Soluble CD30 and ELISA-detected human leukocyte antigen antibodies for the prediction of acute rejection in pediatric renal transplant recipients.

Biomarker-based post-transplant immune monitoring for the prediction of impending graft rejection requires validation in specific patient populations. Serum of 28 pediatric renal transplant recipients within the framework of a well-controlled prospective randomized trial was analyzed pre- and post-transplant for soluble CD30 (sCD30), a biomarker reflecting mainly T-cell reactivity, and anti-human leukocyte antigen (anti-HLA) antibody reactivity, a biomarker for B-cell activation. A sCD30 concentration ≥40.3 U/ml on day 14 was able to discriminate between patients with or without biopsy-proven acute rejection (BPAR) with a sensitivity of 100% and a specificity of 76%. Six of seven patients (86%) with BPAR showed a sCD30 above this cut-off, whereas only 3/21 patients (14%) without BPAR had a sCD30 above this cut-off (P = 0.004). For pre- and post-transplant anti-HLA class II reactivities by enzyme-linked immunosorbent assay, a cut-off value of 140 optical density was able to discriminate rejecters from nonrejecters with a sensitivity of 86% or 71% and a specificity of 81% or 90%, respectively. Withdrawal of steroids was associated with a approximately twofold higher serum sCD30 compared to controls, but did not affect anti-HLA reactivities. An increased post-transplant sCD30 serum concentration and positive pre- and post-transplant anti-HLA class II reactivities are informative biomarkers for impending BPAR in pediatric renal transplant recipients. (TWIST, Clinical Trial No: FG-506-02-43).

No association of kidney graft loss with human leukocyte antigen antibodies detected exclusively by sensitive Luminex single-antigen testing: a Collaborative Transplant Study report.

BACKGROUND: It is unclear whether kidney transplant recipients with preformed donor-specific human leukocyte antigen (HLA) antibodies (DSA) detectable only in the highly sensitive Luminex single-antigen (LSA) assay are at an increased risk of graft failure. METHODS: We studied 3148 patients who received a deceased donor kidney graft between 1996 and 2008 and were enrolled in the prospective serum project of the Collaborative Transplant Study. There were 118 patients with graft loss during the first 3 years after transplantation on whom recipient and donor DNA was available for complete HLA typing. We compared the incidence of LSA-detected DSA in these patients with graft failure and matched controls with functioning grafts. All patients were found negative in the less-sensitive complement-dependent lymphocytotoxicity and enzyme-linked immunosorbent assays. RESULTS: When mean fluorescence intensity (MFI) of greater than or equal to 1000 was used as a cutoff for Luminex positivity, 118 patients with graft loss did not show a higher incidence of DSA against HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, or -DPB1 antigens than 118 matched controls without graft loss (for all loci P not significant). The incidence of strong DSA (MFI ≥2000 or MFI ≥3000) detected only by LSA was low (for all loci between 0% and 5%) and did not identify unacceptable antigens that were relevant for graft loss within the first 3 years after transplantation. CONCLUSION: We conclude that, given currently practiced crossmatch procedures and immunosuppressive regimens, exclusion of donor organs carrying "unacceptable" HLA based exclusively on sensitive LSA antibody testing is not justified.

An integrative approach for the transplantation of high-risk sensitized patients.

BACKGROUND: Sensitized patients have a lower chance of receiving a crossmatch-negative kidney and, if transplanted, are at risk of antibody-mediated allograft rejection. METHODS: For safe and timely transplantation of sensitized patients at our center, we developed an integrative algorithm that includes identification of high-risk patients, good human leukocyte antigen match, inclusion in the Eurotransplant Acceptable Mismatch Program when applicable, apheresis, anti-CD20 therapy, posttransplant antibody monitoring, and protocol biopsies. Thirty-four high-risk recipients of a deceased donor kidney (DDK: n=28) or living donor kidney (LDK: n=6) were transplanted using this algorithm. RESULTS: One-year graft survival, death-censored graft survival, and patient survival rates in DDK recipients were 92.4%, 96.4%, and 95.8%, respectively. No graft loss or patient death was observed in the six LDK patients. Median serum creatinine at 1 year in DDK and LDK recipients was 1.2 and 1.4 mg/dL, respectively. Eleven DDK and three LDK patients experienced at least one biopsy-proven acute rejection episode, mostly showing borderline changes. Antibody-mediated rejection without graft loss was diagnosed in two DDK and one LDK patients. Delayed graft function was observed in 13 DDK and 1 LDK patients. Infectious complications were infrequent. CONCLUSIONS: We describe an algorithm for the categorization and treatment of presensitized high-risk patients. This protocol provides effective prevention of antibody-mediated rejection and is associated with a low rate of side effects and good graft outcome.

Does borderline kidney allograft rejection always require treatment?

BACKGROUND: Borderline rejection (Bord-R) is a frequent diagnosis in renal transplantation, and there is increasing evidence that regulatory T lymphocytes are involved in its pathogenesis. Current histopathologic practice does not differentiate between graft-protecting and -damaging T lymphocytes, and patients with Bord-R routinely receive rejection treatment. We analyzed Treg-associated forkhead box P3 (Foxp3) gene expression in Bord-R and more severe forms of acute rejection episodes (ARE). METHODS: Foxp3 transcripts were measured in 520 serial peripheral blood samples from 177 kidney graft recipients obtained during the first 20 days posttransplantation. RESULTS: The highest Foxp3 transcripts were observed in patients with Bord-R or without rejection and the lowest in patients with ARE. Patients with Bord-R on posttransplant days 5 to 7 showed an increased Foxp3 transcript level of 156%, which increased to 302% by posttransplant days 14 to 16. In contrast, patients with ARE demonstrated significantly lower Foxp3 gene expression than that observed in Bord-R, nonrejectors, or acute tubular necrosis patients (P=0.001, P<0.001, and P=0.005, respectively, on days 11-13). Acute tubular necrosis patients demonstrated intermediately high Foxp3 gene expression. CONCLUSIONS: Our data indicate that increased Treg activity in peripheral blood is a frequent feature of Bord-R. This finding questions the appropriateness of rejection treatment in all patients with the histopathologic diagnosis "Bord-R".

HLA antibodies and the occurrence of early adverse events in the modern era of transplantation: a collaborative transplant study report.

BACKGROUND: Adverse events occurring early after kidney transplantation were reported to influence graft outcome. METHODS: In a prospective multicenter study initiated in 2001, we investigated the relationship between human leukocyte antigen (HLA) alloantibodies, early adverse events, and graft outcome. RESULTS: Pretransplant presence of HLA class I antibodies was associated with a higher rate of no immediate function (NIF) of the graft (odds ratio [OR] 1.78, P=0.023) and acute rejection episodes (ARE) during the first 3 months after transplantation (OR 2.53, P<0.001). NIF and ARE during posttransplant days 15 to 90 were associated with increased risk of graft loss to year 3 (OR 2.06 and 3.75, P=0.006 and P<0.001, respectively). ARE within the first 2 posttransplant weeks did not increase the risk significantly, especially if they occurred in nonsensitized patients without antibodies. Graft survival at 3 years in patients with both NIF and ARE during the first 3 months was significantly lower (81.3%+/-6.2%) than in patients who did not experience NIF or ARE (95.1%+/-1.0%, P<0.001). Importantly, neither NIF nor ARE had an impact on subsequent graft survival if good graft function (serum creatinine <130 mumol/L) was observed at the end of the third month. CONCLUSION: Our results show that NIF and ARE associated with pretransplant antibodies against HLA class I, and they suggest that early diagnosis and treatment of adverse events with the aim of obtaining normal 3-month graft function should be pursued rigorously. Good 3-month graft function is associated with excellent long-term survival, even in patients with pretransplant HLA antibodies and posttransplant adverse events.

Successful deceased-donor kidney transplantation in crossmatch-positive patients with peritransplant plasma exchange and Rituximab.

Sensitized patients awaiting renal transplantation have a markedly reduced probability of receiving a crossmatch (XM)-negative renal allograft, and, even if transplanted with a negative complement-dependent cytotoxicity (CDC)-XM, they are at an increased risk of antibody-mediated humoral rejection with early graft loss. We report here for the first time on the effectiveness of a single pretransplant plasma exchange session in rendering a positive complement-dependent cytotoxicity-XM negative and, in combination with anti-CD20 therapy, allowing successful renal transplantation in two sensitized deceased-donor kidney allograft recipients. In a third patient with high donor-specific reactivity, the therapy was unsuccessful. Plasma exchange treatments were continued during the posttransplant period until stable allograft function was achieved. Both patients showed good graft outcome at 27 and 21 months after transplantation with serum creatinine values of 1.60 and 1.25 mg/dL, respectively.

Successful treatment of chronic antibody-mediated rejection with IVIG and rituximab in pediatric renal transplant recipients.

BACKGROUND: Chronic antibody-mediated rejection (CAMR) of renal allografts has recently been recognized as a defined nosologic entity. The outcome of CAMR is poor; there is no established treatment protocol for this condition. We therefore initiated a pilot study on treatment of CAMR with an antihumoral regimen consisting of high-dose intravenous immunoglobulin (IVIG) and the chimeric anti-CD20 antibody rituximab. METHODS: Six pediatric renal transplant recipients with CAMR received four weekly doses of IVIG (1 g/kg body weight per dose), followed by a single dose of rituximab (375 mg/m2 body surface area) 1 week after the last IVIG infusion. Renal allograft biopsies were evaluated using the Banff '05 classification. Human leukocyte antigen-specific antibodies were detected by panel-reactive lymphocytotoxicity and solid phase ELISA assays. RESULTS: Median glomerular filtration rate during 6 months before intervention dropped by 25 (range, 11-26) mL/min/1.73 m2 (P<0.05) and increased in response to antihumoral therapy by 21 (-14 to +30) 6 months (P<0.05) and by 19 (-14 to +23) mL/min/1.73 m2 12 months (P=0.063) after start of treatment. Glomerular filtration rate improved or stabilized in 4 patients; the two nonresponders had the highest degree of transplant glomerulopathy, the highest degree of C4d deposition in peritubular capillaries and pronounced interstitial inflammation. The treatment regimen was well tolerated. CONCLUSION: This pilot study demonstrates that CAMR in pediatric renal transplant recipients can be treated successfully and safely with a combination of IVIG and rituximab. This observation should encourage more extensive studies to evaluate this new treatment strategy.

Proteasomal chymotrypsin-like peptidase activity is required for essential functions of human monocyte-derived dendritic cells.

The ubiquitin-proteasome pathway is the principal system for extralysosomal protein degradation in eukaryotic cells, and is essential for the regulation and maintenance of basic cellular processes, including differentiation, proliferation, cell cycling, gene transcription and apoptosis. The 26S proteasome, a large multicatalytic protease complex, constitutes the system's proteolytic core machinery that exhibits different proteolytic activities residing in defined proteasomal subunits. We have identified proteasome inhibitors - bortezomib, epoxomicin and lactacystin - which selectively inhibit the proteasomal beta5 subunit-located chymotrypsin-like peptidase activity in human monocyte-derived dendritic cells (DCs). Inhibition of proteasomal chymotrypsin-like peptidase activity in immature and mature DCs impairs the cell-surface expression of CD40, CD86, CD80, human leucocyte antigen (HLA)-DR, CD206 and CD209, induces apoptosis, and impairs maturation of DCs, as demonstrated by decreased cell-surface expression of CD83 and lack of nuclear translocation of RelA and RelB. Inhibition of chymotrypsin-like peptidase activity abrogates macropinocytosis and receptor-mediated endocytosis of macromolecular antigens in immature DCs, and inhibits the synthesis of interleukin (IL)-12p70 and IL-12p40 in mature DCs. As a functional consequence, DCs fail to stimulate allogeneic CD4(+) and CD8(+) T cells and autologous CD4(+) T cells sufficiently in response to inhibition of chymotrypsin-like peptidase activity. Thus, proteasomal chymotrypsin-like peptidase activity is required for essential functions of human DCs, and inhibition of proteasomal chymotrypsin-like peptidase activity by selective inhibitors, or by targeting beta5 subunit expression, may provide a novel therapeutic strategy for suppression of deregulated and unwanted immune responses.