Unit: Necker-Institut Pasteur joint Hepatitis Laboratory

Director: Christian Bréchot, Valérie Thiers

The activities of our laboratory, which includes the National Reference Centre for Hepatitis B and C viruses, focus on implementing of new methodological approaches for the study of hepatitis B and C infections. HBV and HCV are the two principal agents implicated in the development of hepatocellular carcinoma (HCC), in Europe. Working closely with the Inserm 370/Pasteur Joint Research Unit, we are developing an ambitious project, aimed at analysing the gene expression profile (transcriptome and proteome) of microdissected hepatocytes so as to clarify the mechanisms of carcinogenesis in HCV-infected patients. In parallel, the Reference Centre on hepatitis B and C viruses ensures the molecular characterization of specific B and C viral isolates, and investigates the residual risks of transmission of these viruses.

The principal risk factor associated with the onset of HCC is chronic hepatitis resulting from infection with HBV or HCV, although the molecular mechanisms underlying hepatocarcinogenesis are still obscure. The reference techniques for the diagnosis of HCC include serological tumour markers and imaging modalities, but there is an urgent need to identify molecular markers to enable earlier diagnosis and improve the prognosis for HCC. The identification of differentially expressed genes (transcriptome and proteome analysis) during the multi-step process of hepatocarcinogenesis would provide valuable insights into the mechanisms of HCC and the determination of potential markers and drug targets for diagnosis and treatment. The liver is composed of admixtures of numerous cell types and the use of homogenized liver samples for DNA microarrays greatly hampers comparative studies of gene expression. It is therefore essential to obtain pure samples of the cells of interest if we are to acquire meaningful data. Laser-assisted microdissection techniques procure pure populations of hepatocytes from frozen liver sections, and enable the analysis of genome mutations and the development of gene expression programmes on well characterized cells.

1 Study of HCV core gene variants, a potential protein for virus-induced transformation. (R. SOBESKY, V. THIERS).

The core protein of HCV has been reported to fulfil a pleiotropic function in the viral replication cycle, as well as being a component of viral nucleocapsids. Data suggest that the expression of certain HCV proteins, such as core protein, is involved in viral persistence, modulation of the antiviral effect of interferon, cell proliferation and cell viability. The HCV genome exhibits remarkable sequence variation, responsible for viral persistence and resistance to anti-viral therapy. Mutations may accumulate in the core protein during replication of the viral genome which will then modify the biological properties of this protein. In particular, in a few cases of HCV-positive HCC, a difference has been demonstrated between HCV core proteins encoded by sequences isolated from HCC tumour tissues and those from non-tumour counterparts. Moreover, distinct biological effects have been observed for proteins encoded by genomes isolated from tumoral and non-tumoral tissues (Delhem et al, 2001, Oncogene). The aim of our project is thus to extend these observations to a larger number of cases. We aim to characterize full core sequences isolated from microdissected hepatocytes in tumour and non-tumour counterpart tissue from the liver of HCV-infected patients. The impact (if any) of amino acid variations on the secondary structure of the protein will be studied using bioinformatic tools. The phenotype effects of the mutations identified in vivo will be also examined.

2. Transcriptomic analysis of steatotic hepatocytes, isolated by microdissection, during chronic HCV infection (JB TRABUT, R. SOBESKY, V. THIERS)

Macrovesicular steatosis is frequently observed in subjects infected with HCV (around 50% of cases), but not during chronic hepatitis caused by the B virus. Data emerging from the literature suggest that steatosis may, at least in some cases, promote the development of hepatitic fibrosis. The mechanisms underlying its development in HCV infected patients remain uncertain. The aim of our project is to study the molecular mechanisms responsible for the development of steatosis during chronic hepatitis C. Laser microdissection will be performed to obtain homogeneous populations of steatotic and morphologically normal hepatocytes. These will be subjected to a comparative analysis of gene expression utilizing high throughput human micorarrays (Affymetrix). The experimental techniques to obtain high quality RNA from microdissected hepatocytes have been developed and validated by the performance of hybridization on Affymetrix test chips. Liver needle biopsy specimens from patients with chronic HCV infection, with and without steatosis, have been selected (collaboration with the Hepatology unit in Necker Hospital) and are under investigation. (project supported by the French Ministry for Research and Technology, performed in collaboration with the Génopole in Strasbourg).

3. Proteomic expression profiling of microdissected hepatocytes from tumour and non tumour liver tissues. (ARECA network) (A. DOS SANTOS, F. DEMAUGRE,& V. THIERS)

In order to improve the early detection and prognosis of HCC, there is an urgent need to identify molecular markers to detect the disease. The integration of laser-assisted microdissection and proteomic analysis could identify novel protein markers useful for diagnosis and prognosis. Thus, this study will be using laser assisted microdissection to isolate HCC and non-HCC hepatocytes from HCV infected patients, and combine microdissection with two-dimensional electrophoresis (2DE). Protein fingerprinting of matched pairs of HCC and non-HCC tissues will be compared and the differentially expressed proteins identified by mass spectrometry (MALDI-TOF-MS).

We have demonstrated the feasibility of proteomic analysis by achieving identification using mass spectrometry of randomly selected protein spots derived from microdissected cells. (MS performed in collaboration with Inserm U467 and CNRS - ESA 8067). Alternatively, so as to enable a better appraisal of low molecular weight proteins, those recovered will be analyzed using surface-enhanced laser desorption and ionization time of flight (SELDI-TOF) (collaboration with CNRS ESA 8067). (This work is supported by ARC-Inserm)

4. Activities of the National Reference Centre (CNR) for B and C viral hepatitis (F. RIMLINGER, V. THIERS)

The activities of the CNR are closely linked to the research projects being carried out by the laboratory. Molecular data, obtained by phylogenetic analysis, can provide an opportunity to identify the routes of HCV transmission (patient to patient, nosocomial). Detailed analysis of virus nucleotide sequences, in informative viral regions, can provide evidence for a common source of infection in outbreaks of infection or horizontal transmission between pair of individuals. Studies on the nosocomial aspects of hepatitis B and C virus transmission are ongoing, in collaboration with the French Health Surveillance Institute "Institut de Veille Sanitaire". Analyses of the genetic diversity of particular HCV types, in different geographical regions, are performed in collaboration with the HepMed network (EU project, coordinated by B. Larouze) and Pasteur Institute network (PTR project coordinated by P. Mavromara).

Keywords: Molecular epidemiology, HBV; HCV, genetic variability, laser assisted microdissection, HCC, steatosis, transcriptome, proteome

Activity Reports 2003 - Institut Pasteur

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