HEPATOLOGY VoK 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 4 3 9 A

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PREVALENCE OF BCL-2 RECOMBINATION IN HEPATITIS C VIRUS- RELATED MIXED CRYOGLOBULINEMIA WITH OR WITHOUT COM- PLICATING B-CELL LYMPI-IOMA. Patrizio Caini, Francesca Giannelli, Uni- wxsity of Florence, Florence Italy; Clodoveo Ferri, University of Pisa, Pisa Italy; Carlo Giannini, Monica Monti, Laura Gragnani, Elena Di Pietro, Giorgio La Villa, Giacomo Laffi, Paolo Gentilini, Anna L Zignego, University of Flo- rence, Florence Italy

Background. Hepatitis C virus (HCV) infection is strictly associated with mixed cryoglobulinemia, a benign B-cell lymphoproliferative disorder possibly evolving to a frank lymphoma, bcl-2 recombination [t(14;18) translocation], the cytogenetic hallmark of follicular non-Hodgkin lymphoma, has been shown with increased prevalence in HCV infected patients. Aim. To evaluate the prevalence of bcl-2 recombination in a series of HCV-related mixed cryo- globulinemia patients in comparison to patients with chronic HCV infection without mixed cryoglobulinemia. Methods and patients. 37 HCV-related mixed cryoglobulinemia and 101 type C chronic hepatitis without mixed cryo- globulinemia were consecutively recruited. We evaluated clinico-serological characteristics, liver biopsy, bcl-2 recombination and Bcl-2 expression with Bd-2/Bax ratio in total peripheral blood mononuclear cells or in cell subpopu- lations by polymerase chain reaction and western blot analysis, respectively. Sequence analysis of bcl-2/JH junctions was performed in positive samples. Results. bcl-2 recombination was observed in 28/37 (76%) mixed cryoglobu- linemia (65% in type III, and 85% in type II, including 3/4 patients with complicating lymphoma), and in 38/101 (37.6%) chronic hepatitis patients (p<0.001). Sequence analysis showed specificity and, in 2 cases followed over time, clonal expansion of translocated cells. Overexpression of Bcl-2 oncopro- tein with high Bcl-2/Bax ratio were observed in translocated B-cell samples. Conclusions. bcl-2 recombination may represent a cytogenetie marker in type tI mixed cryoglobulinemia as well as a pathogenetic factor involved in the donal B-cell proliferation surrounding this disorder.

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CONTROL OF HEPATITIS C DURING CHRONIC INFECTION IS NOT MEDIATED BY C D 4 + T CELLS. Robert Thimme, The Scripps Research Institute, La Jolla, CA;Jens Bukh, National Institutes of Health, Bethesda, MD; Carola Steiger, The Scripps Research Institute, La Jolla, CA; John Ghrayeb, Centocor, Malveru, PA; Keith A Reimann, Harvard Medical School, Boston, MA; Robert H Purcell, National Institutes of Health, Bethesda, MD; Francis V Chisari, The Scripps Research Institute, La Jolla, CA

We reported last year that viral clearance in HCV-infected chimpanzees was associated with a high peak titer of viral RNA (~ 105-107 GE/ml), vigorous peripheral CD4+ and CD8+ T cell responses, rapid T cell homing to the liver, and an intrahepatic type I cytokine profile, all of which disappeared after viral clearance. In contrast, we showed that the virus persisted at initial peak levels (~104 GE/ml) in chimps whose peripheral HCV-specific T cells failed to accumulate in the liver and, therefore, didn't produce IFNg in the intrahepatic compartment. We have recently studied 2 additional HCV-infected chimps that displayed a third type of outcome. Initially they developed high HCV RNA titers (~106 GE/ml) that they rapidly controlled to ~102 - 103 GE/ml, but eventually they became chronically infected maintaining this low virus titer indefinitely. Like the animals that cleared the virus, these chimps developed an early, strong intrahepatic T cell response and intrahepatic IFNg, but they became chronically infected and the responses persisted along with virus for the duration of the experiment. These results suggest that the intrahepatic inflammatory response was able to control the level of HCV infection in these animals but not eradicate it. Together with our earlier observations, they dem- onstrate that viral load can be either controlled or uncontrolled during chronic infection depending on the vigor of the intrahepatic immune response. This implies the existence of at least two pathways to viral persistence in HCV infection. To determine whether CD4+ or CD8+ cells control HCV at low levels in these two chronically infected animals, we are monitoring the effect of monoclonal antibody-mediated depletion of each T cell subset on viremia. Preliminary results of the anti-CD4 experiment indicate that CD4-depletion was highly efficient (decreasing from 75 to 0.9% of peripheral T cells and total depletion of intrahepatic CD4+ T cells), rapid (occurring in <1 wk), and prolonged (lasting for> lmonth). Importantly, no significant change in vire- mia was observed during this phase, suggesting that CD4+ T cells don't con- trol HCV during persistent infection, even though they are present in the liver. intrahepatic virus-specific CD8+ T cells and antibodies were still detectable during the phase of CD4+ T cell depletion, suggesting that they may control HCV in this setting. CD8-depletion experiments to test this hypothesis will begin soon, and the results will be presented.

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CONSTRAINTS ON HEPATITIS C VIRUS (HCV) NS3 SERINE PROTEASE GENETIC HETEROGENEITY AND EVOLUTION. Muriel Soler, Hopital Henri Mondor, Creteil France; FranCois Penin, IBCP, CNRS-UMR 5086, Lyon France; Stuart C Ray, Johns Hopkins University, Baltimore, MD; Daniel Dhumeaux, Jean-Michel Pawlotsky, Hopital Henri Mondor, Creteil France

The NS3 serine protease catalyses cleavage of nonstructural viral proteins at four downstream sites. The catalytic triad (His-1083, Asp-1107 and Ser-1165) is conserved among all reported HCV sequences as well as in Flaviviruses and Pestiviruses. It is an obvious target for future specifc HCV inhibitors. How- ever, natural NS3 genetic heterogeneity might predispose to viral resistance to snch inhibitors related through selection of resistant variants. RESULTS: We studied the NS3 protease and its evolution in 5 patients infected with HCV genotype lb during treatment with interferon (IFN) alpha and/or ribavirin. A total of 420 NS3 serine protease clones were generated at various time points during follow-up. The NS3 protein had a typical quasispecies distribution, characterized by the coexistence of several distinct but closely related se- qnences in every patient at each time point. The impact of these amino acid variations on NS3 protease structure-function relationships will be presented based on the NS3 protease 3D structure. Overall, genetic and phylogenetic analyses showed: (i) low genetic diversity as compared to regions subjected to strong pressure toward change and tolerant to amino acid substitutions, such as the hypervariable region of envelope glycoprotein E2; (ii) minimal evolution over time in spite of strong pressure toward change related to IFN administra- tion; (iii) use of the VarPlot program to calculate nonsynonymous and synon- yraous distance (dN and dS, respectively) with a large (20-30 codon) sliding window did not reveal regions of strong positive selection (dN-dS > 0). To- gether, these findings suggested that the NS3 protein is highly constrained, probably related to both its structure and function, such as interactions with viral proteins, host proteins, or ions necessary to its function (Zn). However, when a small window was applied with VarPlot, individual sites with positive dN-dS suggesting positive selection were identified. Interestingly, two of 5 study subjects showed positive selection at position 1074, an anchor residue for the HLA A2-restricted immunodominant 1073-1081 epitope. CONCLU- SIONS: NS3 protease, like other HCV proteins, is naturally variable. Amino acid mutations might influence NS3 protease structure and function, but they appear to be strongly constrained by the need for maintaining NS3 function. Host immune responses could however exert some positive pressure toward change, the structural and functional consequences of which are currently being studied.

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HEPATITIS C IMPAIRS CELL CYCLE PROGRESSION - IMPLICATIONS FOR HEPATIC REGENERATION. Arleen E Marshall, Simon M Rushbrook, Lesley S Morris, Alex Freeman, Kate Bird, Ian S Scott, Nicholas Coleman, Graeme J Alexander, University of Cambridge, Cambridge United Kingdom

Background: The mechanisms by which hepatitis C virus (HCV) causes chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC) are unknown. HCV core protein and nonstruetural protein 5A (NS5A) are known to affect cell cycle regulation in vitro. Progression through the cell cycle is dependent on serial activation of cycfn dependent kinases (edks) in association with cydins. Inhibition of cdks occurs via p21, p27, p57 and INK4 proteins (p16, p15, pl8, p19) and prevents cell cycle progression. Ectopic expression of p21 can cause G1 or G2 arrest. Methods: Using immunohistochemistry, we have examined liver biopsies from 50 patients with chronic HCV at all stages of disease for the presence of cell cycle markers: minichromosome maintainance protein 2 (MCM2), a sensitive and specific marker expressed throughout the cell cycle, eycfn DI in G1 phase, cyclin A in S, cyclin B1 in G2 and phospho- histone H3 in mitosis, and p21. HCC and colonic carcinoma served as positive controls and normal liver as a negative control. Results: Throughout the spec- trum of disease, MCM2 expression was increased and correlated with the stage of fibrosis (stage 0, 3-14%; stage 5, 5-40%, p<0.0001, highest level 59%). Cyclin D1 expression was similar to and did not exceed MCM2. In contrast, hepatocyte expression of markers for the later stages of the cell cycle was very low: 0.4-1.6% expressed cycfn A, d0.1% expressed cydin B1 and 0.36-0.5% expressed phosphohistone H3. p21 expression was present in up to 60% of hepatocytes and was closely associated with MCM2 expression. Conclusions: These data indicate that a significant proportion of hepatocytes have entered the cell cycle, but are arrested at G1/S. Changes are seen in alt stages of disease, therefore it is likely to be a consequence of viral infection. As p21 and MCM2 expression are linked, HCV may induce p21. HCV may therefore impair he- patic regeneration.