Ruchuang Ding

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. In the other four recipients, it was possible to discontinue all immunosuppressive therapy 9 to 14 months after the transplantation, and renal function has remained stable for 2.0 to 5.3 years since transplantation. The T cells from these four recipients, tested in vitro, showed donor-specific unresponsiveness and in specimens from allograft biopsies, obtained after withdrawal of immunosuppressive therapy, there were high levels of P3 (FOXP3) messenger RNA (mRNA) but not granzyme B mRNA.

Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine

BACKGROUND Acute rejection is a serious and frequent complication of renal transplantation, and its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for rejection could improve the outcome of transplantation. METHODS We obtained 24 urine specimens from 22 renal-allograft recipients with a biopsy-confirmed episode of acute rejection and 127 samples from 63 recipients without evidence of acute rejection. RNA was isolated from the urinary cells. Messenger RNA (mRNA) encoding the cytotoxic proteins perforin and granzyme B and a constitutively expressed cyclophilin B gene were measured with the use of a competitive, quantitative polymerase chain reaction, and the level of expression was correlated with allograft status. RESULTS The log-transformed mean (+/-SE) levels of perforin mRNA and granzyme B mRNA, which encode cytotoxic proteins, but not the levels of constitutively expressed cyclophiiin B mRNA, were higher in the urinary cells from the 22 patients with a biopsy-confirmed episode of acute rejection than in the 63 recipients without an episode of acute rejection (perforin, 1.4+/-0.3 vs. -0.6+/-0.2 fg per microgram of total RNA; P<0.001; and granzyme B, 1.2+/-0.3 vs. -0.9+/-0.2 fg per microgram of total RNA; P<0.001). Analysis involving the receiver-operating-characteristic curve demonstrated that acute rejection can be predicted with a sensitivity of 83 percent and a specificity of 83 percent with the use of a cutoff value of 0.9 fg of perforin mRNA per microgram of total RNA, and with a sensitivity of 79 percent and a specificity of 77 percent with the use of a cutoff value of 0.4 fg of granzyme B mRNA per microgram of total RNA. Sequential urine samples were obtained from 37 patients during the first nine days after transplantation; and measurements of the levels of mRNA that encoded cytotoxic proteins identified those in whom acute rejection developed. CONCLUSIONS Measurement of mRNA encoding cytotoxic proteins in urinary cells offers a noninvasive means of diagnosing acute rejection of renal allografts.

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). We found a strong association between intragraft expression of miRNAs and messenger RNAs (mRNAs), and that AR, and renal allograft function, could be predicted with a high level of precision using intragraft levels of miRNAs. Our investigation of miRNA expression in normal human peripheral blood mononuclear cells (PBMCs) showed that miRNAs (miR-142–5p, -155, and -223) overexpressed in AR biopsies are highly expressed in PBMCs, and that stimulation with the mitogen phytohaemagglutinin results in an increase in the abundance of miR-155 and a decrease in miR-223 and let-7c. Quantification of miRNAs in primary cultures of human renal epithelial cells (HRECs) showed that miR-30a-3p, -10b, and let-7c are highly expressed in HRECs, and that stimulation results in a decreased expression of miR-30a-3p. Our studies, in addition to suggesting a cellular basis for the altered intragraft expression of miRNAs, propose that miRNA expression patterns may serve as biomarkers of human renal allograft status.

Dendritic cells with TGF-β1 differentiate naïve 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 β-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-β1. These induced, antigen-specific T regs maintain high levels of clonotype-specific T cell receptor expression and exert islet-specific suppression in vitro. When cotransferred with diabetogenic cells into NOD scid recipients, T regs induced with DCs and TGF-β1 prevent the development of diabetes. Furthermore, in overtly NOD mice, these cells are able to significantly protect syngeneic islet grafts from established destructive autoimmunity. These results indicate a role for DCs in the induction of antigen-specific CD4+CD25+Foxp3+ T cells that can inhibit fully developed autoimmunity in a nonlymphopoenic host, providing an important potential strategy for immunotherapy in patients with autoimmune diabetes.

In vivo expression of transforming growth factor-beta1 in humans: stimulation by cyclosporine.

BACKGROUND Transforming growth factor-beta1 (TGF-beta1) is an immunoregulatory and fibrogenic cytokine. In an earlier in vitro study, we demonstrated that cyclosporine (CsA) increases TGF-beta1 transcription rate in human T lymphocytes. Herein, we explored whether CsA augments the in vivo expression of TGF-beta1 in humans. METHODS The inherent difficulty in studying the in vivo effect of CsA in humans was circumvented by investigating stable end-stage renal disease patients who were preconditioned with CsA before their living donor renal transplantation. Sera and peripheral blood mononuclear cells were obtained from CsA-preconditioned patients and quantified for TGF-beta1 expression at the mRNA (by competitive polymerase chain reaction) and protein (sandwich enzyme-linked immunosorbent assay) levels. RESULTS Our studies demonstrated a significant increase in TGF-beta1 expression after CsA therapy. The stimulatory effect was unique to TGF-beta1, and CsA did not increase interleukin (IL)-10, IL-6, IL-2, or tumor necrosis factor-alpha expression. CONCLUSIONS Our first-time demonstration of a TGF-beta1-selective in vivo stimulatory effect of CsA in humans: (1) advances a TGF-beta1-centered hypothesis for the beneficial (immunosuppression) and detrimental (fibrosis, hypertension) effects of CsA use, and (2) broadens the mechanism of immunosuppressive action of CsA to include heightened expression of an endogenous immunosuppressive cytokine.Background.Transforming growth factor-β1(TGF-β1) is an immunoregulatory and fibrogenic cytokine. In an earlier in vitro study, we demonstrated that cyclosporine (CsA) increases TGF-β1transcription rate in human T lymphocytes. Herein, we explored whether CsA augments the in vivo expression of TGF-β1i

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. Messenger RNA (mRNA) levels were measured in urinary cells and correlated with allograft-rejection status with the use of logistic regression. RESULTS A three-gene signature of 18S ribosomal (rRNA)-normalized measures of CD3ε mRNA and interferon-inducible protein 10 (IP-10) mRNA, and 18S rRNA discriminated between biopsy specimens showing acute cellular rejection and those not showing rejection (area under the curve [AUC], 0.85; 95% confidence interval [CI], 0.78 to 0.91; P<0.001 by receiver-operating-characteristic curve analysis). The cross-validation estimate of the AUC was 0.83 by bootstrap resampling, and the Hosmer-Lemeshow test indicated good fit (P=0.77). In an external-validation data set, the AUC was 0.74 (95% CI, 0.61 to 0.86; P<0.001) and did not differ significantly from the AUC in our primary data set (P=0.13). The signature distinguished acute cellular rejection from acute antibody-mediated rejection and borderline rejection (AUC, 0.78; 95% CI, 0.68 to 0.89; P<0.001). It also distinguished patients who received anti-interleukin-2 receptor antibodies from those who received T-cell-depleting antibodies (P<0.001) and was diagnostic of acute cellular rejection in both groups. Urinary tract infection did not affect the signature (P=0.69). The average trajectory of the signature in repeated urine samples remained below the diagnostic threshold for acute cellular rejection in the group of patients with no rejection, but in the group with rejection, there was a sharp rise during the weeks before the biopsy showing rejection (P<0.001). CONCLUSIONS A molecular signature of CD3ε mRNA, IP-10 mRNA, and 18S rRNA levels in urinary cells appears to be diagnostic and prognostic of acute cellular rejection in kidney allografts. (Funded by the National Institutes of Health and others.).

Noninvasive diagnosis of BK virus nephritis by measurement of messenger RNA for BK VP1 virus in urine.

Background. Polyoma virus type BK (BKV) nephritis has emerged as an important cause of renal allograft dysfunction and graft failure. Its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for BKV nephritis could improve clinical outcome. Methods. We obtained 25 urine specimens from 8 renal allograft recipients with biopsy-confirmed BKV nephritis, 31 samples from 28 recipients in whom BKV nephritis was excluded by allograft biopsy, and 74 specimens from 34 patients with stable allograft function. RNA was isolated from the urinary cells and reverse transcribed to complementary DNA. We designed gene-specific oligonucleotide primers and probes for the measurement of messenger RNA (mRNA) encoding BKV VP1 protein and a constitutively expressed 18S ribosomal RNA (rRNA) by realtime quantitative polymerase chain reaction. We explored the hypothesis that BKV VP1 mRNA levels predict BKV nephritis. Results. The levels of BKV VP1 mRNA but not the levels of 18S rRNA predicted BKV nephritis. Analysis involving the receiver operating characteristic curve demonstrated that BKV nephritis can be predicted with a sensitivity of 93.8% and a specificity of 93.9% with the use of a cutoff value of 6.510 5 BKV VP1 mRNA copy number per nanogram of total RNA (P 0.2) for 18S rRNA.

CD103 mRNA levels in urinary cells predict acute rejection of renal allografts1

Background. CD103 is displayed on the cell surface of alloreactive CD8 cytotoxic T lymphocytes (CTLs) and is a critical component for the intraepithelial homing of T cells. Because intratubular localization of mononuclear cells is a feature of acute cellular rejection of renal allografts, we explored the hypothesis that CD103 messenger (m)RNA levels in urinary cells predict acute rejection. Methods. We collected 89 urine specimens from 79 recipients of renal allografts. RNA was isolated from the urinary cells, and we measured CD103 mRNA levels and a constitutively expressed 18S ribosomal (r)RNA with the use of real-time quantitative polymerase chain reaction assay. Results. CD103 mRNA levels, but not 18S rRNA levels, were higher in urinary cells from 30 patients with an episode of acute rejection (32 biopsies and 32 urine samples) compared with the levels in 12 patients with other findings on allograft biopsy (12 biopsies and 12 urine samples), 12 patients with biopsy evidence of chronic allograft nephropathy (12 biopsies and 12 urine samples), and 25 patients with stable graft function after renal transplantation (0 biopsies and 33 urine samples) (P = 0.001; one-way analysis of variance). Acute rejection was predicted with a sensitivity of 59% and a specificity of 75% using natural log-transformed value 8.16 CD103 copies per microgram as the cutoff value (P = 0.001). Conclusion. CD103 mRNA levels in urinary cells are diagnostic of acute rejection of renal allografts. Because CD103 is a cell surface marker of intratubular CD8 CTLs, a noninvasive assessment of cellular traffic into the allograft may be feasible by the measurement of CD103 mRNA levels in urinary cells.

SERINE PROTEINASE INHIBITOR-9, AN ENDOGENOUS BLOCKER OF GRANZYME B/PERFORIN LYTIC PATHWAY, IS HYPEREXPRESSED DURING ACUTE REJECTION OF RENAL ALLOGRAFTS

Background. Serine proteinase inhibitor (PI)-9 with a reactive center P1 (Glu)-P1′ is a natural antagonist of granzyme B and is expressed in high levels in cytotoxic T lymphocytes (CTL). In view of the role of CTL in acute rejection, we explored the hypothesis that PI-9 would be hyperexpressed during acute rejection. Because PI-9 can protect CTL from its own fatal arsenal and potentially enhance the vitality of CTL, we examined whether PI-9 levels correlate with the severity of rejection as well as predict subsequent graft function. Methods. We obtained 95 urine specimens from 87 renal allograft recipients. RNA was isolated from the urinary cells and mRNA encoding PI-9, granzyme B, or perforin and a constitutively expressed 18S rRNA was measured with the use of real-time quantitative polymerase chain reaction assay, and the level of expression was correlated with allograft status. Results. The levels of PI-9 (P =0.001), granzyme B (P <0.0001), and perforin mRNAs (P <0.0001), but not the levels of 18S rRNA (P =0.54), were higher in the urinary cells from the 29 patients with a biopsy-confirmed acute rejection than in the 58 recipients without acute rejection. PI-9 levels were significantly higher in patients with type II or higher acute rejection changes compared with those with less than type II changes (P =0.01). Furthermore, PI-9 levels predicted subsequent graft function (r =0.43, P =0.01). Conclusions. PI-9 mRNA levels in urinary cells are diagnostic of acute rejection, predict renal allograft histology grade, and predict functional outcome following an acute rejection episode.

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. BKV replication was quantified by measurement of urinary cell BKV VP1 mRNA levels using BKV specific primers and TaqMan probe in a real-time quantitative polymerase chain reaction assay. Levels of urinary cell mRNA for granzyme B, CD103, and transforming growth factor-β1 were measured to ascertain whether BKV replication is associated with an inflammatory signature. Results. The prevalence of BKV replication increased over time and was highest at 6 months compared with 1 or 3 months posttransplantation (P<0.001). A logistic regression model analysis demonstrated that steroid maintenance therapy (odds ratio: 8.3, P=0.003) and induction with rabbit anti-human thymocyte globulin (odds ratio: 5.8, P=0.008) were independent risk factors for BKV replication. Neither mycophenolate mofetil dose nor tacrolimus dose or trough levels were different between those with or without BKV replication. The development of acute rejection or antirejection treatment with methylprednisolone did not increase the risk of BKV replication. BKV replication was associated with heightened levels of urinary cell mRNA for granzyme B (P<0.002), CD103 (P<0.005) but not for transforming growth factor-β1 (P>0.05). Conclusions. Steroid maintenance therapy and induction with antithymocyte globulin are independent risk factors for BKV replication in renal allograft recipients treated with tacrolimus and mycophenolate mofetil.