HEPATOLOGY VoL 22, No. 4, P t . 2, 1995 A A S L D A B S T R A C T S 1 3 3 A

1 0 5 CRYOGLOBULINEMIA IN LIVER TRANS~PLANT RECIPIENTS WITH HCV INFECTION. E Qitn~- N Naoumov. E Davies*. B Portmann. Roger Williams. institute of Liver Studies, Dept of Immunology*, Kings College School of Medicine and Dentistry, London, SE5 9PJ, UK.

Background: Cryoglobulins are found in 20-50% of patients with chronic HCV infection and may be associated with more severe liver disease and failure of interferon therapy. The aim of this study was to determine the prevalence and the impact of cryoglobulinemia in liver transplant recipients with HCV infection. Methods: 85 consecutive patients transplanted for hepatitis C-related cirrhosis were followed for 12-60 months. HCV was detected in postOLT serum by PCR and genotyped with LiPA. Post- transplant serum was tested for cryoglobulins and if detected these were typed and quantified. Serial measurements of cryoglobulins were performed pre-OLT and 3, 6 and 12 months post-OLT in 25 patients. Results: Cryoglobullns were detected in 15 patients (18%) after OLT - 5 had type 2 eryoglobniins, 4 had type 3 and typing was not possible in the remaining 6. Cryoglobulins were detected in 35% of patients infected with HCV genotype lb compared to 13% of other genotypes (p<0.05). Clinical vasculitis was not seen in any patient prior to transplant but developed in 5 patients 1-18 months post-OLT (rash in 3, MPGN in 2 and 1 each of CVA, retinal artery occlusion and Raynaads) in association with low serum C4 levels (median 0.09; range 0.07-0.12g/I). In one patient, systemic vasculitis was associated with acute graft dysfunction and histological evidence of vasculitis within the graft. Levels of cryoprecipitate were higher in patients with Clinical vasculitis (2-11 g/l) than in asymptomatic patients (all < lg/I). In 4 patients, the initial detection of cryoglobulins coincided with acute hepatitis in the graft 1-6 months postOLT. 11 / 15 (73 %) of patients with cryoglobulinemia developed CAH in the graft compared to 19170 (27%) of patients without (p < 0.05). In 5/8 patients, cryoglobulinemia disappeared during antiviral therapy accompanied by a decrease in serum HCV RNA. Conclusion: Cryoglobulinemia after OLT is associated with more severe graft damage suggesting a direct role of cryoglobulins in the pathogenesis of graft injury through vasculitis and Kupffer cell activation. Alternatively, severe graft damage may lead to cryoglobulinemia because of the associated reduced hepatic clearance of immune complexes or because of stimulation of the humeral immune response by viral antigens released by hepatocytolysis.

106 SPECIFIC HUMORAL IMMUNE RESPONSE TO HEPATITIS C VIRUS PROTEINS IN RECURRENT HCV INFECTION AFTER LIVER TRANSPLANTATION. El Gane ~. G Maertens 2. KP OianL JYN Lau 3, B Ponmann =. NV Naoumov t. Roger Williams t . Instituteof Liver Studies ~, King's College Hospital, London, UK, Innogenetics 2, Ghent, Belgium, University of Florida 3, Gainesville, USA.

Aim: To investigate the changes in antibody response to individual HCV proteins fullowing liver transplantation (OLT) and their correlation with HCV replication and the evolution of graft damage. Methods: Serum samples were collected pre43LT, I and 12 months post-OLT in 51 consecutive patients undergoing OLT for HCV-cirrhusis. HCV isolates were genotyped with the line probe assay (LiPA). In patients infected with HCV Type 1 (n=30), antibody responses to 8 different HCV proteins were quantitated as follows: antienvelope antibodies with ELISA (using purified vaccinia virus-expressed HCV Type 1- specific El [aa 192-326] and E2 [aa384-673] proteins); antibodies against core 1 and core 2 (non-overlapping 1st and 2od epitope clusters), E2/NS1 (mixture of HVRI peptides), NS4 (mixture of 4A and 4B) and NS5 synthetic peptides and against E.coli recombinant NS3 protein with the INNO-LIA HCV Ab I[l/LIAscan automated package. HCV RNA was quantitated in each sample by the bDNA assay. At 12 months, liver biopsy was performed and serum tested for cryoglobulins. Results: Titers of each HCV antibody fell following OLT (p <0.05): E1 by 36+5%; E2 30+5%; corel 13 +4%; core2 19+5%; E2/NS 1 33+9%; NS3 26+7%; NS4 33+6%; NS5 38+-8%. At 1 month pest OLT, anti-El levels were higher in the 15 patients who developed acute hepatitis (p=0.01). At 12 months, only anti-El and anti-E2 titers increased to pretransplant levels and correlated with serum HCV RNA levels (El: r=0.5, p=0.03; E2: r =0.6, p=0.02). Anti-E1 levels were lower in the 9 patients with cryoglobulinemia (p=0.04) and were higher in patients with CAB in the graft (n--6) than in patients with CPH or normal histology (10.4+3.Su/ml vs 2.9_0.8u/ml. p=0.02). Conclusion: Titers of all HCV antibodies fall in the early post-OLT period, when immunosuppression is maximal and levels of viremia are low. Despite maintenance immunosuppression, titers of anti- envelope antibodies increase thereafter in correlation with viremia, suggesting that the specific humeral immune response to envelope proteins is enhanced by increased HCV replication. However, only anti-El correlates with graft damage and this antibody may be a useful marker in monitoring patients with recurrent HCV infection'.

107 SERUM CYTOKINE LEVELS IN PATtENTS WITH RECURRENT HEPATITIS C AFTER LIVER TRANSPLANTATION (OLT). MW Fried. LC Sesff, L tam. K Gutekunst, T Hodue: TD Bover. Dept of Medicine, Emon/Univ. School of Medicine; Immunology Branch, Centers for Disease Control; Atlanta, GA, and Roche Molecular Systems, Branchburg, NJ.

Previous studies have demonstrated that circulating cytokines may be detected in serum in vadous liver diseases. It has been suggested that these cytoldnes are involved in mediating the immune response to viral infection. The aim of this study was to evaluate the host response to HCV infection in immunosuppressed patients with recurrent HCV after liver transplantation. Methods: Thirty patients with recurrent hepatitis C after liver transplantation (OLT-HCV) and ~1 non-cirrhotic patients with chronic hepatitis C (CHC) with stable liver enzymes were evaluated. All patients were positive for HCV RNA in serum. Post-OLT patients had been transplanted at least 3 months eadier and were on stable doses of immunosuppreseion (cyclosporin or tacrolimus), with stable liver enzymes and no evidence of cellular rejection. Sera was collected, separated, stored frozen at -70 ° C, and thawed once prior to use. Serum tumor necrosis factor (TNF), IL-4, and IL-6 were. measured in duplicate using commercially available EIA (R & D Systems, Minneapolis). Serum HCV RNA levels were measured in the same sample (AMPLICOR HCV MONITOR, Roche Molecular Systems). Cytokine levels were correlated with serum ALT activity, viral load, and immunosuppressive agent.

OLT-HCV • CHC IL-4 (pg/ml) 12,7 + 8.5 10.8 _+ 2.7 (n=10) IL-6 7.2 + 3~9 4.9 _+ 0.5 (n=l 0) TNF 14.6+ 13.2 7.8 + 1" *p=.01 HCV RNA ix 10 s I 6.2 + 4.2 1.3 + 1.1" *p < .005 Results: Mean serum IL-4 and IL-6 levels were similar between the two groups. Serum TNF levels were significantly higher in patients with recurrent hepatitis C after liver transplantation compared to controls. Among the patients with recurrent HCV after transplant, mean serum TNF levels were higher in those treated with cyclospodn vs tacrolimus (17.8 _+ 17.6 vs 11 + 2.2, p=.08). HCV RNA levels were increased in patients with recurrent HCV after OLT as compared to controls, but did not correlate with serum TNF levels. Among the transplanted patients, there was no correlation between serum TNF levels and ALT activities. Conclusions: Recurrent HCV after OLT is associated with increased expression o f TNF in serum. The role of immunoseppreseive agents in altering this response and their effect on disease progression need to be further evaluated.

108 HEPATITIS C VIRUS (HCV) DIVERSITY AND LIVER TRANSPLANTATION (OLTx) S Zhou~ NA Terranlt~ M. Kim, TL Wright. Departments of Medicine, Veterans Administrstion Medical Center and University of Callfomh, San Francisco, CA.

Background: HCV is a heterogeneous virus believed to mutate under "immune pressure". The relationship between viral diversity (or viral quasispecies) and therapeutic interventions has been largely unexplored. We determined viral heterogeneity by two independent methods in a single patient prior to and following OLTx, and prior to and following interferon (IFN) therapy for post, OLTx disease. Methods: HCV quasispecies were measured using single-strunded conformational polymorphism (SSCP) and expressed as bands detected. Viral mutational rate was calCulated from a sequence Of 81 nucleotides within the hypervariable region from multiple clones at each time point. Based upon nucleotide sequences, a master and consensus sequence were assigned. The master sequence (A1) was determined prior to OLTx and all subsequent quasispecies were compared to it. The master sequence was used to define the major species (bold type). Virus less closely related to the master sequence (<90% homology) were defined as minor species. Rate of diversity was calculated as the change in consensus sequence relative .to the master sequence. Results: Patient was infected with type 1 gen0ty .L0e. ' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Time Point Yrs SSCP % Homology tO Rate of HCV RNA

- - ~ o m _ - - Master Sequence ~ OLTX (n=# of e lo~s) (10e3/~0

Pre,OLTX 2 A1 100% (n--4) . . . . < 0 ~ 5 " A2 89.1% (n=l)

Pre-IFN 3.5 3 B1 95.1% (n=l) 24.5 12.3 B2 88.9% (n=2) B3 86.4% (n=2)

End of IFN 4 2 CI 96.3% (n=2) 15.4 6.5 C2 90.2% (n=2)

After IFN 4.5 2 DI 95.1% (n=2) 16.5 D2 88.9% (n=2)

The major species present pre~0LTx l~'Isl[ed throughout (B1,CLD'IiI"0n immunosuppression and prior to IFN, there was considerable viral diversity (B1,2,3). IFN treatment decreased viral diversity (only two major species detectable and both highly homologous to B1) and the rate of viral divergence. Conclusions: Therapeutic interventions affect viral divergence and alter the rate of viral mutation in HCV-infected transplant recipients.6