Jean-Marc Cisterne

Longitudinal Analysis of Hepatitis C Virus Replication and Liver Fibrosis Progression in Renal Transplant Recipients

The pathogenesis of hepatitis C virus (HCV) infection was investigated by analysis of changes in viral and histologic parameters in 36 renal transplant recipients who were infected with HCV before transplantation. Each patient was classified according to development of liver fibrosis as assessed by 2 liver biopsies done 45 and 81 months after transplantation: 13 had progressing liver fibrosis (fibrosers) and 23 did not (nonfibrosers). All developed high-titer posttransplant viremia with a significant increase of 1.2 log RNA copies/mL. There were no significant differences in the increases in serum HCV RNA or genotype distributions in fibrosers and nonfibrosers. The hypervariable region (HVR)-1 of the HCV genome was analyzed by cloning and sequencing 20 clones per sample from 5 fibrosers and 5 nonfibrosers. Comparison of samples revealed that liver fibrosis progression was significantly associated with slower HVR-1 quasispecies diversification, suggesting the selection of more aggressive variants in fibrosers.

Changes in hepatitis C virus RNA viremia concentrations in long-term renal transplant patients after introduction of mycophenolate mofetil.

BACKGROUND Mycophenolate mofetil (MMF) is a potent immunosuppressive agent and might inhibit chronic rejection, at least in primates. The prevalence of chronic hepatitis C virus (HCV) infection is high in renal transplant (RT) patients. To date, it has not been demonstrated whether MMF has any effect upon HCV viremia. METHODS Fourteen long-term HCV(+) RT patients with chronic allograft dysfunction whose maintenance immunosuppression was based on cyclosporine, were given MMF therapy either in place of azathioprine (n=11) or in addition to baseline therapy (n=3). HCV viremia levels were measured by the Amplicor HCV-Monitor RT-PCR assay (Roche Diagnostic Systems) on two separate occasions before the introduction of MMF, and 1 year after changing to MMF or at the last follow-up visit. RESULTS MMF therapy was associated with a significant rise in HCV viremia, i.e., 5.8+/-0.5 vs. 5.2+/-0.7 log copies/ml (P=0.01), although there were no significant changes in liver enzymes. The increase in HCV viremia was not related to HCV genotypes either. At the patient level, HCV RNA concentrations changed in only seven patients (group B), i.e. >1 log copies/ml, whereas it remained stable in the others (group A). Before conversion, the only significant difference between group A and B was the level of HCV RNA, i.e., 5.5+/-0.4 log copies/ml in group A and 4.9+/-0.7 log copies/ml in group B (P=0.05). CONCLUSION Our study suggests that MMF should be used with caution in stable HCV RT patients whose maintenance immunosuppressive therapy is based on cyclosporine, at least in the case of patients with a low HCV RNA titer.

Outcome of patients with antineutrophil cytoplasmic autoantibody—associated vasculitis following cadaveric kidney transplantation

In this retrospective study, we report on the progress of eight patients with biopsy-proven systemic vasculitis following cadaveric renal transplantation. Extrarenal manifestations associated with antineutrophil cytoplasmic autoantibodies (ANCAs) were present in all but one case. All the patients were on cyclosporine A-based immunosuppression, and none of them had active disease at transplantation. The mean time from the last episode of vasculitis to transplantation was 46 months (range, 10 to 132 months). On the day of transplantation, all but one patient had detectable ANCAs, which ranged from 1:100 to 1:2,000 (perinuclear ANCAs [P-ANCAs], four patients; cytoplasmic ANCAs [C-ANCAs], three patients). There were three patients with Wegeners granulomatosis, none of whom relapsed. Of those patients with microscopic polyangiitis (P-ANCAs, three patients; C-ANCAs, two patients), only one presented with relapse episodes after transplantation; the episodes involved the lungs and the kidney graft, and were successfully treated by methylprednisolone pulses. This patient had the highest ANCA titer before (1:2,000) and after (up to 1:10,000) grafting, and received no immunosuppression prior to transplantation since vasculitis was diagnosed after she had developed end-stage renal failure. This study shows that a relapse of ANCA-associated vasculitis following successful cadaveric renal transplantation occurs infrequently in the cyclosporine A era (ie, 12%), despite the persistence of circulating ANCAs in these patients at the time of transplantation, and even afterward in some cases.

Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients.

Recent multicenter, randomized clinical trials have shown that in renal transplant patients tacrolimus (FK506) was more efficient than cyclosporine A (CsA) at preventing acute rejection. In order to try and evaluate whether this difference was related to a different in vivo T‐cell suppression we assessed, in a prospective study, the frequencies of interleukin (IL)‐2‐, IL‐4‐, IL‐5‐, IL‐6‐, IL‐10‐, interferon‐gamma (IFN‐Γ)‐ and double‐positive IL‐2/IFN‐Γ‐producing whole T cells, CD4+ and CD8+ T‐cell subsets by means of cytokine flow cytometry. This was performed after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol myristate acetate (PMA) and ionomycin, in the presence of monensin, in 14 healthy volunteers (controls) and in 14 renal transplant patients. The immunosuppression of the latter was based either on CsA (n=7) or on FK506 (n=7). Cytokine‐expressing T‐cell frequencies were assessed immediately pre‐transplantation (D0), and subsequently 3 months (M3) and 6 months (M6) afterwards in fasting patients prior to the morning intake of the immunosuppressive drug. We found that at D0 the frequencies of IL‐2‐ (22±2% vs. 22.2±2%), IFN‐Γ‐ (26±3% vs. 29±3.4%) and IL‐4‐ (0.8±0.2% vs. 1.4±0.2%)‐expressing T lymphocytes were not significantly different between the controls and the patients, respectively. Conversely, the frequency of IL‐2/IFN‐Γ double positive cells was higher in the latter (9.3±1.6%) than in the controls (5.6±0.8); p=0.06. Finally, on D0 the frequencies of IL‐5‐, IL‐6‐, and IL‐10‐producing T lymphocytes were lower than 1% in both groups, as well as after grafting, i.e. on M3 and M6. As compared to baseline (D0): (a) chronic immunosuppression significantly decreased the frequencies of IL‐2‐, IL‐4‐ and IL‐2/IFN‐Γ‐expressing T cells, whereas those of IFN‐Γ, IL‐5, IL‐6, and IL‐10 were not significantly affected; (b) the frequencies of cytokine‐expressing T cells were not statistically different between M3 and M6; (c) the decrease in the frequencies of IL‐2‐ and IL‐2/IFN‐Γ‐expressing T cells affected CD4+ and CD8+ cells equally; (d) there was a marginal decrease in the frequency of IFN‐Γ‐expressing cells only in the CD4+ subset but not in the CD8 population; and (e) for CsA, but not for FK506, the frequency of the IL‐2‐expressing T cells was negatively correlated with the whole blood trough levels. When we compared the frequencies of cytokine‐expressing cells in FK506‐ and CsA‐treated patients, we found that the frequency of IL‐2‐expressing T cells was significantly lower with FK506 (10.9±1.6%) than with CsA (16.3±1.8%; p=0.03), whereas the frequencies of the other cytokine‐expressing cells were not statistically different between the two groups. In conclusion, our study clearly demonstrated that studied ex vivo, FK506 and CsA decrease the frequencies of cells expressing IL‐2, IL‐4 and IL‐2/IFN‐Γin vivo but do not affect those expressing IFN‐Γ. Meanwhile, the frequency of IL‐2‐producing T cells was more affected with FK506 than with CsA and was negatively correlated with the CsA trough level. Finally, our results regarding IL‐2 might explain to some extent the higher efficiency of FK506 in vivo than CsA.

Impact of hepatitis C virus duration and hepatitis C virus genotypes on renal transplant patients: correlation with clinicopathological features.

BACKGROUND The aim of this study was to evaluate the long-term impact of chronic hepatitis C virus (HCV) infection on the liver in renal transplant patients. METHODS We studied 78 patients for whom at least one posttransplant liver biopsy (LB) was available and for whom the duration of HCV infection was precisely defined. The LB were graded according to a histological activity index, i.e., the Knodell score, divided into the activity score and the fibrosis score. They were also classified as either normal or showing evidence of chronic persistent hepatitis, chronic active hepatitis (CAH), or cirrhosis. RESULTS The study comprised 7 HCV-positive/hepatitis B surface antigen-positive patients (group 1); 4 HCV-positive/RNA-negative patients (group 2); and 67 HCV-positive/RNA-positive patients (group 3). The three groups were comparable according to demographic data and baseline immunosuppression. The median time from transplantation to LB was 38 months (range, 10-306 months). At that time, alanine aminotransferase (ALT) levels had increased in 71.4%, 0%, and 42% of patients from groups 1, 2, and 3, respectively (P=0.07). The total Knodell score showed significantly more severe lesions in group 1 patients (6.2+/-3.2) than in group 2 (1+/-1.2) or in group 3 (4.6+/-2.4) patients (P=0.007). The Knodell score also showed that the fibrosis score was significantly higher in group 1 (2.3+/-1.6) than in group 2 (0) or in group 3 (0.9+/-1.1) patients (P=0.007). Overall, there were 28 cases of CAH (36%) and 4 cases of cirrhosis (5%). We did not observe any correlation between liver histology and characteristics of HCV infection or the type of chronic immunosuppression (double-drug versus triple-drug therapy). However, liver histology (total Knodell score) and the activity score were significantly correlated with ALT levels. Multivariate analysis did identify (i) four independent variables that could explain the degree of liver fibrosis-the sex of the patient, the number of blood units received before transplantation, increased ALT levels at the time of LB, and the occurrence of at least one acute rejection episode (thus the receipt of methylprednisolone pulses); and (ii) two independent variables associated with the occurrence of CAH-the number of blood units before transplantation and increased ALT levels at the time of LB. CONCLUSION This study showed that renal transplant patients infected by HCV for more than 10 years, with a mean posttransplant follow-up of more than 5 years, showed more severe liver lesions when coinfected by hepatitis B virus. Overall, we observed only four cases of cirrhosis (5%) and evidence of histological CAH lesions in 36% of the patients.

Long-term impact of superinfection by hepatitis G virus in hepatitis C virus-positive renal transplant patients.

BACKGROUND The hepatitis G virus (HGV) has been recently cloned. Studies in immunocompetent patients have shown that HGV superinfection in hepatitis C virus (HCV)-positive patients does not affect (i) clinical presentation, HCV RNA level, or response to interferon-alpha therapy; or (ii) the histopathologic severity and characteristics of chronic hepatitis. No data are currently available on the impact of HGV infection on liver histology of renal transplant (RT) patients although the reported prevalence of serum HGV RNA in this population is high, ranging from 14% to 55%. PATIENTS AND METHODS We determined the prevalence of HGV infection in 103 HCV-positive RT patients for whom HGV RNA was retrospectively determined by reverse transcription-polymerase chain reaction before, at the time of, and after transplantation (last follow-up). We evaluated the impact of HGV on liver function tests, liver histology (by means of the Knodell score), and renal parameters such as the prevalence of acute rejection and renal function. RESULTS A total of 29 (28%) of the HCV-positive RT patients had a positive HGV RNA (group 1). The mean duration of HGV infection was at least 119+/-64 months (range: 18-240 months). Group 1 patients were compared to the 74 HGV RNA-negative/HCV-positive RT patients (group 2). Liver histology showed a significantly lower degree of fibrosis in group 1 (0.4+/-0.5) than in group 2 (1+/-1.2; P=0.02); two patients from group 2 but none from group 1 had overt cirrhosis. Conversely, the extent of hepatic inflammation and hepatocellular destruction was not statistically different between the two groups. The number of patients who experienced at least one acute rejection episode was significantly higher in group 1 (69%) than in group 2 (42%; P=0.01). However, the multivariate analysis did not identify the presence of HGV RNA at the time of renal transplantation as an independent factor of acute rejection; conversely, (i) the occurrence of cytomegalovirus infection or disease and (ii) the duration of HCV infection significantly increased the likelihood of having acute rejection. CONCLUSIONS This study shows that: (i) HGV infection was often present when the patients seroconverted for HCV, (ii) HGV RNA-positive/HCV-positive RT patients experienced acute rejection more frequently than HGV RNA-negative/HCV-positive RT patients, and (iii) HGV infection seems to have no detrimental effect upon liver enzymes or liver histology in HCV-positive RT patients.

CMV Prophylaxis in High-Risk Renal Transplant Patients (D+/R-) by Acyclovir with or without Hyperimmune (CMV) Immunoglobulins: A Prospective Study

In this prospective randomized study including 28 patients, we show that, in cytomegalovirus (CMV)-seronegative renal transplant recipients (R-) receiving a CMV-seropositive graft (D+), high doses of acyclovir (ACV, i.e. 3,200 mg/day) during the first 3 months after transplantation were as efficient as hyperimmune CMV immunoglobulins (CMV Igs) plus high doses of ACV regarding the prophylaxis of CMV primoinfection. Fifty-four percent of the patients in the ACV arm and 50% in the other arm presented at least one episode of viremia (n.s.). The incidence of CMV disease was 31% in the ACV group and 20% in the ACV + CMV Ig group (n.s.). By comparison with historical controls (no prophylaxis), we found that ACV with or without CMV Ig significantly delayed and significantly decreased the rate of CMV disease, although the severity score was not statistically different. Moreover, high doses of ACV were far less expensive than their combination with hyperimmune CMV Igs. Thus, until oral ganciclovir is available for the prophylaxis of primary CMV infection in renal transplant patients, we recommend the use of high doses of ACV for the first 3 months after transplantation in high-risk renal transplant patients, i.e. D+/R-.

Serological Markers of Autoimmunity in Renal Transplant Patients before and after Alpha-interferon Therapy for Chronic Hepatitis C

Chronic hepatitis C is the major cause of chronic liver disease after successful cadaveric renal transplantation. The aims of this prospective, open study were to assess in such a population, firstly the prevalence of different organ-specific and nonspecific antibodies and related disorders, and secondly their outcome after inteferon-alpha therapy as well as the incidence of new immunologic disorders under and after this therapy. In 15 cadaveric renal transplant patients (10 men, 5 women, ages 29-65 years) with chronic hepatitis C and histological features of chronic active hepatitis, undergoing chronic immunosuppression (ciclosporine A with or without steroid and azathioprine) and treated with recombinant alpha 2b-interferon (IFN alpha) (mean duration 142 +/- 35 days), we assessed before and after this therapy the serum levels of cryoglobulinemia, rheumatoid factors (RF), thyroid-stimulating hormone (TSH), free thyroxine (fT4), and antinuclear (ANA), antismooth muscle (ASMA), antimitochondrial (AMA), anti-LKM1, antimicrosomal thyroid (MCA), antithyroglobulin (TGA) autoantibodies. At the start of IFN alpha therapy, 14 of 15 patients had detectable autoantibodies (RF: 9; ANA > 1/50: 8; ASMA > 1/50: 4; other autoantibodies: 0); 1 had cryoglobulinemia. At the end of therapy the cryoglobulinemia had disappeared, the preexisting autoantibodies remained present in all patients but 2; 3 patients had developed MCA without evidence of clinical or biological thyroid abnormalities and 3 others had developed either RF (1) or ANA (1) or ASMA (1), without any related symptoms. One patient developed transient type II diabetes mellitus without anti-Langerhans beta-cell antibodies. Finally, the occurrence of autoantibodies in our patients was associated either with HLA DR3 or DR4 or DR7 phenotypes. We found that the prevalence of extrahepatic immunologic abnormalities was high in renal transplant patients with chronic hepatitis C and no exacerbation was observed during of after IFN alpha therapy. The most frequent autoantibody appearing after IFN alpha therapy was MCA although without thyroid abnormalities.

Treatment of Chronic Hepatitis B and C with Alpha Interferon in a Renal Transplant Patient

We report the case of a 51-year-old renal transplant patient, treated by interferon alpha (5MUI, three times a week) since he presented a coinfection by hepatitis B (HBV) and hepatitis C (HCV) virus for more than 7 years, associated with a chronic increase in serum alanine aminotransferase (ALT) levels and a chronic active hepatitis. The 4-month treatment was associated with a sustained normalization of ALT, a disappearance of HBV replication and a transient clearance of HCV viremia. Side effects were moderate and included thrombopenia (90,000/mm3), leucopenia (2200/mm3), an increase in serum creatinine (178 mumol/l). The withdrawal of alpha interferon was associated with the correction of these parameters. No rejection was observed on kidney biopsy. Meanwhile, liver histology was not affected by the treatment. To date, nineteen months after the end of alpha interferon therapy HBV DNA was still negative; ALT remained normal despite the early recurrence of HCV viremia; this emphasized the fact that HBV infection was certainly the most important factor involved in the patients chronic hepatitis. It is concluded that alpha interferon therapy is able to decline HBV replication for a prolonged period in renal transplant patient although its use should be performed with caution due to the potential renal side effects.

Predicting Factors of Long-Term Results of OKT3 Therapy for Steroid Resistant Acute Rejection following Cadaveric Renal Transplantation

In this retrospective study, we evaluated the histological and biological predictors of long-term response of renal transplant (RT) patients treated with orthoclone OKT3 for steroid resistant acute rejection (AR). Seventy-three patients, aged 37 ± 12 years, were included in this study between March 1987 and December 1996. All the patients but one had received sequential quadruple immunosuppression (polyclonal antilymphocyte globulins; steroids; azathioprine, and cyclosporin A). OKT3 (5 mg/day for 10 days) was administered for biopsy-proven steroid resistant AR i.e., after 3 consecutive pulses of methylprednisolone (10 mg/kg each). This was the first AR in 46 cases, the second AR in 22 cases and the third AR in 4 cases. Renal histology (Banff) showed borderline (BL) changes in 18 patients, grade I AR in 28 patients; grade II AR in 22 patients, and grade III AR in 5 patients. When treatment with OKT3 commenced (107 ± 18 days post-transplantation) the mean serum creatinine (SCr) level was 325 ± 195 μmol/l; this had decreased to 191 ± 106 μmol/l by the end of OKT3 therapy. The immediate response to OKT3 therapy i.e., within the first month, was not dependent on the histological score. Twenty-six patients (35%) subsequently experienced at least one more AR episode of whom 4 were retreated with OKT3. The overall patient’s survival was 94.5% at last follow-up. The overall cumulative graft survival was 64.5% at 2 years, 52.5% at 5 years, and 40.5% at 8 years. The graft survival (5 years) tended to depend on the initial histological score, i.e. BL 30%; grade I 66%; grades II and III 55.5% (p = 0.08). In a multiple logistic regression analysis we tried to identify independent factors that would predict that a graft would still be functioning at least 2 years after OKT3 therapy. We therefore analyzed the following parameters: donor and recipient’s age; gender; cold ischemia time; HLA matching; panel reactive antibodies (PRA) prior to grafting; previous transplantation(s); total number of AR episodes; the time of onset of the AR treated by OKT3 compared to the other AR; the time of onset of the AR treated by OKT3; SCr levels at days 0, 10 and 30 after OKT3 therapy; histological score (Banff) i.e., the magnitude of AR and the presence or absence of chronic lesions. The only independent factors which would predict that a graft was still functioning 2 years after OKT3 therapy were: PRA <25% (Odds ratio (OR) 7.68 (1.15–51.3); p = 0.035); a grade I AR (OR 10.52 (1.18–93.5); p = 0.035); SCr level 1 month after OKT3 therapy (OR 0.935 (0.87–1.002); p = 0.05). HLA matching and the presence of histological chronic lesions were nearly significant (p = 0.06 and 0.09 respectively). In conclusion, this retrospective study shows that independent predictors of the long-term response to OKT3 therapy for AR in RT patients are the magnitude of pre-transplant PRA, the histological score, and the SCr level one month after OKT3 therapy.