 80% in chronic hepatitis D. We have long been interested in understanding the mechanisms underlying the different outcomes observed in patients with chronic viral hepatitis. Presently, it is not possible to predict which individuals will sustain more dire outcomes, although age at infection has been shown to have a significant impact on the pace of disease progression in chronic hepatitis C, which is more rapid in the elderly. Likewise, male gender is associated with a worst outcome. To investigate the role of age and gender in liver fibrosis progression, we have completed an extensive study of gene expression analysis in normal livers obtained from individuals in different age groups. We are now determining whether specific gene signatures associated with innate or adaptive immune response, increased fibrinogenesis, or decreased fibrinolysis are modulated according to age or if there are significant differerences in gene expression between males and females. The results of this study may help to understand the influence of age and gender in the natural history of chronic hepatitis C, as well as in other forms of chronic liver diseases. Cirrhosis may be a stable disease for decades in some patients, whereas in others it may lead to liver-related death for decompensation or hepatocellular carcinoma (HCC). The reasons why some patients eventually die of complications of liver cirrhosis and some dont are presently unknown. To address these important questions, we have designed and completed a study in which a large number of liver specimens rigorously selected to represent clean cases of cirrhosis of different etiologies (HBV, HBV+HDV, HCV, HCV+alcohol, alcohol, autoimmune) were analyzed by microarray. We have found that each subtype of cirrhosis is characterized by a specific gene expression signature, with a striking and unexpected heterogeneity even between biologically related conditions such as HBV vs. HBV+HDV cirrhosis. Gene expression profiling will be integrated by virological studies using confocal microscopy and sequencing analysis. 3. Pathogenesis of HCC and search for biomarkers for the early detection of HCC. The clinical heterogeneity of HCC and the lack of good diagnostic markers and treatment strategies have made this disease one of the most important challenges for the scientific community. The presence of cirrhosis of any etiology is the strongest risk factor for the development of HCC. Although the major etiologic agents of HCC have been identified, the mechanisms of hepatocarcinogenesis are unclear and genetic predisposition has rarely been described. In collaboration with Dr. Zamboni, from the Liver Transplantation Center in Cagliari, Italy, we have designed a study in which multiple biopsies obtained from a single liver have been studied. We have collected more than 500 liver specimens from patients with HCC and about 200 have already been subjected to microarray analysis.The study is currently in progress. A thorough characterization of genes associated with liver cirrhosis will be critical to identify which genes are involved in the progression of cirrhosis toward HCC. 4. Search for new hepatitis agents. The cause of primary biliary (PBC)cirrhosis is unknown,but because of the presence of autoantibodies against mitochondrial protein in 90% of the patients, PBC is generally thought to be an autoimmune disease. However, an infectious agent has not been excluded. We have identified a Sardinian patient with early PBC (stage 1).
The aim of this study is to attempt to transmit an infectious agent from PBC to chimpanzees. We are currently following a chimpanzee that was inoculated with serum from the patient with PBC (week 77 post-inoculation). Interestingly, we have observed ultrastructural alterations of the mitochondria by EM, as well as portal inflammatory infiltrates by light microscopy. We are planning to examine all the weekly liver biopsies by microarray to see whether there are changes in genes related to innate immunity, which may reflect the nonspecific, but universal, immune response elicited by an infectious agent, genes related to mithocondria or other cellular genes. If there is evidence of an infectious agent, we will attempt to to identify the putative agent by molecular techniques.
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