an isocaloric control diet, were exposed to gut ischemia for 30 rain. followed by 60 min of reperfusion. Intravital videomicroscopy was used to monitor leukocyte recruitment in hepatic microcirculation and the number of nonperfused hepatic sinusoids (NPS). Plasma ALT levels were measured 6 hr aRer the onset of reperfusion. Results: In control rats, gut I/R elicited increases in the number of stationary leukocytes, NPS and plasma ALT levels. In EtOH-fed rats, the gut t/R-induced increases in NPS (control; 22.5_+0.8, EtOH: 11.6+_1.1, %, p<O.Ol) and leukostasis were blunted in the midzonal region (control; 12.6-+0.6, EtOH: 8.0_+0.8, per field, p<O.01), while exaggerated leukostasis was noted in the pericentral region (control; 4.3+_0.8, EtOH: 7.1 -+0.8) and terminal hepatic venules (THV) (control; 4.0_+0.6, EtOH: 13.3_+0.7, per field). Chronic EtOR consumption also enhanced the gut I/R-induced increase in plasma ALT levels (control; 115_+ 12 I U/I, EtOH: 263_+48 IU/I). The exaggerated responses to gut I/R normally seen in EtOH-fed rats, were largely prevented by pretreatment with a blocking anti ICAM-1 monoclonal antibody (leukostasis in the pericentral region; 5.0_+0.7, THV; 5.5_+0.7, per field, ALT levels; 149_+12 IU/I). Conclusions: These results suggest that chronic EtOH consumption enhances the gut I/R induced hepatic microvascular dysfunction in the pericentraf region and THV by an enhanced expression of/CAM-f, which may contribute to the corresponding enhancement of liver (hepatocellular) injury.

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Early Graft Function In Related And Unrelated Living Donor Liver Tram~pleMation (LDLT). Venkataraman Ramachandran, Jeffrey A. Lowell, Surendra Shenoy, Todd K. Howard, Washington Univ, Saint Louis, MO

Graft function in the first week after transplantation is directly dependent on the amount of liver tissue transplanted and the preservation injury sustained by the graft. LDLT provides for the minimum cold preservation time from the healthiest donors. Unrelated donor livers are potentially subject to early rejection. We have analyzed the early graft function after LDLT. Methods: 15 adult patients undergoing LDLT over a 4-yeer period were studied. Donors were extensively evaluated to rule out co-morbid conditions and to define the arterial and billary anatomy. The donor right lobe was used for orthotopic transplantation. Outcome parameters, graft to body weight %(GBW), transaminase levels in the first week, prothrombin time and early rejection episodes were analyzed Results: 8/15 adults had genetically related donors (4 sons, 2 sisters, 1 daughter, 1 brother), 6 had unrelated donors (3 husbands, 4 friends). Two LD LTs did not proceed to completion due to technical reasons and received cadaver transplants. Mean donor age, patient age, Childs-Pugh score, were similar in the related and unrelated donor groups. Mean GBW was 1.10 for the whole group, 1.2 for unrelated and .96 for the related group. In both groups peak AST was seen on the first day (mean 385) and showed excellent resolution by day 7 (mean 87). All patients were extubated by 48 hours with excellent synthetic function (mean protime 15.7 improving to12.6.in 72 hours). 2 patients in each group had empirical treatment for mild rejection in the first month. 3 patients died from sepsis in the first month and another after 11 months from sepsis and secondary biliary cirrhosis. 4/15 (25%) had biliary complications during a mean follow up 15.6 months. Summary: Early graft function is excellent after LDLT with mean GBW of 1.1. Sepsis early post LDLT is the commonest cause for mortality (20%). Billary complications are common. Related and unrelated donor livers show little difference in function or incidence of rejection. Prophylaxes of bacterial and viral infections need to be addressed to improve outcomes.

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Donor Morbidity Associated With Living Donor Liver Transplantation Kimberly L. Beavers, Jeffrey H. Fair, Robert S. Sandier, Roshan Shrestha, Univ of North Carolina at Chapel Hill, Chapel Hill, NC

Background: Since the introduction of living donor liver transplantation (LDLT) in 1989 nearly 2oog LDLT have been successfully performed worldwide. Previous data regarding living donor (LD) complications report morbidity rates of 0-20%. The donor perspective of acceptable complications has not been previously addressed but must be understood to improve quality of care. The aim of this study was to assess the morbidity associated with LOLT from the donor perspective. Methods: We conducted a cross-sectional study of all LD at our transplant center from September 1996 to October 2000. Subjects completed a questionnaire designed specifically to assess the process, consent, surgery, follow-up, and satisfaction of liver dona- tion, as well as the SF-12, a well-validated health assessment tool. Data analysis was performed using STATA software, version 6 (College Station, IX). Results: Of 27 subjects eligible to participate in the study 26 (96%) participated. The donor population included 70% women, mean age 35 (range 18-50); 44% donated to adult recipients. Mean follow-up was 21 months (range 1.5-48). Mean hospital stay was 8 days (range 3-14). Twenty-three percent of donors reported an event they deemed an immediate complication, of which 60% were recorded in the medical record. Delay in return to normal bowel function was the most frequently reported complication; others included pain, brachial plexus injury, blood clot, incisional infection, pleura/effusion, sore throat, foot parastbesias, persistent short term memory loss, chronic fatigue, "fluid between lung and liver", and bile leak. Complications requiring readmission were reported by 23% and include biloma, fever, pancreatltis, Ioculated pleural effusion, emesis, chronic abdominal pain. Thirty-two percent experienced more pain than anticipated, and 35% reported a larger surgical scar than expected. Mean recovery time was 12 weeks (range 1- 52) as reported by donors. Ninety-two percent of donors returned to their pro- donation occupation. There was no statistically significant difference between donor SF-12 scores (52.77 + / - 1.57) and general US population norms (50.12 + / - 9.45, p=0.14). Conclusions: When assessed from the donor perspective, morbidity in the first year following LDLT may be higher than previously thought. A national registry is needed to assess precise short and long-term morbidity and target improved quality of care.

131

Leptin and Hepatic Innate Immu~'~f Shiqi Yang, Huizhi Lin, Johns Hopkins Univ, Baltimore, MO; Robert Schwenk, Ursula Krytch, Walter Reed Army institute for Research, Washington, DC; Anna Mae Diehl, Johns Hopkins Univ, Baltimore, MO

BACKGROUND: Liver macrophages, i.e.,Kupffer cells (KC), have receptors for leptin (ob),a sateity factor that is reduced or inactive in obesity. Leptin modulates hepatic inflammation and wound healing. For example,obese leptin-deficient ob/ob mice are sensitized to endotoxin- mediated hepatitis but protected from cirrhosis. Cytokines produced by KC regulate the function and viability of neighboring stellate cells and lymphocytes, including CD4NKT cells. Stellate cell activation promotes cirrhosis, while CD4NKT cell depletion exacerbates endotoxin- hepatitis. Our PURPOSES were to determine it production of cytokines by ob/ob KC is abnormal and if leptin treatment corrects this or reverses the depletion of CD4NKT cells that exists in oblob livers. METHODS: ob/ob mice received leptin (n = 24) or saline (n = 12) continuously from subcu minipumps for 2 wks. KC or total liver mononuclaar cells were isolated from these mice and from 36 age-, gender-, and strain-matched, saline-treated lean mice. Pooled KC (from 6 mice/group/study x 3 studies) were plated on plastic dishes and incubated overnight in serum-containing medium with or without leptin (500 ng/ml). The next morning KC and medium were harvested before (0) or 1.5 or 6 h after exposure to lipopolysacchafide (LPS)(1 ug/ml). RNAase protection analysis and ELISA were done to measure cytokine mRNAs and proteins. The percentage of CD4NKT cells in the liver mononuclear cell preparations were quantified by FACS. RESULTS: Compared to KC from lean mice,KC from saline-treated ob/ ob mice expressed lower levels of several TNF~-inducible cytokines (ILl,&IL6,1L10,IL15) basal/y, but more TNFc~, IL1/3, IL6, ILIO and IL15 after LPS treatment. KC that were exposed to leptin in vitro had increased expression of TNFcr and some TNF~regulated genes (ILlO, /L6, ILlp)paselly, but attenuated induction of TNF~ and TNF-related cytokines after LPS. In v/vo legtin treatment also inhibited LPS induction of several TNF-inducible cytokines (IL6, IL10,IL15). In addition, it decreased basal TNF,z expression. Saline-treated ob/ob mice had 10 fold fewer hepatic CD4NKr cells than lean mice and after leptin treatment, hepatic CD4NKT cells increased almost 3 fold. CONCLUSIONS: Leptin-deficient mice have abnormalities of the hepatic innate immune system, including basal CD4NKT cell depletion and enhanced KC production of TNFot and related cytokines after LPS challenge. Leptin tends to correct both abnormalities but the mechanisms involved may be indirect given the discordances between leptin's in vivo and in vitro effects.

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Molecular Mechanism For AaR-ApoptoRc Actions Of A Non-Toxic Heat Shock Protein 70 Inducer, Geranylgeronylacetone, In Hepato~-yles Kazuhito Rokutan, Shizuo Ikeyama, Kenji Kusumoto, Sch of Medicine, Ufliv of Tokushima, Tokushima Japan; Shigetada Teshima, Sch of Medicine, Univ of Tokushima, Teshima Japan; Tomoko Kawai, Tsukasa Kawahara, Takeshi Nikawa, Sch of Medicine, Univ of Tokushima, Tokushima Japan; Kelya Nakamura, Kyoto Rainess Hosp, Kyoto Japan

BACKGROUND: An acyclic polyisoprenoid, geranylgeranylacetone (GGA), has been shown to induce high resistance against hepatocyte injury under various stressful conditions both in vivo and in vitro. This compound is now recognized as a non-toxic heat shock protein (HSP) 70 inducer. In this study, we examined the mechnisms by which GGA activates the transcription of the HSP7O gene in hepatocytes and how GGA exerts anti-apoptotic actions on the cells. METHODS: Primary cultures of rat hepatocytes were used in this study. Activation of heat shock transcription factor 1 (HSF1) was monitored by phosphorylation and nuclear translocation of HSF1, and DNA-binding activity of HSF1 was measured by gel mobility shift assay with the heat shock element oligonucleotide. The level of HSP70 mRNA was measured by Northern blotting. The levels of HSF1, HSP90, HSP70, and HSP27 were measured by Western blot analysis with respective antibodies. The activation of apoptotic pathways was assessed by monitoring phosphorylation of c-Jun N-terminal kinases (JNKs), formation of apoptosome, activation of caspases 3 and 9. Apoptotic cells were detected by DNA ladder formation and nuclear morphology. RESULTS: Compared with the action on gastric mucosal cells, GGA itself was not a potent inducer for HSP7O in hepatocytes; treatment of hepatocytes with 1 p.M GGA incresed the levels of HSP70 and HsPgO about f .5-fold. However, treatment of hepatocytes with 1 ~ GGA for 2 h primed hepatocytes for enhanced induction of HSP70, but not HSP9O and HSP27, in response to subsequent exposure to 100 mM ethanol or 0.5 mM hydrogen peroxide. The nuclear translocation and phosphorylation of HSF1, heat shock element-binding of HSF1, HSPTO mRNA expression, and HSP70 accumulation were markedly up-regulated, suggesting that GGA directly acts on HSFI. This enhanced HSP70 induction blocked the activation of JNKs 1 and 2, recruitment of caspase 9 to the Apaf-1 apoptosome, activation of caspase 3, and DNA ladder formation, resulting in the suppression of hepatocyte apoptosis caused by the insults. CONCLUSIONS: GGA appeared to interfere multiple apoptotic pathways mainly by priming hepatocytes for enhanced HSP70 expression rather than by directly stimulat- ing HSP70 induction. Our results suggest that GGA may have a potential benefit for treatment of ~iver diseases, such as alcoholic and Jschemla-reperfusion injuries as well as liver transplant- ation.

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Cox-2 Inhibits Fas-modiatnd Apoptosis of Cholangiocarcinoma Cells Ugochukwu C. Nzeako, Sloven F. Bronk, Gregory J. Gores, Mayo Clin, Rochester, MN

Fas expression has been shown to prevent cholangiocarcinoma tumor growth in xenogratts. These data suggest Fas regulates the progression of cholangiocarcinoma by inducing apoptosis of malignant cells. However, many human cholangiocarcinomas express Fas suggesting these cancers have developed mechanisms to inhibit Fas signaJing. Cyclooxygenaee-2 (Cox-2) is a potential candidate for inhibition of Fas-signaling because it has been shown to inhibit apop- tosis. Thus, our AIM was to test the HYPOTHESIS that cox-2 expression inhibits Fas-mediated apoptosis. METHODS: Two human cholangiocarcinoma cell lines which differ only in their expression of cox-2 were used for these studies: 1) KMBC cells which do not express cox- 2; and ii) KMBC-C cells which constitutively express cox-2. For some experiments, the

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