Evaluation of Immunocompetence and Biomarkers of Tolerance in Chimeric and Immunosuppression-free Kidney Allograft Recipients.

Background: Thirty-seven patients have received a living-donor kidney transplant in a phase 2 study designed to induce tolerance with facilitated allogeneic hematopoietic stem cell transplant. The study protocol is based on tolerogenic CD8 + /T-cell receptor - facilitating cells (FCR001; also including hematopoietic stem cells and αβ-T-cell receptor + T cells) and low-dose, nonmyeloablative conditioning. Persistent chimerism allowing full immunosuppression (IS) withdrawal was achieved in 26 patients (time off IS 36-123 mo).

Urinary cell mRNA profiling of kidney allograft recipients: Development of a portable protocol for noninvasive diagnosis of T cell mediated rejection and BK virus nephropathy.

Background: We developed urinary cell mRNA profiling for noninvasive diagnosis of acute T cell mediated rejection (TCMR) and BK virus nephropathy (BKVN), two significant post-transplant complications. Our profiling protocol for the multicenter Clinical Trial of Transplantation-04 (CTOT-04) study consisted of centrifugation of urine to prepare cell pellets, washes, addition of an RNA preservative, storage at 80C and shipment in cold containers to our Gene Expression Monitoring (GEM) Core for RNA isolation and quantification of mRNA in RT-qPCR assays. To simplify profiling, we developed a filter-based protocol (ZFBP) that eliminated the need for centrifugation, RNA preservative, storage at 80C, and shipment in cold containers for mRNA profiling.

Development of a Bak gene based standard curve for absolute quantification of BK virus in real time quantitative PCR assay and noninvasive diagnosis of BK virus nephropathy in kidney allograft recipients.

Background: BK virus nephropathy (BKVN) is a frequent and serious post-transplant complication and undermines realization of the full benefits of kidney transplantation. We developed a Bak amplicon-based standard curve for absolute quantification of BKV VP1 mRNA copy number in the real time quantitative PCR (RT-qPCR) assay and investigated the performance characteristics of this novel assay.

Methods: We determined analytical specificity, sensitivity, and precision of our 73 bp mouse Bak amplicon based standard curve for absolute quantification of BKV VP1 mRNA in RT-qPCR assays.

Urinary cell mRNA profiling of kidney allograft recipients: A systematic investigation of a filtration based protocol for the simplification of urine processing.

Background: Kidney transplantation is a life-restorative therapy, but immune rejection undermines allograft survival. Urinary cell mRNA profiles offer a noninvasive means of diagnosing kidney allograft rejection, but urine processing protocols have logistical constraints. We aimed to determine whether the centrifugation-based method for urinary cell mRNA profiling could be replaced with a simpler filtration-based method without undermining quality.

Validation of a noninvasive prognostic signature for allograft failure following BK virus associated nephropathy.

Identifying kidney transplant recipients at risk for graft failure following BK virus nephropathy (BKVN) may allow personalization of therapy. We have reported that a noninvasive composite signature of urinary cell level of plasminogen activator inhibitor-1(PAI-1) mRNA and serum creatinine level, measured at the time of BKVN diagnosis, is prognostic of graft failure. In this investigation, we determined whether the composite signature is prognostic of graft failure in an independent cohort of 25 patients with BKVN.

FOXP3 mRNA Profile Prognostic of Acute T Cell-mediated Rejection and Human Kidney Allograft Survival.

Background: T cell-mediated rejection (TCMR) is the most frequent type of acute rejection and is associated with kidney allograft failure. Almost 40% of TCMR episodes are nonresponsive to therapy, and molecular mechanisms for the nonresponsiveness are unknown. Our single-center study identified that urinary cell FOXP3 mRNA abundance predicts TCMR reversibility and allograft survival.

Urinary Cell Transcriptome Profiling and Identification of ITM2A, SLAMF6, and IKZF3 as Biomarkers of Acute Rejection in Human Kidney Allografts.

Unlabelled: Identification of a shared gene expression pattern between T cell-mediated rejection (TCMR) and antibody-mediated rejection (AMR) in human kidney allografts may help prioritize targets for the treatment of both types of acute rejection.

Methods: We performed RNA sequencing and bioinformatics of genome-wide transcriptome profiles of urinary cells to identify novel mRNAs shared between TCMR and AMR and of mechanistic relevance. Customized RT-QPCR assays were then used to validate their abundance in urinary cells.

Urine Metabolite Profiles Predictive of Human Kidney Allograft Status.

Noninvasive diagnosis and prognostication of acute cellular rejection in the kidney allograft may help realize the full benefits of kidney transplantation. To investigate whether urine metabolites predict kidney allograft status, we determined levels of 749 metabolites in 1516 urine samples from 241 kidney graft recipients enrolled in the prospective multicenter Clinical Trials in Organ Transplantation-04 study. A metabolite signature of the ratio of 3-sialyllactose to xanthosine in biopsy specimen-matched urine supernatants best discriminated acute cellular rejection biopsy specimens from specimens without rejection.

Allograft rejection and tubulointerstitial fibrosis in human kidney allografts: interrogation by urinary cell mRNA profiling.

Because the kidney allograft has the potential to function as an in-vivo flow cytometer and facilitate the access of immune cells and kidney parenchymal cells in to the urinary space, we hypothesized that mRNA profiling of urinary cells offers a noninvasive means of assessing the kidney allograft status. We overcame the inherent challenges of urinary cell mRNA profiling by developing pre-amplification protocols to compensate for low RNA yield from urinary cells and by developing robust protocols for absolute quantification mRNAs using RT-PCR assays. Armed with these tools, we undertook first single-center studies urinary cell mRNA profiling and then embarked on the multicenter Clinical Trials in Organ Transplantation-04 study of kidney transplant recipients.

Urinary cell mRNA profiles and differential diagnosis of acute kidney graft dysfunction.

Noninvasive tests to differentiate the basis for acute dysfunction of the kidney allograft are preferable to invasive allograft biopsies. We measured absolute levels of 26 prespecified mRNAs in urine samples collected from kidney graft recipients at the time of for-cause biopsy for acute allograft dysfunction and investigated whether differential diagnosis of acute graft dysfunction is feasible using urinary cell mRNA profiles. We profiled 52 urine samples from 52 patients with biopsy specimens indicating acute rejection (26 acute T cell-mediated rejection and 26 acute antibody-mediated rejection) and 32 urine samples from 32 patients with acute tubular injury without acute rejection.

Urinary cell mRNA profiles predictive of human kidney allograft status.

Kidney allograft status is currently characterized using the invasive percutaneous needle core biopsy procedure. The procedure has become safer over the years, but challenges and complications still exist including sampling error, interobserver variability, bleeding, arteriovenous fistula, graft loss, and even death. Because the most common type of acute rejection is distinguished by inflammatory cells exiting the intravascular compartment and gaining access to the renal tubular space, we reasoned that a kidney allograft may function as an in vivo flow cytometer and sort cells involved in rejection into urine.

Urinary-cell mRNA profile and acute cellular rejection in kidney allografts.

Background: The standard test for the diagnosis of acute rejection in kidney transplants is the renal biopsy. Noninvasive tests would be preferable.

Methods: We prospectively collected 4300 urine specimens from 485 kidney-graft recipients from day 3 through month 12 after transplantation.

Noninvasive prognostication of polyomavirus BK virus-associated nephropathy.

Background: BK virus-associated nephropathy (BKVN) is associated with an increased risk of graft failure.

Methods: Levels of mRNAs encoding proteins implicated in inflammation and fibrosis were measured in urine collected at the time of biopsy diagnosis of BKVN in 29 kidney graft recipients and analyzed for prognosticating graft failure using logistic regression.

Results: Ten of 29 BKVN patients had graft failure within 36 months of BKVN diagnosis and the remaining 19 patients did not.

HIV-infected kidney graft recipients managed with an early corticosteroid withdrawal protocol: clinical outcomes and messenger RNA profiles.

Background: The outcome of HIV-infected kidney transplant recipients managed with an early corticosteroid withdrawal protocol is not known.

Methods: Eleven consecutive HIV-infected patients with undetectable plasma HIV RNA and more than 200/mm CD4 T cells underwent deceased-donor (n=8) or living-donor (n=3) kidney transplantation at our center. All were managed with an early corticosteroid withdrawal protocol; 9 of 11 received antithymocyte globulin and 2 received basiliximab induction.

Discovery and validation of a molecular signature for the noninvasive diagnosis of human renal allograft fibrosis.

Background: Tubulointerstitial fibrosis (fibrosis), a histologic feature associated with a failing kidney allograft, is diagnosed using the invasive allograft biopsy. A noninvasive diagnostic test for fibrosis may help improve allograft outcome.

Methods: We obtained 114 urine specimens from 114 renal allograft recipients: 48 from 48 recipients with fibrosis in their biopsy results and 66 from 66 recipients with normal biopsy results.

Dendritic cells with TGF-β1 and IL-2 differentiate naive CD4+ T cells into alloantigen-specific and allograft protective Foxp3+ regulatory T cells.

Background: Naturally occurring, thymic-derived Foxp3+CD25+CD4+ regulatory T cells (nTregs) are pivotal for the maintenance of self-tolerance. nTregs, however, are sparse and lack alloantigen specificity, and these properties pose challenges for their use in clinical transplantation.

Methods: We established mixed leukocyte reaction (MLR) with dendritic cells (DCs) as stimulators and CD4+ T cells as responders and supplemented the MLR with IL-2 and TGF-β1 and investigated whether DCs+IL-2+TGF-β1 differentiate the polyclonal CD4+ cells into alloantigen-specific and allograft protective Tregs.

Urinary cell levels of mRNA for OX40, OX40L, PD-1, PD-L1, or PD-L2 and acute rejection of human renal allografts.

Background: The positive costimulatory proteins OX40 and OX40L and negative regulatory proteins programmed death (PD)-1, PD ligand 1, and PD ligand 2 have emerged as significant regulators of acute rejection in experimental transplantation models.

Methods: We obtained 21 urine specimens from 21 renal allograft recipients with graft dysfunction and biopsy-confirmed acute rejection and 25 specimens from 25 recipients with stable graft function and normal biopsy results (stable). Urinary cell levels of mRNAs were measured using real-time quantitative polymerase chain reaction assays, and the levels were correlated with allograft status and outcomes.

Diannexin decreases inflammatory cell infiltration into the islet graft, reduces β-cell apoptosis, and improves early graft function.

Background: A major unmet challenge is to reduce the islet mass needed for insulin independence in type 1 diabetic recipients after islet transplantation. The recombinant homodimer of human annexin V, diannexin, has completed a Phase II Clinical Trial in Kidney Transplantation (NCT00615966).

Methods: We developed a marginal islet mass transplantation model (10-12 islets per gram of recipient body weight) and investigated whether diannexin prevents β-cell apoptosis and improves islet graft function.

Validation of noninvasive diagnosis of BK virus nephropathy and identification of prognostic biomarkers.

Background: BK virus nephropathy (BKVN) may cause renal allograft dysfunction and failure. The gold standard test is kidney biopsy, which is invasive and costly. A noninvasive, accurate biomarker for diagnosis of BKVN and prognostication of allograft function after BKVN infection may improve allograft survival.

Transforming growth factor beta and excess burden of renal disease.

End-stage renal disease (ESRD) is more frequent in African Americans (blacks) compared to whites. Because renal fibrosis is a correlate of progressive renal failure and a dominant feature of ESRD, and because transforming growth factor beta 1 (TGF-beta1) can induce fibrosis and renal insufficiency, we hypothesized that TGF-beta1 hyperexpression is more frequent in blacks compared to whites. We measured circulating levels of TGF-beta1 in black and white patients with ESRD, hypertension, and in normal patients.

MicroRNA expression profiles predictive of human renal allograft status.

Immune rejection of organ transplants is a life-threatening complication and is exemplified by alterations in the expression of protein-encoding genes. Because microRNAs (miRNAs) regulate the expression of genes implicated in adaptive immunity, we investigated whether acute rejection (AR) is associated with alterations in miRNA expression within allografts and whether expression profiles are diagnostic of AR and predict allograft function. Seven of 33 renal allograft biopsies (12 AR and 21 normal) were profiled using microfluidic cards containing 365 mature human miRNAs (training set), and a subset of differentially expressed miRNAs were quantified in the remaining 26 allograft biopsies (validation set).

Epidemiology of BK virus in renal allograft recipients: independent risk factors for BK virus replication.

Background: Identification of risk factors for BK virus (BKV) replication may improve transplant outcome. We investigated the impact of immunosuppressive drugs on the prevalence of BKV replication in recipients of human renal allografts.

Methods: One hundred twenty renal allograft recipients were studied prospectively at 1, 3, and 6 months posttransplantation to identify risk factors for BKV replication.

HLA-mismatched renal transplantation without maintenance immunosuppression.

Five patients with end-stage renal disease received combined bone marrow and kidney transplants from HLA single-haplotype mismatched living related donors, with the use of a nonmyeloablative preparative regimen. Transient chimerism and reversible capillary leak syndrome developed in all recipients. Irreversible humoral rejection occurred in one patient.

Hyperexpression of Foxp3 and IDO during acute rejection of islet allografts.

Background: We investigated the hypothesis that Foxp3+ cells are an integral component of antiallograft immunity but are dominated by pathogenic effectors.

Methods: Wild-type H-2b C57BL/6 (B6) mice or B6 mice with a targeted disruption of c-Rel gene (c-Rel-/-) were used as recipients of islet grafts from allogeneic DBA/2 (H-2d) mice or syngeneic B6 mice. We developed kinetic quantitative polymerase chain reaction assays and measured intragraft expression of mRNA for Foxp3, IDO, cytolytic molecules, proinflammatory cytokines, and chemokines/receptors.

Dendritic cells with TGF-beta1 differentiate naive CD4+CD25- T cells into islet-protective Foxp3+ regulatory T cells.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (T regs) are important for preventing autoimmune diabetes and are either thymic-derived (natural) or differentiated in the periphery outside the thymus (induced). Here we show that beta-cell peptide-pulsed dendritic cells (DCs) from nonobese diabetic (NOD) mice can effectively induce CD4(+)CD25(+)Foxp3(+) T cells from naïve islet-specific CD4(+)CD25(-) T cells in the presence of TGF-beta1. These induced, antigen-specific T regs maintain high levels of clonotype-specific T cell receptor expression and exert islet-specific suppression in vitro.