HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995 AASLD ABSTRACTS 389A

1129 IMPROVED DETECTION OF ANTI-ECVANTIBODY BY WESTERN BLOT USING A TRUNCATED GST-CORE FUSION PROTEIN. AP Andonov and HE Jacobsen. Laboratory Centre for Disease Control, Health Canada, Ottawa, Ontario.

The glutathione S-transferase (GST) gene fusion system was used to express a truncated construct encoding amino acids 1-118 within hepatitis C virus (HCV) core (C) protein (l- 191aa). The size of the fusion protein corresponded to the calculated combined molecular mass of the 29 kDa GST protein and the approx. 15 kDa HCV C protein. Antibody reactivity to the recombinant HCV core fusion protein was analysed by Western blotting (WB) in 245 serum samples. The basic perfomance of WB was similar to CHIRON RIBA 3.0;16 RIBA 3.0 reactive samples were all strong positive with HCV C fusion protein (100%) and 42 OUt of 43 RIBA-3 indeterminate samples that reacted only to c22p peptide were strong positive also by WB (97.7%). To evaluate borderline reactivity 30 RIBA 3,0 negative samples in which the c22p strip pattern was scored as +/-were randomly selected. Of these one third (33.3%) were found to be positive by WB with HCV C fusion protein. In addition, 6/36 (16.6%) RIBA 3.0 indeterminate c33c(+), c22p(-) were found to be reactive by WB with HCV C fusion protein. Reactivity to this antigen was also observed in five out of 78 ABBOTT HCV EIA-2.0 negative samples (6.4%). Three of these were patients in a hemodialysis unit in which a HCV outbreak has been reported. These results suggest that the GST-HCVC fusion protein used in Western blot improves detection of anti-HCV core antibodies.

1130 A N T I B I O T I C T H E R A P Y F O R B A C T E R I A L F L O R A IN T H E R E C I P I E N T GUT ATI 'ENUATES L I V E R INJURY F O L L O W - ING O R T H O T O P I C L I V E R TRANSPLANTATION (OLTX) IN RATS M. Arai 2. S. Moehida I. A. Ohno 2 and IC Fuiiwara 1 13rd Dept. of Internal Medicine, Saitama Medical School, Saitama, Zlst Dept. of Internal MediCine, University of Tokyo, Tokyo, Japan

Microcirculatory dis turbance due t o s inusoidal f ibrin deposi t ion provokes hepatic necrosis following OLTX in rats. Such fibrin deposi- tion may develop as a consequence of tissue factor activity increasing in Kupffer ceils after activation. Gut-derived substances might contrib- ute to this activation because the congestion in the intestinal wall due to the occlusion of portal vein flow, an unavoidable process dur ing OLTX, can facilitate the absorption of these substances from the gut to increase tissue factor activity in Kupffer cells. Thus, we investigated the effects of antibiotic therapy for bacterial flora in the recipient gut on liver injury and coagulability in hepatic sinusoids following OLTX in rats. METHODS & RESULTS Recipient rats received an oral daily dose of 1.25 X 106 uni t /kg polymyxirr B sulfate (PMB) for 7 days preceding OLTX. OLTX was performed according to the method of Kamada using nontreated rat liver preserved in UW solution at I°C for 18 hr as a transplant, Experiment 1 Serum concentrations of ALT and TNFct increased markedly in recipients 24 hr following OLTX. Such an increase, however, was significantly attenuated in PMB-treated recipients compared to the control. Experiment 2 Tissue factor activity was significantly increased in Kupffer cells isolated from recipients 1 hr after OLTX compared to those from normal rats. Such an increase was significantly attenuated in PMB-treated recipients compared to the control. Experiment 3 The endotoxin concentration in the portal vein immedia te ly after the ahepatie period of OLTX was s ignif icant ly decreased in PMB-treated recipients Compared to the control In PMB- treated recipients, PMB was not detected in the portal vein during these Pneriods. £LO.N.CLIJSI_OA~ Hypercoagulability in the hepatic sinusoids

duced by gut-derived substances such as endotoxin may cause liver injury after OLTX in rats. Antibiot ic therapy for bacterial flora in the recipient gut would be useful for protection against early graft failure following OLTX.

1131 CLINICAL, BIOLOGICAL AND HISTOLOGICAL FEATURES OF ANTI- HBe POSITIVE AND HBe Ag POSITIVE PATIENTS WITH CHRONIC ACTIVE HEPATITIS B IN PORTUGAL. I Areias: I Pedroto. T Freitas. R Cerqueira. R Teixeira. A M Saraiva. Dept of Gastroenterology, Hospital Geral de Santo Ant6nio, Oporto, Portugal

Some patients with chronic active B hepatitis have an atipical biological form including anti-HBe positive and HBV DNA positive. This form is frequently observed in the Mediterranean area and is due to genomic mutation in the pre-C genome. The AIM of this study was to assess clinical, biological antihistological characteristics of this form in a presumely low endemic area. PATIENTS AND METHODS - Analysis of epitiemiological, clinical, biological and histological features was performed in two groups of patients with a HBV DNA positive chronic B hepatitis according to HBe status. There were 50 patients with anti-HBe positive (group I) and 32 with HBe Ag positive (group II). All patients were prospectively followed during 9 months. RESULTS Table 1. Clinical, biological and histological features of anti-HBe positive and HBe Ag positive patients

Group I (n=50) Group H (n=32) p

Age 44+12 36+10 <0.01 Sex (M/F) 31/19 20/12 NS Acute hepatitis history (%) 20 52 <0.05 Oldness of hepatitis (years) 7 5 <0.05 ALT (UI/L)* 96+10 117+12 NS HBV DNA levels (pg/ml)* 25_+10 146+107 <0.005 Cirrhosis (%) 32 12.5 <0.05

*mean+.SD

CONCLUSIONS - These results suggest that chronic B hepatitis with anti-HBe positive has epidemiological particularities but is also more severe than is classical form.

1132 CRLT-q. J Armendariz-Borunda, C Guron, AR Rinc6n, A Panduro. J Sever end 0 Cam~ollo. Institute of Molecular Biology in Medicine, CUCS-University of Guadalajara, Mexico end VAMC, Memphis, TN.

Kupffer cells undergo a process of activation in the liver of rats treated with CC14~ These cells are-- responsible for a massive production in situ of proinfla~matory end fibrogenic cytokines playing a pivotal role in the development of liver fibrosis. This study had two goals, First: to determinate if entisense DNA oligonucleotides could specifically inhibit TGF-fl synthesis by hepatic cells in vitro, ~ : to determine if entisense DNA can be specifically directed in ~ivo to block Kupffer cells-gene expression. Kupffer ceils were isolated from SD rats livers 48 h after acute CCI 4 intoxication by collagenase/pronase digestion, centrifugation and preferential adherence to glass surfaces. Kupffer cells obtained this way, produced and secreted active TGF-8 abundantly. Cells were incubated overnight in DM~4 supplemented with t 0% FCS. A 23-base phosphorothioate-DNA oligonucleotide (S-Oligo DNA) designed to hibridize to the AUG translation initiation codon of murine TGF-8 mRNA (5'- CCC CGA GGG CGG CAT GGG GGA GG- 3') was synthesized. Media was removed end replaced with DMEM supplemented with 2% lactalbemin hydrolizate end the S-Oligo DNA was added at 0, 0.1 end 10 ~M. Cells were incubated for additional 24 h and then the serum-free supernatans were collected for TGF-8 biological assays (Growth-inhibition of Mv I Lu mink lung epithelial cells). Parallel cultures were treated with an irrelevant oligonucleotide end processed in the same way. The S-01igo DNA specifically inhibited TGF-8 production by Kupffer cells in a dose-dependent fashion as compared with the irrelevant oligo. For the second goal, horseradish peroxidase (HRP) was used. HRP is rich in mannose groups and is taken up by Kupffer cells and endothelial cells in vive in a specific manner. HRP was conjugated with polylysine end an eucaryotic vector containing TGF-8 cDNA in reverse orientation. This complex was injected intravenously in the rats to look for specific uptake by the Kupffer cells. The HRP-poIylysine-cDNA complex was detected inside the sinusoidal cells by specific histological reaction. We propose to use this strategy to regulate gene expression by Kupffer cells in vivo.