key: cord-023033-tgt69ir6 authors: nan title: Poster Session (pp. 78A–178A) date: 2006-02-10 journal: Hepatology DOI: 10.1002/hep.1840380503 sha: doc_id: 23033 cord_uid: tgt69ir6 nan , ALA-60 (n=4), TYR-77 (N=2), GLY-42 ( n = l ) and LYS-89 ( n = l ) . The mean time from onset of symptoms to diagnosis was 3.4 years (0-10) and patients were listed for OLT an average of 10.8 months (0-60) after diagnosis. The mean time from listing to OLT was 8.2 months (1-18). Five patients died after OLT, during a mean follow-up of 3.5 years (4 months -6 years). One-year patient survival was 100% and threeyear patient survival was 92%. One patient required retransplantation for hepatic artery thrombosis. Neurologic symptoms were the initial clinical manifestation in the majority of patients (7/12), followed by cardiopulmonary (4112) and gastrointestinal symptoms (1112). Most patients experienced multiple symptoms. Subjective evolution of symptoms as assessed by chart review demonstrated that symptoms referable to amyloidosis worsened after OLT in seven patients, and improved or stabilized in five. Following OLT, neuropathy symptoms improved in four patients, worsened in seven patients and were unchanged in one patient. Pre and post-OLT nerve conduction velocities were available for 5 patients and the post-OLT studies showed progression of disease in 3, improvement in 1 and were unchanged in 1 patient. Prior to OLT, all twelve patients had an increased IVST on echocardiogram. Post OLT cardiac symptoms improved in six patients (five of whom also had cardiac transplantation), worsened in three patients, and were unchanged in three patients (one of whom also had cardiac transplantation BACKGR0UND :Accurate delineation of the scope and magnitude of peri-operative donor risk is necessary to better allow for informed consent, to maximize the potential for donor safety, for comparative outcome analysis and ultimately to serve as a key determinant of the utility of adult-to-adult living donor liver transplantation (AALDLT). However, at present, there is lack of uniformity regarding what constitutes a complication in this setting. Moreover, a system to stratify adverse events with respect to their life altering (quantity or quality) impact is lacking. AIMS: 1) to define a graded, inclusive classification schema for both early (E) and late (L) adverse operation-related events in live liver donors a n d 2) to apply this system to a retrospective review of events in individuals undergoing partial hepatectomy for live liver donation at our center. Two patients (6.5%) suffered cut-surface bile leaks and one (3.2%) a right colon injury (Grade 3E complications). One patient (3.2%) developed a transient bilateral ulnar neuropathy (Grade 2E). Two patients (6.5%) were readmitted within 30 days of operation for nausea and dyspnea respectively (Grade 1E). One patient underwent repair of an incisional hernia 6 months post donation (Grade 2L). One patient suffered positioning-related brachial plexopathy (Grade 3L). CONCLUSIONS: The definition and adoption of a graded, scale-based system of operation-related adverse events in live liver donors will allow for an inclusive, consistent and universally applicable method to collect, analyze and report donor complications. All AALDLT programs must be encouraged to fully review and report their donor related morbidity, ideally through the creation of a national donor registry Initiation of antiviral therapy in the early post-transplant period, prior to overt evidence of HCV recurrence (i.e. preemptive therapy) may enhance rates of HCV eradication. Aims: To determine the efficacy and tolerability of preemptive IFN versus IFNlRBV in anti-HCV positive LT patients. Methods: Consecutive and eligible LT recipients from a single center were enrolled. The goal was to initiate treatment within 6 wks of LT. Patients were randomized to IFN or IFN plus RBV (400mg daily X 2wks, then 800mg daily X 2 wks, then 1.0-1.2g daily based upon body weight 75 kg). Induction therapy with daily IFN was used for the first 8 wks (1.5MU daily X 2wks, then 3MU daily X 6 wks) followed by 3 MU TIW (N=39) or peg-IFN 1.5 uglkglwk (N=10) for 40 wks. Key inclusion criteria were stable clinical status, Cr45,OOO and WBC>3.0. Dose reductions for side effects, especially cytopenias were standardized. Growth factors were given for neutropenia (ANC<1000) and anemia (Hgb<9.0gldL) beginning 7l2001. Virological (VR) and biochemical responses (BR) were evaluated at end-of-treatment (ET) and 6 mos post-treatment (sustained virological (SVR) and biochemical (SBR) responses (table below) were associated with response. Histological disease was mild in the majority of patients at treatment end (78% stage 0 fibrosis and 72% grade 1 or less necroinflammatory activity). Conclusions: Preemptive antiviral therapy was applicable to -60% of transplanted patients. Biochemical responses were frequent but SVRs uncommon. Treatment discontinuation and lowering of RBV doses due to side effects likely reduced SVRs and may have limited our ability to detect differences between treatment groups. The only predictor of SVR was a negative qHCVRNA pre-treatment, which implies that patients with low VL early post-LT may be the best candidates for preemptive therapy. Compared to historical reports, the severity of histological disease was very mild at 1-year post-LT in the majority of treated patients, suggesting preemptive antiviral therapy may provide important histological benefits. The cadaveric organ shortage has led to the development of alternative strategies for organ transplantation. Living donor liver transplant (LDLT) is one strategy that has offered many individuals transplantation before they die or become too sick for transplant.Chronic hepatitis C (HCV) is the leading indication for liver transplantation. Preliminary results from small single center studies suggest that recurrent HCV is more common after LDLT compared to cadaveric transplant with concern that graft survival may be lower after LDLT. &: To compare patient and graft survival in HCV recipients who undergo LDLT to cadaveric liver transplant recipients. Methods: We analyzed the United Network for Organ Sharing (UNOS) liver transplant database from January, 1999 -December, 2002. Inclusion criteria included liver transplant recipients 218 years old transplanted from 1999-2002 with the transplant diagnosis of chronic hepatitis C. Exclusion criteria included subjects who were hepatitis B surface antigen positive, history of prior organ transplant, concurrent kidney or heart transplant. The logrank test was used to compare survival between the two groups. Results: From 1999-2002 6.6% of adult liver transplants were LDLT. 279 LDLT recipients and 3,955 cadaveric recipients transplanted for end stage liver disease from HCV were identified. The results are shown in the Table: LDLT ( A greater proportion of LDLT recipients were female and LDLT recipients were less ill at the time of transplant.1 and 3 year patient and graft survival were not significantly different between the 2 groups, (p=O.11). If only the pre-MELD era is considered (01l99 thru 2/02) 1 year graft survival for LDLT (n=240) and CLT (n=3322) are 78% and 83%, respectively, p=O.lO. Conclusions: Our analysis demonstrates that short-term patient and graft survival are equivalent in LDLT and cadaveric recipients with HCV suggesting recurrent hepatitis C does not adversely affect survival over this time period. LDLT recipients are less ill at transplantation which did not confer a short-term survival advantage. Continued follow-up should determine the impact of less advanced liver disease at the time of transplant and recurrent hepatitis C after transplant on long-term graft and patient survival in LDLT recipients. Disclosures: Introduction: Histologic injury due to recurrent HCV has been reported in up to 90% of HCV infected patients who undergo liver transplantation with a cadaveric graft. However, the natural history of HCV following living donor liver transplantation (LDLT) is not as well described. Anecdotal evidence suggests that HCV recurrence may be more severe in LDLT recipients. We hypothesize that post-operative liver regeneration in the partial graft procured from living donors may facilitate HCV replication, or increase the vulnerability of hepatocytes to infection. Methods: We performed a retrospective analysis comparing outcomes, and the incidence, timing, and severity of histologic recurrence of HCV following transplantation in patients who underwent living donor liver transplant compared to recipients of cadaveric organs. Between 12/98 and 3/02,68 HCV infected adult patients (age > 18 years old) underwent liver transplantation for HCV associated cirrhosis. 45 patients received a cadaveric graft (CAD) and 23 received a graft from a living donor (LDLT). Mean time of patient follow up was 25 months, with a range of 6 to 39 months. Elevated serum transaminases, positive HCV RNA, and liver biopsy consistent with histologic evidence of HCV defined recurrence. Cholestatic Hepatitis C (CHC) was confirmed if all of the following criteria were met: serum total bilirubin greater than lOmg/dl with no evidence of extra-hepatic biliary obstruction on ultrasound and cholangiography, and histologic features on liver biopsy consisting of portal expansion, ductular proliferation, and bile stasis with or without hepatocyte ballooning. Immunosuppression, definition and treatment of rejection were standardized for both CAD and LDLT. Results: When comparing CAD to LDLT, both the incidence of HCV recurrence and time to recurrence were not different. The overall incidence of "severe" sequelae of HCV recurrence, either cholestatic hepatitis, grade 111-IV inflammation, and/or HCV induced graft failure requiring re-transplantation was also not different when comparing CAD to LDLT. However, when comparing CAD versus LDLT, no CAD patient developed cholestatic hepatitis C, compared to 17% of LDLT who developed this complication (p = 0.001). Conclusions: In this patient population, the timing and OVERALL incidence of HCV recurrence were not different when comparing CAD versus LDLT, but the incidence of cholestatic hepatitis was significantly greater in patients with HCV who underwent LDLT. Further multicenter studies are warranted to determine the incidence and risk factors for cholestatic hepatitis C following liver transplantation. UCSF, Sun Francisco, CA; Fabien Zoulim, INSERM, Lyon, France; Carol L Brosgart, Michael Wulfsohn, Michael D Miller, Shelly Xiong, Gilead Sciences, Inc., Foster City, CA Background Lamivudine (LAM) resistance occurs in approximately 40% of chronic hepatitis B (CHB) patients after 2 years of LAM monotherapy. In contrast, resistance to adefovir dipivoxil (ADV) occurs infrequently with 1.6% of CHB patients developing the adefovir resistance mutation rtN236T after 2 years of ADV therapy. Pre-existing LAM resistance mutations or concurrent use of immunosuppressive therapy by LT patients may increase the risk of resistance to ADV. Objective: To determine the incidence of ADV resistance in a clinical trial of liver transplantation (pre and post) patients with LAM-resistant HBV treated with ADV for 96 weeks (LAM therapy was maintained in most patients). Methods: The HBV reverse transcriptase domain was sequenced for LT patients with detectable HBV DNA by PCR (>lo00 copieslml) after 96 weeks of ADV therapy (n=114). In vih-o drug susceptibility was determined following transfection of HepG2 cells with patient-derived HBV clones from baseline and week 96 serum samples. Results: The rtN236T mutation wa!j observed in 21114 patients (1.8%) at week 96. LAM had been discontinued at weeks 16 and 28 after initiation of ADV in these two patients respectively. The baseline LAM-resistant YMDD mutation reverted to wildtype prior to week 96 in both patients. Emergence of rtN236T was associated with rebound in serum HBV DNA and ALT elevation in both patients. In vitro phenotypic analysis showed approximately 4-fold reduced susceptibility to adefovir with rtN236T. However, these adefovir-resistant HBV clones were fully susceptible to LAM and entecavir in vitro. LAM therapy was re-initiated, in addition to the ongoing ADV therapy, after emergence of rtN236T in these patients resulting in a > 2.9 loglo copies1mL reduction in serum HBV DNA in both patients. One patient also had a significant reduction (>80%) in ALT within 5 months of LAM treatment. No other mutations potentially associated with adefovir resistance were detected. Conclusions: Emergence of the adefovir resistance mutation rtN236T was observed infrequently after 2 years in liver transplantation patients (1.8%, 2/114) infected with LAM-resistant HBV, similar to observations in treatment naYve non-liver transplantation patients. The ADV-resistant HBV was sensitive to LAM and addition of LAM resulted in clinical stabiliation in both patients. Local liver immune responses are thought to play a major role in chronic autoimmune diseases directed at biliary epithelium.Using the apical sodium dependent bile acid transporter (ASBT) promoter to drive biliary epithelial cell -specific expression of a membrane form of ovalbumin (OVA), we have previously developed OVA-BIL transgenic mice. Because these mice are tolerant to OVA, we use OVA-specific T cells from OT-1 and OT-I1 transgenic mice, restricted by MHC class I and class 11, respectively, with well defined peptide epitopes specific for OVA to induce biliary damage in a dose dependent manner. AIM: 1) To determine the liver mononuclear cell populations (MNC) involved in necroinflammatory disease, and 2) to determine where adoptively transferred cells home and proliferate. METHODS: 10 million OT-I and 2 million OT-I1 naYve T cells were adoptively transferred to OVA-BIL mice by intraperitoneal injection. At days 0, 5, and 7, liver MNC were isolated by collagenase digestion, purified by discontinuous Percoll gradient centrifugation, and analyzed by flow cytometry. Tail bleeds were performed at days 0, 3, 5, and 7 to follow serum ALT. In a subset of experiments, OT-1 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and analyzed on day 3 and 5 by flow cytometry after adoptive transfer with unlabeled OT-I1 T cells into OVA-BIL mice. RESULTS: OVA-BIL mice develop normally without evidence of disease up to 2 years. After adoptive transfer of OVA-specific T cells, there was a marked increase in serum ALT. CD8+ OT-I T cells were required for liver damage and OVA-specific CD4+ T cells markedly augmented this inflammation. Adoptive transfer of OT-I1 CD4 T cells alone did not induce liver injury. There was extensive portal inflammation in every portal triad, centered around the bile ducts with infiltrating lymphocytes in the bile duct epithelia, apoptotic cells, loss of biliary epithelial cells as well as interface hepatitis. Liver MNC were abundant in OVA-BIL mice and increased after adoptive transfer of OVA-specific T cells. Serum ALT peaked at day 5 (mean 263 IUlml), coincident with liver MNC peak. CFSE labeling studies revealed robust homing of adoptively transferred OT-I CD8 cells to the liver, but not to the spleen, of OVA-BIL mice. The OVA-specific CD8 cells, but not CD4, NK1.l, or CD19 cells, underwent cell division. CONCLU-SION: Recognition of biliary epithelial antigen in OVA-BIL mice induces a necroinflammatory response in the liver as assessed by serum ALT, liver MNC numbers and immunohistochemistry. The magnitude of this response correlates with influx of nahe OVAspecific cytotoxic T cells, which are activated and divide in the liver but not in the spleen. T cell recognition of antigen expressed on bile duct epithelium occurs rapidly, causes biliary specific inflammation with interface hepatitis, which may be a model of autoimmune bile duct injury or cholangiopathy. The etiology of autoimmune hepatitis (AIH) is only poorly understood although the major autoantigens, such as cytochrome P450 2D6 (CYP2D6), could be identified and immunodominant epitopes have been mapped. One major reason for this lack of comprehension is the fact that there are currently only few valid animal models for AIH and none of them involves the autoantigen targeted in humans. Thus, we generated an animal model for human AIH using a natural autoantigen (CYP2D6). Self-tolerance in transgenic mice that express human CYP2D6 in the liver was challenged by infecting these CYP2D6-mice with an Adenovirus-CYP2D6 vector (Ad-2D6) in order to deliver large amounts of the critical antigen to the liver and to provide a inflammatory environment that would favour autoimmunity. Infection with an empty Adenovirus vector resulted in a transient form of focal necrosis 4 days after infection that subsequently disappeared after 2 weeks. In contrast, infection with Ad-2D6 resulted in extended and persistent infiltration of CD4, CD8 lymphocytes as well as macrophages resulting in confluentlbridging necrosis at week 10 post-infection. In addition, the overall morphology of the liver was massively disturbed after Ad-2D6 infection. At week 10 postinfection, the liver was approximately half the size of the control and its lobules were fused together and rounded up. First indications of fibrosis and hyperplasic nodules become apparent. The overall architecture of the liver was disrupted I disorganized and the liver parenchyma was partially collapsed. Furthermore, theliver of Ad-2D6 infected mice was surrounded by multiple layers of connective tissue as seen in some stages of liver cirrhosis. Additional signs of cirrhosis started to become apparent in the form of nodules that are entrapped in fibrous tissue. In addition, accumulation of infiltrating cells are visible directly under the liver capsule. These observations indicate that the CYP2D6-mouse displays a persistent form of hepatitis after infection with Ad-2D6 that may form the basis for a novel model system which would allow to study mechanisms involved in the initiation, propagation and precipitation of autoimmune processes involved in human autoimmune hepatitis. Disclosures: Urs Christen -No relationships to disclose Eric F Johnson -No relationships to disclose Michael P Manns -No relationships to disclose Antje Rhode -No relationships to disclose Matthias G von Herrath -No relationships to disclose Herkel, Peter R Galle, Edgar Schmitt, Ansgar W Lohse, Johannes Gutenberg University, Mainz, Germany Background: In clinical hepatitis, hepatocytes express MHC class I1 molecules; we recently reported that MHC I1 expressing hepatocytes can function as antigen presenting cells that stimulate specific CD4 T cells (Hepatology 2003; 37: 1079-85) . To understand the relevance of hepatocellular antigen presentation for hepatic immunity, we now examined whether hepatocytes may induce the differentiation of primary CD4 T cells. Because inflammatory CD4 T cells in the liver are most likely primed by dendritic cells of the draining lymph nodes, we also examined whether hepatocytes may also re-differentiate dendritic cell-primed CD4 T cells. Methods: Primary CD4 T cells from Ovalbumin-specific T cell receptor-transgenic mice were stimulated by antigen presenting hepatocytes from hepatocyte-specific CIITA-transgenic mice or splenic dendritic cells.The response type was determined by measuring secreted interferon-gamma (IFN) and interleukin-4 (IL-4). Results: We found that antigen presenting hepatocytes by default induced differentiation of primary CD4 T cells to TH2 cells (IFN: 200 Ulml; 1L-4: 1800 Ulml). In contrast, primary CD4 T cells stimulated by dendritic cells differentiated to Thl effector cells (IFN: 2400 Ulml; 1L-4 20 Ulml). However, these dendritic cellprimed Thl type cells, when re-stimulated by hepatocytes, had a decreased capacity to produce IFN (580 Ulml vs. 2800 Ulml after dentritic cell stimulation); and after repeated re-stimulation by hepatocytes, the dendritic-cell primed T cells even differentiated into Th2 cells (IFN: 112 Ulml; IL-4: 1270 Ulml). Conclusions: These data show that antigen presenting hepatocytes induce Th2 differentiation of undifferentiated CD4 T cells. Most notably, even dendritic cell-primed CD4 T cells that are committed to Thl differentiation could be reverted by hepatocytes to Th2 type. Thus, hepatocytes seem to have an extraordinary capacity to promote antiinflammatory hepatic immune responses. It is therefore conceivable that antigen presentation by hepatocytes associated with clinical hepatitis, in the absence of pro-inflammatory stimuli, seems to downregulate inflammatory infiltrating T cells. Background NKT cells are a unique subset of regulatory lymphocytes with important immune modulatory effects. These cells recognize exogenous glycolipids anchored by a ceramide tail to the MHC-like CDld molecule, expressed by various antigen presenting cells. Glycosylceramides, including the marine sponge-derived a-galactosylceramide can activate NKT cells, leading to exacerbation of hepatitis. Concanavalin A induces immune mediated hepatitis in which NKT cells are key participants. Glucocerebroside (GC) is a naturally occurring glycolipid. Aims: To determine the immune modulatory effect of GC in a murine model of Con A hepatitis. Methods: Five groups of BalblC mice were studied Group A and B mice were treated with GC (1.0 pg IP) two hours prior to and two hours following administration of 500 pg Con A, respectively; group C mice were treated with Con A alone; group D mice were treated with GC alone; group E mice did not receive any treatment. The degree of liver damage was evaluated by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, and by liver histology. The immunmodulatory effect of GC was determined by FACS analysis of intrahepatic and intrasplenic lymphocytes for NKT markers, and by ELISA measurements of serum IFNy, IL2, IL4, IL10, and IL12. The effect of GC on NKT lymphocyte proliferation was assessed in vitro. Results: Treatment with GC markedly reduced serum AST and ALT levels in group A compared to group C (143 vs. 600 IU in group A and group C, respectively, for AST; 57 vs. 801 IU in group A and group C, respectively, for ALT, p<0.05). Administration of GC alone did not change AST or ALT levels compared to naive controls. Histological sections of livers from group A mice revealed markedly attenuated damage compared to sections from group C livers, in which massive hepatocyte necrosis was present. The beneficial effect of GC on immune mediated hepatitis was associated with a 20% decrease in the intrahepatic NKT lymphocyte number, and with significant lowering of serum IFNy levels (3725 vs. 5620 pglml in groups A and C, respectively, p<0.05); administration of GC alone led to increased serum IL-12 levels (573 pglml vs. 92 pglml in group D vs. group E, respectively, p<0.05). In vitro, administration of GC decreased NKT cell pro-liferation by 42% in the presence of dendritic cells, but not in their absence. Conclusions: Administration of GC led to significant amelioration of Con A hepatitis that was associated with a dendritic cell-dependent decrease of NKT cell proliferation in vitro, and with decreased intrahepatic NKT lymphocytes and serum IFNy levels in vivo. These results suggest that the effect of GC may be mediated by inhibition of intrahepatic NKT cells, resulting from competitive displacement of activating elements from the CDld molecule on antigen presenting cells. Glucocerebroside, a naturally occurring glycolipid, holds promise as a new immunomodulatory agent, with a possible role in treatment of autoimmune hepatitis and other immune-mediated liver disorders. Disclosures: Background CD4+ cells constitutively expressing CD25 have a regulatory function, their experimental removal leading to spontaneous autoimmune disease in normal rodents. CD4+CD25+ regulatory T cells suppress both TH1 and TH2 responses, competing with effector CD4+ cells in recognizing the same peptide antigens. Their ability of expansion is key to the maintenance of tolerance. Autoimmune hepatitis type 2 (AIH-2) is characterized by T cell immune responses against 6 well-defined regions on cytochrome P4502D6, the autoantigen of AIH-2. Aims: To investigate the ability of expansion of CD4+CD25+ after exposure to non-antigen-specific and CYPZD6-specific stimuli in AIH-2. Methods: CD4+CD25+ T cells were analysed by triple colour flow-cytometry of freshlcryopreserved peripheral blood mononuclear cells (PBMCs) befare and after culture in the presence of a T cell expander capable of maintaining the original T cell function (CD3KD28 Dynabeads T cell expander, Dynal Biotech, Norway) and of 28 synthetic CYP2D6 peptides (10 pMol), spanning the 6 antigenic regions known to induce proliferative CD4+ responses in AIH-2. Patients and controls: Nine patients with AIH-2 (7 female, median age 11.4 yrs, range 2.5 to 21 yrs) were investigated, 3 at diagnosis and 6 while on sustained remission. Nine healthy laboratory workers served as normal controls. Results: Before stimulation, the level of CD4+CD25+ T cells was significantly lower in AIH-2 patients (3.59 2 1.08) than in normal controls (6.39 f 0.87; p=O.O2), a difference present both at diagnosis (2.24 ? 0.56, p<0.005) and during remission (4.25 2 0.35, p=O.Ol). Following exposure to the T cell expander, the level of CD4+CD25+ T cells increased 4.5 times (28.6 2 30.8) in normal controls, but only 1.85 times in AM-2 patients (6.62 ? 1.85, p=O.Ol). Upon exposure to cyMD6 peptides, CD4+CD25+ T cells remained numerically unchanged, in contrast to the PBMCs from the same patients giving a strong proliferative response (up to 6.9 times). Summary & Conclusion: Our data show a numerical and functional impairment of regulatory T cells in AM-2. This defect is likely to be key to the initiation and perpetuation of the autoaggressive process in this condition. Orth, Mark A McNiven, Nicholas F LaRusso, Mayo Medical School, Clinic and Foundation, Rochester, MN Cryptosporidium parvum (CP) opportunistically infects intestinal and biliary epithelia causing worldwide morbidity, especially in patients with AIDS. Epithelial invasion by CP involves host cell membrane alterations resulting in a parasitophorous vacuole that envelops the parasite and a dense-band within the host cell underlying the attachment site; both processes require host cell actin remodeling. Since recent studies in other systems have demonstrated that Cdc42, an actin-associated GTP-binding protein, plays a central role in microbial-induced actin remodeling, we tested the HYPOTHESIS that CP activates host cell Cdc42 and its downstream effectors to induce actin rearrangement and microbial invasion. METHODS: We exposed cholangiocytes derived from normal human liver and immortalized by SV40 transformation to freshly excysted CP sporozoites and applied molecular and morphofogical approaches to test our hypothesis. RESULTS By immunofluorescent and immunoelectron microscopy, we found accumulation of Cdc42 in cholangiocytes at the parasite-host cell interface during CP invasion. We confirmed activation of Cdc42 in infected cholangiocytes by immunoprecipitation using an antibody to PAK4, a downstream effector molecule that binds exclusively to the activated form of Cdc42. Phosphatidylinositol 3-kinase (PI-3K), a membrane-associated kinase associated with Cdc42 activation, was also recruited to the site of attachment, as were N-WASP, PAK4 and p34-Arc, downstream effectors of Cdc42. Inhibition of PI-3K by wortmannin or LY294002 blocked CP-induced Cdc42 accumulation. While overexpression in cholangiocytes of a constitutively active mutant of Cdc42 promoted CP invasion (up to 50%), overexpression of function-deficient mutants of PI-3K, Cdc42 or of the WA fragment of N-WASP inhibited CP invasion by 60 -80 %. Moreover, inhibition of host cell Cdc42 activation by dominant negative mutation inhibited CP-induced actin accumulation, and parasitophorous vacuole and dense-band formation at the attachment site. CONCLUSIONS: These combined data suggest that CP invasion of cholangiocytes (and perhaps other target epithelia) results from the organism's ability to activate a complex host cell Cdc42 signaling pathway that induces host cell actin remodeling at the attachment site. BACKGROUND: Hepatitis B (HBV) and Hepatitis C (HCV) infections are a world-wide health concern. Studies of these infections have been hampered by a lack of a convenient animal model. We previously have shown that immunocompetent rats tolerized and transplanted with human hepatocytes can support HBV infection for at least 15 weeks. AIM: To demonstrate that the immunocompetent rat which has been tolerized and transplanted with human hepatocytes can be used to sustain and study HCV infection. METHODS: Sprague-Dawley rats were injected in utero at 16-17 days gestation with one hundred thousand Huh 7 cells (human hepatocyte cell line) to tolerized them to human hepatocytes. One week after birth, the tolerized newborns were intrasplenically transplanted with 5 million Huh 7 cells. One week after transplantation, rodents were inoculated with one hundred thousand HCV RNA copies, genotype l b human serum. Animals were sacrificed at 6 and 12 weeks. The presence of human cells was determined by immunofluorescent staining of cryosectioned liver tissue using antibodies to human albumin. PCR and RT-PCR was used to confirm the presence of human albumin in liver tissue. The presence of HCV was assayed using an antibody to NS5A viral protein, and visualized using a rhodamine labeled secondary antibody. HCV infection in the liver was confirmed by the presence of negative strand RNA using nested PCR. RESULTS: Functional Huh7 cells in the chimeric livers were confirmed by the presence of human albumin mRNA and DNA using RT-PCR and PCR. The presence of human albumin protein in the liver was further demonstrated by immunofluorescence of human albumin in frozen liver sections. Eighty-one percent (n=57) of the tolerized, Huh 7 transplanted rodents were positive for human albumin in the livers. Seventy-two percent (n=36) of the chimeric animals infected with HCV were positive for the presence of NS5A HCV protein from immunofluorescence studies. Livers of control rats that were only tolerized and control rats that were tolerized and transplanted with Huh7 cells, but not inoculated with HCV were negative for NS5A (n=12). HCV viral replication could be demonstrated in livers of tolerized, transplanted and HCV infected rats by the presence of HCV RNA bands of the correct size from nested PCR using negative strand specific primers. CONCLUSION Immunocompetent rats tolerized and transplanted with human hepatocytes can be a useful laboratory model to study HCV infection. ( We have previously observed that allogeneic hepatocellular transplants are highly immunogenic and are resistant to immunosuppressive therapy with a variety of agents. Donor specific transfusion in combination with anti-CD40L mAb is highly effective in inducing prolonged survival of skin and myoblasts and inducing indefinite and donor-specific tolerance to heart and islet allografts. The proposed mechanism of action for DST and anti-CD40L mAb suggests peripheral deletion of alloreactive CD8+ T cells and induction of a regulatory CD4+ T cell population. Resistance to induction of indefinite acceptance has been attributed to peripheral reappearance of alloreactive CD8+ T cells. In preliminary studies, we observed that DST and anti-CD40L mAb prolonged FVBlN hepatocyte (HC) survival in C57E3L/6 mice to median survival time (MST) of 88 days and induced indefinite acceptance (>90 days) in 43% of mice. HC rejection occurs by CD4-dependent and (CD4-independent) CD8-dependent pathways. This model permits evaluation on these two pathways separately. The current studies address the hypothesis that DST and anti-CD40L mAb induces prolonged HC allograft acceptance by inducing immunoregulation of both alloreactive CD4+ and CD8+ T cells. Methods: FVBlN (hAlAT-FVBIN, H-24) HCs were transplanted into CD4 KO, CD8 KO, and C57BL/6 (all H-2b) mice. Hc survival was monitored by detection of reporter hAlAT protein in serum by ELISA. For comparison, donor-matched islets were transplanted into streptozotocin-induced diabetic CD8 KO (H-2b) mice, and survival was monitored by blood glucose. Recipient mice received peritransplant administration of DST ( 1 0~1 0~ FVBlN splenocytes, day -7) and anti-CD4OL mAb (1.0 mg, ip, d-7, -4, 0, 4) . Some CD8 KO hosts were reconstituted CD8+ T cells (2x107 iv, d-10). Results: When the CD8-dependent rejection was studied in isolation (CD4 KO mice), DST and anti-CD4OL mAb prolonged HC survival to MST of 35 days (N=4) compared to 10 days in untreated CD4 KO (N=7); however, this was not significantly different from anti-CD40L mAb alone. When CD4-dependent rejection was studied in isolation (CD8 KO mice), HCs were unexpectedly rejected with MST of 7 days (N=8) despite DST and anti-CD40L mAb treatment. In contrast, islets of the same strain were accepted indefinitely in CD8 KO mice (N=5, MST>70 days). Since this siraiegy effectively controlled HC rejection in C57BL16 mice which have both CD4-and CD8-dependent pathways available, the collective results suggested that perhaps host CD8+ T cells were necessary for the beneficial effects of the DST and anti-CD40L mAb. To determine whether CD8+ T cells were required for induction of longterm HC survival with DST and anti-CD40L mAb, CD8 KO HC recipients were reconstituted with CD8+ T cells prior to DST and anti-CD4OL mAb treatment. CD8reconstituted CD8 KOs accepted HCs for MST of 56 days. Conclusion: The effects of DST and anti-CD40L mAb on combined CD4-and CD8-initated HC rejection are more pronounced than on either pathway alone. CD8+ T cells appear to be required for induction of longterm HC survival under cover DST and anti-CD40L mAb, which offers an apparently distinct mechanism compared to the existing paradigm for the mechanism of action of DST and anti-CD40L mAb. Engraftment of transplanted cells in the liver is affected by cellcell interactions involving hepatic endothelial cells and Kupffer cells. The proximity of transplanted cells to HSC suggested that HSC could promote cell adhesion and extracellular matrix remodeling during cell engraftment. To investigate cell-cell interactions in the liver, we analyzed HSC activation in DPPN-rats transplanted intrasplenically with F344 hepatocytes. Analysis of tissues by immunostaining from animals 6h, 24h, 3d and 7d after cell transplantation showed appearance of desmin +ve HSC in periportal areas, reaching a peak on day 3 (4-10 fold increase in desmin f v e HSC in cell recipients, p