April 2000 We investigated the role of NS3 on IL-18 production in the elucidation of a primary HCV-specific immune response. NS3 upregulated IL-18 mRNA expression in mature dendritic cells (DC), which constitute the most potent antigen-presenting cells. Furthermore, NS3 increased IL-18 release from premature (maximum antigen-uptake) as well as and mature DCs. In experiments co-culturing autologous CD4+ T helper cells and NS3-primed DCs we mimicked the initiation of a primary immune response. We observed a substantial increase in IL-18 release by NS3-primed DCs. Herein we provide evidence that DCs are a source of IL-18 and we propose that IL-18 derived from NS3-primed DCs might be a major contributor for the elucidation of a Thl immune response against hepatitis C virus. 187 AMINO ACID SEQUENCE OF PUTATIVE DSRNA-DEPENDENT PROTEIN KINASE-EUKARYOTIC INITIATION FACTOR 2A PHOSPHORYLATION HOMOLOGY DOMAIN OF HEPATITIS C VIRUS AND INTERFERON SENSITIVITY. Hideki Watanabe, Kazuyoshi Nagayama, Nobuyuki Enomoto, Masayuki Kurosaki, Yuka Miyasaka, Shin-Han Yu, Naoya Sakamoto, Takaaki Ikeda, Namiki Izumi, Chifumi Sato, Tokyo Med and Dental Univ, Tokyo, Japan; Yokosuka Kyousai Gen Hosp, Yokosuka, Japan; Musashino Red Cross Hosp, Musashino, Japan. BACKGROUNDS AND AIM: Recently, the E2 protein of hepatitis C virus (HCV) was reported to inhibit the kinase activity of the dsRNA- dependent protein kinase (PKR) and to block its inhibitory effect on protein synthesis and cell growth, suggesting that the E2 protein is involved in the mechanism of interferon (IFN) resistance. The E2 protein was shown to interact with PKR through an element homologous to the phosphorylation site of the PKR and eukaryotic protein synthesis initiation factor subunit 2 (eIF2a) (PKR-eIF2aphosphorylation homology domain (PePHD». There- fore, it is possible that the amino acid sequence of PePHD influences IFN sensitivity. The aim of this study was to clarify whether the variation of the amino acid sequence in PePHD affects IFN efficacy or not. PATIENTS AND METHODS: The amino acid sequence of PePHD (aa. 658-670) was determined in randomly selected 75 patients with genotype I b HCV (HCV-I b) infection. All patients received IFN-aor I3therapy(total amount: 480-1060 MU) for six months. Sustained response (SR) was defined as negative serum HCV-RNA and normal serum ALT levels for 6 months after the end ofIFN treatment. HCV-RNA was extracted from pretreatment serum, followed by reverse transcription and PCR amplification. Subse- quently, amino acid sequences of PePHD and the flanking region were determined by direct sequencing. RESULTS: SR was achieved in 23 patients (31%). In 21 of the 23 SR patients (91%) PePHD was identical to that of HCV-J, the prototype of HCV-Ib. The other two SR patients showed one amino acid substitution in PePHD. On the other hand, 48 out of the 52 non-SR patients (92%) PePHD was same as HCV-J. Only four non-SR patients showed amino acid substitutions in PePHD. Among them, three patients had one substitution and the other had three. Therefore, the response rate to IFN therapy was similar between those with the prototype PePHD and PePHD with amino acid substitutions (21/69 (30%) vs. 2/6 (33%), p=0.99 by Fisher's exact test). CONCLUSIONS: Although the E2-PKRleIF2ainteraction through PePHD was demonstrated in an in vitro study, our analysis of clinical samples revealed no relationship between the amino acid sequence of PePHD and IFN sensitivity. PePHD polymorphism was not suggested to play a role in the heterogeneous efficacy of IFN therapy among patients infected with HCV-Ib. 188 LIVER CIRRHOSIS IN CHRONIC HEPATITIS C PATIENTS: AN ASSOCIATION WITH POLYMORPHISMS IN THE TRANSFORM- ING GROWTH FACTOR (TGF)·Bl GENE. Jeff J. Germer, Pedro G. Vidigal, Nizar N. Zein, Mayo Clin, Rochester, MN; Mayo Clin, Rochetser, MN. Background: Hepatic injury results in rapid increase in TGF-131 production within the liver. TGF-131 is profibrotic cytokine that appears to be an important factor in mediating response to hepatic injury. Two polymor- phisms have been identified within the leader sequence of the gene at positions +869 and +915. The substitution at +869 results in the change Leu-e-Pro and may result in disrupted protein transport while the polymorphism at +915 resulting in the change Arg-s-Pro, has been shown to be associated with decreased levels of TGF-I3!. Aim: To evaluate genetic polymorphisms at positions +869 and +915 of the TGF-131 gene and their relationship to cirrhosis in pts with chronic HCV. Method: We studied 33 chronic HCV patients and 37 racially matched controls. All HCV pts had a liver biopsy performed and the degree of fibrosis determined. HCV RNA titers were measured by a commercially available quantitative RT-PCR assay and HCV genotype was determined by direct sequence analysis of the 5 1UTR. The presence of polymorph isms within the leader sequence was determined by PCR amplification of a 275 bp product extending from nucleotide position +691 to +965. Amplifica- tion products were purified and sequenced directly using the ABI 377 DNA Sequencer. Result: Of 33 HCV pts, 18 (54%) were determined to be (TIC) heterozygous and 4 (12%) were found to be (CIC) homozygous at position +869. The prevalence of these substitutions in HeV pts was similar to that of healthy controls (21/37, P= 0.5 and 4/37, P=0.9 respec- tively). Similarly, 6 of 33 (18%) pts were (G/C) heterozygous at position +915 which was similar to the frequency in healthy controls (3137, P=0.2). No (C/C) homozygotes were identified at position +915 in either AASLDA939 group. The results suggest that these specific polymorphisms are not likely to play an important role in the development of chronic HCV after exposure. However, pts who were (TIT) homozygous at position +869 were less likely to have liver cirrhosis than either (CIT) heterozygotes or (C/C) homozygotes (0/11 vs 4122, P=0.03), and more likely to have HCV RNA titers < 2 million copies/mL (5/11 vs 1/18, P=O.03). The association between the polymorphism at position +869 and liver cirrhosis was independent of that between the polymorphism and HCV RNA titer. Conclusion: The polymorphism at position +869 of the TGF-131 gene may be associated with a decreased likelihood for the development of liver cirrhosis in patients with chronic HCV as well as a decreased viral titer. 189 IS THERE A CORRELATION BETWEEN HEPATIC STEATOSIS, FIBROSIS, BODY MASS INDEX AND VIRAL LEVELS IN AMER- ICANS WITH CHRONIC HEPATITIS C? Ann L. Silverman, Stuart C. Gordon, David 1. Ternes, Heather H. Wolak, Mamtha Balasubramaniam, Neal S. Goldstein, William Beaumont Hosp, Royal Oak, MI. A correlation with hepatic steatosis, body mass index (BMI) and fibrosis was described among Australian hepatitis C carriers (Hepatology 1999;29: 1215). Among Italian patients (Hepatology 1999;30:1530), a correlation was found between HCV genotype 3a and steatosis, independent of BMI. It was recently proposed (PNAS 1999;96:12766) that one potential mech- anism for HCV entry into cells was via the LDL receptor, leading to the hypothesis that higher HCV RNA levels might be associated with in- creased hepatic steatosis. We sought to determine a potential association between hepatic steatosis, fibrosis, BMI and viral load in an American cohort of HCV carriers. Methods: We studied the records of 49 untreated nonalcoholic patients with chronic HCV; subjects with high and low viral loads were selected for review. 7149 did not have biopsies done at our institution and 1/49 did not have a recorded weight. The remaining 41 patients were designated H (high viral levels), n=27, (>2.4 x 10 6 copiesl ml; mean, 17.2 x 10 6 ) and L (low viral levels), n=14, «1.5 x 10 6 copieslml; mean, 6.7 x 10 5 ). Quantitative HCV RNA determinations were based on the NGI assay. Steatosis was graded on a scale of 0-3, corre- sponding to none to marked. Fibrosis was scored using the Metavir Fibrosis Scale. Results: There was no difference in steatosis, hepatic fibrosis, or genotype between the Hand L groups. In the total cohort, 5 patients were genotype 3a; 4/5 had little or no (grade 0-1) steatosis, and 1/5 had grade 3 steatosis. Considering the 41 patients as one cohort, we found that there was no correlation between BMI, degree of steatosis, or hepatic fibrosis. Conclusions: In an American cohort of HCV carriers, we found that viral load does not correlate with steatosis, BMI or fibrosis. Overall, there was no association between BMI and fibrosis or steatosis. 190 DO MUTATIONS IN CD81 AFFECT CLINICAL OUTCOME IN PATIENTS WITH CHRONIC HEPATITIS C INFECTION? Simon C. Hellier, Manon Martin-Ginolhac, Rodney E. Phillips, Roger W. Chapman, Paul Klenerman, John Radcliffe Hosp, Oxford, United King- dom. Introduction: Hepatitis C Virus (HCV) causes chronic infection in greater than 70% of those infected. Of these 25% will go on to develop significant liver disease, while others get minimal damage. It is probable that there are host genetic factors that influence clearance of the virus and the rate of liver disease progression in HCV. Recently it has been shown that HCV binds to the major extracellular loop of the transmembrane protein CD8!. Al- though it is unlikely that CD81 is the only cell surface receptor involved in binding of HCV and its entrance into cells, it is possible that functional polymorphisms within the gene may affect disease progression. This is seen in HIV-I where functional polymorphisms in chemokine receptor CCR5, a co-receptor for viral entry, have been shown to affect disease outcome. Our aim was to identify polymorphisms in the major extracellular loop of CD81, assessing correlation with clinically determined phenotypes. Methods: We recruited 21 patients from the Viral Hepatitis clinic in Oxford. 14 were chronically infected with HCV, 8 with mild and 6 with moderate or severe liver disease, as determined by liver histology. The remaining 7 had cleared the virus spontaneously. Human genomic DNA was extracted from whole blood. We performed PCR reactions for the 3 exons that encode the major extracellular loop and the products were cloned and sequenced in the lab using an automated sequencer with fluorescein staining. Results: In Exon 5, 5 patients had sporadic mutations that were not reproduced between patients. 3 mutations induced an amino acid substitution, while 2 were conservative. In Exon 6, no reproducible mutations were detected. In Exon 7, in 2 patients we identified a 3bp deletion and a point mutation that led to an amino acid substitution within the transmembrane region. Conclusion: We found that the major extracel- lular loop of CD81 was well conserved as previously reported. We iden- tified a point mutation and a 3bp deletion in the transmembrane potion of Exon 7 that was reproducible between patients. However there was no clear correlation between this mutation and disease phenotype.