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The challenge of progressive hepatitis C following liver transplantation
Liver Transplantation
Volume 12, Issue 1, Pages 19-21
Jan 2006
Gary L. Davis
Baylor Regional Transplant Institute, Baylor University Medical Center, Dallas, TX
The complications of cirrhosis due to chronic hepatitis C are the leading indications for liver transplantation in the United States and currently account for more than 40% of cases.[1] This proportion has risen steadily over the 15 yr since the hepatitis C virus (HCV) was identified and it is projected that the number will continue to rise for another 10 to 20 yr.[1-3] The overwhelming majority of patients who present for liver transplantation have either never been treated with antiviral therapy, have been unable to tolerate it, or have failed to respond. Thus, viremia persists after transplantation in nearly all patients and commonly results in recurrent liver injury.[4] Progression of liver injury after transplant is frighteningly rapid in comparison to immune competent patients with cirrhosis developing in 10 to 25% within 5 yr.[4][5] In perhaps the most distressing report, Berenguer et al.[6] reported that half of patients have bridging fibrosis or cirrhosis within 5 yr of the date of transplant. A large proportion of cirrhotic patients will develop decompensation and death within 1 yr.[6] In contrast, only 20% of immune competent patients develop cirrhosis after 20 yr and only 3 to 6% of these decompensate each year; most remain well compensated without significant threat to their survival.[7] Given the number of patients and the dire implications of disease recurrence, chronic hepatitis C has become perhaps the most difficult problem currently facing physicians who manage liver transplant recipients.
Management of persistent HCV infection and recurrent chronic hepatitis is difficult. Hyperimmune anti-HCV immune globulin preparations have not yet been shown to be effective in this setting as they have been in hepatitis B.[8][9] Antiviral therapy with interferon-based regimens, as expected, is relatively ineffective in these patients since many have previously failed treatment, most are genotype 1 with high viral levels, and many have significant cytopenia due to hypersplenism and medication effects.[10-12] Preemptive therapy with interferon-based therapy administered shortly after transplantation is especially difficult because of these issues, the debility of many patients, and the potential for drug interactions.[12] Furthermore, this requires treatment of all HCV infected patients including the half who are not destined to have rapidly progressive graft injury. Thus, most hepatologists prefer to wait at least 6 to 12 months until patients have recovered from transplant and surveillance liver biopsies have identified those with progressive disease before considering antiviral treatment. Nonetheless, treatment still remains a substantial commitment for both patient and physician. Approximately 70% of cases require dose reduction; ribavirin, in particular, is poorly tolerated in these patients, who almost always have some degree of renal impairment from their calcineurin inhibitor.[10][12] Despite these challenges, many centers have reported a surprisingly high 20 to 30% sustained viral clearance rate.[10][12] Of course, considering the difficulty of treatment in this setting, these responses are far from what we would hope for. Considerable effort is being invested in investigating other ways to improve response to antiviral therapy in these patients. Recent reports of potent HCV protease and polymerase inhibitors raise the possibility that these might increase the effectiveness of interferon-based treatment in the transplant setting, though these studies are undoubtedly some distance in the future.
Another potential way to influence the course of posttransplant HCV infection might be to alter the immunosuppressive regimen. The more rapid progression of HCV recurrence in recent years has been attributed to more potent immunosuppressive drugs that are employed at and around the time of transplantation.[4][5] Indeed, both OKT3 and high-dose corticosteroid pulse therapy used to treat acute cellular rejection have been shown to accelerate HCV recurrence and graft injury.[13][14] However, it does not appear that chronic steroid administration has a similar effect.[5] A pilot study and a more recent large multicenter randomized study have reported that steroid-free immunosuppression does not appear to reduce HCV recurrence in short-term follow-up.[15][16] Neither mycophenolate mofetil nor azathioprine have been shown to have a consistent effect on HCV recurrence.[17][18] Although it has been suggested that the change in preference from cyclosporine to tacrolimus by most liver transplant centers in the late 1990s might explain the apparent rapidity of HCV disease in recent years, the data do not appear to support this. The choice of calcineurin inhibitor has not been shown to significantly influence HCV levels, severity of disease recurrence, or the course of recurrent infection in either prospective or retrospective reports.[5][18-21] Indeed, one might imagine that the higher chance of early rejection with cyclosporine might require more steroid boluses and thereby lead to more rapid progression of HCV disease,[22] though this has not been reported. Despite the absence of clinical data to support one calcineurin inhibitor over another in HCV patients, recent in vitro studies have shown that cyclosporine inhibits HCV replication, independent of interferon induction, in a cell-based replicon model.[23] This has led to renewed interest in studying the potential benefit of primary cyclosporine-based immunosuppression following liver transplantation, particularly in its ability to facilitate the response to antiviral therapy. In this issue, Firpi et al.[24] from the University of Florida confirm the in vitro effect of cyclosporine on HCV replication in the replicon. They also report that patients with HCV recurrence who were receiving cyclosporine were almost twice as likely to clear HCV with interferon-based therapy as those receiving tacrolimus. These results are intriguing, but are preliminary and must be interpreted with caution. First, previous studies, including a prior report from the same authors, have not shown such a difference in responses to antivirals or have found low responses in cyclosporine treated patients.[10-12][25-28] However, the studies are highly selected and any difference might have been masked by this and by the high proportion of patients who discontinue therapy. Furthermore, many studies have not looked for a potential effect of the immunosuppressive regimen on treatment response.[29-31] Second, the current study was retrospective and the cyclosporine and tacrolimus groups were neither selected nor treated concurrently.[24] Third, there are some clinically significant differences in the treatment groups that make interpretation of these results difficult. Nonetheless, these results deserve attention and need to be addressed in a prospective study.
Hepatitis C is the major indication for liver transplantation today and it will become even more common in the future. Recurrent chronic hepatitis C is a frequent and serious problem after transplant, causes significant morbidity, and results in graft loss in about 10% of patients. Effective treatment of HCV infection in these patients is extremely difficult and time-consuming, and not often effective in altering the course of recurrent disease. The need for more a more effective way to prevent reinfection of the graft or to at least reduce progression of liver injury is absolutely critical. Prospective studies are urgently needed to optimize donor selection to minimize the severity of recurrence, delineate the best tolerated immunosuppressive regimens, clarify the timing and role of antiviral therapy, and investigate new therapies such as hyperimmune anti-HCV immune globulin, HCV protease or polymerase inhibitors, and antifibrotic agents. We have a long way to go.

Cyclosporine suppresses hepatitis C virus in vitro and increases the chance of a sustained virological response after liver transplantation
Liver Transplantation
Volume 12, Issue 1, Pages 51-57
Jan 2006
Roberto J. Firpi 1 *, Haizhen Zhu 2, Giuseppe Morelli 1, Manal F. Abdelmalek 1, Consuelo Soldevila-Pico 1, Victor I. Machicao 1, Roniel Cabrera 1, Alan I. Reed 3, Chen Liu 2, David R. Nelson 1 1Division of Gastroenterology, Hepatology and Nutrition Section of Hepatobiliary Diseases, University of Florida, Gainesville, FL 2Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 3Department of Surgery, University of Florida, Gainesville, FL
Cyclosporine is an immunosuppressive agent widely used in the management of liver transplant recipients. Cyclosporine has been shown to have antiviral activities against HIV, herpes simplex, and vaccinia viruses. The aim of this study was to determine the effect of Cyclosporine in viral clearance in the liver transplant recipients during therapy with combination of interferon and ribavirin, and to determine the anti-viral potential of Cyclosporine in vitro. Immunosuppression consisted of either Cyclosporine or Tacrolimus-based therapy. Both groups received therapy with interferon and ribavirin for 48 weeks when evidence of progressive histologic disease was determined. We found that subjects on Cyclosporine-based immunosuppression (n = 56) had a higher sustained virological response of 46% compared to 27% in the patients on Tacrolimus-based therapy (n=59, P = 0.03). In vitro studies were performed to evaluate the antiviral effect of Cyclosporine in the replicon system. These studies showed that Cyclosporine inhibits hepatitis C viral replication in a dose-dependent manner. Combination of Cyclosporine with interferon showed additive effect, and its function is independent of interferon signaling pathways. In conclusion, Cyclosporine may offer an advantage to Tacrolimus in those patients undergoing interferon-based therapy and should be studied in a prospective randomized trial.
Viral Response
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.
Article Text
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.
Interferon-Based therapy
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 ug 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 ug 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/uL, the interferon was reduced to 1.5 MU three times a week/PEG interferon to 135 g/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.
End Point
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.
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.
In-Vitro Analysis
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-a, followed by real time PCR analysis. As shown in Figure 3, addition of CsA and IFN- 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- 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.
In-Vivo Analysis
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.
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