April 1995 AASLD A l113

THE HUMORAL IMMUNE RESPONSE TO HCV CORRELATES WITH THE OUTCOME FROM INTERFERON TREATMENT H. F. LrhL C. Elst¢, G. Michel*, K.-H. Meyer zum Biisehenfelde, G. Crerken. I. Dept. Int. Med., Univ. Malnz, Malnz, * Abbott Eurp. Res. Dee., Wiesbaden, Germany

To assess whether the quantitative and qualitative anti-HCV production correlates with the outcome from antiviral treatment and may serve as predictive factor. Methods: 18 selected viremic patients with histologically proven chronic active hepatitis C were treated with 3x3 to 3x6 miu weekly interferon-or (IFN) and were followed-up after treatment for 6 to 60 months. Sera were collected at different times andtitered quantitatively with the Matrix EIA (Abbott) for antibodies to structural and non-structural HCV antigens. In addition, circulating immune complexes bound to Raji cells were measured quantitatively by flow cytometry analysis. Results: 9/18 patients showed a sustained complete response to IFN therapy (CR) with normalised transaminases and HCV-RNA elimination for at least six months In these patients the humored immtm¢ response against c22-3, c33c and el00 antigens were down-regulatad simultaneously to decreased virus expression. 6/18 patients were non-responders with persisting HCV-RNA and elevated transaminases after treatment (NR). In these patients the humoral immune response persisted quantitativdy. Additional 3 Ni l showed relapsing disease activity after withdrawal of IFN. In one of these anti-c22-3 production occurred simultaneously, whereas in the two others anti-c22-3 production p r e c ~ l e d the rising transaminases for months. Before treatment, 8/9 CR showed a predominant immune response to c33e antigen whereas in the 9 NR c22-3 was the most immunogeneic HCV antigen. Furtherrnore, all the 18 patients showed significant circulating immune complexes onc~ in their course that did not correlate with disease activity or therapy outcome. Conclusions: The persistent HCV specific humoral immune response, i. e. to HCV core antigen, correlated with disease activity, viremia and negative outcome from therapy. Some patients showed increased anfi-HCV antibody production prior to disease reactivation. In CR NS3-derived c33c antigen is the most immunng¢~ic. The hurnoral immune response was down-regulated when HCV-RNA was diminated. Furthermore, immune complexes did not correlate with the clinical course. Therefore, anti-HCV antibodies may be pathoganetically relevant and may serve as response predictive factors.

O EVIDI~NCE AGAINST HCV BEING A CYTOPATHIC VIRUS. A ~ Lok. TM Chart. M Ilrdea: R Sanchez-Pescador. Tulane Univ & VA Medical Center, New Orleans, LA; Univ of Hong Kong, Hong Kong; and Chiron Corp, Emeryville, CA.

The p redominan t mechanism of HCV induced hver injury remains unclear. The aims of this s tudy were to de termine (1) if there is a correlat ion between serum HCV RNA and ALT levels, and (2) the effect of immunosuppress ive therapy on serum HCV RNA a n d ALT levels in pa t ients with chronic HCV infection.

Serial serum samples from 8 immunocompeten t (IC) and 8 renal t ransplan t (RT) pat ients were tested for quant i ta t ive HCV RNA levels by bDNA assay. A total of 147 samples collected over a 1-4.5 y r follow-up per iod were tested. Correlation between serum HCV RNA and ALT levels was de te rmined by analyzing serial values from each pa t ien t using the Pearson test.

5 paO.ents received interferon (IFN) therapy, 2 had susta ined biochemical response. A d i rec t correlat ion between serum HCV RNA and ALT levels was observed dur ing biochemical response to t rea tment as well as post - t rea tment relapse (p values 0.05- 0.0003). No corre la t ion between serum HCV RNA and ALT levels was found dur ing the pre- t rea tment obervat ion per iod in these 5 pat ients and in the other 11 Untreated pat ients (p values 0.97- 0.06).

Among the 11 pat ients who did not receive IFN therapy, the mean serum HCV RNA level in the RT pat ients was 4-fold h igher (1.4 x 107+4.0 x 106vs 3.6 x 106+1.3 x 106 Eq/ml) but the mean ALT level Was 40% lower than the IC pat ients (67+18 vs 116±18 U/l). The RT pat ients had greater var ia t ion in HCV RNA levels (mean: 3 2± 10 vs 14±6 fold changes) than the IC pa t i en t s dur ing the course of follow-up.

~ummarv : Except dur ing IFN treatment , there was no correlat ion between sermn HCV RNA and ALT levels in both immunocompeten t and immunosuppressed pat ients with chronic HCV infection. The RT pat ients had higher serum HCV RNA but lower ALT levels compared to the IC ones. Our da ta argue against HCV being a cyopathic virus.

@CYCLOSPORIN-A (CsA) DOES NOT INHIBIT TRANSCYTOTIC VESICULAR TRANSPORT: A MORPHOMETR1C ANALYSIS L. Lora, *E. Mazzon, C. Carlotto, *C. Milanesi, R. Naccarato, D. Martines. Gastroenterology Department and *Biology Department of Padua University - Italy -

Inhibition of hepatoeytary vesicular transport, induced by CsA, has been observed in a bile-fistula rat model. The aim of this study was to verify the influence of CsA (l oM) on transcytotic vesicular pathways in perfused rat liver. This was achieved measunng the biliary excretion of horseradish peroxydase (HRP) and examming HRP- labeled vesicles in the perisinusoidal (PS) and pericanabeular (PC) areas, using ultrastructural morphometric analysis. Male Sprague Dawley rat livers were perfused with Krebs Henseleit buffer (albumin 1%, RBC 20% and ammoacid mixture). Taurodehydrocholate (l Mmol/min) was coinfused into portal vein. 1 MM of CsA; dissolved in Creanophor-EL (CsA livers) or the vehicle alone (CEL livers) was added to the medium. Two perfusion protocols were used in the study. First, to examine the pattern ofHRP bili~y excretion, HRP (25 mg) was given as a 1-mm pulse, under single pass conditions, after 30 min of recirculating perfusion. Bile samples were collected to measure HRP output. Second, to visualize HRP in hepatocytes and to study "rapid" and "late" transcytotic vesicular pathways, a 1 -mm pulse of high dose of HRP (500 and 200 rag, respectively) was given. Two and 18 min after a single-pass perfusion the livers were fixed with 2.5% glutaraldehyde-0.8% paraformaldehyde in 0.1 mM cacodilate buffer. The total pericanalicular area, the area and the number of HRP-containing structures were quantified morphometrieally in liver samples by Ibas Kontrun seamantematic analyzer on electron micrographs obtained by a Zeiss 10B transmission electron microscope at 80 kV. RESULTS The appearance of the second biliary HRP peak (transcellular vesicular pathway) was observed at 15 min in CsA and also in CEL livers. The area under the second peak of biliary HRP excretion curve was similar in CsA and CEL livers (399±13.8 vs 38.6±159 ng expressed as Mean±SE of 5-6 observations). Morphometric analysis confirmed that CsA perfusion did not affect neither percent area (Tab) or density (data not shown) of HRP labeled vesicles, both in pericanalicular (PC) and in perisinusoidal (PS) area at 2 min (rapid pathway) as well as atl8 nun (late pathway). Transcellular vesicular pathways

2 nun after 500-mg HRP 18 min after 200-rag HRP

n = 3 PC % area PS % area PC % area PS % area

CEL Livers 0.47 4- 0.35 0.59 ± 0.03 139 ± 0.21 0.09 4- 0.05

CsA Livers 0.34±0.09 0.65±0.16 1.27±0.44 0.10±0.05 CONCLUSIONS: These results indicate that CsA does not inhibit transcytotic vesicle pathways and therefore cholestasis is not related to transcenular vesicular transport alterations.

OBiochemical Character izat ion and Molecular Regulat ion of a New Human Hepat ic Quinone Reductase. H. Lou, M. Villalvazo and A~ Stolz. USC School of Medicine, Los Angeles, CA 90033.

Quinones are potentially noxious compounds that may undergo one electron reduction by microsomal NADH oxidoreductases forming highly reactive semiquinone metabolites. Two electron reduction generating stable hydroquinones catalyzed by NADPH Quinone Oxidoreductase (NQO), also known as DT diaphorase, has been implicated as the major detoxification route for these toxic quinones. We now report that the cytosolic Human Hepatic Bile Acid Binder (HBAB) which we identified by its high affinity binding can catalyze quinone reduction and is induced in both human hepatoma (HepG2) and colon carcinoma (HT29) cell lines by agents capable of inducing NQO. Methods: Recombinant expressed HBAB was expressed and purified as described (JBC 2688:10448). HepG2 and HT29 cells were grown in DMEM and 10% Fetal Calf Serum (FCS) and treated for 24 hours with Ethacrynic Acid (EA), a known inducer of NQO. HBAB steady state mRNA was determined by northern blot analysis and hybridization quantitated by Ambesis Beta scanner. Relative transcriptional rate for HBAB was compared to the non-inducible geae, ~-actin by nuclear runoff technique. Results: Recombinant HBAB reduced the following quinone substrates listed with consumption of NADPH(uM)/min/mg protein: 1,4 beazoquinone (100uM) - 0.86, Phenanthrene-9,10-quinone (25uM) - 0.56, Adriamycin (200uM) - 0.10. We observed no NADPH consumption in the absence of substrate or enzyme. Redox cycling with molecular oxygen was found with Phenanthrene-9,10-quinone as evidence by greater consumption of NADPH then mass of substrate. This redox cycling was not inhibited by Superoxide Dismutase (SOD). Dicumoral, a recognized inhibitor of NQO was also able to inhibit HBAB reductase activity by greater then 50%. We then evaluated regulation of HBAB in cell lines in response to EA. EA caused a dose and time dependent induction of HBAB steady state mRNA levels in HepG2 and HT29 cells line (4 and 16 fold respectively) associated with a comparable increase in HBAB mass as determined by western blotting. Using nuclear runoff in HepG2 and HT29 cell lines, a 4 and 3.5 fold increase in transcriptional rates was observed as compared to ~-actin. This induction was inhibited by Cycloheximide indicating that protein synthesis is required for geoe induction. No effect on HBAB steady state mRNA in absence of inducer was found. Conclusion: HBAB is a multifunctional protein capable of binding bile acids and reducing quinones. HBAB is induced by EA and requires protein synthesis. Differential regulation in these two cell lines will be beneficial for defining cis acting elements governing gene expression.