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End-stage liver disease associated with hepatitis C (HCV) infection is the most common indication for liver transplantation (LT) in the United States.1 In contrast to the other leading indications for LT, recurrence of HCV infection, as measured by detection of HCV RNA by PCR, is nearly universal.2, 3 Although patients undergoing transplantation for HCV have been reported to have patient and graft survival comparable to most other indications,4, 5 recurrence of HCV is a substantial source of morbidity, mortality, and graft loss.6, 7 In a recent retrospective cohort study of over 11,000 transplant recipients, HCV infection as an indication for LT was associated with significantly impaired patient and allograft survival.8 Viral recurrence occurs universally; however, recurrence is apparent histologically in only about 50% of HCV-infected grafts and progression to allograft failure leading to death or graft loss occurs in approximately 10% by the fifth post-operative year.9–11 When recurrent HCV leads to decompensated cirrhosis, retransplantation is often denied due to very poor survival.12 HCV-related disease progression is accelerated in immunocompromised compared to immunocompetent patients with a progressive increase in patients who have recently undergone LT, although the reasons for this worsening outcome are under question.13 Possible reasons for this disturbing trend include the use of more potent immunosuppressive agents and more marginal donors (older).14 The two most frequently used immunosuppressive drugs are Cyclosporin (CsA) and Tacrolimus (TAC). Of interest, the usage of TAC has increased from 0% before 1996 to nearly 80% after 1999. It is intriguing to hypothesize that alterations in immunosuppressive regimens may impact on disease recurrence and response to antiviral therapy.
Limitations in treatment of patients with chronic HCV have led to multiple trials to understand the role of immunosuppression in disease progression and response to antiviral therapy. OKT3, IL-2r antibodies (Abs) steroids, and others have been reported to accelerate HCV, but there is no apparent relationship with the primary immunosuppressive regimen of TAC or CsA.15–17 Currently, TAC is the primary immunosuppression agent used in the majority of liver transplant recipients. However, CsA may have some theoretical benefits in the HCV population. CsA has been shown to have antiviral activities against human immunodeficiency virus type I,18 herpes simplex virus,19 and vaccinia virus.20 A recent report by Wastashi et al. showed that CsA has a strong suppressive effect on HCV replication using the HCV replicon cell culture system.21 This reduction was not observed with other immunosuppression like TAC. This CsA effect was independent of its immunosuppresant function. In another recent report, CsA appears to have a beneficial impact on HCV antiviral therapy when combined with interferon-based regimens.22 There is also indirect evidence that CsA may augment the activity of interferon against HCV.23 However, several conflicting reports regarding the efficacy of CsA in the liver transplant population have also been published and there remains significant uncertainty regarding the potential role of CsA in the HCV transplant recipient.24–26. A better understanding of immunotherapy and its relation to resolution of the infection may help in the design of better therapies for the control of HCV infection in this population.
In this study, we have analyzed the impact of CsA on HCV replication and response to interferon-based therapy within out liver transplant population. Our data confirms an in-vitro antiviral effect for CsA and suggests a role for its use for liver transplant patients undergoing antiviral therapy for HCV.
The HCV replicon cell line, GSB1, was treated with varying doses of CsA for 48 hours. Total cellular RNA was isolated from cells as described before.15 First-strand cDNAs were synthesized from total cellular RNA by reverse transcription (20 μl of reaction volume) using the Superscript II (50 U reverse transcriptase per reaction) first-strand synthesis for RT-PCR kit (Invitrogen, Carlsbad, CA) primed with oligo (dT)12–18 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Fluorophore-labeled LUX primers and their unlabeled counterparts were obtained from Invitrogen. Reactions were conducted in a 96-well spectrofluorometric thermal cycler (ABI PRISM 7700 Sequence detector system, Applied Biosystems, Foster City, CA). Fluorescence was monitored during every PCR cycle at the annealing step. The primers are for HCV, 5′ -CGCTCAATGCCTGGAGATTTG-3′, 5′ -GCACTCGCAAGCACCCTATC-3′; for GADPH, 5′ -TGCTGGCGCTGAGTACGTC-3′, 5′ -GTGCAGGAGGCATTGCTGA-3′; PCR conditions were as follows: 50°C, 2 min; 95°C, 10 min; (95°C, 15 sec; 60°C, 1 min) × 40 cycles. For determination of the interferon-stimulated gene 6-16 (G1P3), similar RT-PCR analysis was used, except for the primers specific for G1P3.
This retrospective analysis of our study cohort was identified from the University of Florida LT database. Information was collected according to the Institutional Review Board at the University of Florida, Gainesville, FL. A total of 906 liver transplants were performed at our institution from 1991 to November 2002; 358 adult liver transplants were performed for the indication of HCV cirrhosis. All other forms of liver disease were excluded from the analysis. Only patients started on interferon [standard or Pegylated (PEG)] and ribavirin were included in the analysis.
Standard immunosuppression consisted of Cyclosporine (Gengraf or Neoral) or Tacrolimus in combination with prednisone. The use of TAC in our center was initiated in 1997. TAC use has gradually increases over the past 8 yr and now is used as primary calcineurin inhibitor, if tolerated. Patients initially receiving CsA were treated with a dose 2.0-4.0 mg/kg/day orally in two divided doses with target trough whole blood concentrations of 200-250 ng/ml for the first month post-transplant followed by 150-200 ng/ml thereafter. Patients initially receiving TAC were treated with a dose of 0.08-0.12 mg/kg/day orally in two divided doses with target trough whole blood concentrations of 10-15 ng/ml for the first month post-transplant followed by 5-10 ng/ml thereafter. Immunosuppression was typically tapered to monotherapy (TAC or CsA) within 4-6 months of transplantation. There was no difference in the average steroid dose protocol between the 2 groups, as reflected in the similar acute cellular rejection rates seen in both groups and protocol steroid taper regimens that were identical. Prednisone was discontinued between month 4-6 of transplantation as per our transplant protocol for HCV patients.
Combination therapy with interferon and ribavirin was utilized in those patients with significant fibrosis (Ishak fibrosis stage ≥ 3) on protocol or indication liver biopsy. Therapy was initiated at half dose [1.5 MU interferon alfa-2b SQ thrice weekly (tiw)/PEG interferon alfa-2a 135 μg weekly and ribavirin 400-600 mg daily] for 2 weeks, and if tolerated, the dose was increased to full dose (interferon alfa-2b 3 MU SC tiw/PEG interferon alfa-2a 180 μg weekly and ribavirin 800-1,200 mg daily). Ribavirin dosage was based on weight; patients < 75 kg received 800 mg and > 75 kg 1,000 mg. Hemoglobin, white blood cell count, and platelet count were monitored weekly for the first four weeks and then monthly thereafter. Dose reduction was performed as follows: if PMN < 750 or platelets < 50,000/μL, the interferon was reduced to 1.5 MU three times a week/PEG interferon to 135 μg/week; if PMN < 500 or platelets < 30,000, therapy was stopped; hemoglobin < 10 mg/dl, ribavirin was reduced to 600 mg per day; if hemoglobin < 8 mg/dl the ribavirin was discontinued. Erythropoietin was used to prevent ribavirin discontinuation when possible. Granulocyte colony stimulating factor was not used to treat cytopenias. Therapy was discontinued in any patient who developed moderate to severe rejection, systemic bacterial infection, severe neuropsychiatric symptoms, or symptomatic anemia. The intended duration of treatment was 48 weeks for genotype 1 and 24 weeks for genotype 2 and 3.
HCV RNA Testing
Serum HCV RNA values were measured six months after completion of interferon-based therapy to assess for the presence of a sustained virological response (SVR) in those patients with a negative HCV at the end of treatment. Subsequently, an HCV RNA titer was obtained annually. The HCV RNA titer was determined using a branched DNA signal amplification assay (Quantiplex HCV RNA; Chiron Corporation, Emeryville, CA). Serum samples that tested negative by the branched DNA assay were analyzed using RT-PCR with a sensitivity of about 10-200 copies (Qualitative Amplicore HCV Test; Roche, Mississauga, Ontario, Canada).
Protocol liver biopsies were performed in our institution at month 4, yearly, or when clinically indicated after liver transplantation. Recurrent HCV disease was scored for inflammation and fibrosis, using the modified Knodell scoring system of Ishak.16 Patient treated were those with Ishak score ≥ 3.
The primary end points were to determine the effect of CsA in viral clearance in the liver transplant recipients with recurrence HCV during therapy with combination of interferon and ribavirin, and to determine the anti-HCV potential of CsA in the replicon cell culture system.
Fisher Exact probability test for 2 × 2 contingency table was used for the comparison of the two outcome variables. Statistical analysis was performed with statistical software (SPSS 10.1 for Windows 95/98; SPSS Inc., Chicago, IL). Results for all in-vitro experiments represent triplicate determinations. Results are represented as means ± SD. Results were analyzed with SDS 2.0 software from Applied Biosystems.
Anti-HCV Activity of CsA
The HCV replicon cell line was used to assess the effects of different doses of CsA on the intracellular replication of HCV. GSB1 cells were treated with different concentration of CsA for 48 hours. Figure 1 depicts the HCV replicon RNA replication inhibition by CsA in a dose-dependent manner. By this in-vitro assay, 250 ng/ml of CsA (comparable to therapeutic levels achievable in patients) leads to a 20% reduction in HCV replication. No effect on viral replication was observed when the HCV replicon cells were treated with comparable concentrations of TAC (see Fig. 2).
CsA effect on IFN Antiviral Activity
To test the effect of CsA on IFN antiviral activity, the replicon cells were treated with both CsA and IFN-α, followed by real time PCR analysis. As shown in Figure 3, addition of CsA and IFN-α shows an additive antiviral effect, but not synergistic effect, suggesting an independent antiviral mechanism. To test this hypothesis, examination of the IFN-stimulated gene G1P3 expression was performed after cells were treated with IFN-α and G1P3, either alone or in combination. As shown in Figure 4, the CsA did not have any significant effect on IFN-induced antiviral pathway.
HCV Transplant Population
Between 1991 and 2002, 842 adult liver transplants were performed at the University of Florida. Of a total of 842 LT, 358 (40%) liver transplants were performed secondary to HCV cirrhosis. Of the 358 HCV infected LT recipients, 107 (30%) received interferon-based therapy before transplant. The median time from LT to initiation of therapy was 2.6 ± 1.2 yr for the TAC group and 4.9 ± 1.1 yr for the CsA group. Antiviral therapy after transplant was initiated for Ishak fibrosis stage ≥ 3 or HCV-related cholestasis. A total of 115 (32%) patients received treatment with interferon-based therapy after LT.
Characteristics of Study Groups
The general characteristics of the two study groups are shown in Table 1. There were no statistically significant differences between the CsA and TAC group.
Table 1. General Characteristics of Study Groups
Abbreviations: F, Female; M, Male; C, Caucasian; H, Hispanic; AA, African American; P, Pacific Islander; BMI, Body Mass Index; HCV, Hepatitis C Virus; LT, Liver Transplantation.
The study population consisted of 115 patients that were treated with interferon-based therapy. From the 115 patients, 56 patients received CsA and 59 patients received TAC-based immunosuppression. The mean duration of the treatment was 48 weeks. The end-of-treatment response (ETR) was 40% in the TAC group compared to the CsA group that achieved an ETR of 60%. In the CsA group, 26 out of 56 (46%) patients achieved a sustained virological response; while 16 out of 59 (27%) patients on TAC achieved a SVR (P = 0.03). The patients who were treated with CsA-based immunosuppression achieved a higher SVR compared to those treated with TAC-based therapy. The pre-treatment viral load was slightly higher in TAC group, but this did not reach statistical significance (2.0 × 106 IU/ml vs. 1.5 × 106 IU/ml) (NS). The percent of patients with genotypes 2 and 3 was also not significantly different as seen in Table 1. The rate of SVRs based on genotypes for the TAC and CsA groups is shown per each group, respectively: 16% and 44 % for Genotypes 1, 80 % and 67% for Genotypes 2, 80% and 40% for Genotypes 3, and no patients for the genotypes 4. Dose reduction of IFN or ribavirin due to cytopenias was necessary in 66% of the patients on CsA and in 82% on TAC (P =0.1). Twenty patients (6 on CsA and 14 on TAC) had to stop therapy due to side effects including cytopenias and depression (P = 0.3). Sixty-one percent of the CsA patients and 50% in the TAC group received 80% interferon or peginterferon plus 80% ribavirin for more than 80% of the expected duration of therapy (P = 0.2). Thirteen patients of the 56 on CsA (23%) and 6 patients of the 59 (10%) have died, mostly due to infections and HCV complications. However, although only one death was thought related to IFN treatment side effect (chronic rejection) (see Table 2). This difference in survival between the 2 groups was statistically significant (P = 0.05), although was likely related to the longer follow-up in the CsA group.
Table 2. Cause of Death in Both Study Groups
Abbreviations: HCV, hepatitis C virus infection; CVA, Cerebrovascular Accident.
HCV infection is of major concern after LT due to universal recurrence, more rapid fibrosis progression, and potential graft failure. Given the dismal outcomes with retransplantation for HCV, all efforts to limit HCV recurrence and liver injury need to be aggressively pursued after LT. The impact of the primary immunosuppression regimen on disease and treatment outcomes has not been well defined. Our non-concurrent cohort study suggests that CsA may play a beneficial role as primary immunosuppression for patients transplanted for HCV infection and may offer an advantage to TAC in those patients undergoing IFN-based therapy. A recent study by Martin et al. showed no significant differences between TAC and CsA on histologic HCV recurrence after LT.17 Kakumu and colleagues reported that the administration of CsA monotherapy can suppress aminotransferases levels but not HCV RNA. Unlike patients on corticosteroids, an increase of HCV RNA was not observed.27 Our in-vitro data suggests that CsA may confer a patient advantage from an HCV replication standpoint, although there is yet no clinical data to support these in-vitro findings. However, it may also be that the clinical and/or viral benefit may be confined to those patients undergoing combination therapy with interferon and ribavirin, which was not addressed in this former study.
There is some evidence that combination of IFN and CsA may increase the activity of IFN against the HCV infection, but there is no published data about the effect of CsA on HCV infected patients receiving treatment post-LT. In our analysis, patients receiving combination therapy with interferon and ribavirin on CsA as basic immunosuppression achieved a higher SVR than those patients on TAC-based immunosuppression. There is no doubt that this SVR is higher compared to the literature, but this study represents a preliminary pilot study. The high SVR may be related to an antiviral effect of CsA or other unknown factors. We were unable to find any significant differences in patients characteristics between the two groups, including dose reductions, viral load, genotype, BMI, or percentage of patients who had failed interferon-based therapies before transplantation.
In addition, we were able to confirm that CsA has antiviral activity in cell culture systems and that the mechanism does not appear to act via standard interferon pathways. We demonstrated that CsA inhibits HCV RNA replication in a dose-dependent manner. At comparable therapeutic levels of 250 ng/ml, CsA can suppress viral replication by 20%. Of note, there was no evidence of cell toxicity at these doses, as determined by both morphology and cell count. Combination of CsA and IFN achieve better antiviral effect than either CsA or IFN alone, suggesting an additive or synergistic effect. Analysis of the IFN-stimulated gene G1P3 showed that CsA had no effect on this classic IFN antiviral pathway. This data indicates that the CsA antiviral pathway is different from classic IFN pathways. This represents a novel antiviral property of CsA that requires further investigation. Two recent Japanese studies have also reported an anti-viral effect of CsA using the replicon cell culture system.9, 18 Nakagawa treated HCV replicon cells with CsA showing suppression of viral replication in a dose-dependent manner. In another study, Watashi and colleagues treated HCV replicon cells with CsA and decreased specific HCV proteins and HCV RNA levels. Together, these studies demonstrate anti-HCV activity in vitro.
Owing to the accelerated rate of disease progression and graft failure after LT in HCV patients, it will be very important to determine the ideal immunosuppression regimen. Our data suggests a potential advantage of CsA vs. TAC in HCV patients undergoing antiviral therapy. A prospective randomized comparative trial between CsA and TAC is warranted to further evaluate these observations.