Considerable progress has been made in the control and treatment of chronic viral hepatitis, but the success is still limited and further progress will also depend on a more thorough knowledge of the mechanisms of pathogenesis. Since joining the Laboratory of Infectious Diseases early this year, I have continued studies on the mechanisms of pathogenesis of chronic viral hepatitis, especially chronic hepatitis C, shifting the focus of research to the molecular level. Presently, it is not possible to predict which HCV-infected individuals will sustain more dire outcomes and what are the factors that determine the rate and speed of disease progression. Although the determinants of disease severity are not yet well defined, a major determinant of the different rate of disease progression is the speed of liver fibrosis progression. Progressive liver fibrosis is the main cause of organ failure in chronic liver diseases of any etiology. Moreover, the association of HCV, apparently a non-oncogenic virus, with the development of HCC has redirected the attention to the role of inflammation and fibrosis in the pathogenesis of this tumor. Cirrhosis, the end-stage consequence of hepatic fibrosis, has generally been considered irreversible. However, recent evidence, including a prospective 20 years-long-term study from our group, has demonstrated that hepatic fibrosis and even cirrhosis may be reversible, thus challenging a longstanding dogma and paving the way for developing antifibrotic therapies. Although the development of cirrhosis in chronic hepatitis C may take decades, in chronic hepatitis D it may develop within 15 months from the onset of acute hepatitis. The biological reasons for such a """"""""fulminant"""""""" fibrosis course are unknown, although they underline the variability in the natural history of liver fibrosis. ? ? 1. Molecular analysis of the determinants of liver fibrosis progression. Progressive liver fibrosis is the main cause of organ failure in chronic liver diseases of any etiology. The major goal of this project is to study the molecular mechanisms that determine the pace of progression of liver fibrosis or, conversely, regression of liver fibrosis. Although age at infection has been shown in almost all published studies to have an important impact on progression of chronic hepatitis C, which is more rapid in the elderly, the mechanisms responsible for the influence of age on fibrosis progression are not known. In an attempt to define the role of age in fibrosis progression, we have studied, by microarray analysis, the gene expression profiles of liver obtained from individuals with a normal liver to evaluate whether comparison of gene expression profiles among individuals grouped according to age (less than 20 years, 21-30, 31-40, 41-50, and over 51) could reveal different patterns. Interestingly, we have found significant differences in gene expression profiles according to age, and we are now investigating whether specific genes associated with innate or adaptive immune responses, increased fibrinogenesis, or decreased fibrinolysis are modulated according to age. In parallel, we are studying by microarray a series of patients selected according to their different rate of liver fibrosis progression associated with chronic hepatitis C, as well as with other forms of chronic liver disease of viral or non-viral etiology that exhibit a significant difference in the course of hepatic fibrosis ranging from a non-progressive to a """"""""fulminant"""""""" fibrosis. We have detected differences in global gene expression profiles of liver tissue from patients with different stages of liver fibrosis. We are now dissecting the global response to see whether differences in gene expression profiles might involve genes related to cell-matrix interactions, cellular immune responses, or matrix degradation. The results of this analysis might uncover patterns of disease in which hundreds of genes are jointly regulated. ? 2. Molecular analysis of the determinants of liver fibrosis regression. We are very interested in studying the molecular mechanisms of regression of liver fibrosis.We know almost nothing about the genetic determinants of fibrosis regression. Taking advantage of a unique series of patients with chronic viral hepatitis who responded to alpha-interferon therapy with different degrees of regression of liver fibrosis, we have started to analyze serial gene expression profiles of liver tissue from patients with regression of liver fibrosis from the most advanced stage of fibrosis (stage 4 or cirrhosis) to stage 0, which corresponds to the absence of liver fibrosis, to define the molecular characteristics of fibrosis regression and to test the predictive value of gene expression profiles. Studies in animal models suggest that more prolonged injury leads to increasingly thickened fibrotic septae, with enhanced cross-linking.These septae become insoluble and may be more resistant to proteolysis by metalloproteinases, limiting a complete regression. Understanding this issue is of major clinical relevance because, presently,in humans it is not known whether cirrhosis reaches a threshold after which it becomes irreversible.In parallel, we are also performing serial assessments of specific cytokine/chemokine levels in plasma and liver tissue. ? 3. Molecular pathogenesis of hepatocellular carcinoma ? Chronic infection with HCV is a major risk factor for the development of HCC, a tumor that represents a major global health problem, being the fifth most common cancer. Its prevalence has markedly increased in several developed countries, including the U.S. where the incidence of HCC doubled between 1975 and 1998,with the highest increase (25%) between 1996 and 1998. Recent data suggest that HCV infection accounts for most of the increasing incidence of HCC in the U.S. Given the current prevalence of HCV infection among persons 30 to 50 years of age, estimates of the burden of HCC suggest that its incidence and mortality rates are likely to double over the next 10-20 years. ? HCC is associated with multiple risk factors. Cirrhosis represents the highest predisposing factor since in 80% of the cases HCC arises from cirrhotic livers. Among HCV-infected persons, once cirrhosis is established, HCC develops at an annual rate of 1% to 4%.Several lines of evidence suggest that hepatocarcinogenesis begins in preneoplastic lesions, such as macroregenerative nodules and low-grade dysplastic or high-grade dysplastic nodules, which occur with the highest frequency in chronic liver diseases most strongly associated with the development of HCC. These observations suggest a multi-step carcinogenesis model, as postulated for many other human cancers. However, the molecular mechanisms of hepatocarcinogenesis and the molecular genetics of HCC are still undefined. The most extensive studies have been performed in fully developed HCC, whereas our knowledge of the preneoplastic lesions is still very limited.? Since the presence of cirrhosis is the strongest risk factor for the development of HCC, it will be critical to determine which genes are involved in the progression of HCC and to identify gene markers capable of discriminating cancer or pre-cancer from non-cancer lesions.For this purpose, we are performing gene expression profiles of carefully selected patient livers explanted from patients undergoing liver transplantation for end-stage liver disease, including HCC. Patients with both viral and non-viral end-stage liver disease are being studied. Understanding the genetic basis of the earliest changes in hepatocellular carcinogenesis is fundamental in order to obtain a comprehensive picture of HCC progression from its precursors.In this respect, gene expression profiling of carefully selected patient material represents at present the most powerful technique for addressing these issue
