Charles T. Van Buren

Immunosuppressive effects and safety of a sirolimus/cyclosporine combination regimen for renal transplantation

Background. Sirolimus, a novel immunosuppressant that inhibits cytokine-driven cell proliferation and maturation, prolongs allograft survival in animal models. After a phase I trial in stable renal transplant recipients documented that cyclosporine and sirolimus have few overlapping toxicities, we conducted an open-label, single-center, phase I/II dose-escalation trial to examine the safety and efficacy of this drug combination. Methods. Forty mismatched living-donor renal transplant recipients were sequentially assigned to receive escalating initial doses of sirolimus (0.5-7.0 mg/m 2 /day), in addition to courses of prednisone and a concentration-controlled regimen of cyclosporine. We conducted surveillance for drug-induced side effects among sirolimus-treated patients and compared their incidence of acute rejection episodes as well as mean laboratory values with those of a historical cohort of 65 consecutive, immediately precedent, demographically similar recipients treated with the same concentration-controlled regimen of cyclosporine and tapering doses of prednisone. Results. The addition of sirolimus reduced the overall incidence of acute allograft rejection episodes to 7.5% from 32% in the immediately precedent cyclosporine/prednisone-treated patients. At 18- to 47-month follow-up periods, both treatment groups displayed similar rates of patient and graft survival, as well as morbid complications. Although sirolimus-treated patients displayed comparatively lower platelet and white blood cell counts and higher levels of serum cholesterol and triglycerides, sirolimus did not augment the nephrotoxic or hypertensive proclivities of cyclosporine. The degree of change in the laboratory values was more directly associated with whole blood trough drug concentrations than with doses of sirolimus. Conclusions. Sirolimus potentiates the immunosuppressive effects of a cyclosporine-based regimen by reducing the rate of acute rejection episodes.

The Presence of Cyclosporine in Body Tissues and Fluids During Pregnancy

A term pregnancy, labor, and delivery is reported in a cyclosporine- and prednisone-treated female cadaveric renal allograft recipient. A male child, small for gestational age at 2370 g, was born at 38 weeks of gestation with neither congenital anomalies nor nephrotoxicity or hepatotoxicity. Cyclosporine (CSA) concentrations as determined by a radioimmunoassay are reported in maternal and fetal tissues. CSA is present in the fetal circulation during gestation at similar concentrations to those in the mother. Fetal serum at birth displayed 25% suppression of a third-party mixed lymphocyte culture (MLC) compared with control incubations. CSA was present in maternal breast milk; therefore, breastfeeding of children by CSA-treated mothers should be avoided.

Low Incidence of Malignancy among Sirolimus/ Cyclosporine-Treated Renal Transplant Recipients

Background. Malignancies, a well-known complication of immunosuppressive therapy in renal transplant recipients, represent an important cause of long-term morbidity and mortality. One approach to addressing this problem is identifying agents that display antineoplastic properties concomitant with their immunosuppressive effects. Methods. We examined the neoplasms among 1008 renal transplant recipients treated at a single center with sirolimus-cyclosporine ± prednisone. Results. Clinical and laboratory data, including 62.3±26.1 months follow-up (range 27.1–131), revealed 36 tumors in 35 patients (3.6%) presenting at 32.5±29.8 months. The 2.4% incidence of skin tumors, the most common neoplasms, was 1.58-fold greater than the general U.S. population. In addition to a 0.4% incidence of posttransplant lymphoproliferative disorders (PTLD) and a 0.2% incidence of renal cell carcinomas, we observed single cases of breast, bladder, endometrial, lung, and brain neoplasms as well as leukemia. The mean trough drug concentrations at the time of diagnosis in affected recipients were within our putative target ranges. In addition to eleven graft losses due to death with a functioning kidney, two were related to chronic rejection following reduced immunosuppression, and one, therapeutic nephrectomy for PTLD. Five of twelve deaths were caused by malignancies; four others among 1008 patients over the entire follow-up were attributed to cardiovascular events; one, to respiratory failure; and two, at distant locations to unknown causes. Conclusions. The sirolimus-cyclosporine ± prednisone combination appears likely to be associated with a reduced incidence of tumors.

Effect of Nucleotide Restriction and Supplementation on Resistance to Experimental Murine Candidiasis

The influence of dietary nucleotides on susceptibility to candidiasis in mice was studied using two criteria: animal survival and recovery of viable Candida albicans organisms from the kidney and spleen. One-month-old mice were placed on one of five diets with varying nucleotide content. The results show that mice maintained on a nucleotide-free diet (NF) exhibit a significantly decreased mean survival time and a significantly increased viable organism recovery in the spleen following intravenous injection of graded inocula of C. albicans compared to mice fed diets containing RNA or uracil as a nucleotide source.

Dietary nucleotides, a requirement for helper/inducer T lymphocytes.

Previous investigations have revealed that dietary nucleotide restriction delays the onset of primary murine cardiac allograft rejection and acute graft-versus-host disease followed H-2-incompatible bone marrow transplantation, suppresses sensitization to intradermally injected antigens and suppresses in vivo and in vitro lymphocyte proliferation to alloantigen or lectin stimulation. To determine the mechanisms responsible for these phenomena, BALB/c mice were placed on chow (F), nucleotide free (NF) diet, or NF diet supplemented with 0.25% RNA (NFR), with 0.6% adenine (NFA), or with 0.06% uracil (NFU). Following four weeks of dietary equilibrium, splenic lymphocytes harvested from naive or immunostimulated mice in the various dietary groups were stained with monoclonal antibodies directed Lyt 1, Lyt 2, 3, or surface mouse immunoglobulin (IgG) surface markers. While naive animals demonstrated no differences in lymphocyte subpopulations between groups, following complete Freunds adjuvant (CFA) stimulation, splenic lymphocytes for NF mice demonstrated 27.3\pm1.7% Lyt 1+ cells compared with F (32.6\pm.04%) and NFR mice (33.2\pm1.2%) (P<0.02). Restriction of dietary nucleotides affected not only phenotypes of T lymphocytes, but also T cell function. Following conconavalin A stimulation of irradiated splenic lymphocytes, IL-2 production was decreased in NF mice compared with the F control group (P<0.01). The RNA-repleted diet maintained normal IL-2 production, while addition of adenine or uracil alone did not. Finally, NF diets adversely affected host resistance to the opportunistic pathogen Candida albicans. Following inoculation with 0.25x106 organisms NF or NFA-fed hosts succumbed more rapidly than F, NFR, or NFU fed hosts (P<0.001). These data suggest that helper/inducer T lymphocytes require exogenous nucleotides to respond normally following immune stimulation. Uracil may be the critical substrate, based upon the studies of Candida resistance. By understanding the metabolic basis of NFD-induced immunosuppression, the role of dietary nucleotides in combatting infection and alloantigen rejection can be more clearly defined.

Immunopharmacological monitoring of cyclosporin A-treated recipients of cadaveric kidney allografts

Twelve cadaveric kidney allograft recipients, who were established preoperatively to be strong responders, were treated with cyclosporin A (Cy A) and subjected to postoperative monitoring of drug levels and immune performances. The Cy A-treated recipients were compared with 72 historical (36 strong and 36 weak immune responders) and 18 current, strong responder, azathioprine-treated control patients. Estimation of Cy A levels in plasma and whole blood revealed that 75% of the drug at trough and 44% at peak was cell bound. Concomitant radioimmunoassay (RIA) and high performance liquid chromatography (HPLC) determinations on whole blood yielded concordant values. Trough levels above 200 ng/ ml in plasma and 600 ng/ml in whole blood were associated with toxic manifestations. Although absolute peak levels were not helpful, calculation of peak to trough ratios yielded values which when less than 3.0 predicted toxicity. Post-transplant immune monitoring showed administration of Cy A to be associated with fewer (1) rejection episodes; (2) nonspecific immune events; and (3) donor-specific in vitro reactions than were observed after treatment with azathioprine. Although the activity of peripheral blood mononuclear cells as natural killers of K562 target cells was not affected by Cy A treatment, their capacity to suppress the generation of a third-party mixed lymphocyte culture was enhanced to the same degree as cells from azathioprine-treated patients. Enumeration of peripheral blood lymphocyte T cell subpopulations using monoclonal xeonoantisera revealed (1) the total number of T cells to be unaffected by administration of either Cy A or azathioprine and (2) a reduction in the ratio of helper-inducer to suppressor-cytotoxic cells specificially in Cy A-treated recipients compared with normal individuals, hemodialysis patients, or azathioprine-treated recipients. Although pharmacological monitoring of blood levels may be useful to discern patients at high risk for toxic complications, the achievement of maximal therapeutic efficacy may depend upon identifying and quantitating the cellular target responsible for the disruption of immune homeostasis observed during Cy A administration.

Improved graft survival for flow cytometry and antihuman globulin crossmatch-negative retransplant recipients.

We compared our standard NIH (extended incubation) crossmatch (XM) with antihuman globulin (AHG) and flow cytometry XMs and correlated the results with rejection episodes and graft survivals. For 89 CsA-Pred, primary renal allograft recipients, AHG and/or FCXM results did not improve on the NIH-XM-negative (NEG) graft survival results, whether testing pretransplant or historical (Hx) sera. Similarly, there was no association of a positive (POS) AHG or FCXM with increased rejection episodes in these primary recipients. However, for retransplant (Re-Tx) recipients a neg AHG or FCXM did discriminate fewer rejections and an improved graft survival compared with the NIH-XM-neg. results. The overall one-year graft survival for the 47 Re-Tx recipients studied herein was 66% (based on a neg pre-Tx NIH-XM). Pre-Tx AHG-NEG, Re-Tx recipients displayed an improved graft survival compared with NIH-XM NEG recipients (77% vs. 66%, P less than 0.05) and with AHG-POS recipients (77% vs. 47%, P less than 0.05). Similarly, pre-Tx, FCXM-NEG, Re-Tx recipients displayed improved graft survivals compared with NIH-XM-NEG recipients (83% vs. 66%, P less than 0.05) and FCXM-POS recipients (83% vs. 48%, P less than 0.05). Re-Tx recipients displaying a POS AHG and/or FCXM experienced a significantly greater number of rejections than NEG-XM recipients (P less than 0.05, respectively). The AHG and FCXM results correlated with rejections and graft survivals whether testing pre-Tx or Hx high-PRA sera. Re-Tx recipients who were AHG-XM-NEG but FCXM-POS, experienced more rejection episodes than recipients who displayed a negative XM reactivity for both AHG and FCXM (P less than 0.02), but with no resulting differences in graft survival. HLA matching, pre-Tx blood transfusions and PRA did not impact on these crossmatch and graft survival results. Use of AHG and/or FCXMs for Re-Tx, but not primary, recipients should help to improve graft survival for these high-risk recipients.

Risk factors for impaired wound healing in sirolimus‐treated renal transplant recipients

Abstract:  Aim:  As sirolimus has been implicated in impaired wound healing, the aim of this study was to evaluate risk factors for wound complications after renal transplantation in patients treated with this drug de novo.

Correlation of ELISA-detected IgG and IgA anti-HLA antibodies in pretransplant sera with renal allograft rejection.

The present study compared the occurrence of rejection episodes during the first twelve posttransplant (Tx) months and the 1-, 2-, and 3-year graft survivals among recipients stratified by the percent panel reactive antibody (% PRA) of pre-Tx sera as detected using either an antihuman globulin determined PRA (AHG-% PRA) or an ELISA methodology detecting IgG reactive against soluble HLA class I antigens (% PRA-STAT). There was a significant correlation between AHG-PRA greater than or equal to 10% and a PRA-STAT greater than or equal to 10% (P<0.001). However, among 200 sera displaying an AHG-PRA greater than or equal to 10% (mean 57 +/- 2l%), only 69% (138/200) displayed a PRA-STAT greater than or equal to 10%. With further study the discrepant finding, of 62 sera that were AHG-PRA greater than or equal to 10% but PRA-STAT <10%, was due to the presence of IgM and/or IgG non-MHC reactivity. In contrast, among 293 sera displaying an AHG-PRA < 100% (mean 3 +/- 2%), 15% (43/293) displayed a PRA-STAT greater than or equal to 10%. There was no correlation between AHG-% PRA and rejection episodes occurring during the first twelve post Tx months. In contrast, however, there was a highly significant correlation between PRA-STAT greater than or equal to 10% and the occurrence of rejection episodes during the first twelve post-Tx months (P < 0.001). Patients with PRA-STAT greater than of equal to 10% experienced a 70% rejection frequency compared with the 35% rejection frequency for patients with PRA-STAT sera < 10% (P<0.001). A significant correlation was observed between the presence of IgG-1 and rejection (P<0.01) but not IgG-subclasses 2, 3, or 4. Of particular interest was the observation in 11 patients that the presence of ELISA-detected IgA anti-HLA class I antigen (ELISA-IgA PRA greater than or equal to 10%) was associated with a significantly reduced rejection risk compared with sera where only PRA-STAT greater than or equal to 10% was present (27% vs. 70% incidence of rejection episodes, P<0.01). Finally, patients displaying pretransplant PRA-STAT results < 10% experienced significantly improved l-, 2-, and 3- year graft survivals of 85% vs. 74%, 82% vs. 70% and 81% vs. 67%, respectively (P<0.01 for each time point), compared with patients displaying PRA-STAT results greater than or equal to 10%. These data suggest that the use of the ELISA methodology to detect IgG reactivity against soluble HLA class I antigens (PRA-STAT) may allow for the determination of a more clinically informative % PRA than the AHG-% PRA. Moreover, the presence of ELISA-detected IgA anti-HLA may act to inhibit rejection mechanisms associated with ELISA-detected IgG anti-HLA greater than or equal to 10%.

De novo hemolytic uremic syndrome after kidney transplantation in patients treated with cyclosporine-sirolimus combination.

Objective. We sought to examine factors that predisposed 1.5% (10/672) of renal transplant recipients treated with a cyclosporine (CsA)/sirolimus (SRL)/steroid immunosuppressive regimen to develop hemolytic uremic syndrome (HUS). Methods. Two cohorts of recipients were treated for 1–212 months (mean: 25.0±26.4, median: 18.1) with concentration-control CsA regimens based upon either area under the concentration-time curve (AUC; n=412 patients) or trough measurements (C0; n=260 patients). Results. The only demographic feature more common to affected patients was an original glomerulopathic disease in 7 patients, 4 of whom had displayed IgA glomerulonephritis. All 10 affected patients showed a clinical picture of hemolysis with schistocytes, thrombocytopenia (nadir: 35,000±19,600 platelets/mm3), as well as elevated serum levels of lactate dehydrogenase (1697±1427 IU) and creatinine (Scr; 2.05±1.52 mg/dL prediagnosis to 5.13±2.43 mg/dL at diagnosis). Seven patients experienced adverse events concomitant with the bout of HUS, namely, acute rejection episodes prior to (n=2) or during (n=3), and 2 patients, infections (Herpes simplex and pancolitis). The mean values of daily steroid dose and the immunosuppressive drug C0 values were above the putative therapeutic targets: namely, CsA C0=294.9±153.2 ng/ml versus 150±50 ng/ml and SRL C0=20.1±14.0 ng/ml versus 10±5 ng/ml, respectively. The therapeutic approach included discontinuation of CsA in 9/10, which was transient in 6/9; discontinuation of SRL in all 10, which was transient in 3, OKT3 for concurrent rejection in 3, and plasmapheresis in 5 patients. At 24 weeks postdiagnosis 9/10 patients have well-functioning kidneys with a mean Scr value of 1.6±0.59 mg/dL. One patient who underwent transplant nephrectomy subsequently succumbed due to a cluster of refractory thrombocytopenia, Aspergillus infection, and multiorgan failure. Conclusion. This initial experience suggests that a time-limited and reversible de novo HUS syndrome may be less frequent and milder among renal transplant recipients treated with SRL-based immunosuppression.