A l 1 9 2 AASLD ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4

• BILE SALTS D E T E R M I N E N U C L E A T I O N B E H A V I O U R O F C H O L E S T E R O L C R Y S T A L S IN M O D E L BILE. K.J. van Erpecum, P. Portincasa, M. Gadellaa, B.J.M. van de Heijning, G.P. vanBnrge-Hnnegou- wen, W. Renooij t. Depts of Gastroenterology and Surgery t University Hospi- tal Utrecht, The Netherlands.

Nucleation of cholesterol crystals in bile occurs from vesicles with a high cholesterol-phospholipid (chol/pl) ratio. Besides the well-km)wn triclinic (tricl) crystals, various crystal shapes were recently discovered such as arcs and needles (AN), spirals (S), tubules (T) and aggregates (AG). It is not known what determines crystal shape. We compared nucleation from isolated vesicles (chol/pl ratio 1.6) after addition of various bile salts (final B.S. cone. 30 m M ) w i t h nucleation from whole model biles (TLCo 10g/dl, CSI 1.4) containing the corresponding bile salt. Nucleation was scored during 10 days using a semiquantitative score (1+ to 4 + ) and polarizing microscopy. As shown by ultracentrifugati0n whole biles containing the hydrophobic bile Salt taurodeoxycholate (TDC) had a significantly higher vesicolar chol/pl ratio (3.14-0.2 vs 1.64-0.1) compared to model biles containing the more hydrophilic taurocholate (TC). Various crystal forms (AN, S, T, Tricl, AG) appeared within 1-3 days in TDC-whole biles, with rapid progression to 4 + score. In contrast, only AN, Tricl and AG appeared in TC-whole bile after 6-8 days with s l o w progression to 1+ . Results after addition of bile salts to isolated vesicles were remarkably similar. Integrated 10-day score (max. 40) for TDC-whole bile and after addition of TDC to vesicles 'were: AN 21.3 and 17.3; S 1 and 1; T 18.3 and ~18; Tricl 24.6 and 19.5; AG 19.3 and 19.5 resp. Values for TC were: AN 2.7 and 1.3; Tricl 3.2 and 9.7; AG 0.7 and 1.6 resp. The hydrophilic bile salts tauroursodeoxycholic acid (TUDC) and taurohyodeoxycholic acid (THDC) never induced nucleation from vesicles, whereas corresponding whole biles nucleated only sporadically at prolonged observation. When THDC-TDC or TUDC-TDC mixtures (ratio I/2 and 2/1) were added to vesicles, nucleation was inhibited progressively at increasing proportion THDC and TUDC. TUDC-whole biles had a low vesicular chol/pl ratio: 0.94-0.1. Conclusions: bile salts dictate nucleation behaviour of cholesterol crystals in model bile. Potential mechanisms include modulation of vesicular chol/pl ratio or direct bile salt-vesicle interactions.

• INFLUENCE OF DIETARY CHOLATE AND URSODEOXYCHOLATE ON LIVER PATHOLOGY IN MDR2 KNOCKOUT MICE. C.M.J. van Nieuwkerkl'2, R. Ottenhoff 1, M. v. Wijland 1, K.P Dingemans 2, M.A. van den Bergh Weerman 2, G.N.j. Tytgat ~, G.J.A. Offerhaus 2, A.K. Groen ~, R.P.J. Oude Elferink ~. Depts. of Gastroenterology & Hepatology t and Pathology 2, Academic Medical Center, Amsterdam, The Netherlands

The mouse mdr2 gene encodes a P-glycoprotein that is mainly expressed in the canalicular membrane of the hepatocyte. Mice in which this gene has been inactivated lack biliary phospholipid secretion and develop non-suppurative cholangitis with portal inflammation and ductular proliferation. (Mauad nt al, Am J Patho11994;145:1237-45). Aim: to compare the effect of dietary cholate (CA) and ursodeoxycholate (UDCA) on the development of liverpathology in mdr2 knockout mice. Methods: 63 mice homozygous ( + / + ) or (4-) for the mdr2 gene were fed with either purified control diet or diet supplemented with CA (0.1%) or UDCA (0.5%) for 3, 6 or 22 weeks after weaning. Subsequently, bile was collected after cannulation of the gallbladder for a period of 2 hrs after which the liver was resected for histological analysis. Analysis of liver histology included eosinophilic bodies, portal inflammation, ductular proliferation, and mitotic activity and was semi-quantitatively scored from 0-3 for each parameter. Results: The bile salt pool of ( + / + ) and (-/-) mice on control diet consisted mainly of muricholate (MCA; 70-+ 14%) and cholate conjugates (CA; 29_+ 14%). This changed to 87_+ 10% CA and 81 _+7% UDCA On CA- and UDCA-containing diets resp., the remainder being MCA. The CA diet induced mild liver pathology in ( + / + ) mice (median 1.5; range 1-2), hut caused death in 3 of the 12 (-/-) mice and pronounced pathology in the surviving nine (median 9; range 9-9). Serum alkaline phosphatase did not change in ( + / + ) but increased 14- fold in (-/-) mice after 22 weeks on a CA diet. Dietary UDCA had no effect on liver histology and serum enzymes in ( + / + ) but decreased liverpathology significantly in (-/-) mice (7;5-9 on control diet --* 3.5;2-4 on UDCA diet). Both duetular proliferation and portal inflammation decreased and this decrease was strongest at 22 weeks. Improvement of alkaline phosphatase on UDCA diet was less obvious. Conclusions: The endogenous bile salt pool of mice is hydrophilic which is probably the reason why pathology in (-/-) mice starts mildly and progresses slowly. Replacement of MCA by CA induces severe liver pathology. Addition of UDCA, which slightly increases the hydrophilicity of the bile salt pool, improved liver histology and particularly decreased portal inflammation.

• EVIDENCE THAT THE DURATION OF IFN THERAPY SHOULD BE TAILORED TO THE VIROLOGIC RESPONSE TO TREATMENT USING MORE THAN ONE IN VIVO RESERVOIR OF INFECTION David Van Thiel, Lois Friedlander, Tarek Hassanein, Ahmet Gurakar, Harlan Wright, P. J. Molloy, R. J. Kania and H. Faruki, Oklahoma Transplant Institute, Baptist Medical Center of Oklahoma, Oklahoma City, Oklahoma; West Penn Hospital, Pittsburgh, PA; and, Central Blood Bank of Pittsburgh, Pittsburgh, PA

With IFN givan at a dose of 3 MU TIW as treatment for chronic HCV, an initial 20-40% response rate, defined as a normal serum ALT level with 50% of the responders relapsing within 3-6 months is reported. The ideal dose, duration and end point for treating HCV related liver disease with IFN is however not known. As a result the present study was performed. A group of 31 HCV positive individuals were treated with 5 MU of IFN TIW for 6 months and then had their therapy terminated. A second group consisting of 14 was treated with 5 MU IFN administered daily but had their therapy discontinued only if their serum was HCV-RNA negative by PCR on 3 consecutive month!y studies, at which time a liver biopsy for HCV-RNA was obtained and was also found to be negative for HCV-RNA. Responses were classified using 2 different criteria: one was using the serum ALT level at the end of therapy and after 6 months of fo|lbwup; the second was defined as being HCV-RNA negative by PCR in serum and #BMC's 6 months after discontinuing IFN therapy. The entry WBC (xl03) and plate[~t count (x103), serum ALT and liver biopsy results for the 2 groups were as follows: 5.64-0.3 vs. 6.7+0.5; 1924-11 vs. 2304-20; 2584-32 vs. 1724-32 and CPH 11, CAH 11 and CAH + cirrhosis 9 vs. CPH I0, CAH 3 and CAH + cirrhosis I respectiyely. The mean duration of therapy for the 2 groups was 6 months vs. 9.3 4- 1.7 months (range 4.5-18 months) while the mean total dose of IFN administered was 360 ~ U vs 1335 MU (range 560-2520; median 1300). Of the 30 subjects treated for 6 months, an ,~LT response (normal ALT) Was achieved in 13 (43%)i After an addf~ional 6 months of follow-up, only 2 (6.6%) still had a normal ALT. None were vireoiogic responders. Of the 14 subjects treated until their serum PBMC and liver biopsies were negative for HCV-RNA, all 14 (100%) could be defined as ALT resi~onders at the end of therapy. One month after discontinuing therapy, 50% relapsed and were HCV-RNA positive in semm. The other 7 have been HCV-RNA negative for 8-12 months. All 7 relapsers in this second group have been retreated with IFN an, d become HCV-RNA negative immediately with the reinitiation of IFN therapy.

It can be concluded from this study that: 1) IFN treatment should be continued without respect to the absolute length of time of tbe~'apy but instead should be continued until viral clearance occurs in all 3 in vivo reservoirs over an extended period of time; and, 2) current recommendations for IFN treatment are inadequate.

LIVER TRANSPLANTATION FOR ISOLATED PRIMARY BILIARY CIRRHOSIS 0PBC) AND PRIMARY BILIARY CIRRHOSIS WITH ASSOCIATED AUTOIMMUNE DISEASE (THE PBC OVERLAP SYNDROME): IS THERE A DI~'I~RENCE IN PROGNOSIS? D. H. Van Thiel, T. Hassanein, A. Gusakar, H. I. Wright, B. Nour, E. Katz. Oklahoma Transplant Institute, Baptist Medical Canter of Oklahoma, Oklahoma City, Oklahoma 73112.

From a large data bank of liver transplant recipients available to the investigators, 163 individuals with classical PBC without any associated markers of autoimmune diseasewere identified. From this same data base, individuals with PBC with one or more serologic markers of autoimmune disease consisting of ANA positivity (n=21), anti-smooth muscle antibody positivity (n=33), HLAB8 positive (n=19), HLA Dr3 or Dr4 positive (n=51), any combination of 2 of these putative autoimnnme markers (n =23) and any combination of 3 of these autoimmune markers (n=7) were identified as patients with the PBC-CAH overlap syndrome. Kaplan Meier life table survival curves were calculated from the data available for each group.

The laboratory data at the time of transplant were similar (not significantly different) in the various study groups for platelet count, total bilirubin level, alkaline phusphatase, ALT, AST, liver volume by CT and UNOS score. All patients were treated post-trausplantation with either a CyA or FK 506 based immunosuppmssinn regimen.

Despite the degree of liver disease manifested by these patients prior m OLTx, the survival of each group was excellent and did not vary significantly ranging from a high of 97% for the Dr3, 4 positive group and a low of 93% for the B8 positive group at 5 years foUowing OLTx. Based upon this data, it can be concluded that: 1) the prognosis for susvival following liver transplantation for PBC is excellent; 2) markers of autoimmune disease in patients with PBC do not affect the long-term survival of individuals transplanted for PBC.