April 1995 AASLD Al181 • INTERLEUKIN-6 PLASMA LEVEL IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS AND OTHER CHRONIC LIVER DISEASES F. Szalav ,B. Bfiki, K. Rfisky 1st Dept of Medicine Semmelweis Medical University and Research Laboratory of Diagnosticum Rt. Budapest, Hungary Interleukin-6 plasma level was determined in 119 patients with chronic liver disease (20 primary biliary cirrhosis, 24 CAH and 75 alcoholic cirrhosis) and in 103 healthy controls by ELISA kit of the Diagnosticum Rt. The patients were grouped according to the aetiology and stage of the disease. Elevated interleukin-6 level was found in 48 out of 119 patients with liver disease. The IL-6 level was higher in 4 out of 20 patients with PBC, in 6 out of 24 CAH and in 38 out of 75 patients with alcoholic cirrhosis. The highest level range values were found in patients with alcoholic cirrhosis accompanied by other acute inflammatory process or acute shub of the disease as acute alcoholic hepatitis. There was no correlation between the IL-6 plasma level and the serum bilirubin,AST,ALT.ALP, gamma-GT, albumin, prothrombin time, cholinesterase activity and immunglobulin levels. Follow up investigations proved a significant fluctuation of IL-6 level within the same patient during the course of the disease. The increased IL,6 level may be the result of increased production as well as of decreased elimination by the damaged parenchyma of the liver. This work has been supported by grant ETT T 02 379/93 IN VlVO GENE THERAPY FOR BILIRUBIN-UDP-GLUCURONOSYLTRANS- FERASE DEFICIENCY: AMELIORATION OF JAUNDICE IN GUNN RATS AFTER EXSANGUINEOUS PERFUSIONOF RECOMBINANT RETROVIRUS. K. Tada ~'2, V.R. Prasad2'3, P.J. Bosma4, M. Heards, D. Neufield L2` N. Roy Chowdhury 1'2'6, J. Roy Chowdhury 1"2'6. Dept. of 1Medicine, 2Marion Bessin Liver Research Center, aMicrobiology and Immunology, and eMolcu- lar Genetics, Albert Einstein College of Medicine, Bronx, NY; 4Department of Gastroenterology and Liver Diseases, Academic Medical Center, Amster- dam, The Netherlands; and SLaboratoire Retrovirus et Transfert Genetique, Institut Pasteur, Paris, France. Crigler-Najjar syndrome type I is a potentially lethal genetic disorder characterized by a lack of hepatic bilirubin-UDP-glucuronosyltransferase (B- UGT) activity. Because, liver transplantation is the only definitive treatment for this disease, we have been developing methods for gene therapy. Toward this goat, we constructed a replication-deficient recombinant eco- tropic retrovirus (MFG-S hl3-UGTQ capable of transferring the coding region for human B-UGT isoform 1, the only UGT isoform that significantly con- tributes to bilirubin glucuronidation in human liver. For producing a high- tiler recombinant retrovirus, the ecotropic producer cells (CRE) were cocuF tured with an amphotropic packaging cell line (CRIP). Viral tiler was meas- ured by a specific competitive RT-PCR. Infection of Gunn rat hepatocytes that were conditionally immortalized by transduction with a temperature sensitive mutant SV40 virus T antigen resulted in expression of human B- UGT-1 in 15% of the cells, as determined by immunofluorescence analysis using an isoform-specific anti-peptide antibody• To transfer the gene into the Gunn rat liver in vivo, Gunn rats were subjected to 66% hepatectomy. The portal vein, the hepatic artery and the inferior vena cava above and below the hepatic vein was transiently clamped and the liver was perfused with 50 ml of the viral supernatant containing 8 pg/ml polybrene in 10 min. Following this period the circulation was reestablished. For determination of transduction efficiency of this method, two rats were infused with a lacZ recombinant retrovirus, which resulted in the expression of bacterial B- galactosidase activity in 1.4% cells. The Gunn rats that were perfused with the 13-UGT~-reoombinant virus exhibited a progressive decline of serum bilirubin concentrations by 30% and 41%, respectively, of the initial levels in three weeks. Conclusion: Exsanguineous liver perfusion with a high-titer recombinant retrovirus expressing human B-UGT has resulted in the signif- icant amelioration of jaundice, indicat ng the potentia applicability of this reagent in gene therapy for patients with Crigler-Najjar syndrome type l. RELATIONSHIP BETWEEN ANTIVIRAL ACTIVITY CAUSED BY INTERFERON AND HEPATITIS C VIRUS IN PATIENTS WITH HEPATITIS C. T. Tak0da, T. Kuroki, Y. Moriyoshi, S. Nishiguchi, S. Nakajima, S. Shiomi, K. Kobayashi. Third Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan. Background It has been reported that the viral factors such as HCV genotype and HCVRNA titer are the important factors for determing the effects of interferon (IFN) therapy• However, the antiviral state brought about IFN is mediated by induced enzymes, of which 2',5'-oligoadenylate synthetase (2-5AS) is particularly important, We noticed that the same dose of IFN led to different levels of 2-5AS activity in different patients• Therefore, we studied the relationship between the induction of 2-5AS induced by IFN and the titer in the serum,and genotype of hepatitis C virus (HCV). Subjects and Methods Patients with chronic hepatitis C (CH-C) before therapy were subjected to this study. HCVRNA in the serum was quantitatively measured by the competitive RT-PCR method. The HCV genotype was determined according to the method by Okamoto, et al. The extent of 2-5AS induction, which seems to reflect the response of the individual to IFN, was expressed as the ratio of 2-5AS activity in peripheral blood mononuclear cells (PBMC) in vitro with 1031U/ml IFN-o~ to its activity in untreated PBMC. 2-5AS activity was assayed with an RIA kit (Eiken, Tokyo). In the patients infected with genotype II HCV, 2-5AS was induced less in high titers of HCVRNA than in lower titers (Table 1). In the patients with same level tilers (less than 4 log copies/50gt serum) of infected HCVRNA, 2-5AS induction was higher in genotype II than in genotype Ill (Table 2). < Table 1 > (genotype II) HCVRNA liters (log copies/50~l serum) _<2 2 - 4 4_< 2-5AS induction (fold) 4.0 +2.8 2.7 + 1.5 2.1 + 0.8 a < Table 2 > genotype II genotype III 2-5AS induction (fold~ 3.2 + 1,8 2.1 + 1.1 b __ •" p < 0.02 compared with lowest tiler, ~ p < 0.05. Conelu_sion_s These results means infected titer and genotype of HCV might effect response to IFN in patients with CH-C. • ESTRADIOL-17~-GLUCURONIDE (EzI7G) IS PHYSIOLO- GICALLY EXCRETED INTO BILE BY A CANALICULAR ORGANIC ANION CARRIER FOR BSP, NOT BY P- GLYCOPROTEIN (P-GP). J~L TAKLFx,AWA= R. YAMAZAKI, N. SANO, Y. TAKAMORI, S. TOMITA, K. KITAURA, M. YAMANAKA Department of Medicine, Teikyo University School of Medicine, Tokyo E217G is a cholestatic agent and is considered to be related to the pathogenesis of intrahepatic cholestasis of pregnancy. Recent report suggested that biliary E217G excretion is mediated by the multidrag resistance (MDR) gene product, P-GP, according to the study with canalicular membrane vesicles and an MDR sarcorrm cell line (Cancer Res 53:5382,1993). However, we previously reported that E217G-induced eholestasis did not occur due to the impaired biliary E217G excrelion in EHBR, a hyperbilirubinemic mutant SD rat (SDR) (J Hepatol 17:241, 1993). In the present study, we investigated the mechanism of the biliary excretion of E217G and estradiol (E2) metabolites in rats. Biliary excretion of tracer doses of 13H]E217G and [14C]F~or [3H]taurocholate (C-tau) and l]4C]vinblastine (VLB), a P-GP substrate, intravenously administered as a bolus to bile-drained male SDR or EHBR (270 g) was studied. Biliar~ excretion of radioisotopes is expressed as M±SD of % dose during 60 min. Biliary excretion of E217G (12+2%) and E2 (20-a:5%) in EHBR was markedly delayed than that of E,z 17G (92±3%) and E2 (48±8%) in SDR. Analysis of the bile after [14C]F~injection by TLC-radiochromatoscanner revealed more polar E2 conjugates in SDR than in EHBR. In contrast, the excretion of C-tan and VLB was only slightly delayed in EHBR. Although phenothiazine treatment (5 mg/100g ip for 3 days) to induce the expression of P-GP increased biliary VLB excretion from 14±2% to 18+2%, it did not affect biliary excretion of a tracer dose of [3HIE217G. However, phenothiazine treatment inhibited the cholestasis induced by E217G infused at the rate of 0.075/~mol/min/100g for 20 min (bile flow was increased from 0.7±0.9 to 3.8± 1.8/~l/min/lO0g at 100 rnin) and increased biliary E217G excretion during 100 min from 25~15% to 69-a:21%. BSP (0.2 /zmol/min/10Og) infusion markedly inhibited the biliary excretion of E217G (24+5%) and E2 (23±4%), whereas DBSP at the same infusion rate had no effect (81±4% for E217G and 45±4% for E2). VLB (1 mg/100g ip) had no effect on biliary F-,217Gexcretion. These data indicate that E217G is excreted into bile by a canalicular organic anion carrier for BSP, not for DBSP, under physiological condition, and that P-GP has a role on E217G excretion only with its high-dose load.