Thursday, October 31, 2013

Trends in Immune Cell Function Assay and Donor-Specific HLA Antibodies in Kidney Transplantation: A 3-Year Prospective Study

The immune cell function assay (ICFA) and de novo anti-donor-specific HLA antibodies (DSA) have been proposed as assays for immune monitoring in renal transplantation, but longitudinal studies examining the modification of both parameters over time and their relation with clinical events are lacking. We prospectively measured longitudinal changes in ICFA and DSA levels in 55 kidney transplant recipients over 3-year follow-up (534 visits) and analyzed their relation with the risk of developing acute rejections or infections. Seven patients (12.7%) developed biopsy-proven acute rejection, and 20 (36.4%) developed viral infections. At 3 years posttransplant, 28% of the patients had developed de novo DSA. ICFA levels peaked at 1–2 months posttransplant (p = 0.005) and leveled off thereafter. They were not associated with the risk of acute rejections, viral infections or development of de novo DSA. Instead, the incidence of de novo DSA was higher in patients who previously had viral infections (adjusted-odds ratio of de novo DSA associated with prior infections: 6.03 [95% CI, 1.64–22.06; p = 0.007]). Our prospective, longitudinal study does not support using ICFA to quantify the immune risk in kidney transplantation. Further studies are needed to confirm the relationship between viral infections and the subsequent development of de novo DSA (read more)

Wednesday, October 30, 2013

A Comprehensive and Quantitative Analysis of the Major Specificities in Rabbit Antithymocyte Globulin Preparations

Antithymocyte globulin (ATG) preparations are used for treatment and prevention of graft rejection episodes, graft versus host disease and aplastic anemia. The immunomodulatory and immuosuppressive properties of ATGs are mediated by their interaction with a large variety of antigens expressed on immune and nonimmune cell populations. We have conducted a comprehensive analysis on antibody specificities contained in rabbit ATGs in clinical use, ATG-Fresenius (ATG-F) and Thymoglobulin (THG). We have used retroviral expression cloning to identify novel ATG antigens and demonstrate that together with ATG antigens described earlier, these molecules account for the majority of ATG antibodies directed to human cells. Moreover, we have employed cell lines engineered to express antigens at high levels to quantify the antibodies directed to each ATG antigen. We have used cell lines expressing the T cell receptor complex, CD2 and CD28 to remove antibodies to these antigens from ATG preparations and demonstrate that this treatment abrogated the ability of ATGs to induce activation and forkhead box P3 expression in T cells. Comprehensive information and differences on the antigens targeted by ATG-F and THG as well as novel approaches to assess their functional properties are the basis for a better understanding of their immunomodulatory capacities and might eventually translate into improved ATG-based regimen (read more).

Saturday, October 26, 2013

Class II HLA Epitope Matching—A Strategy to Minimize De Novo Donor-Specific Antibody Development and Improve Outcomes

De novo donor-specific antibody (dnDSA) develops in 15–25% of renal transplant recipients within 5 years of transplantation and is associated with 40% lower graft survival at 10 years. HLA epitope matching is a novel strategy that may minimize dnDSA development. HLAMatchmaker software was used to characterize epitope mismatches at 395 potential HLA-DR/DQ/DP conformational epitopes for 286 donor–recipient pairs. Epitope specificities were assigned using single antigen HLA bead analysis and correlated with known monoclonal alloantibody epitope targets. Locus-specific epitope mismatches were more numerous in patients who developed HLA-DRdnDSA alone (21.4 vs. 13.2, p < 0.02) or HLA-DQ dnDSA alone (27.5 vs. 17.3, p < 0.001). An optimal threshold for epitope mismatches (10 for HLA-DR, 17 for HLA-DQ) was defined that was associated with minimal development of Class II dnDSA. Applying these thresholds, zero and 2.7% of patients developed dnDSA against HLA-DR and HLA-DQ, respectively, after a median of 6.9 years. Epitope specificity analysis revealed that 3 HLA-DR and 3 HLA-DQ epitopes were independent multivariate predictors of Class II dnDSA. HLA-DR and DQ epitope matching outperforms traditional low-resolution antigen-based matching and has the potential to minimize the risk of de novo Class II DSA development, thereby improving long-term graft outcome (read more)

Friday, October 25, 2013

HLA-B*13:01 and the Dapsone Hypersensitivity Syndrome

BACKGROUND : Dapsone is used in the treatment of infections and inflammatory diseases. The dapsone hypersensitivity syndrome, which is associated with a reported mortality of 9.9%, develops in about 0.5 to 3.6% of persons treated with the drug. Currently, no tests are available to predict the risk of the dapsone hypersensitivity syndrome.
METHODS : We performed a genomewide association study involving 872 participants who had received dapsone as part of multidrug therapy for leprosy (39 participants with the dapsone hypersensitivity syndrome and 833 controls), using log-additive tests of single-nucleotide polymorphisms (SNPs) and imputed HLA molecules. For a replication analysis, we genotyped 24 SNPs in an additional 31 participants with the dapsone hypersensitivity syndrome and 1089 controls and performed next-generation sequencing for HLA-B and HLA-C typing at four-digit resolution in an independent series of 37 participants with the dapsone hypersensitivity syndrome and 201 controls.
RESULTS : Genomewide association analysis showed that SNP rs2844573, located between the HLA-B and MICA loci, was significantly associated with the dapsone hypersensitivity syndrome among patients with leprosy (odds ratio, 6.18; P=3.84×10−13). HLA-B*13:01was confirmed to be a risk factor for the dapsone hypersensitivity syndrome (odds ratio, 20.53; P=6.84×10−25). The presence of HLA-B*13:01 had a sensitivity of 85.5% and a specificity of 85.7% as a predictor of the dapsone hypersensitivity syndrome, and its absence was associated with a reduction in risk by a factor of 7 (from 1.4% to 0.2%). HLA-B*13:01 is present in about 2 to 20% of Chinese persons, 1.5% of Japanese persons, 1 to 12% of Indians, and 2 to 4% of Southeast Asians but is largely absent in Europeans and Africans.
CONCLUSIONS : HLA-B*13:01 was associated with the development of the dapsone hypersensitivity syndrome among patients with leprosy. (Funded by the National Natural Science Foundation of China and others.) (read more)

Identification of a cellular ligand for the natural cytotoxicity receptor NKp44

With an array of activating and inhibitory receptors, natural killer (NK) cells are involved in the eradication of infected, transformed, and tumor cells. NKp44 is a member of the natural cytotoxicity receptor family, which is exclusively expressed on activated NK cells. Here, we identify natural cytotoxicity receptor NKp44 (NKp44L), a novel isoform of the mixed-lineage leukemia-5 protein, as a cellular ligand for NKp44. Unlike the other MLL family members, NKp44L is excluded from the nucleus, but expressed at the cell-surface level; its subcellular localization is being associated with the presence of a specific C-terminal motif. Strikingly, NKp44L has not been detected on circulating cells isolated from healthy individuals, but it is expressed on a large panel of the tumor and transformed cells. The sharply decreased NK lysis activity induced by anti-NKp44L antibodies directly demonstrates the role of NKp44L in cytotoxicity. Taken together, these results show that NKp44L could be critical for NK cell-mediated innate immunity. The identification and cellular distribution of NKp44L highlight the role of this self-molecule as a danger signal to alert the NK cell network (read more)

Identification of a KIR antisense lncRNA expressed by progenitor cells

Human NK cells express cell surface class I MHC receptors (killer cell immunoglobulin-like receptor, KIR) in a probabilistic manner. Previous studies have shown that a distal promoter acts in conjunction with a proximal bidirectional promoter to control the selective activation of KIRgenes. We report here the presence of an intron 2 promoter in several KIRgenes that produce a spliced antisense transcript. This long noncoding RNA (lncRNA) transcript contains antisense sequence complementary to KIR-coding exons 1 and 2 as well as the proximal promoter region of the KIRgenes. The antisense promoter contains myeloid zinc finger 1 (MZF-1)-binding sites, a transcription factor found in hematopoietic progenitors and myeloid precursors. The KIR antisense lncRNA was detected only in progenitor cells or pluripotent cell lines, suggesting a function that is specific for stem cells. Overexpression of MZF-1 in developing NK cells led to decreased KIR expression, consistent with a role for the KIR antisense lncRNA in silencing KIR gene expression early in development (read more)

Friday, October 18, 2013

Inhibitory Effects of Belatacept on Allospecific Regulatory T-Cell Generation in Humans

Background : It is unclear if new costimulatory blockade agents, such as the cytotoxic T lymphocyte–associated antigen 4-Ig molecule belatacept (BEL), promote or inhibit the potential for immunologic tolerance in transplantation. We therefore tested the in vitro effects of BEL on human regulatory T cells (Tregs) in mixed lymphocyte reactions (MLR) alone and in combination with maintenance agents used in transplant recipients.
Methods : BEL, mycophenolic acid (MPA), and sirolimus, either alone or in combination, were added to healthy volunteer Treg-MLR, testing (a) 3H-TdR incorporation for inhibition of lymphoproliferation and (b) flow cytometry to analyze for newly generated CD4+CD25highFOXP3+ Tregs in carboxyfluorescein succinimidyl ester–labeled MLR responders. In addition, the modulatory effects of putative Tregs generated in the presence of these drugs were also tested using the lymphoproliferation and flow cytometric assays. 
Results : In comparison with medium controls, BEL dose-dependently inhibited both lymphoproliferation and Treg generation in human leukocyte antigen DR matched and mismatched MLRs either alone or in combination with MPA or sirolimus. However, MPA alone inhibited lymphoproliferation but significantly enhanced Treg generation at subtherapeutic concentrations (P<0.01). In addition, purified CD4+CD127- cells generated in MLR in the presence of MPA and added as third component modulators in fresh MLRs significantly enhanced newly developed Tregs in the proliferating responder cells compared with those generated with BEL or medium controls. 
Conclusions : BEL alone and in combination with agents used in transplant recipients inhibits the in vitro generation of human Tregs. BEL might therefore be a less optimal agent for tolerance induction in human organ transplantation (read more)

Pretransplantation GAD-Autoantibody Status to Guide Prophylactic Antibody Induction Therapy in Simultaneous Pancreas and Kidney Transplantation

Background : Daclizumab and antithymocyte globulin (ATG) have been shown to reduce allograft rejection. We assessed the safety and efficacy of daclizumab or ATG prophylaxis in combination with triple immunotherapy in simultaneous pancreas-kidney transplant (SPKT) recipients.
Methods : Thirty-nine type 1 diabetic patients scheduled for primary SPKT were randomized to receive prophylactic therapy with either daclizumab or ATG. A group of 27 patients without prophylactic antibodies was used for retrospective comparison. All patients received cyclosporine and mycophenolate mofetil and gradually tapered prednisone. Autoantibodies and cellular autoreactivity were measured to assess recurrent autoreactive responses. ResultsBaseline and transplant characteristics were comparable among groups. Both daclizumab and ATG therapy resulted in a significant reduction in acute rejection episodes. The incidence of rejection episodes was significantly higher in pretransplantation GAD autoantibody-positive daclizumab-treated recipients compared with GAD autoantibody-negative or ATG-treated recipients. IA-2 islet autoantibodies showed no association with rejection. There were no significant differences between the groups for in vitro autoreactivity, clinical outcome, or functional parameters.
Conclusions : Daclizumab or ATG combined with a maintenance immunosuppressive regime consisting of cyclosporine, mycophenolate mofetil, and prednisolone were well tolerated and equally effective in reducing the incidence of acute rejection episodes in SPKT recipients. Up to 3 years, no adverse sequelae of the immunoprophylaxis or clinical and ex vivo recurrent autoimmunity were observed. We propose that the pretransplantation existence of GAD65 autoantibodies serves as a marker guiding the choice for prophylactic therapy in pancreas transplantation (read more)

Thursday, October 17, 2013

Impact of de novo donor-specific HLA antibodies detected by Luminex solid phase assay after transplantation in a group of 88 consecutive living donor renal transplantations

Background : De novo donor-specific HLA antibodies (DSA) after renal transplantation are known to be correlated with poor graft outcome and the development of acute and chronic rejection. Currently, data for the influence ofde novo DSA in patient cohorts including only living donor renal transplantations (LDRT) are limited.
Methods : A consecutive cohort of 88 LDRT was tested for the occurrence of de novo DSA by utilizing the highly sensitive Luminex solid phase assay for antibody detection. Data were analyzed for risk factors of de novoDSA development and correlated with acute rejection (AR) and graft function.
Results : Patients with de novo DSA [31 (35%)] showed a trend for inferior graft function [Mean creatinine change (mg/dL/year) after the first year: 0.15 DSA (+) vs 0.02 DSA (-) (p= 0.10)] and a higher rate of AR episodes, especially in case of de novo DSA of both class I and II [6 (55%), (p= 0.05)]. Antibody mediated rejection (AMR) appeared in five patients and was significantly correlated with de novo DSA (p= 0.05).
Conclusions : Monitoring for de novo DSA after LDRT may help to identify patients at risk for declining renal function. Especially patients with simultaneous presence of de novo DSA class I and class II are at a high risk to suffer AR episodes (read more)

Wednesday, October 16, 2013

A common rejection module (CRM) for acute rejection across multiple organs identifies novel therapeutics for organ transplantation

Using meta-analysis of eight independent transplant datasets (236 graft biopsy samples) from four organs, we identified a common rejection module (CRM) consisting of 11 genes that were significantly overexpressed in acute rejection (AR) across all transplanted organs. The CRM genes could diagnose AR with high specificity and sensitivity in three additional independent cohorts (794 samples). In another two independent cohorts (151 renal transplant biopsies), the CRM genes correlated with the extent of graft injury and predicted future injury to a graft using protocol biopsies. Inferred drug mechanisms from the literature suggested that two FDA-approved drugs (atorvastatin and dasatinib), approved for nontransplant indications, could regulate specific CRM genes and reduce the number of graft-infiltrating cells during AR. We treated mice with HLA-mismatched mouse cardiac transplant with atorvastatin and dasatinib and showed reduction of the CRMgenes, significant reduction of graft-infiltrating cells, and extended graft survival. We further validated the beneficial effect of atorvastatin on graft survival by retrospective analysis of electronic medical records of a single-center cohort of 2,515 renal transplant patients followed for up to 22 yr. In conclusion, we identified a CRM in transplantation that provides new opportunities for diagnosis, drug repositioning, and rational drug design (read more)

Monday, October 14, 2013

Donor specific HLA antibodies: Evaluating the risk for graft loss in renal transplant recipients with isotype switch from complement fixing IgG1/IgG3 to non-complement fixing IgG2/IgG4 anti HLA alloantibodies

HLA alloantibodies have a multitude of damaging effects on the allograft, both complement(C’) activation and Fc independent ones. To date, the clinical significance of non C’ fixing (NCF) HLA donor specific antibodies (DSA) is still unclear. In this study, we investigated whether renal transplant recipients with NCF-DSA subclasses (IgG2/IgG4,IgA1/IgA2) are at higher risk of graft loss compared to patients with exclusively C’ fixing (IgG1/IgG3). Blood samples from 274 patients were analyzed for HLA IgG and IgA subclasses using a modified single antigen bead assay. We identified 50(18.2%) patients with circulating NCF antibodies either DSA (n=17) or against third party HLA (n=33). NCF DSA were preferentially of IgG2/IgG4 isotype (11/17) and were mainly directed against HLA class II (13/17). NCF DSA were present as a mixture with strong C’ fixing IgG1/IgG3. Graft survival was similar between patients with exclusively C’ fixing antibodies and those with a mixture panel (log rang test p=0.162), and also among patients with different immunoglobulin isotype and subclasse (long rank test, p=0.732). We conclude that expansion of DSA to NCF subclasses post renal transplantation does not seem to be associated with worse graft survival as compared to the presence of exclusive C’ fixing subclasses (read more)

Saturday, October 12, 2013

HLA-F and MHC Class I Open Conformers Are Ligands for NK Cell Ig-like Receptors

Killer Ig-like receptors (KIRs) are innate immune receptors expressed by NK and T cells classically associated with the detection of missing self through loss of their respective MHC ligand. Some KIR specificities for allelic classical class I MHC (MHC-I) have been described, whereas other KIR receptor–ligand relationships, including those associated with nonclassical MHC-I, have yet to be clearly defined. We report in this article that KIR3DL2 and KIR2DS4 and the nonclassical Ag HLA-F, expressed as a free form devoid of peptide, physically and functionally interact. These interactions extend to include classical MHC-I open conformers as ligands, defining new relationships between KIR receptors and MHC-I. The data collectively suggest a broader, previously unrecognized interaction between MHC-I open conformers—including prototypical HLA-F—and KIR receptors, acting in an immunoregulatory capacity centered on the inflammatory response (read more)

Clinical impact of the baseline donor-specific anti-HLA antibody measured by Luminex single antigen assay in living donor kidney transplant recipients after desensitization therapy

The aim of this study is to investigate the clinical impact of donor-specific anti-HLA-antibody (HLA-DSA) baseline levels, measured using the Luminex single-antigen assay (LSA), in living-donor kidney-transplantation (LDKT). Total 129 cases of LDKT were divided into 4 groups according to baseline mean fluorescence intensity (MFI) HLA-DSA values: Strong (n=6), >10,000; Moderate (n=8), 5,000–10,000; Weak (n=11), 1,000–5,000, Negative (n=104), <1,000. Pre-transplant desensitization (DSZ) was performed to decrease the MFI to weak or negative values before KT. Clinical outcomes in the 4 groups were compared. After DSZ, HLA-DSA decreased to weak or negative levels in all patients; Acute rejections developed more frequently in strong group (5/6 (83.3%)) compared to other three groups (P<0.05) and especially acute antibody-mediated rejection (AAMR) developed almost exclusively in strong group (4/6 (66.7%)). Strong HLA-DSA levels at baseline were more predictive of AAMR than either type of XM (complement-dependent lymphocytotoxicity or flow cytometry) in ROC analysis. Allograft function in this group showed significant deterioration during follow-up compared to the other groups. In conclusion, strong HLA-DSA levels at baseline are associated with worse allograft outcome even after successful desensitization; therefore, strict monitoring and strong maintenance immunosuppression may be required in such patients (read more)

International Kidney Paired Donation

We report a 10-way domino transplant, which was initiated in September 2009 and concluded in July 2010. The uniqueness of this chain was that it included the international exchange of kidneys between the United States and Canada. The chain was initiated by an altruistic donor and included three hospitals, four flights, one international exchange, and one pediatric recipient. The international donor and recipient procedures were performed at McGill University (Montreal, Quebec, Canada) and Johns Hopkins University (Baltimore, MD). Both organs were transported via chartered international flights with cold ischemia times of less than 6 hr, which included not only procurement and travel time but also time for customs clearance. Donor-related expenses were charged at the institution where donation occurred (read more)

Rapid Generation of EBV-Specific Cytotoxic T Lymphocytes Resistant to Calcineurin Inhibitors for Adoptive Immunotherapy

Epstein–Barr virus (EBV)-associated posttransplant lymphoproliferative disorder (PTLD) remains a major cause of morbidity and mortality after hematopoietic stem cell (HSCT) or solid organ transplant (SOT). Strategies to reconstitute immunity by adoptive transfer of EBV-specific cytotoxic T lymphocyte (CTL) therapy while highly effective in the HSCT setting where immunosuppression can be withdrawn have been less successful in the SOT setting where continued immunosuppression therapy is necessary. Additionally, the complexity and time taken to generate EBV-CTLs for adoptive transfer limit the clinical applicability. We have developed a system for the rapid generation of EBV-CTLs resistant to immunosuppression based on selection of interferon-gamma (IFN-γ) secreting EBV-CTLs and retroviral transduction with a calcineurin B mutant. With this methodology, EBV-CTLs resistant to the calcineurin inhibitor Tacrolimus (TAC) can be produced in 14 days. These CTLs show high specificity for EBV with negligible alloreactivity in both proliferation and cytotoxicity assays and are able to proliferate and secrete IFN-γ in response to antigen stimulation in the presence of therapeutic doses of TAC. This strategy will substantially facilitate clinical application of this approach for the treatment of PTLD in SOT recipients (read more)

Postoperative management of severe acute anemia in a Jehovah's Witness

Background : Low levels of hemoglobin (Hb) are not rare in patients who refuse blood components but this case is unique due to the severity of anemia and the possibility that her previous episode of acute normovolemic hemodilution has influenced her outcome.
Case Report : We report an incident involving acute blood loss after surgery with an extremely low hematocrit. Despite her Hb levels (2.8 g/dL) she remained lucid, expressing her wish not to receive transfusion. When the patient lost consciousness (Hb, 1.4 g/dL) she was promptly sedated, curarized, and put onto mechanical controlled ventilation. Aggressive erythropoietin therapy increased the patient's Hb level by 240% in 10 days, despite a high platelet count. This case demonstrates that critical levels of oxygen delivery may be lower than previously thought possible.
Conclusion : This case is an example of the resilience of the human body in an extreme circumstance. It might be the most severe case of anemia that a person has survived without any sequelae (read more)

Tuesday, October 1, 2013

Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival

Background : Anti-HLA antibodies hamper successful transplantation, and activation of the complement cascade is involved in antibody-mediated rejection. We investigated whether the complement-binding capacity of anti-HLA antibodies plays a role in kidney-allograft failure.
Methods : We enrolled patients who received kidney allografts at two transplantation centers in Paris between January 1, 2005, and January 1, 2011, in a population-based study. Patients were screened for the presence of circulating donor-specific anti-HLA antibodies and their complement-binding capacity. Graft injury phenotype and the time to kidney-allograft loss were assessed.
Results : The primary analysis included 1016 patients. Patients with complement-binding donor-specific anti-HLA antibodies after transplantation had the lowest 5-year rate of graft survival (54%), as compared with patients with non–complement-binding donor-specific anti-HLA antibodies (93%) and patients without donor-specific anti-HLA antibodies (94%) (P<0.001 for both comparisons). The presence of complement-binding donor-specific anti-HLA antibodies after transplantation was associated with a risk of graft loss that was more than quadrupled (hazard ratio, 4.78; 95% confidence interval [CI], 2.69 to 8.49) when adjusted for clinical, functional, histologic, and immunologic factors. These antibodies were also associated with an increased rate of antibody-mediated rejection, a more severe graft injury phenotype with more extensive microvascular inflammation, and increased deposition of complement fraction C4d within graft capillaries. Adding complement-binding donorspecific anti-HLA antibodies to a traditional risk model improved the stratification of patients at risk for graft failure (continuous net reclassification improvement, 0.75; 95% CI, 0.54 to 0.97).(read more)

Acute Cellular and Antibody-Mediated Rejection of the Pancreas Allograft: Incidence, Risk Factors and Outcomes

Antibody-mediated rejection (AMR) after pancreas transplantation is a recently identified entity. We describe the incidence of, risk factors for, and outcomes after AMR, and the correlation of C4d immunostaining and donor-specific antibody (DSA) in the diagnosis of AMR. We retrospectively analyzed 162 pancreas transplants in 159 patients who underwent 94 pancreas allograft biopsies between 2006 and 2009. Univariate and multivariate analyses were performed to evaluate risk factors for pancreas graft AMR. One-year rejection rates and survival after rejection were calculated by Kaplan–Meier methods. AMR occurred in 10% of patients by 1-year posttransplant. Multivariate risk factors identified for AMR include nonprimary simultaneous pancreas–kidney (SPK) transplant, primary solitary pancreas (PAN) transplant and race mismatch. After pancreas rejection, patient survival was 100% but 20% (8 of 41) of pancreas grafts failed within 1 year. Graft survival after acute cellular rejection (ACR), AMR and mixed rejection was similar. Of biopsies that stained >5% C4d, 80% were associated with increased Class I DSA. In summary, AMR occurs at a measurable rate after pancreas transplantation, and the diagnosis should be actively sought using C4d staining and DSA levels in patients with graft dysfunction, especially after nonprimary SPK and primary PAN transplantation (read more)

RIPK3-Mediated Necroptosis Promotes Donor Kidney Inflammatory Injury and Reduces Allograft Survival

Kidney transplant injury occurs with ischemia and alloimmunity. Members of the receptor interacting protein kinase family (RIPK1,3) are key regulators of “necroptosis,” a newly recognized, regulated form of necrosis. Necroptosis and apoptosis death appear to be counterbalanced as caspase-8 inhibition can divert death from apoptosis to necrosis. Inhibition of necroptosis in donor organs to limit injury has not been studied in transplant models. In this study, necroptosis was triggered in caspase inhibited tubular epithelial cells (TEC) exposed to tumor necrosis factor alpha in vitro, while RIPK1 inhibition with necrostatin-1 or use of RIPK3−/−TEC, prevented necroptosis. In vivo, short hairpin RNA silencing of caspase-8 in donor B6 mouse kidneys increased necroptosis, enhanced high-mobility group box 1 release, reduced renal function and accelerated rejection when transplanted into BALB/c recipients. Using ethidium homodimer perfusion to assess necrosisin vivo, necrosis was abrogated in RIPK3−/− kidneys postischemia. Following transplantation, recipients receiving RIPK3−/− kidneys had longer survival (p = 0.002) and improved renal function (p = 0.03) when compared to controls. In summary, we show for the first time that RIPK3-mediated necroptosis in donor kidneys can promote inflammatory injury, and has a major impact on renal ischemia–reperfusion injury and transplant survival. We suggest inhibition of necroptosis in donor organs may similarly provide a major clinical benefit (read more)

Kidney Intragraft Donor-Specific Antibodies as Determinant of Antibody-Mediated Lesions and Poor Graft Outcome

Allograft pathology, antibody–tissue interaction as demonstrated by C4d deposition and serological evidence of donor-specific antibodies (DSA) are the cardinal diagnostic features of antibody-mediated lesions (AML) in kidney transplantation. However, discrepancy between histological and serological findings is common, and more reliable diagnostic tools are called for. Here, we asked whether the in situ detection of DSA could serve as marker for AML. To that end, we applied the anti-HLA single antigen flow bead assay to eluates from 51 needle core graft biopsies performed for cause. Intragraft antibody profiles were correlated to serum DSA (sDSA), histological data and transplant outcome. The prevalence and the mean number of intragraft DSA (gDSA) were lower than that of sDSA (15/51 gDSA+ vs. 37/51 sDSA+ patients; 1.64 gDSA vs. 2.24 sDSA per patient). DSA were detected in all anti-HLA antibody-positive biopsies (15/15). The presence of gDSA was significantly associated with (1) microcirculation lesions taken individually (g, cg) and analyzed in functional clusters (ptc + g + cg > 0, cg + mm > 0), (2) C4d positivity and (3) a worse short-term transplant outcome (p = 0.05). These associations were not found for patients presenting only sDSA. Taken together, these results indicate that gDSA is a severity marker of antibody-mediated pathogenic process (read more)