RESUMO
Early subclinical inflammation in kidney transplants is associated with later graft fibrosis and dysfunction. Regulatory T cells (Tregs) can reverse established inflammation in animal models. We conducted a pilot safety and feasibility trial of autologous Treg cell therapy in three kidney transplant recipients with subclinical inflammation noted on 6-month surveillance biopsies. Tregs were purified from peripheral blood and polyclonally expanded ex vivo using medium containing deuterated glucose to label the cells. All patients received a single infusion of ~320 × 106 (319, 321, and 363.8 × 106 ) expanded Tregs. Persistence of the infused Tregs was tracked. Graft inflammation was monitored with follow-up biopsies and urinary biomarkers. Nearly 1 × 109 (0.932, 0.956, 1.565 × 109 ) Tregs were successfully manufactured for each patient. There were no infusion reactions or serious therapy-related adverse events. The infused cells demonstrated patterns of persistence and stability similar to those observed in non-immunosuppressed subjects receiving the same dose of Tregs. Isolation and expansion of Tregs is feasible in kidney transplant patients on immunosuppression. Infusion of these cells was safe and well tolerated. Future trials will test the efficacy of polyclonal and donor alloantigen-reactive Tregs for the treatment of inflammation in kidney transplants.
Assuntos
Rejeição de Enxerto/terapia , Inflamação/terapia , Falência Renal Crônica/cirurgia , Transplante de Rim/efeitos adversos , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Idoso , Feminino , Seguimentos , Taxa de Filtração Glomerular , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Humanos , Inflamação/etiologia , Inflamação/patologia , Isoantígenos , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Complicações Pós-Operatórias , Prognóstico , Fatores de Risco , Doadores de Tecidos , Adulto JovemRESUMO
Gene expression profiling of transplant recipient blood and urine can potentially be used to monitor graft function, but the multitude of protocols in use make sharing data and comparing results from different laboratories difficult. The goal of this study was to evaluate the performance of current methods of RNA isolation, reverse transcription and quantitative polymerase chain reaction (qPCR) and to test whether multiple centers using a standardized protocol can obtain the same results. Samples, reagents and detailed instructions were distributed to six participating sites that performed RNA isolation, reverse transcription and qPCR for 18S, PRF, GZB, IL8, CXCL9 and CXCL10 as instructed. All data were analyzed at a single site. All sites demonstrated proficiency in RNA isolation and qPCR analysis. Gene expression measurements for all targets and samples had correlations >0.938. The coefficient of variation of fold-changes between pairs of samples was less than 40%. All sites were able to accurately quantify a control sample of known concentration within a factor of 1.5. Collectively, we have formulated and validated detailed methods for measuring gene expression in blood and urine that can yield consistent results in multiple laboratories.
Assuntos
Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica , Transplante de Rim , RNA Mensageiro/análise , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Perfilação da Expressão Gênica/métodos , Humanos , Limite de Detecção , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/genética , Sensibilidade e Especificidade , Transplante HomólogoRESUMO
To determine whether steroid avoidance in pediatric kidney transplantation is safe and efficacious, a randomized, multicenter trial was performed in 12 pediatric kidney transplant centers. One hundred thirty children receiving primary kidney transplants were randomized to steroid-free (SF) or steroid-based (SB) immunosuppression, with concomitant tacrolimus, mycophenolate and standard dose daclizumab (SB group) or extended dose daclizumab (SF group). Follow-up was 3 years posttransplant. Standardized height Z-score change after 3 years follow-up was -0.99 ± 2.20 in SF versus -0.93 ± 1.11 in SB; p = 0.825. In subgroup analysis, recipients under 5 years of age showed improved linear growth with SF compared to SB treatment (change in standardized height Z-score at 3 years -0.43 ± 1.15 vs. -1.07 ± 1.14; p = 0.019). There were no differences in the rates of biopsy-proven acute rejection at 3 years after transplantation (16.7% in SF vs. 17.1% in SB; p = 0.94). Patient survival was 100% in both arms; graft survival was 95% in the SF and 90% in the SB arms (p = 0.30) at 3 years follow-up. Over the 3 year follow-up period, the SF group showed lower systolic BP (p = 0.017) and lower cholesterol levels (p = 0.034). In conclusion, complete steroid avoidance is safe and effective in unsensitized children receiving primary kidney transplants.
Assuntos
Imunossupressores/administração & dosagem , Transplante de Rim , Esteroides/administração & dosagem , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Masculino , Adulto JovemRESUMO
Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross-validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q-PCR analysis of a five gene-set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training-set (n = 47) and validated on independent test-set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non-AR phenotypes with 91% sensitivity and 90% specificity. The 5-gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.
Assuntos
Rejeição de Enxerto/diagnóstico , Transplante de Rim , Doença Aguda , Rejeição de Enxerto/sangue , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
We report 1-year outcomes of a randomized study of Rituximab versus standard-of-care immunosuppression (Thymoglobulin and/or pulse steroids) for treatment of biopsy confirmed, acute transplant rejection with B-cell infiltrates, in 20 consecutive recipients (2-23 years). Graft biopsies, with Banff and CADI scores, CD20 and C4d stains, were performed at rejection and 1 and 6 months later. Peripheral blood CMV, EBV and BK viral loads, graft function, DSA, immunoglobulins, serum humanized antichimeric antibody (HACA) and Rituximab, and lymphocyte counts were monitored until 1 year posttreatment. Rituximab infusions were given with a high index of safety without HACA development and increased infections complications. Rituximab therapy resulted in complete tissue B-cell depletion and rapid peripheral B-cell depletion. Peripheral CD19 cells recovered at a mean time of approximately 12 months. There were some benefits for the recovery of graft function (p = 0.026) and improvement of biopsy rejection scores at both the 1- (p = 0.0003) and 6-month (p < 0.0001) follow-up biopsies. Reappearance of C4d deposition was not seen on follow-up biopsies after Rituximab therapy, but was seen in 30% of control patients. There was no change in DSA in either group, independent of rejection resolution. This study reports safety and suggests further investigation of Rituximab as an adjunctive treatment for B-cell-mediated graft rejection.