Julie M. Yabu

C1q-fixing human leukocyte antigen antibodies are specific for predicting transplant glomerulopathy and late graft failure after kidney transplantation.

Background. Human leukocyte antigen (HLA) antibodies, especially those that fix complement, are associated with antibody-mediated rejection and graft failure. The C1q assay on single antigen beads detects a subset of HLA antibodies that can fix complement and precede C4d deposition. The aim of this study was to determine whether C1q-fixing antibodies distinguish de novo donor-specific antibodies (DSA) that are clinically relevant and harmful. Methods. We retrospectively studied 31 of 274 kidney transplant recipients who had pretransplant and concurrent biopsy and serum specimens, 13 with C4d-positive and 18 with C4d-negative staining. We measured IgG and C1q DSA pretransplant and at the time of biopsy using single antigen bead assays. We identified 13 recipients who developed de novo DSA by IgG or C1q and examined associations with C4d deposition, transplant glomerulopathy, and graft failure. Results. Testing for DSA by IgG is more sensitive for C4d deposition (IgG: 100%, 95% confidence interval [CI] 0.60–1; C1q: 75%, 95% CI 0.36–0.96). Testing for DSA by C1q is more specific for transplant glomerulopathy (C1q: 81%, 95% CI 0.57–0.94; IgG: 67%, 95% CI 0.43–0.85) and graft loss (C1q: 79%, 95% CI 0.54–0.93; IgG: 63%, 95% CI 0.39–0.83). Absence of de novo DSA by IgG and C1q has a high negative predictive value for the absence of C4d deposition (IgG: 100%, 95% CI 0.73–1; C1q: 88%, 95% CI 0.62–0.98), transplant glomerulopathy (IgG: 100%, 95% CI 0.73–1; C1q: 100%, 95% CI 0.77–1), and graft failure (IgG: 86%, 95% CI 0.56–0.97; C1q: 88%, 95% CI 0.62–0.98). Conclusion. Monitoring patients with the C1q assay, which detects antibodies that fix complement, offers a minimally invasive means of identifying patients at risk for transplant glomerulopathy and graft loss.

Sensitization from transfusion in patients awaiting primary kidney transplant

BACKGROUND Sensitization to human leukocyte antigen (HLA) from red blood cell (RBC) transfusion is poorly quantified and is based on outdated, insensitive methods. The objective was to evaluate the effect of transfusion on the breadth, magnitude and specificity of HLA antibody formation using sensitive and specific methods. METHODS Transfusion, demographic and clinical data from the US Renal Data System were obtained for patients on dialysis awaiting primary kidney transplant who had ≥ 2 HLA antibody measurements using the Luminex single-antigen bead assay. One cohort included patients with a transfusion (n = 50) between two antibody measurements matched with up to four nontransfused patients (n = 155) by age, sex, race and vintage (time on dialysis). A second crossover cohort (n = 25) included patients with multiple antibody measurements before and after transfusion. We studied changes in HLA antibody mean fluorescence intensity (MFI) and calculated panel reactive antibody (cPRA). RESULTS In the matched cohort, 10 of 50 (20%) transfused versus 6 of 155 (4%) nontransfused patients had a ≥ 10 HLA antibodies increase of >3000 MFI (P = 0.0006); 6 of 50 (12%) transfused patients had a ≥ 30 antibodies increase (P = 0.0007). In the crossover cohort, the number of HLA antibodies increasing >1000 and >3000 MFI was higher in the transfused versus the control period, P = 0.03 and P = 0.008, respectively. Using a ≥ 3000 MFI threshold, cPRA significantly increased in both matched (P = 0.01) and crossover (P = 0.002) transfused patients. CONCLUSIONS Among prospective primary kidney transplant recipients, RBC transfusion results in clinically significant increases in HLA antibody strength and breadth, which adversely affect the opportunity for future transplant.

Kidney Transplantation: The Ideal Immunosuppression Regimen

Kidney transplantation today has excellent short-term outcomes, but long-term graft survival has not improved in a parallel fashion. The goal of immunosuppressive therapy is to balance the beneficial effects of reducing acute rejection while minimizing adverse effects from oversuppression including the development of infections, malignancy, and cardiovascular risk factors. In general, current immunosuppressive protocols use combinations of immunosuppressive agents with different mechanisms of action to maximize efficacy and minimize the toxicity of each drug. During the past decade, there has been a growing interest in identifying regimens that permit the minimization of calcineurin inhibitors or corticosteroids in an attempt to decrease nephrotoxicity and metabolic side effects. The emergence of new immunosuppressive agents and tolerance protocols appear promising as a means to deliver immunosuppression without long-term toxicity. Ultimately, the goal of prescribing immunosuppression is to transition from empiric therapy to one of individualized therapy.

Desensitization combined with paired exchange leads to successful transplantation in highly sensitized kidney transplant recipients: strategy and report of five cases.

Sensitization remains a major barrier to kidney transplantation. Sensitized patients comprise 30% of the kidney transplant waiting list but fewer than 15% of highly sensitized patients are transplanted each year. Options for highly sensitized patients with an immunologically incompatible living donor include desensitization or kidney paired donation (KPD). However, these options when used alone may still not be sufficient to allow a compatible transplant for recipients who are broadly sensitized with cumulative calculated panel-reactive antibody (cPRA) > 95%. We describe in this report the combined use of both desensitization and KPD to maximize the likelihood of finding a compatible match with a more immunologically favorable donor through a kidney exchange program. This combined approach was used in five very highly sensitized patients, all with cPRA 100%, who ultimately received compatible living and deceased donor kidney transplants. We conclude that early enrollment in paired kidney donor exchange and tailored desensitization protocols are key strategies to improve care and rates of kidney transplantation in highly sensitized patients.

Calcitonin gene-related peptide stimulates intracellular camp via a protein kinase C- controlled mechanism in human ocular ciliary epithelial cells

Calcitonin gene-related peptides I and II (CGRP I and II) were found to stimulate cAMP levels by approximately 4-6 fold in human nonpigmented ciliary epithelial cells with half-maximal effective concentrations of 20 x 10(-10) and 3 x 10(-10) M, respectively. Prior exposure of cells to 6 x 10(-7) M phorbol 12-myristate, 13-acetate for 15 min resulted in a 40-50% inhibition of CGRP II-dependent cAMP stimulation. Phorbol didecanoate and dioctanoylglycerol also effectively inhibited, whereas 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, had no effect. Staurosporine, a protein kinase C inhibitor, blocked the inhibition of cAMP formation by phorbol esters. cAMP stimulation by forskolin or cholera toxin was not inhibited by phorbol esters, suggesting that neither a Gs protein nor adenylyl cyclase is the site of inhibition by protein kinase C. These data therefore suggest that CGRP receptors are required for inhibition of adenylate cyclase by protein kinase C.

Stimulation of inositol phosphate formation in cultured human retinal pigment epithelium.

Several hormones, neurotransmitters, and neuropeptides were screened for the ability to stimulate inositol phosphate formation in cultured human retinal epithelial (RPE) cells. Carbachol, vasopressin and thrombin were found to be effective. Treatment of RPE cells with all three agents produced increases in inositol monophosphate, inositol bisphosphate and inositol trisphosphate in the presence of 10 mM LiCl. Carbachol stimulated a 4-fold increase in the total of inositol phosphates at 1 mM. Studies with cholinergic antagonists showed a rank order of 4 DAMP greater than QNX greater than pirenzepine greater than methoctramine, suggesting the presence of M3 muscarinic receptors. Vasopressin gave a 2.5-fold stimulation at 10 microM. Agonists of vasopressin were also tested and gave differential responses. Studies using a V1 agonist (PIOVP) and a V2 agonist (DAVP) showed DAVP matching the level of stimulation elicited by vasopressin whereas treatment with PIOVP only reached 50% of the vasopressin response. These data suggested the presence of V2 receptors in the RPE cells. Several proteases were tested for their ability to stimulate RPE inositol phosphates. Thrombin caused a 7-fold increase in inositol phosphate formation at 1 U/ml, whereas trypsin and plasmin elicited smaller responses (approximately 2-fold). The thrombin effect was blocked by the thrombin-specific inhibitor, hirudin, but not by other protease inhibitors. Several mediators of inflammation such as bradykinin, histamine and serotonin were also tested, and they were ineffective in stimulating inositol phosphate turnover in the RPE cells.

Immune Profiles to Predict Response to Desensitization Therapy in Highly HLA-Sensitized Kidney Transplant Candidates.

Background Kidney transplantation is the most effective treatment for end-stage kidney disease. Sensitization, the formation of human leukocyte antigen (HLA) antibodies, remains a major barrier to successful kidney transplantation. Despite the implementation of desensitization strategies, many candidates fail to respond. Current progress is hindered by the lack of biomarkers to predict response and to guide therapy. Our objective was to determine whether differences in immune and gene profiles may help identify which candidates will respond to desensitization therapy. Methods and Findings Single-cell mass cytometry by time-of-flight (CyTOF) phenotyping, gene arrays, and phosphoepitope flow cytometry were performed in a study of 20 highly sensitized kidney transplant candidates undergoing desensitization therapy. Responders to desensitization therapy were defined as 5% or greater decrease in cumulative calculated panel reactive antibody (cPRA) levels, and non-responders had 0% decrease in cPRA. Using a decision tree analysis, we found that a combination of transitional B cell and regulatory T cell (Treg) frequencies at baseline before initiation of desensitization therapy could distinguish responders from non-responders. Using a support vector machine (SVM) and longitudinal data, TRAF3IP3 transcripts and HLA-DR-CD38+CD4+ T cells could also distinguish responders from non-responders. Combining all assays in a multivariate analysis and elastic net regression model with 72 analytes, we identified seven that were highly interrelated and eleven that predicted response to desensitization therapy. Conclusions Measuring baseline and longitudinal immune and gene profiles could provide a useful strategy to distinguish responders from non-responders to desensitization therapy. This study presents the integration of novel translational studies including CyTOF immunophenotyping in a multivariate analysis model that has potential applications to predict response to desensitization, select candidates, and personalize medicine to ultimately improve overall outcomes in highly sensitized kidney transplant candidates.

ABO-incompatible living donor kidney transplantation without post-transplant therapeutic plasma exchange.

Blood group incompatibility remains a significant barrier to kidney transplantation. Approximately, one‐third of donors are blood group incompatible with their intended recipient. Options for these donor‐recipient pairs include blood group incompatible transplantation or kidney paired donation. However, the optimal protocol for blood group incompatible transplantation is unknown. Protocols differ in techniques to remove ABO antibodies, titer targets, and immunosuppression regimens. In addition, the mechanisms of graft accommodation to blood group antigens remain poorly understood. We describe a blood group incompatible protocol using pretransplant therapeutic plasma exchange (TPE), high‐dose intravenous immunoglobulin, and rituximab in addition to prednisone, mycophenolate mofetil, and tacrolimus. In this protocol, we do not exclude patients based on a high initial titer and do not implement post‐transplant TPE. All 16 patients who underwent this protocol received a living donor transplant with 100% patient and graft survival, and no reported episodes of antibody‐mediated rejection to date with a median follow‐up of 2.6 years (range 0.75–4.7 years). We conclude that blood group incompatible transplantation can be achieved without post‐transplant TPE. J. Clin. Apheresis 30:340–346, 2015.

B cell repertoires in HLA-sensitized kidney transplant candidates undergoing desensitization therapy

BackgroundKidney transplantation is the most effective treatment for end-stage renal disease. Sensitization refers to pre-existing antibodies against human leukocyte antigen (HLA) protein and remains a major barrier to successful transplantation. Despite implementation of desensitization strategies, many candidates fail to respond. Our objective was to determine whether measuring B cell repertoires could differentiate candidates that respond to desensitization therapy.MethodsWe developed an assay based on high-throughput DNA sequencing of the variable domain of the heavy chain of immunoglobulin genes to measure changes in B cell repertoires in 19 highly HLA-sensitized kidney transplant candidates undergoing desensitization and 7 controls with low to moderate HLA sensitization levels. Responders to desensitization had a decrease of 5% points or greater in cumulated calculated panel reactive antibody (cPRA) levels, and non-responders had no decrease in cPRA.ResultsDominant B cell clones were not observed in highly sensitized candidates, suggesting that the B cells responsible for sensitization are either not present in peripheral blood or present at comparable levels to other circulating B cells. Candidates that responded to desensitization therapy had pre-treatment repertoires composed of a larger fraction of class-switched (IgG and IgA) isotypes compared to non-responding candidates. After B cell depleting therapy, the proportion of switched isotypes increased and the mutation frequencies of the remaining non-switched isotypes (IgM and IgD) increased in both responders and non-responders, perhaps representing a shift in the repertoire towards memory B cells or plasmablasts. Conversely, after transplantation, non-switched isotypes with fewer mutations increased, suggesting a shift in the repertoire towards naïve B cells.ConclusionsRelative abundance of different B cell isotypes is strongly perturbed by desensitization therapy and transplantation, potentially reflecting changes in the relative abundance of memory and naïve B cell compartments. Candidates that responded to therapy experienced similar changes to those that did not respond. Further studies are required to understand differences between these two groups of highly sensitized kidney transplant candidates.

Cytomegalovirus in the transplanted kidney: a report of two cases and review of prophylaxis

Cytomegalovirus (CMV) disease is a leading cause of infectious morbidity and mortality in patients with a kidney transplant. Before the advent of specific prophylaxis and therapy, more than half of the patients who had not been exposed to CMV prior to kidney transplantation developed primary disease and ~20% of patients with prior evidence of exposure developed reactivation disease [1]. Mortality estimates from CMV disease exceeded 30% [2]. Although use of prophylaxis or preemptive treatment of detectable viral load has drastically reduced its burden, CMV remains a common clinical problem with a wide variety of presentations. To highlight the importance of clinical vigilance for CMV even in the current era of effective prophylaxis, we describe two adult kidney transplant recipients with invasive CMV disease. Both patients were diagnosed with CMV disease after they underwent allograft biopsy, which demonstrated the rare finding of CMV inclusions in the kidney allograft.