Gregor Bond

Complement component C3 activation: the leading cause of the prozone phenomenon affecting HLA antibody detection on single-antigen beads.

Background Luminex-based anti-HLA IgG detection on single-antigen flow beads (SAFB) represents a valuable tool for characterization of allosensitization patterns. Assay interpretation, however, may be impeded by false-low test results caused by the prozone effect. Recent experimental data have related this artifact to direct blockade of IgG detection by complement component C1 as a possible candidate mechanism. Methods To dissect a causative role of C1 complex formation and subsequent steps of classical complement activation, native or modified sera obtained from transplant candidates with HLA class I sensitization were evaluated applying SAFB-based IgG, IgM, C1q, C1r, C1s, or C4 and C3 split product detection, respectively. Results Evaluating a total of 1,164 single-antigen reactions, serum dilution (1:10) revealed an 11% incidence of the prozone effect as defined by a greater than 100% increase in IgG mean fluorescence intensity. Prozoning was found to be related to the amount of antibody-triggered C1q, C4 or C3 split product deposition, and was eliminated by serum modifications affecting the integrity of the C1 complex (dithiotreitol, ethylenediaminetetraacetic acid, heat inactivation). Remarkably, the same effect was achieved without C1 disintegration, either by serum treatment with methylamine to block C4 and C3 split product binding or by cobra venom factor to trigger C3 consumption. Conclusions Our results reinforce a central role of C1 as a trigger of prozoning. However, we provide evidence that C1 may exert its effects only indirectly, namely via inducing C3 fragment deposition, which by coating of the bead surface may block the binding of IgG detection reagents.

Diagnostic Contribution of Donor-specific Antibody Characteristics to Uncover Late Silent Antibody-mediated Rejection—results of a Cross-sectional Screening Study

Background Circulating donor-specific antibodies (DSA) detected on bead arrays may not inevitably indicate ongoing antibody-mediated rejection (AMR). Here, we investigated whether detection of complement-fixation, in parallel to IgG mean fluorescence intensity (MFI), allows for improved prediction of AMR. Methods Our study included 86 DSA+ kidney transplant recipients subjected to protocol biopsy, who were identified upon cross-sectional antibody screening of 741 recipients with stable graft function at 6 months or longer after transplantation. IgG MFI was analyzed after elimination of prozone effect, and complement-fixation was determined using C1q, C4d, or C3d assays. Results Among DSA+ study patients, 44 recipients (51%) had AMR, 24 of them showing C4d-positive rejection. Although DSA number or HLA class specificity were not different, patients with AMR or C4d + AMR showed significantly higher IgG, C1q, and C3d DSA MFI than nonrejecting or C4d-negative patients, respectively. Overall, the predictive value of DSA characteristics was moderate, whereby the highest accuracy was computed for peak IgG MFI (AMR, 0.73; C4d + AMR, 0.71). Combined analysis of antibody characteristics in multivariate models did not improve AMR prediction. Conclusions We estimate a 50% prevalence of silent AMR in DSA+ long-term recipients and conclude that assessment of IgG MFI may add predictive accuracy, without an independent diagnostic advantage of detecting complement-fixation.

Capillary C4d and Kidney Allograft Outcome in Relation to Morphologic Lesions Suggestive of Antibody-Mediated Rejection

BACKGROUND AND OBJECTIVES Recent studies highlighting a role of C4d- antibody-mediated rejection (ABMR) have debated whether C4d staining has independent value as a rejection marker. Considering the presumed role of complement as an important effector of graft injury, this study hypothesized that capillary C4d, a footprint of antibody-triggered complement activation, indicates a particularly severe manifestation of ABMR. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This large retrospective clinicopathologic study sought to assess the clinical predictive value of C4d staining in relation to ABMR morphology. Overall, 885 renal allograft recipients who underwent transplantation between 1999 and 2006 (median duration of follow-up, 63.3 [interquartile range, 40.6-93.5] months; 206 graft losses) were included if they had had one or more indication biopsies. A total of 1976 biopsy specimens were reevaluated for capillary C4d staining (C4d data were available for 825 patients) and distinct morphologic lesions suggestive of ABMR, including glomerulitis, peritubular capillaritis, capillary microthrombi, transplant glomerulopathy, and severe intimal arteritis. RESULTS C4d+ patients, with or without ABMR features, had worse death-censored 8-year graft survival (53% or 67%) than C4d- patients (66% or 81%; P<0.001). In Cox regression analysis, C4d was associated with a risk of graft loss independently of baseline confounders and ABMR morphology (hazard ratio, 1.85 [95% confidence interval, 1.34 to 2.57]; P<0.001). The risk was higher than that observed for C4d- patients, a finding that reached statistical significance in patients showing fewer than two different ABMR lesions. Moreover, in a mixed model, C4d was independently associated with a steeper decline of eGFR (slope per year, -8.23±3.97 ml/min per 1.73 m(2); P<0.001). CONCLUSIONS These results suggest that detection of intragraft complement activation has strong independent value as an additional indicator of ABMR associated with adverse kidney transplant outcomes.

Deceased donor kidney transplantation across donor-specific antibody barriers: predictors of antibody-mediated rejection

BACKGROUND Apheresis-based desensitization allows for successful transplantation across major immunological barriers. For donor-specific antibody (DSA)- and/or crossmatch-positive transplantation, however, it has been shown that even intense immunomodulation may not completely prevent antibody-mediated rejection (ABMR). METHODS In this study, we evaluated transplant outcomes in 101 DSA+ deceased donor kidney transplant recipients (transplantation between 2009 and 2013; median follow-up: 24 months) who were subjected to immunoadsorption (IA)-based desensitization. Treatment included a single pre-transplant IA session, followed by anti-lymphocyte antibody and serial post-transplant IA. In 27 cases, a positive complement-dependent cytotoxicity crossmatch (CDCXM) was rendered negative immediately before transplantation. Seventy-four of the DSA+ recipients had a negative CDCXM already before IA. RESULTS Three-year death-censored graft survival in DSA+ patients was significantly worse than in 513 DSA- recipients transplanted during the same period (79 versus 88%, P = 0.008). Thirty-three DSA+ recipients (33%) had ABMR. While a positive baseline CDCXM showed only a trend towards higher ABMR rates (41 versus 30% in CDCXM- recipients, P = 0.2), DSA mean fluorescence intensity (MFI) in single bead assays significantly associated with rejection, showing 20 versus 71% ABMR rates at <5000 versus >15 000 peak DSA MFI. The predictive value of MFI was moderate, with the highest accuracy at a median of 13 300 MFI (after cross-validation: 0.72). Other baseline variables, including CDC assay results, human leukocyte antigen mismatch, prior transplantation or type of induction treatment, did not add independent predictive information. CONCLUSIONS IA-based desensitization failed to prevent ABMR in a considerable number of DSA+ recipients. Assessing DSA MFI may help stratify risk of rejection, supporting its use as a guide to organ allocation and individualized treatment.

Torque Teno Virus Load - Inverse Association with Antibody-Mediated Rejection after Kidney Transplantation.

BackgroundAntibody-mediated rejection (AMR) represents one of the cardinal causes of late allograft loss after kidney transplantation, and there is great need for noninvasive tools improving early diagnosis of this rejection type. One promising strategy might be the quantification of peripheral blood DNA levels of the highly prevalent and apathogenic Torque Teno virus (TTV), which might mirror the overall level of immunosuppression and thus help determine the risk of alloimmune response. MethodsTo assess the association between TTV load in the peripheral blood and AMR, 715 kidney transplant recipients (median, 6.3 years posttransplantation) were subjected to a systematical cross-sectional AMR screening and, in parallel, TTV quantification. ResultsEighty-six of these recipients had donor-specific antibodies and underwent protocol biopsy, AMR-positive patients (n = 46) showed only 25% of the TTV levels measured in patients without AMR (P = 0.003). In a generalized linear model, higher TTV levels were associated with a decreased risk for AMR after adjustment for potential confounders (risk ratio 0.94 per TTV log level; 95% confidence interval 0.90-0.99; P = 0.02). ConclusionsFuture studies will have to clarify whether longitudinal assessment of TTV load might predict AMR risk and help guide the type and intensity of immunosuppression to prevent antibody-mediated graft injury.

A Randomized Trial of Bortezomib in Late Antibody-Mediated Kidney Transplant Rejection

Late antibody-mediated rejection (ABMR) is a leading cause of kidney allograft failure. Uncontrolled studies have suggested efficacy of the proteasome inhibitor bortezomib, but no systematic trial has been undertaken to support its use in ABMR. In this randomized, placebo-controlled trial (the Bortezomib in Late Antibody-Mediated Kidney Transplant Rejection [BORTEJECT] Trial), we investigated whether two cycles of bortezomib (each cycle: 1.3 mg/m2 intravenously on days 1, 4, 8, and 11) prevent GFR decline by halting the progression of late donor-specific antibody (DSA)-positive ABMR. Forty-four DSA-positive kidney transplant recipients with characteristic ABMR morphology (median time after transplant, 5.0 years; pretransplant DSA documented in 19 recipients), who were identified on cross-sectional screening of 741 patients, were randomly assigned to receive bortezomib (n=21) or placebo (n=23). The 0.5-ml/min per 1.73 m2 per year (95% confidence interval, -4.8 to 5.8) difference detected between bortezomib and placebo in eGFR slope (primary end point) was not significant (P=0.86). We detected no significant differences between bortezomib- and placebo-treated groups in median measured GFR at 24 months (33 versus 42 ml/min per 1.73 m2; P=0.31), 2-year graft survival (81% versus 96%; P=0.12), urinary protein concentration, DSA levels, or morphologic or molecular rejection phenotypes in 24-month follow-up biopsy specimens. Bortezomib, however, associated with gastrointestinal and hematologic toxicity. In conclusion, our trial failed to show that bortezomib prevents GFR loss, improves histologic or molecular disease features, or reduces DSA, despite significant toxicity. Our results reinforce the need for systematic trials to dissect the efficiency and safety of new treatments for late ABMR.

The diffuse extent of peritubular capillaritis in renal allograft rejection is an independent risk factor for graft loss

By the Banff classification, the score of peritubular capillaritis, its extent, and its cellular composition should normally be reported in renal allograft pathology. While the score represents an important diagnostic and prognostic variable, the clinical value of capillaritis extent or composition has yet to be resolved. In a retrospective study of 749 renal transplant recipients subjected to 1322 indication biopsies, we found that prevalence scores of 1, 2, or 3 in the biopsy specimens were 10.7, 11.6, and 2.6%, respectively. Focal and diffuse peritubular capillaritis (inflammation over 50% of cortical peritubular capillaries) was diagnosed in 10.5 or 14.4% of cases, respectively. Mononuclear, granulocytic, and mixed peritubular capillaritis was present in 13.1, 3.3, and 8.5%, respectively. While peritubular capillaritis without further subclassification was not related to higher allograft loss rates, a score of 3 (hazard ratio 2.57 (CI: 1.25-5.28)) and diffuse peritubular capillaritis (1.67 (1.1-2.54)) were significant impartial risk factors for allograft loss. Diffuse peritubular capillaritis was independently associated with features of chronic antibody-mediated rejection and greater eGFR decline after 3 years. In contrast, detailed report of leukocytic composition in peritubular capillaritis did not confer additional prognostic information. Thus, in contrast to typing the infiltrating inflammatory cells, the score and extent of peritubular capillaritis in kidney allograft pathology is essential to assess transplant prognosis.

Longitudinal assessment of HLA and MIC-A antibodies in uneventful pregnancies and pregnancies complicated by preeclampsia or gestational diabetes

The significance of antibodies directed against paternal epitopes in the context of obstetric disorders is discussed controversially. In this study anti-HLA and anti-MIC-A antibodies were analysed in sera of women with uneventful pregnancy (n = 101), preeclampsia (PE, n = 55) and gestational diabetes (GDM, n = 36) using antigen specific microbeads. While two thirds of the women with uneventful pregnancy or GDM were HLA and MIC-A antibody positive in gestational week 11 to 13 with a modest increase towards the end of pregnancy, women with PE showed an inverse kinetic: 90% were HLA antibody positive in gestational week 11 to 13 and only 10% showed HLA reactivities at the end of the pregnancy. HLA antibody binding strength was more pronounced in gestational week 14 to 17 in patients with PE compared to women with uneventful pregnancy (maximum median fluorescence intensity of the highest ranked positive bead 7403, IQR 2193–7938 vs. 1093, IQR 395–5689; p = 0.04) and was able to predict PE with an AUC of 0.80 (95% CI 0.67–0.93; p = 0.002). Our data suggest a pathophysiological involvement of HLA antibodies in PE. HLA antibody quantification in early pregnancy may provide a useful tool to increase diagnostic awareness in women prone to develop PE.

Quantification of Torque Teno Virus Viremia as a Prospective Biomarker for Infectious Disease in Kidney Allograft Recipients

Quantification of the apathogenic and ubiquitous torque teno virus (TTV) in the peripheral blood predicts infectious disease in recipients of a kidney allograft. TTV might be a potential tool to tailor immunosuppressive drug therapy to prevent infections after kidney transplantation.

Allograft rejection is associated with development of functional IgE specific for donor MHC antigens

Background: Donor‐specific antibodies of the IgG isotype are measured routinely for diagnostic purposes in renal transplant recipients and are associated with antibody‐mediated rejection and long‐term graft loss. Objective: This study aimed to investigate whether MHC‐specific antibodies of the IgE isotype are induced during allograft rejection. Methods: Anti‐MHC/HLA IgE levels were measured in sera of mice grafted with skin or heart transplants from various donor strains and in sera of kidney transplant patients with high levels of HLA IgG. Mediator release was triggered in vitro by stimulating basophils that were coated with murine or human IgE‐positive serum, respectively, with specific recombinant MHC/HLA antigens. Kidney tissue samples obtained from organ donors were analyzed by using flow cytometry for cells expressing the high‐affinity receptor for IgE (Fc&egr;RI). Results: Donor MHC class I– and MHC class II–specific IgE was found on acute rejection of skin and heart grafts in several murine strain combinations, as well as during chronic antibody‐mediated heart graft rejection. Anti‐HLA IgE, including donor HLA class I and II specificities, was identified in a group of sensitized transplant recipients. Murine and human anti‐MHC/HLA IgE triggered mediator release in coated basophils on stimulation with specific MHC/HLA antigens. HLA‐specific IgE was not linked to atopy, and allergen‐specific IgE present in allergic patients did not cross‐react with HLA antigens. Fc&egr;RI+ cells were found in the human renal cortex and medulla and provide targets for HLA‐specific IgE. Conclusion: These results demonstrate that MHC/HLA‐specific IgE develops during an alloresponse and is functional in mediating effector mechanisms. GRAPHICAL ABSTRACT Figure. No caption available.