Cytokine polymorphisms do not influence acute rejection in renal transplantation under tacrolimus-based immunosuppression

INTRODUCTION: Acute rejection remains an important cause of graft loss after renal transplantation. It has been suggested that cytokine genotyping may play a predictive role in identifying individuals who are at higher risk of acute rejection with a view to individualizing their immunosuppression. The aim of this study was to investigate any possible associations between acute rejection and certain cytokine polymorphisms. METHODS: We genotyped 91 cadaveric renal transplant recipients on tacrolimus-based immunosuppression and 84 of their donors. The cytokine polymorphisms studied were the following: tumor necrosis factor (TNF)-alpha-1032 T/C, TNF-alpha-865 C/A, TNF-alpha-859 G/A, interleukin (IL)1-R1-970 C/T, IL-10 haplotype [-1082, -819, -592], and IL-6-174 C/G. RESULTS: We found no association between any polymorphism and the incidence of acute rejection. This was true for both the recipient and donor population. CONCLUSION: Cytokine polymorphisms did not influence acute rejection in our study. We conclude that in the modern era of immunosuppression cytokine genotyping is not a significant predictor of acute rejection in renal transplantation.     

Steroid sparing in renal transplantation with tacrolimus and mycophenolate mofetil: three-year results

Although renal transplantation with a 7-day steroid-sparing regimen, tacrolimus and mycophenolate, is associated with good short-term outcomes, late allograft dysfunction and failure remain concerns. In this study 101 consecutive patients underwent renal transplantation using this immunosuppressive regimen. In addition, anti-CD25 monoclonal antibody was used in 25 high-risk patients (regrafts, two-antigen human leukocyte antigen (HLA)-DR mismatch or sensitized with anti-HLA panel reactivity >30%). After a median follow-up of 39 months (range 29 to 49), overall patient survival is 98%, with two cardiac deaths. Three other graft losses occurred, one each to early venous thrombosis, polyoma viral nephropathy, and late rejection due to noncompliance. Therefore, overall graft survival is 95%. The acute rejection rate at 6 and 12 months was 19% (no rejection occurred between months 6 and 12). Late rejection was uncommon, with only two further episodes beyond 12 months. Mean creatinine at 12 months was 144 micromol/L and mean estimated glomerular filtration rate (GFR) of 55 mL/min. Graft function was stable at 3 years with a mean creatinine of 142 micromol/L and mean estimated GFR 56 mL/min. During the study, five patients developed posttransplant diabetes mellitus (two cases beyond 12 months). Tissue-invasive cytomegalovirus disease and BK viral nephropathy each occurred in three patients, with all episodes in the first 12 months. Mean weight gain is 3.3 kg and mean blood pressure is 135/81 on an average of 1.5 antihypertensive agents. This steroid-avoidance regimen is associated with excellent medium-term patient and graft outcomes and a low incidence of side effects.     

The prenatal identification of fetal compatibility in neonatal alloimmune thrombocytopenia using amniotic fluid and variable number of tandem repeat (VNTR) analysis

Summary. Most severe episodes of neonatal alloimmune thrombocytopenic purpura (NATP) are caused by antiplatelet alloantibodies against the HPA-la (Pl^A1) antigen. However, half of subsequent fetuses produced from a HPA-la/b father (genotypic frequency 28%) will result in a child who is not affected. Some investigators manage NATP by confirming the fetal platelet phenotype using percutaneous umbilical cord sampling, a procedure that carries a low but real risk of fetal morbidity and mortality. More recently, physicians determine the fetal platelet antigen genotype using DNA derived from amniotic fluid or chorionic villus samples. All therapy is withdrawn for a fetus who genotypes as HPA-lb/b. However, since the fetus is the same genotype as the mother, there can be uncertainty about the origin of the genetic material and thus the validity of the fetal genotype. The inappropriate withdrawal of therapy for a erroneously genotyped fetus could be fatal, and consequently many physicians advocate fetal HPA-1 phenotyping with confirmation using percutaneous umbilical blood sampling. In this report we describe the management of two pregnancies with previously affected infants due to anti-HP A-la alloantibodies. Both husbands were HPA-la/b. For the current pregnancies, amniotic fluid was collected at 20 or 29 weeks of gestation, and the platelet genotype indicated that the fetuses were HPA-lb/b. The fetal origin of the amniotic fluid derived DNA was confirmed by the forensic technique of DNA profiling using variable number of tandem repeat (VNTR) analysis. All therapy was withdrawn, percutaneous umbilical blood sampling was not performed, and both women vaginally delivered healthy non-thrombocytopenic infants. The application of platelet alloantigen genotyping using DNA from amniotic fluid cells identified the HPA-lb/b fetus, and VNTR analysis confirmed that the tissue was fetal derived, thus avoiding the necessity for percutaneous umbilical blood sampling. The use of this approach in patients at risk will avoid additional investigation and treatment in approximately one-seventh of all NATP pregnancies involving the HPA-la antigen.     

Impaired Direct Priming of CD8 T Cells by Donor-Derived Cytomegalovirus Following Kidney Transplantation

Cytomegalovirus (CMV) infection increases the risk of complications after renal transplantation, but the mechanisms controlling donor-derived infection are not adequately characterized. Here, we assessed the risk of clinically significant CMV disease in donor-seropositive, recipient-seropositive (D+R+) renal transplantation and examined recipients’ CMV antigen-specific cellular immune responses primed directly by donor cells. In a retrospective cohort of 569 patients administered standardized basiliximab-tacrolimus-mycophenolate-corticosteroid immunosuppressive therapy, CMV disease rates increased in D+R+ serostatus pairings compared with D−R+ pairings (hazard ratio [HR], 2.61; 95% confidence interval [CI], 1.36 to 5.01; P=0.004) and associated with increased donor-recipient HLA mismatch in the D+R+ group (HR [per class 1 mismatch], 1.43; 95% CI, 1.12 to 1.82]; P=0.02). D+R+ and D+R− transplants in which the donor and recipient differentially expressed at least one HLA class I allele were followed prospectively from the time of transplantation. During the first year after transplantation, four of eight seropositive recipients and one of three seronegative recipients displayed peripheral blood CD8+ T cell responses to CMV presented by recipient-specific HLA. Notably, no recipients mounted responses to CMV presented by donor-specific HLA, despite the detection of CMV antigen expression in all seropositive donor organs examined (n=10), suggesting that the allograft of Class I HLA-mismatched seropositive donors is inaccessible to CD8+ T cell responses. Finally, pretransplant assays of anti-CMV cellular immunity predicted post-transplant CMV replication less accurately in D+R+ pairings than in D−R+ pairings, possibly reflecting in vitro assay specificity for recipient, rather than donor, HLA. These findings are relevant to the clinical management and immunologic understanding of donor-transmitted viral infection.Cytomegalovirus (CMV) infection remains an important complication of kidney transplantation, being associated with increased graft failure rates, morbidity, and mortality. Although the cellular immune response to CMV, especially the CD8+ T cell response, is of primary importance in controlling infection,^1–3 the CMV-specific antibody serostatus of donor (D) and recipient (R) is a useful surrogate to aid in risk stratification because it identifies individuals with latent CMV infection that may subsequently reactivate. High disease rates in primary infection (D+R− transplantation) are well recognized, and requirement for antiviral prophylaxis in this context is largely uncontested. CMV disease is also seen in seropositive recipients (R+) and may occur irrespective of donor serostatus. Seropositive recipients are often grouped together as “intermediate risk,” irrespective of donor serostatus. However, historical evidence suggests that D+R+ transplantation is associated with increased disease rates compared with D−R+ transplantation.⁴ Also, two recent trials^5,6 and a single-center study⁷ showed higher infection rates in D+R+ than in D−R+ transplantation. Because these latter studies focused on (often asymptomatic) CMV infection, rather than symptomatic disease, and because all used antiviral prophylaxis or pre-emptive therapy for CMV infection, it remains unclear whether disease rates differ between D+R+ and D−R+ transplantation, particularly under contemporary immunosuppression without antiviral prophylaxis.After D+R+ transplantation, some CMV cases may result from donor-derived infection, although the mechanism behind this is incompletely understood.^4,8 Although lack of protective immunity to newly infecting strains may be important,⁹ other mechanisms may also play a role. For example, HLA mismatch between donor and recipient may result in failure of “cognate” immunity to control viral infection in donor tissue. In this regard, clinical data are conflicting, with studies from the 1980s and 1990s suggesting increased CMV disease rates with increased HLA class II mismatch^10,11 but others suggesting reduced rates in this setting.^12,13 One of these studies also identified HLA class I mismatch as a risk factor for disease.¹¹The initial purpose of this study was to evaluate the effect of donor CMV serostatus on disease risk in seropositive recipients and whether HLA class I mismatch modifies the risk. We then used HLA-peptide tetramers to determine the specificity of cellular immunity to defined CMV peptides presented through donor or recipient HLA alleles and examined the development of T cell responses (or lack of them) against CMV primed “directly” on donor cells. These findings should be of value in the clinical management of CMV infection in this setting and are of considerable interest in further the understanding of the mechanism of viral infection in solid organ transplantation.     

Validity of glycated haemoglobin to diagnose new onset diabetes after transplantation

Diagnosing new onset diabetes after transplantation (NODAT) by glycated haemoglobin (HbA1c) has not been validated against the gold‐standard oral glucose tolerance test (OGTT). We analysed the predictive and optimum value of HbA1c to diagnose NODAT amongst nondiabetic renal transplant recipients. Assessment of glucose metabolism (OGTT and HbA1c) was prospectively undertaken at 3 and 12 months post‐transplantation in 71 nondiabetic renal transplant recipients. Receiver operator characteristic (ROC) curve analyses were performed to determine accuracy, sensitivity, specificity and area under curve (c‐statistic). Incidence of NODAT at 3 and 12 months post‐transplantation was 14.3% and 9.5% respectively. At 3 months, optimum HbA1c cut‐off value for predicting NODAT based on fasting glucose was 7.35 [AUC 1.00 (sensitivity 100.0%, specificity 100.0%, P = 0.004)] and for postprandial glucose‐defined NODAT was 6.20 [AUC 0.98 (sensitivity 100.0%, specificity 88.9%, P < 0.001)]. At 12 months, optimum HbA1c cut‐off value for both fasting‐ and postprandial glucose‐defined NODAT was 6.45 [AUC 0.92 (sensitivity 100.0%, specificity 87.5%, P = 0.048) and AUC 0.84 (sensitivity 75.0%, specificity 89.5%, P = 0.026) respectively]. Concordance between diagnosis of NODAT (OGTT+, HbA1c+) and nondiagnosis of NODAT (OGTT?, HbA1c?) was 88.9% and 98.7% respectively. To conclude, HbA1c (≥6.5%) can be utilized to diagnose NODAT beyond 3 months post‐transplantation but the OGTT remains the gold‐standard tool.     

Cytomegalovirus‐Associated CD4+CD28null Cells in NKG2D‐Dependent Glomerular Endothelial Injury and Kidney Allograft Dysfunction

Emerging data suggest that expansion of a circulating population of atypical, cytotoxic CD4⁺ T cells lacking costimulatory CD28 (CD4⁺CD28^null cells) is associated with latent cytomegalovirus (CMV) infection. The purpose of the current study was to increase the understanding of the relevance of these cells in 100 unselected kidney transplant recipients followed prospectively for a median of 54 months. Multicolor flow cytometry of peripheral blood mononuclear cells before transplantation and serially posttransplantation was undertaken. CD4⁺CD28^null cells were found predominantly in CMV‐seropositive patients and expanded in the posttransplantation period. These cells were predominantly effector‐memory phenotype and expressed markers of endothelial homing (CX3CR1) and cytotoxicity (NKG2D and perforin). Isolated CD4⁺CD27^?CD28^null cells proliferated in response to peripheral blood mononuclear cells previously exposed to CMV‐derived (but not HLA‐derived) antigens and following such priming incubation with glomerular endothelium resulted in signs of endothelial damage and apoptosis (release of fractalkine and von Willebrand factor; increased caspase 3 expression). This effect was mitigated by NKG2D‐blocking antibody. Increased CD4⁺CD28^null cell frequencies were associated with delayed graft function and lower estimated glomerular filtration rate at end follow‐up. This study suggests an important role for this atypical cytotoxic CD4⁺CD28^null cell subset in kidney transplantation and points to strategies that may minimize the impact on clinical outcomes.     

Modeling graft loss in patients with donor‐specific antibody at baseline using the Birmingham‐Mayo (BirMay) predictor: Implications for clinical trials

Predicting which renal allografts will fail and the likely cause of failure is important in clinical trial design to either enrich patient populations to be or as surrogate efficacy endpoints for trials aimed at improving long‐term graft survival. This study tests our previous Birmingham‐Mayo model (termed the BirMay Predictor) developed in a low‐risk kidney transplant population in order to predict the outcome of patients with donor specific alloantibody (DSA) at the time of transplantation and identify new factors to improve graft loss prediction in DSA+ patients. We wanted define ways to enrich the population for future therapeutic intervention trials. The discovery set included 147 patients from Mayo Cohort and the validation set included 111 patients from the Paris Cohort—all of whom had DSA at the time of transplantation. The BirMay predictor performed well predicting 5‐year outcome well in DSA+ patients (Mayo C statistic = 0.784 and Paris C statistic = 0.860). Developing a new model did not improve on this performance. A high negative predictive value of greater than 90% in both cohorts excluded allografts not destined to fail within 5 years. We conclude that graft‐survival models including histology predict graft loss well, both in DSA+ cohorts as well as DSA‐ patients.     

Tacrolimus monitoring in renal transplantation: a comparison between high-performance liquid chromatography and immunoassay

It is recommended that specific methods of tacrolimus monitoring rather than immunoassays, which overestimate tacrolimus levels, should be used in transplant recipients. Direct comparison of these techniques, however, has not been conducted in renal transplantation. In this study, 40 renal transplant recipients with tacrolimus monitoring by microparticle enzyme immunoassay (MEIA; target trough level 10 to 15 ng/mL) were compared with 40 patients monitored by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS; target trough level 8 to 13 ng/mL). All patients received anti CD25 antibody induction and mycophenolate mofetil in a steroid-sparing protocol. No differences were seen between MEIA and HPLC-MS groups in patient demographics. All patients were followed for 6 months. Patient survival was 100% in both groups; graft survival was 100% in the MEIA group and 97.5% in the HPLC-MS group. The groups did not differ in the number of dose changes required in the first 6 months or in the number of patients displaying tacrolimus levels within target range at 3 and 6 months. Delayed graft function occurred in 14 patients in the MEIA group and 12 patients in the HPLC-MS group (P = NS). Biopsy-proven acute rejection occurred in four patients in the MEIA group and one patient in the HPLC-MS group (P < .2). No differences were seen for the following parameters at 3 or 6 months: biopsy-proven tacrolimus nephrotoxicity, serum creatinine or estimated creatinine clearance, systolic or diastolic blood pressure, cholesterol, cytomegalovirus disease, posttransplant diabetes, or tremor. This study suggests that renal transplantation with HPLC-MS monitoring of tacrolimus is safe and effective.     

Determinants of mycophenolic acid levels after renal transplantation

There are data suggesting an association between mycophenolic acid (MPA) levels and acute rejection and toxicity in renal transplant recipients treated with mycophenolate mofetil (MMF), and therefore, knowledge of factors determining MPA levels may aid in accurate adjustment of MMF dosage. A total of 4970 samples taken 12 hours postdose were analyzed for MPA by immunoassay at regular intervals from the first week posttransplantation in 117 renal transplant patients immunosuppressed with MMF and tacrolimus in a steroid-sparing regimen (prednisolone for the first 7 days only). MPA levels rose in the first 3 months and stabilized thereafter; dose-normalized MPA levels rose throughout the first 12 months and subsequently stabilized. Multivariate analysis by means of a population-averaged linear regression showed positive associations between MPA level and total daily dose (P < 0.001) but not individual dose or total daily dose corrected for body weight. Positive associations were also seen with serum albumin (P = 0.01), tacrolimus trough level (P = 0.01), and female gender (P = 0.002). The association with tacrolimus levels diminished with time. Negative associations were seen between MPA level and higher estimated creatinine clearance (P < 0.001), and also with increasing alanine transaminase levels (P = 0.002), the use of oral antibiotics (P < 0.001), and infective diarrhea (P < 0.001). The latter findings may be related to changes in enterohepatic recirculation of MPA. Many clinical variables show associations with trough MPA levels. An understanding of these factors may aid therapeutic monitoring of MMF.