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sdonor gender, donor age, donor height, African American donor, and donor DM. Whencontrolling for the year of LT and transplant center in addition to the issues discussed above,it was found that HCV had a signif'ly increased risk of graft failure in both strata of # LTsreceived. The hazard ratio (HR) for the effect of HCV on the 1o LT increased signif'ly overtime (p<.0001). At 1yr: HR=1.55 (95%CI 1.5-1.6), 3yrs: HR=1.82 (95%CI 1.7-2.0), 5yrs:HR=1.96 (95%CI 1.8-2.1) 7yrs: HR=2.06 (95%CI 1.9-2.2). The HR for the effect of HCVon Re-LT did not, however, vary signif'ly over time and was 1.5 (95%CI 1.2-1.8, p<.0001).After 1,512 days, the effect of HCV on 1o LT was signif'ly greater than that on Re-LT (p<.05).CONCLUSIONS: HCV status was found to be associated with worse graft survival for 1o

LT (HR increased over time) and for 1st re-LT after controlling for known covariates associatedw/ graft survival and pts who had Re-LT. The HR increased signif'ly over time for 1o LTfor the effect of HCV on 1o LT and did not vary signif'ly over time for effect of HCV onRe-LT. After 1,512 days, the effect of HCV on 1o LT was signif'ly greater than that on Re-LT. An analysis with a larger proportion of Re-LTs with longer follow-up is recommended.

845

Use of Telaprevir Plus PEG Interferon/Ribavirin for Null Responders Post OLTWith Advanced Fibrosis/Cholestatic Hepatitis CPaul Y. Kwo, Marwan Ghabril, Marco A. Lacerda, Rakesh Vinayek, Jonathan A. Fridell, A.Joseph Tector, Rodrigo M. Vianna

Hepatitis C is the most common indication for OLT worldwide and aggressive recurrenceof hepatitis C remains problematic with low SVR rates when therapy with PEGIFN/RBV(PR) is administered. To date, there is limited data on treatment of G1 HCV infection withDAA therapy in combination with PR and there are substantial drug-drug interactions withtelaprevir(TVR)/calcineurin inhibitors (CNIs). Our AIM was to review our experience withtelaprevir addition to PR in treatment of aggressive hepatitis C in null responders to PRpost OLT. Methods: Adult patients who developed recurrent HCV infection following OLTwith null response to PR for at least 12 weeks (< 2 log reduction) post-OLT received 4week lead-in PEG-IFN alfa-2b (1.0 μg/kg/wk) plus RBV (600-1000 mg/day) followed byaddition of telaprevir 750 q8. All patients who were not on cyclosporine modified (CYA)were converted to CYA from tacrolimus prior to initiation of HCV therapy. On the first dayof TVR, patients received 25 mg CYA with trough levels titrated to 100 ng/ml . HCV RNAmeasured every 4 weeks by Cobas TaqMan HCV Test. Results. Seven patients (3 M, 4 F),mean age 56 years, have been treated thus far, 4 patients have received 12 weeks of TVR afterlead-in. Three were < 1 year post-OLT, 5 had cirrhosis and two bridging fibrosis.Treatmentparamenters are described in table. All patients required ribavirin dose reduction, EPO, ¾GSF, mean packed-red-blood-cell requirement was 10 U per pt during TVR treatment. Onepatient who reached futility at week 4 of TVR therapy has continued PR and has viral levelof HCV RNA <25 IU/ml, undetectable at week 24.Mild-moderate rash noted in all pts with1 patient having perianal pain requiring narcotics. No supra/sub-therapeutic CYA levelsencountered, though 2 patients were hospitalized for anemia and one for decompensatedcirrhosis. Conclusions. Conversion to CYA followed by four week PR lead-in with additionof telaprevir for 12 weeks can lead to significant clearance rates in null responders withadvanced fibrosis though high rates of anemia/RBV dose reduction, growth factor, andtransfusion requirements were noted. CYA Interactions were easily managed by CYA doseadjustment. Twelve week TVR treatment data will be available for 7 patients in 5/2012.HCV RNA, RBV, CYA, Hgb weeks 0-16

846

Is the Primary Immunosuppressive Drug (Cyclosporin a or Tacrolimus)Playing a Role on the Response to Antiviral Treatment for Post-TransplantHCV Recurrence?Vittoria Vero, Senzolo Marco, Luisa Pasulo, Francesca Romana Ponziani, Raffaella Viganò,Maria Marino, Maria F. Donato, Maria Rendina, Pierluigi Toniutto, Matteo Cescon, PatriziaBurra, Lucia Miglioresi, Daniele Di Paolo, Valerio Giannelli, Antonio Gasbarrini, StefanoFagiuoli

Background: HCV-related cirrhosis is the most common indication for liver transplantation.Standard immunosuppression, based on Calcineurin inhibitors (CNI) may also affect HCVreplication and response to antiviral therapy. Aim: to evaluate the impact of CNI on SVR ina population of HCV transplanted patients undergoing antiviral therapy for HCV recurrence.Patients and methods: A multicenter database of 12 Italian Centres was set up to carry ona retrospective analysis of 464 liver transplant recipients, treated for HCV recurrence, from1992 to 2008. Patients were considered eligible for combination interferon plus ribavirin-based therapy according to defined criteria .Antiviral treatment was aimed for 48 weeksregardless of viral genotype (73,9% genotype 1); median follow up was 87± 45 months.Immunosuppressive therapy was based on cyclosporine in 39% of cases, on tacrolimus in56,9%. Results: SVR rate was 34,1%. EOT was significantly higher in the Cyclosporinegroup (64%) compared with the Tacrolimus (54,5%) (p= 0,04): A longer interval betweenOLT and starting of antiviral therapy (32,7 vs 19,2 months), higher daily dose of Ribavirin(659,9 versus 561,9 mg) were associated with virological response in the Cyclosporin group.Acute and chronic rejection rate (p=0,536 and p=0,585 respectively) and pre-treatmentstaging score, were no different between the two groups. No difference in SVR rate and inpatients survival was observed (88% survival in Cyclosporin group vs 87%). At multivariateanalysis Cyclosporine was confirmed as an independent significant predictor of EOT (p=

S-934AASLD Abstracts

0,04) , regardless of viremia, donor and recipient features, genotype, distance from OLT,olt-recurrence interval, fibrosis stage and treatment dose and duration. Conclusions: EOTresponse to antiviral treatment for post-OLT HCV recurrence is significantly higher amongCyclosporin treated recipients, however no differences in SVR and patient survival wasobserved.

926

Bid and Bim are Essential Regulators Involving the Intrinsic Pathway ofApoptosis in Hepatocytes in the Absence of Anti-Apoptotic BCL-2 FamilyProteinsTakahiro Kodama, Hayato Hikita, Tsukasa Kawaguchi, Satoshi Tanaka, HinakoTsunematsu, Kumiko Nishio, Takatoshi Nawa, Minoru Shigekawa, Satoshi Shimizu, WeiLi, Takuya Miyagi, Atsushi Hosui, Tomohide Tatsumi, Tatsuya Kanto, Naoki Hiramatsu,Tetsuo Takehara

Hepatocyte apoptosis via the intrinsic pathway, also known as the mitochondrial pathway,is involved in the pathophysiology of various liver diseases and regulated by a fine balancebetween anti- and pro-apoptotic core Bcl-2 family proteins. We previously reported thathepatocyte-specific disruption of anti-apoptotic Bcl-2 family members, Bcl-xL or Mcl-1,caused spontaneous hepatocyte apoptosis, which was completely prevented in the additionaldeficiency of Bak and Bax, downstream apoptosis executioners (Hikita H, Kodama T, et al.,Hepatology 2009). Meanwhile, BH3-only proteins are known to function as a pro-apoptoticsensor and precede this intrinsic apoptosis, but their actual involvement and requisites forhepatocyte intrinsic apoptosis machinery remained unclear. In the present study, we exam-ined whether hepatocyte apoptosis via the intrinsic pathway can be induced by just a lackof Bcl-xL or/and Mcl-1, or requires the BH3-only proteins In Vivo. Hepatocyte apoptosis inthe absence of Bcl-xL was significantly ameliorated by an additional deficiency of Bid orBim and completely prevented by both as evidenced by TUNEL staining of liver sectionsand serum alanine transaminase (ALT) levels (Bcl-xL-/-, 537 ± 184 IU/L; Bcl-xL-/-Bid-/-, 64± 31 IU/L; Bcl-xL-/-Bim-/-, 114 ± 61 IU/L; Bcl-xL-/-Bid-/-Bim-/-, 27 ± 3 IU/L). In consistentwith these findings, Bid/Bim double knockout mice did not show any hepatocyte apoptosiscompared to Bim knockout mice upon administration of ABT-737 (100 mg/kg), which couldinhibit Bcl-xL (Serum ALT levels: Bim-/-, 83 ± 33 IU/L; Bid-/-Bim-/-, 31 ± 15 IU/L). In Vivoadministration of mcl-1 siRNA decreased Mcl-1 expression in the liver but did not causehepatocyte apoptosis in the Bid/Bim double knockout mice. In addition, Bid/Bim/Bcl-xLtriple knockout mice could survive even upon siRNA-mediated In Vivo knockdown of Mcl-1 in the liver, in sharp contrast to our previous report that inhibition of both Bcl-xL andMcl-1 led to lethal hepatocyte apoptosis In Vivo (Hikita H, Kodama T, et al., Hepatology2010). These findings indicated that the BH3-only proteins, Bid and Bim, are essential forhepatocyte apoptosis via the intrinsic pathway in the absence of anti-apoptotic Bcl-2 familyproteins, suggesting clinical implication that inhibition of these BH3-only proteins may beuseful for preventing unfavorable hepatocyte apoptosis involved in a variety of liver diseases.

927

Antiapoptotic Effect of Insulin on Activated Hepatic Stellate Cells isDependent on AKT and P70s6kCindy X. Cai, Hema Buddha, Amara J. Nidimusili, Vanita T. Talkad, Bruce R. Bacon,Brent A. Tetri

Background: Hyperinsulinemia is a hallmark feature of nonalcoholic steatohepatitis (NASH).Insulin might play a role in NASH-related hepatic fibrosis and cirrhosis. Hepatic stellatecells (HSC) are central to the development and maintenance of fibrosis. We previouslyshowed that insulin has differential profibrogenic effects in the initiation and perpetuationphase of HSC activation via the insulin-activated PI3K-Akt-p70S6K pathway. We investigatedwhether insulin may also be involved in regulating the resolution phase of activated HSC,mainly HSC apoptosis, a potential target for antifibrotic therapy. Aims: To investigate themolecular mechanisms of insulin-mediated antiapoptotic effect on activated HSC. Methods:Primary rat HSCs and the human immortalized HSC cell line, LX-2 were used in this study.Primary HSCs were isolated by sequential pronase and collagenase digestion, separated bydensity gradient centrifugation. Apoptosis was induced by proteasome inhibitor MG132 andtumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Cells were treated withMG132 or TRAIL in the absence or presence of insulin (20 μU/ml, 100 μU/ml, 1 mU/ml)followed by analysis of Annexin V FITC labeling, nuclear fragmentation/condensation, andimmunoblotting for caspase 3, IκBα, phosphorylated Akt and p70S6K. Nuclear translocationof NFκB was visualized by immunofluorescence. Cell proliferation was measured by BrdUincorporation and collagen I was measured by ELISA. Some cells were pretreated with theinsulin receptor tyrosine kinase inhibitor AG1024, PI3K inhibitor LY294002 or the mamma-lian target of rapamycin 1 (mTOR1) inhibitor rapamycin. Results: MG132 and TRAIL inducedapoptosis in both human and rat HSCs, as detected by increased Annexin V labeling, nuclearfragmentation/condensation, and activation of caspase 3. Insulin dose-dependently inhibitedMG132 and TRAIL-induced apoptosis, while inducing phosphorylation of Akt and p70S6K.Insulin-mediated inhibition of MG132-induced HSC apoptosis also involves attenuation ofIκBα and enhanced nuclear translocation of NFκB. Furthermore, insulin potentiated cellproliferation and collagen I secretion. AG1024, LY294002 and rapamycin all inhibitedinsulin-induced cell proliferation, collagen I secretion and inhibition of HSC apoptosis.Conclusions: Insulin is a profibrogenic factor for HSC by enhancing cell proliferation,collagen I secretion and inhibition of apoptosis. One of the anti-apoptotic effects of insulininvolves activation of the NFκB pathway. Importantly, the insulin-mediated antiapoptoticeffect is AKt and p70S6K dependent. Thus, an insulin-mediated PI3K-Akt-p70S6K pathwayis involved not only in the initiation and perpetuation phase but also in the resolutionphase of HSC activation. This suggests potential therapeutic targets for inhibiting hepaticfibrogenesis in NASH.