Differential regulation of interleukin-18 in chronic HCV infection

HEPATOLOGYVol. 34, NO. 4, Pt. 2, 2001

AASLD ABSTRACTS

555A

1531

1532

TRANSCRIPTIONALACTIVATION BY HEPATITIS C VIRUS (HCV) NON STRUCTURAL 5A (NS5A) PROTEIN IN TUMORAL AND NON TUMORAL LIVER TISSUES OF PATIENTS WITH HCV-RELATED HEPATOCELLULAR CARCINOMA. Muriel Pellerin, Hopital Henri Mondor, Creteil France; lVlarie-Claude Yakicier, Inserm U434, Paris France; Francois Penin, IBCP, CNRS UMR 5086, Lyon France; Daniel Dhumeaux, Hopital Henri Mondor, Creteil France; Pierre Laurent-Puig, Jessica Zucman-Rossi, INSERM U434, Paris France; Jean-Michel Pawlotsky, Hopital Henri Mondor, Creteil France

DIFFERENTIAL REGULATION OF INTERLEUKIN-18 IN CHRONIC HCV INFECTION. Petra Traber, Department of Gastroenterology, University Hospital Mannheim, Mannheim Germany; Karin Rossol-Haseroth, Department of Gastroenterology, University Hospital Mannheim, Heidelberg Germany; Birgit Kallinowski, Department of Gastroenterology, University Hospital Heidelberg, Heidelberg Germany; Tobias Manigold, Ulrich Boecker, Jingsan Chen, Manfred V Singer, Siegbert Rossol, Department of Gastroenterology, University Hospital Mannheim, Mannheim Germany

NSSA lacking its 146 N-terminal amino acids and fused to a DNA-binding domain strongly activates transcription in yeast and mammalian cells. Recent data suggested that transcriptional activation by a cleaved form of NS5A could take place in the nucleus of infected hepatocytes in vivo. HCV exists within its host as pools of genetically distinct but closely related variants referred to as quasispecies. We have previously shown that NS5A transcriptional activation is conserved, sequence-dependent and quasispecies-regulated. METHODS: In order to determine whether or not NS5A transcriptional activation could play a role in HCV-associated hepatocarcinogenesis, we compared transcriptional activation by NS5A quasispecies variants isolated from tumoral and non-tumoral liver, respectively, in two patients with chronic hepatitis C (genotype ].b) and hepatocellular carcinoma. NS5A quasispecies were genetically characterized in both compartments. A total of 120 NS5A clones were generated and each different NS5A variant sequence was transfected into yeast to express NS5A fused to GAL4 DNA-binding domain. Transcriptional activation was measured in yeast using beta-galactosidase reporter gene expression under the control of GAL4 binding sites. RESULTS: NS5A had a quasispecies distribution in both tumoral and non tumoral liver tissues. Extensive genetic and phylogenetic analyses showed no compartmentalization of NSSA quasispecies sequences in tumoral and non tumoral tissues, respectively. The levels of transcriptional activation significantly differed among the different NS5A quasispecies variants, and the differences were related to differences in the charges of the residues. Overall, no functional difference was seen between tumoral and non-tumoral quasispecies variants, respectively. CONCLUSIONS: HCV quasispecies do not compartmentalize in tumoral and non tumoral liver tissues, respectively. The lack of functional compartmentalization of NS5A transcriptional activation suggests that this viral protein function does not play a major role in late caminogenetic events. These results do not rule out a role for NS5A transcriptional activation in earlier events involved in triggering the carcinogenetic process.

Chronic hepatitis C virus (HCV) infection is characterised by differences in T helper (TH) i and 2 cytokines and a predominant TH1 response (IFN-gamma) was shown to contribute to a sucessful treatment with a consecutive HCV eradication but also to a gradual hepatic injury. IL-18, a recently discovered proinflammatory cytokine produced in monocytic ceils (Kupffer ceils, macrophages) potentially induces IFN-gamma and shares further immune functions with IL-12. The aim of the current sudy was to analyze IL-18 synthesis in different compartments of HCV infected patients and to determine the correlation with antiviral strategies. 54 patients with chronic hepatitis C (histology of chronic hepatitis; HCV-RNA +tive, GT1 = 20, GT2 = 19, GT3 = 6; 29 male, 25 female, age 24-67 years) were included in the study and compared to 72 healthy controls. IL-18, IL-12, IFN-gamma were determined in sera (all patients), peripheral blood cell culture (PBMC; n = 26, stimulated with LPS or SAC) and liver specimens (n = 28) by ELISA or mRNA specific RT-PCR. Patients received antiviral therapy (IFN monotherapy or IFN + Ribavirin) and were grouped as sustained responder (SR; n = 12), non-responder (NR; n = 27) or relapsing patients (RP; n = 15). Serum levels of IL-18 in HCV-RNA + tive patients were enhanced compared to healthy controls with no difference according to genotype, viremia or transaminases. Sustained responder showed IL-18 levels comparable to controls, whereas non-responder and patients with relapse demonstrated the highest values (p<0.001). IL-18 measurement before treatment was not correlated with a prognostic value for individual patients. Konstitutive or stimulated expression of IL-18 in isolated PBMC in vitro showed no differences for patients vs controls or for any of the treatment groups. In contrast, hepatic IL-18 mRNA was significantly induced in patients with chronic HCV infection (p<0.05) and correlates with the severity of liver injury showing maximum expression in patients with HCV associated liver cirrhosis(p<0.01). In conclusion the current study demonstrates that induction of the proinflammatory cytokine IL-18 in chronic HCV infection is mostly hepatic and reflect local bystander cell activity.

1533

1534

DOES THYROID DYSFUNCTION IN CHRONIC HEPATITIS C PATIENTS INFLUENCE TREATMENT OUTCOMES?. Raksha Jain, University of Texas MedicaI School-Houston, Houston, TX; Nyingi Kemmer, University of Texas Medical Branch-Galveston, Galveston, TX; Mihir Patel, AIice Chuang, Victor Ankoma-Sey, University of Texas Medical School-Houston, Houston, TX

IMMUNOLOGIC-SERUM MANIFESTATIONS OF CHRONIC HEPATITIS C. Clementi Carlo, Italian Association Viral Hepatitis prevention and CareCOPEV Lazio-Italy, Rome Italy; Lecce Rosina, Dept of Infectious Diseses-Univ La Sapienza, Rome Italy; Genderini Maria Pia, Dept Terapia Medical-Univ La Sapienza, Rome Italy; Soccorsi Fabrizio, Ital Ass Viral Hepatitis Prevention and Care- Copev Lazio, Rome Italy

Chronic hepatitis C virus infection is frequently associated with autoimmune thyroid disease. Therapy with interferon either results in de novo induction of autoimmunity or exacerbation of pre-existing quiescent autoimmune thyroid disease. The impact of thyroid dysfunction on the outcome of interferon-based therapy has not been reported. Aim: To determine if thyroid dysfunction either pre-treatment or during treatment with interferon/ribavarin influences the outcome of therapy in patients with chronic hepatitis C viral infection. Methods: We retrospectively reviewed demographic and laboratory data (ALT, AST, BiIirubin, alkaline phosphate, HCV RNA level and genotype, TSH, T3/T4 and Free thyroxine index) from the medical records of patients diagnosed with well compensated chronic hepatitis C infection who had completed combination therapy ofinterferon-a2b and ribavarin at the Texas Liver Center, University of Texas Health Science Center, Houston between September 1999 and March 200i. Thyroid dysfunction was defined as an abnormal TSH and T3/T4. Cases were compared to age and body mass index matched controls. Treatment outcome was defined as, sustained virological and biochemical response 6 months post therapy. The Fischer's exact test for qualitative data and Student's T-test for quantitative data were used to compare clinical and laboratory variables between the two groups of patients (a p-value < 0.05 was considered statistically significant). SAS 6.12 for window NT was used to perform analysees. Results: Our study population consisted of 40 patients. 28 (Gp A-controls) had normal thyroid function tests (pre/during therapy) and 12 (Gp B-cases) had thyroid dysfunction (we/during therapy). There was no difference in ethnicity, baseline ALT, AST, total bilirubin, alkaline phosphate, HCV RNA level and genotype between both groups. There was a statistically significant difference in prevalence of female gender between cases and controls (36% in Gp A vs. 58% in Gp B, p < 0.05). A sustained virological and biochemical response was seen in 7 controls (Gp A-25 %) and in 3 cases (Gp B-25%) (p = NS). Conclusion: Although thyroid dysfunction is commonly seen in patients with chronic hepatitis C infection, it does not seem to influence the outcome of therapy with interferon and ribavarin. A prospective trial will be required to assess the long-term outcome and natural history of chronic hepatitis C patients with autoimmune thyroid dysfunction.

Background: Screening serological autoimmunity precisely, before subjecting patients infected by Chronic Hepetitis C to a-IFN treatment is actually an usual procedure. This is necessary since immunity biochemical alterations are observed in 25-30% of patients: these are sometimes indistinguishable from those observed in autoimmune hepatitis. The presence of these modifications are mostly interpreted as epiphenomena associated with the infective disease and for the majority of cases they are in a low concentration and are represented by autoantibodies not organ-specific and species-specific without an}, pathogenic relevance. Aim: Aim of this work is, above all, to evidence the autoantibodies presence in relation to histological damage and to viral genotype and eventually to considerate ce-IFN therapy with/without RBV in those subjects. Patients and methods: not organ specific autoantibodies (aAN,aAM,aMS), anticardiolipin (aCL) and antiplatelets (aPL), in addition to reumathoid factor (RF) and Cryoglobuline have been researched in 77 patients of a medium average of 47,5 years (range 26-47), infected by HCV and positive to a biochemical and histologic diagnosis. Viral genotypes have been searched too and patients have been parted into groups with reference to histology in conformity with Knodell: 1 steatosis; 9 PCH (IK I-3/0); 31 ACH1 (IK 4-7/0); 26ACH2 (1K 8-12/1-3); 9AC. Results: various autoantibodies presence, all in a low concentration, has been related to sex and the presence of autoimmune serological evidences prevailed among women even if without a statistical value p=ns(Chi-Sqnares).Percentages are variable from a minimum of 3,8% aAM to a maximum of 51,6% RF ; other parametres present intermediate values. By relating serological autoimmune modifications to histological results, it comes out that higher percentages are obtained in conditions of heavy damage: RF is positive in 20,7% of ACH2 cases, in 11,6% of the AC cases, in 14% of the ACH1 cases and in 3,2% of the PCH. Other parametres reveal lower percentages but follow that condition: a higher incidence in subjeers with advanced damage. Correlation to genotype is not statistically relevant. Conclusions: Chronical liver infections, as in chronical hepatitis HCV correlated, involve a continuous exposure of hepatocytes to damaging noxa. This produces a remarkable lymphocytic infiltrate with the presence of substances able to activate lymphocytic clones (PBA-PolycionalB cell Activators) that permit autoantibodies organo-specific to appear without any clinic evidence, but that sometimes can cause anatomo-clinic alterations as vasculitis, membranoproliferative glomerulonephrites etc. in subjects predisposed. Lymphocytic infiltrate presence around vessels, directly connected with sinusoides and therefore with endothelial cells, caused indeed an initial angiogenic stimulation and fibroblastic proliferation and then, in a long period, connective tissue formations and deposits. Inflammatory micro-ambient, which is peculiar to chronical hepatitis, and even more cirrhosis conditions are determining factors of hepatocarcinoma development (HCC). HCC development is certainly a long lasting process during which numerous co-oncogenic evems accumulate and are all necessary to complete the muhifactor and muhifocal processes that cause hepatocytic transformation. As a result, the presence of immunologic serum evidences at a low concentration in chronic hepatitis HCV do not exclude the possibility of a a-IFN therapy for its anti-angiogenetic characteristics.