Early Acute Allograft Rejection Research Articles

68Ga]DOTA-TATE PET for the detection of early transplant rejection in a heterotopic allograft heart transplantation model of the rat: a pilot study.

The most important cause of heart transplant loss is early acute allograft rejection, caused by the infiltration of lymphocytes, development of edema and myocardial necrosis. It has been propagated that [68Ga]DOTA-TATE PET might be suitable to quantify the presence of SSTR over-expressing lymphocytes. With heterotopic allogenic heart transplant models in the rat readily available, we aimed to investigate, if monitoring and quantification of acute allograft rejection after heterotopic allogenic heart transplantation was feasible by non-invasive serial [68Ga]DOTA-TATE PET. Seventeen Lewis rats (9 for serial PET imaging, 8 for histological correlation) received allogenic heterotopic heart transplants from 17 Brown-Norway rats. On days 4, 6 and 7 a [68Ga]DOTA-TATE PET scan was performed. Imaging of acute transplant rejection until 7 days after allogenic heart transplantation in the rat is feasible. Heterotopic allografts showed significantly increased tracer uptake on day 4 until day 7 after transplantation, reflecting the process of histologically detected myocardial lymphocytic infiltration. Both the area of infarction and the amount of necrosis increased over the course of 7 days, with necrosis reaching statistical significance. We purport that the detected PET signal is primarily a specific marker of lymphocyte infiltration and only to a lesser extent an unspecific marker of infarction and necrosis. Thus, [68Ga]DOTA-TATE PET might be a suitable tool for serial imaging and quantification of lymphocyte infiltration as a direct mediator of acute allograft rejection at an early stage after heart transplantation.

Multiple-biomarkers provide powerful prediction of early acute renal allograft rejection by combination of serum fractalkine, IFN-γ and IP-10

Biomarkers are urgently required for predicting rejection so that anti-rejection treatment can be taken early to protect the allograft from irreversible damage. We hypothesized that the combination of circulating fractalkine, IFN-γ and IP-10 might serve as effective biomarkers for predicting early acute renal allograft rejection. We conducted a retrospective study of 87 subjects, who were classified into acute rejection group (ARG; n = 38) and non-rejection group (NRG; n = 49). Serum fractalkine, IFN-γ and IP-10 levels were measured by Luminex. The levels of fractalkine on day 0 and 7th day, IP-10 on 4th and 7th day, and IFN-γ on 7th day in ARG was significantly higher than that in NRG. Kaplan–Meier survival analysis highlighted the higher-levels groups of fractalkine on day 0, 4th and 7th day, IFN-γ on day 0, 1st, 4th, and 7th day and IP-10 on the 4th and 7th day in rejection-free survival probability were significantly lower than low-levels groups. ROC analyses highlight the superiority of fractalkine on day 0, IP-10 on day 0, 4th and 7th day, and IFN-γ on day 0, 1st and 7th day in prediction of acute rejection. We found the combination of fractalkine on day 0, IP-10 on 7th day and IFN-γ on 7th day had the highest AUC (0.866) for predicting rejection with a sensitivity of 86.8% and a specificity of 89.8%. Our findings demonstrated a more powerful prediction of early acute renal allograft rejection during the first month after transplantation by combination of multiple-biomarkers of fractalkine, IFN-γ and IP-10, and the results might help stratify the immunologic risk of acute allograft rejection in recipients.

The role of IL-23/IL-17 axis in human kidney allograft rejection.

Th17 cell subset has been implicated in autoimmune diseases, tumor immunity and, transplant rejection. In order to investigate the role of IL-17/IL-23 pathway in allograft outcome, intragraft expression of IL-17mRNA and single nucleotide polymorphisms (SNPs) of IL-17A, IL-17F, IL-17RC, and IL23R genes were evaluated with a quantification of IL-17A, IL-17F, and IL-23 plasma levels. This study revealed that recipients with acute rejection (AR) had a significant increase in IL-17AmRNA expression levels after transplantation compared to controls (P=0.037). Moreover, IL-17A plasma levels were significantly higher in AR group; pretransplantation (Day-1 [D-1]): P=0.00022 and posttransplantation (Day 7 [D7]): P<10-14 . IL-17F and IL-23 plasma levels were significantly higher in AR at D7 only (47.86vs. 22.99pg/ml; and 33.82vs. 18.811pg/ml; P=0.015 and P<10-17 , respectively). Using receiver-operating characteristic curves, D7 IL-17A and IL-23 plasma levels exhibited excellent sensitivities and specificities for predicting AR. Genetic study revealed no association between IL-17A, IL-17F, IL-17RC, and IL23R studied SNPs and AR. Nevertheless, a significant improvement of graft survival was found in kidney transplant recipients carrying IL-17F-rs763780*A/A, IL-17RC*G/G, and *G/A genotypes. Besides, IL-17AmRNA levels were significantly higher in patients carrying the IL-23R*G/G genotype comparatively to those with *G/A genotype. Based on these findings, significant increase of IL-17A mRNA and protein levels in AR recipients that are genetically controlled highlights the role of this cytokine that can be a useful clinical biomarker to predict early acute renal allograft rejection.

Evidence-Based Review and Discussion Points

Evidence-Based Review and Discussion Points

A Systematic Review about an Advance in Cyclosporine Monitoring in Kidney Transplant Recipients

Context: An immunosuppressive drug, Cyclosporine (CsA), has been commonly used in kidney transplants. A safe dosing of CsA often causes nephrotoxity, bone marrow toxicity, and infection. Pharmacokinetic characteristics of CsA and CsA marker relation to the immune suppression have not been completely described in clinical practices yet. Objectives: This review summarizes our achievements on pharmacokinetic and CsA level Marker in kidney transplant patients. Search methods and Selection criteria: A literature review was done using the National Center for Biotechnology Information’s Pubmed Medline, Ovid Medline and Embase, and Iranian registries (Iranmedex, SID, MagIran, and IranDoc). Titles and abstracts were then reviewed to select studies based upon the predefined inclusion criteria. Our study defined a population of adult solid organ transplant recipients receiving the cyclosporine that Cyclosporine monitoring was done. Data Collection and Analysis: Two reviewers independently appraised the quality of each trial and extracted the data from the included trials. Results: CsA pharmacokinetics is very different between kidney transplant recipients, in order to CsA profiling, no pharmacodynamic tools has been confirmed yet in clinical practices and the best way to individualize calcineurin inhibitor therapy is still a controversial issue. C0 levels do not exactly predict the CsA level or rejection risk, patient monitored by C2 levels have upper doses of CsA and have a lower frequency of early acute allograft rejection than patients profiled with C0 and although CA is highly heterogeneous closely post-transplant and seems to be unhelpful early after post-transplant it is more favorable after first months after. Conclusions: Establishing a biological CsA marker may be helpful in clinical decisions on the dose. It seemed to be logical that we should re-inspect the possibility of using them as a supplementary tool towards better therapeutic drug monitoring of cyclosporine or it needs to be reevaluated and needs to find a new target for a therapeutic plan in kidney transplant patients.

Inhibition of histone methyltransferase EZH2 ameliorates early acute renal allograft rejection in rats.

BackgroundAlthough histone methyltransferases EZH2 has been proved to have significant regulatory effect on the immune rejection after hematopoietic stem cell transplantation, its role in solid-organ transplantation remains uncovered. In this study, we investigate whether histone methylation regulation can impact renal allograft rejection in rat models.ResultsAllogeneic rat renal transplantation model (Wistar to Lewis) was established, and the recipients were administrated with EZH2 inhibitor DZNep after transplantation. Renal allografts and peripheral blood were collected on day 5 after transplantation for histological examination and mechanism investigation. We found that inhibition of EZH2 by DZNep after transplantation significantly ameliorated acute rejection (AR), with decreased histological injury and reduced inflammatory infiltration in renal allografts. Attenuation of AR was due to the prohibited activation of alloreactive T cells, the subsequent impaired production of inflammatory cytokines, and also the elevated apoptosis of alloreactive T cells in both renal allografts and periphery. However, inhibition of EZH2 did not increase the regulatory T cells during the AR.ConclusionsDisruption of EZH2 by DZNep suppressed the immune responses of alloreactive T cells and ameliorated AR of renal allografts. This suggests a therapeutic potential of targeting histone methyltransferases EZH2 in treating allograft rejection after solid organ transplantation.

Loss of CD28 on Peripheral T Cells Decreases the Risk for Early Acute Rejection after Kidney Transplantation.

BackgroundEnd-stage renal disease patients have a dysfunctional, prematurely aged peripheral T-cell system. Here we hypothesized that the degree of premature T-cell ageing before kidney transplantation predicts the risk for early acute allograft rejection (EAR).Methods222 living donor kidney transplant recipients were prospectively analyzed. EAR was defined as biopsy proven acute allograft rejection within 3 months after kidney transplantation. The differentiation status of circulating T cells, the relative telomere length and the number of CD31+ naive T cells were determined as T-cell ageing parameters.ResultsOf the 222 patients analyzed, 30 (14%) developed an EAR. The donor age and the historical panel reactive antibody score were significantly higher (p = 0.024 and p = 0.039 respectively) and the number of related donor kidney transplantation was significantly lower (p = 0.018) in the EAR group. EAR-patients showed lower CD4+CD28null T-cell numbers (p<0.01) and the same trend was observed for CD8+CD28null T-cell numbers (p = 0.08). No differences regarding the other ageing parameters were found. A multivariate Cox regression analysis showed that higher CD4+CD28null T-cell numbers was associated with a lower risk for EAR (HR: 0.65, p = 0.028). In vitro, a significant lower percentage of alloreactive T cells was observed within CD28null T cells (p<0.001).ConclusionImmunological ageing-related expansion of highly differentiated CD28null T cells is associated with a lower risk for EAR.

Low-serum testosterone levels pre-liver transplantation are associated with reduced rates of early acute allograft rejection in men.

Low pretransplant serum testosterone has recently been associated with increased mortality in men awaiting liver transplantation, but the potential impact on rejection has not yet been investigated. Pretransplantation serum testosterone, SHBG, and other variables were collected on 190 consecutive men who received a liver transplant between 2007 and 2013. Rates of subsequent acute cellular rejection were recorded. Multivariable analysis was performed to define variables associated with rejection and other clinically important end points. Thirty (16%) of 190 men experienced acute cellular rejection. Lower pretransplant testosterone was associated with lower rejection rates, -7% (95% confidence interval [CI], -2% to -12%) per nmol/L decrease in total testosterone and -4% (95% CI, -0.5% to -7%) per 10 pmol/L decrease in free testosterone. Total testosterone (correlation 0.29, P=0.04) and free testosterone (correlation 0.37, P=0.01) correlated significantly with the histological severity of rejection. Older age at transplant (+5% [95% CI, 9%-2%]) per year, and nonautoimmune etiology of liver disease (OR, 1.0 for autoimmune, 0.22 [95% CI, 0.07-0.73] for hepatitis C virus, and 0.58 [95% CI, 0.21-1.71] for other etiologies) were also associated with decreased rejection risk. In a generalized linear model including the covariates testosterone, SHBG, age, etiology, and MELD, total testosterone remained a significant predictor of rejection (adjusted OR, 1.06; P=0.03), as did age at transplant (OR, 0.95; P=0.01). Low preliver transplant serum testosterone independently predicts a decreased risk of acute allograft rejection. Whether testosterone is a marker of disease-associated immune dysfunction or has immune-modulatory effects requires further study.

PD30-09 TRANSURETHRAL RESECTION OR INCISION OF THE PROSTATE AFTER RENAL TRANSPLANTATION: IS THERE A SAFE TIME FOR THE PROCEDURE?

improvements in graft survival following kidney transplantation. However, there are no non-invasive diagnostic parameters available that enable early and reliable detection of acute rejection. The objective of our study was to establish a urinary protein profile suitable to determine rejectionspecific changes following kidney transplantation and to predict early acute rejection at a postoperative stage. Furthermore, we focused on identification of candidate proteins for the use as biomarkers in clinical practice for diagnosis of acute rejection. METHODS: Urine samples of 116 kidney recipients were included. Rejection was proven by biopsy (n1⁄458) and stable transplant function was monitored for at least 2 years (n1⁄458). Postoperative urine samples were collected between 3rd and 10th day following transplantation. Urinary protein profiles were analyzed by SELDI-TOF-MS. Multiplex-Fluorescence-2DE and Peptide Mass Fingerprinting were used for identification of candidate proteins. Urinary concentration of candidate proteins was validated by ELISA. RESULTS: A protein signature including 4 masses differentiated acute rejection from stable transplant patients at the postoperative stage with 73% sensitivity and 88% specificity. Alpha-1-microglobulin (A1MG) and Haptoglobin (Hp) were identified as putative biomarkers for acute rejection. Protein levels were significantly higher in postoperative urine samples from patients with upcoming rejection than in stable transplant patients (A1MG: 29.13 mg/ml vs. 22.06 mg/ml, p1⁄40.001; Hp: 628.34 ng/ml vs. 248.57 ng/ml, p1⁄40.003). The combination of both proteins enabled the diagnosis of early rejection with 85% sensitivity and 80% specificity. CONCLUSIONS: Protein profiling using mass spectrometry is suitable for non-invasive detection of rejection-specific changes following kidney transplantation. A specific protein profile enables prediction of early acute allograft rejection in the immediate postoperative period. A1MG and Hp appear to be reliable rejection biomarkers. Further analyses have to show if early diagnosis is possible for patients with allograft rejection that occurs several month after transplantation by analyzing changes in urine protein pattern in regular intervals.

Urine protein profiling identified alpha-1-microglobulin and haptoglobin as biomarkers for early diagnosis of acute allograft rejection following kidney transplantation

Early diagnosis of acute rejection and effective immunosuppressive therapy lead to improvement in graft survival following kidney transplantation. In this study, we aimed to establish a urinary protein profile suitable to distinguish between patients with rejection and stable graft function and to predict acute rejection based on postoperatively collected urine samples. A further objective was to identify candidate proteins for the use as biomarkers in clinical practice. Urine samples of 116 kidney recipients were included. Rejection was proven by biopsy (n = 58), and stable transplant function was monitored for at least 2 years (n = 58). Postoperative urine samples were collected between 3rd and 10th day following transplantation. Urinary protein profiles were obtained by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Protein identification and validation were performed using multiplex fluorescence 2DE, peptide mass fingerprinting and enzyme-linked immunosorbent assay. A protein profile including four mass peaks differentiated acute rejection from stable transplants at the time point of rejection and at the postoperative state with 73 % sensitivity and 88 % specificity. Alpha-1-microglobulin (A1MG) and Haptoglobin (Hp) were identified as putative rejection biomarkers. Protein levels were significantly higher in postoperative urine from patients with rejection (A1MG 29.13 vs. 22.06 μg/ml, p = 0.001; Hp 628.34 vs. 248.57 ng/ml, p = 0.003). The combination of both proteins enabled the diagnosis of early rejection with 85 % sensitivity and 80 % specificity. Protein profiling using mass spectrometry is suitable for noninvasive detection of rejection-specific changes following kidney transplantation. A specific protein profile enables the prediction of early acute allograft rejection in the immediate postoperative period. A1MG and Hp appear to be reliable rejection biomarkers.

Association between a TGFBR2 Gene Polymorphism (rs2228048, Asn389Asn) and Acute Rejection in Korean Kidney Transplantation Recipients

Transforming growth factor-β (TGF-β) signaling transduction initiates TGF-β activation, resulting in activation of TGF-β receptor II (TGFBR2). Any quantitative and qualitative changes in TGFBR2 are expected to affect the TGF-β signaling pathway, which occupies a central position with respect to the regulation of cell growth, differentiation, apoptosis, immune reaction, angiogenesis and extracellular matrix formation. Recent studies have shown that TGF-β1 gene polymorphisms may confer susceptibility to early acute and chronic allograft rejection in kidney transplantation recipients. In this study, we assessed whether polymorphisms of the TGFBR2 gene were associated with susceptibility to kidney transplantation rejection. A total of 347 renal allograft recipients transplanted at three centers in Korea were analyzed. Three SNPs (rs764522, rs3087465, rs2228048) of the TGFBR2 gene were genotyped from genomic DNA with direct sequencing. Multiple logistic regression models (codominant, dominant, recessive, and log-additive) were performed to evaluate odds ratios (ORs), 95% confidence intervals (CIs), and p-values. A total of 63 patients (18%) developed acute rejection (AR). There were no significant differences in age, sex, number of HLA mismatches, cause of renal failure, or immunosuppressant regimen between the AR and non-AR group. The synonymous SNP rs2228048 was significantly associated with AR (p = 0.020 in recessive model, and p = 0.036 in log-additive model. The allele frequencies of rs2228048 were different between the AR and non-AR group (p = 0.026). These results suggest that the synonymous TGFBR2 gene SNP rs2228048 may be associated with the development of AR in Korean kidney transplantation recipients.Authors Yeong-Hoon Kim and Tae Hee Kim contributed equally to this work and are considered co-first authors.

Kidney injury molecule-1 and osteopontin: New markers for prediction of early kidney transplant rejection

BackgroundKidney injury molecule-1 (KIM-1) and osteopontin (OPN) play important roles in immune regulation. We hypothesized that serum KIM-1 and OPN might serve as biomarkers for predicting early acute rejection after kidney transplantation (KTx). MethodsWe conducted a single-center study of 155 subjects, who were classified into acute rejection group (ARG, n=32), non-rejection group (NRG, n=45) and healthy controls (HC, n=78). Serum KIM-1 and OPN levels were measured by Luminex. ResultsThe pre-transplant levels of serum KIM-1 and OPN in all KTx recipients were higher than those of HC (P<0.01). Compared with NRG, ARG showed significantly high serum levels of KIM-1 on day 0 (pre-KTx) and on the 1st, 4th, and 7th post-KTx days, and significantly high OPN levels on day 0 and the 7th day. Kaplan–Meier survival analysis showed that the higher levels of KIM-1 on day 0, the 1st and 4th days and OPN on day 0 and the 7th day were significantly associated with the lower probabilities of rejection-free survival. ROC analyses highlight the superiority of KIM-1 on the 1st day and OPN on the 7th day over those on other post-KTx days in prediction of acute rejection episodes. Multivariate logistic analysis revealed that the serum KIM-1 levels on the 1st post-KTx day and the OPN level on the 7th day were independent and powerful predictors of acute rejection episodes. An optimal predictive model was built by combining KIM-1 on the 1st day and OPN on the 7th day, and this model had the highest AUC (0.922). ConclusionsThis study was the first to demonstrate that serum KIM-1 and OPN may be the promising and elegant markers for prediction of early acute kidney allograft rejection.

Pretransplant Low CD3+CD25high Cell Counts or a Low CD3+CD25high/CD3+HLA-DR+ Ratio Are Associated With an Increased Risk to Acute Renal Allograft Rejection

Detection of markers predicting allograft rejection is important for risk assessment before kidney transplantation, as well as to minimize posttransplantation immunosuppression. We studied the expression of CD25, HLA-DR, CD134, CD62L, and CD44 by flow cytometry in CD4, CD8, and CD3 cells, from pretransplant blood samples from 91 transplanted patients accounting for 16 episodes of acute renal rejection in the first month after transplantation. None of the activation markers showed a significant association to acute rejection. Early rejectors showed less pretransplant CD3CD25 cells than nonrejectors (0.79%±0.50% vs. 1.51%±0.79% of CD3 cells; P=0.001) and a lower CD3CD25/CD3HLA-DR ratio (0.043±0.034 vs. 0.111±0.079; P<0.00001). When levels of CD25 cells fell below 0.7% of CD3 cells, the odds ratio of suffering an episode of acute rejection was 105 fold (95% confidence interval: 11.41-966.43, P<0.0001), with a sensitivity (true-positive results) of 0.63 and a specificity (true-negative results) of 0.98 for predicting the risk of acute rejection. Furthermore, when the CD3CD25/CD3HLA-DR ratio fell below 0.04, the odds ratio of suffering an episode of acute rejection was 7.71 fold (95% confidence interval: 2.29-25.97, P=0.001), with a sensitivity of 0.56 and a specificity of 0.86 for predicting risk of acute rejection. Our results suggest that low pretransplant levels of CD3CD25 cells or a low CD3CD25/CD3HLA-DR ratio could identify those patients with an increased risk of early acute allograft rejection. If these data can be independently confirmed, pretransplant CD3CD25 cells and the CD3CD25/CD3HLA-DR ratio might provide additional information for risk assessment before kidney transplantation.

2251 PROTEIN SIGNATURE IN URINE INDICATES REJECTION AFTER KIDNEY TRANSPLANTATION AT AN EARLY POSTOPERATIVE STATE

You have accessJournal of UrologyTransplantation & Vascular Surgery: Renal Transplantation, Vascular Surgery1 Apr 20112251 PROTEIN SIGNATURE IN URINE INDICATES REJECTION AFTER KIDNEY TRANSPLANTATION AT AN EARLY POSTOPERATIVE STATE Stefanie Finke, Beatrice Stubendorff, Torsten Gruschwitz, Undine Ott, Thomas Steiner, Heiko Wunderlich, Ferdinand von Eggeling, Marc-Oliver Grimm, and Kerstin Junker Stefanie FinkeStefanie Finke Jena, Germany More articles by this author , Beatrice StubendorffBeatrice Stubendorff Jena, Germany More articles by this author , Torsten GruschwitzTorsten Gruschwitz Jena, Germany More articles by this author , Undine OttUndine Ott Jena, Germany More articles by this author , Thomas SteinerThomas Steiner Jena, Germany More articles by this author , Heiko WunderlichHeiko Wunderlich Jena, Germany More articles by this author , Ferdinand von EggelingFerdinand von Eggeling Jena, Germany More articles by this author , Marc-Oliver GrimmMarc-Oliver Grimm Jena, Germany More articles by this author , and Kerstin JunkerKerstin Junker Jena, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2493AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES An effective immunosuppressive therapy leads to improvements in kidney graft survival after transplantation. However, successful treatment requires an early detection of allograft rejection. Currently no non-invasive diagnostic tools exist which allow for early and reliable detection of acute rejections after kidney transplantation. In a previous study we identified a specific urine protein pattern that correlates with rejection at the time of biopsy. This pattern enables a differentiation between patients with and without allograft rejection. In the current investigations we examined if allograft rejection is already detectable within few days after transplantation based on our identified protein signature. METHODS 52 urine samples (collected between postoperative day 5 and 10) from patients with and without allograft rejection (26 each) were analysed by SELDI-TOF-MS technology using two different ProteinChip® surfaces (Q10 and CM10). Bioinformatic analysis was conducted using software XLMiner. Based on the previously defined protein signature, samples were assigned to “rejection” and “no rejection” respectively. The timeframe between postoperative sample collection and rejection was 9 days to 14 months. RESULTS Using our predefined urine protein pattern as predictor for rejection resulted in an accuracy of 63%. In case of 42% of patients with proven rejection the protein signature was able to predict the allograft rejection at an early postoperative state. Considering the time point of rejection, in case of 8 of 11 patients that were assigned correctly to undergo a rejection, the period until rejection was less than 30 days. Vice versa, in 11 of 15 patients those were false negative (rejection was not predicted), the period until rejection was 1 to 14 months. CONCLUSIONS The identified specific urine protein pattern enables the prediction of early acute allograft rejection already a few days after kidney transplantation if rejection occurs during the first 30 days. Further analyses have to show if early and reliable diagnosis is possible for patients with allograft rejection that occurs several month after transplantation by analyzing changes in urine protein pattern in regular intervals. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e903 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Stefanie Finke Jena, Germany More articles by this author Beatrice Stubendorff Jena, Germany More articles by this author Torsten Gruschwitz Jena, Germany More articles by this author Undine Ott Jena, Germany More articles by this author Thomas Steiner Jena, Germany More articles by this author Heiko Wunderlich Jena, Germany More articles by this author Ferdinand von Eggeling Jena, Germany More articles by this author Marc-Oliver Grimm Jena, Germany More articles by this author Kerstin Junker Jena, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of Sample Timing and Treatment on Gene Expression in Early Acute Renal Allograft Rejection

We have shown that genomic biomarkers in peripheral blood provide evidence of early graft rejection and may offer an important option for posttransplant monitoring, and we are working to improve this signature to maximize assay performance. This clinical refinement study (n=79) used gene expression profiling in a case-control design to compare whole blood samples between normal subjects (n=20) and patients with (n=20) or without (n=39) biopsy-confirmed acute rejection (BCAR). Gene expression in peripheral blood from subjects with BCAR before treatment differed significantly from that of normal subjects and transplant recipients without BCAR. Hierarchical clustering and principal component analysis showed that samples obtained 1 to 5 days after the start of treatment of BCAR were segregated across both groups before treatment or without BCAR and that this was closely related to the time lag between treatment and sampling. Genes differentially expressed during BCAR included FKSG49, LMAN2, NFYC, LIMK2, JUNB, NASP, MALAT1, ITGAX, HLA-J, FKBP1A, and RBMS1, and gene ontology analysis highlighted changes in networks related to cytoskeletal reorganization, apoptosis, and immune signaling, whereas after treatment change highlighted pathways of cellular metabolism, cell-cycle regulation, DNA damage, and apoptosis. Gene expression in the peripheral blood is associated with BCAR, and the pattern of expression changes rapidly after treatment. This may offer a potential tool for diagnosis of rejection and immunologic monitoring of response to treatment, which is now being evaluated in a large multicenter international study.

Functional Genomic Analysis of Peripheral Blood During Early Acute Renal Allograft Rejection

Functional Genomic Analysis of Peripheral Blood During Early Acute Renal Allograft Rejection

High-mobility group box 1 promotes early acute allograft rejection by enhancing IL-6-dependent Th17 alloreactive response

Previously, we reported that extracellular high-mobility group box 1 (HMGB1) functions as an innate alarmin implicated in cardiac allograft acute rejection. We now present evidence suggesting that HMGB1 is pivotal in inducing interleukin-17 (IL-17)-producing alloreactive T cells by stimulating dendritic cells secretion of IL-6. Those IL-17+ T cells are likely to be the major effector cells responsible for the early stage of cardiac allograft rejection through mediating an influx of neutrophils into allografts, and therefore, blockade of IL-17A significantly prolonged murine cardiac allograft survival. In contrast to the classical model for a dominant role of IFN-γ+-Th1 cells have in acute allograft rejection, our data suggest that IFN-γ+-Th1 cells are responsible for the late stage of graft destruction by inducing monocyte infiltration when IL-17+ T-cell response recedes. Blockade of HMGB1 significantly decreased splenic alloreactive Th17 cells and IFN-γ-producing CD8+ T cells in the recipients, leading to less infiltration of neutrophils along with lower IL-6 and IL-17 expression levels in the grafts as well as prolongation of cardiac allograft survival. Together, these data support a novel model in which HMGB1 induces IL-17-producing alloreactive T cells to mediate early stage of allograft rejection, whereas IFN-γ-producing alloreactive Th1 cells provoke graft destruction after Th17 response recedes.

Proteomic Signatures in Plasma during Early Acute Renal Allograft Rejection

Acute graft rejection is an important clinical problem in renal transplantation and an adverse predictor for long term graft survival. Plasma biomarkers may offer an important option for post-transplant monitoring and permit timely and effective therapeutic intervention to minimize graft damage. This case-control discovery study (n = 32) used isobaric tagging for relative and absolute protein quantification (iTRAQ) technology to quantitate plasma protein relative concentrations in precise cohorts of patients with and without biopsy-confirmed acute rejection (BCAR). Plasma samples were depleted of the 14 most abundant plasma proteins to enhance detection sensitivity. A total of 18 plasma proteins that encompassed processes related to inflammation, complement activation, blood coagulation, and wound repair exhibited significantly different relative concentrations between patient cohorts with and without BCAR (p value <0.05). Twelve proteins with a fold-change ≥1.15 were selected for diagnostic purposes: seven were increased (titin, lipopolysaccharide-binding protein, peptidase inhibitor 16, complement factor D, mannose-binding lectin, protein Z-dependent protease and β2-microglobulin) and five were decreased (kininogen-1, afamin, serine protease inhibitor, phosphatidylcholine-sterol acyltransferase, and sex hormone-binding globulin) in patients with BCAR. The first three principal components of these proteins showed clear separation of cohorts with and without BCAR. Performance improved with the inclusion of sequential proteins, reaching a primary asymptote after the first three (titin, kininogen-1, and lipopolysaccharide-binding protein). Longitudinal monitoring over the first 3 months post-transplant based on ratios of these three proteins showed clear discrimination between the two patient cohorts at time of rejection. The score then declined to baseline following treatment and resolution of the rejection episode and remained comparable between cases and controls throughout the period of quiescent follow-up. Results were validated using ELISA where possible, and initial cross-validation estimated a sensitivity of 80% and specificity of 90% for classification of BCAR based on a four-protein ELISA classifier. This study provides evidence that protein concentrations in plasma may provide a relevant measure for the occurrence of BCAR and offers a potential tool for immunologic monitoring.

Triple drug Therapy for the Prevention of Early Acute Renal Allograft Rejection in Dogs

A triple drug therapy with emulsified cyclosporine, mycophenolate mofetil and predonisolone was used for 90 days for the prevention of acute rejection in six renal transplanted beagles. Five dogs survived for 90 days, but one dog died suddenly from unknown reason with a functioning graft 9 days after transplantation. Allografts of three of the five survived dogs showed no evidence of acute rejection based on Banff'07 histopathological classification. The other two dogs showed acute allograft rejection on histopathological evaluation and urinary tract infection at the end of the study. None of the dogs showed adverse effects such as hepatotoxicity and myelosuppression. At necropsy, abnormal cystic dilatation of the crypts with secretory accumulation was found in the duodenum mucosa in all dogs survived for 90 days. With further research, this triple drug immunosuppressive therapy could become a potential alternative of the classical immunosuppressive protocol (cyclosporine, azathiopurine and predonisolone) for the prevention of acute allograft rejection in dogs.

Functional Genomic Analysis of Peripheral Blood During Early Acute Renal Allograft Rejection

Acute graft rejection is an important clinical problem in renal transplantation and an adverse predictor for long-term graft survival. Peripheral blood biomarkers that provide evidence of early graft rejection may offer an important option for posttransplant monitoring, optimize the utility of graft biopsy, and permit timely and effective therapeutic intervention to minimize the graft damage. In this feasibility study (n=58), we have used gene expression profiling in a case-control design to compare whole blood samples between normal subjects (n=20) and patients with (n=11) or without (n=22) biopsy-confirmed acute rejection (BCAR) or borderline changes (n=5). A total of 183 probe sets representing 160 genes were differentially expressed (false discovery rate [FDR] <0.01) between subjects with or without BCAR, from which linear discriminant analysis and cross-validation identified an initial gene signature of 24 probe sets, and a more refined set of 11 probe sets found to classify subject samples correctly. Cross-validation suggested an out-of-sample sensitivity of 73% and specificity of 91% for identification of samples with or without BCAR. An increase in classifier gene expression correlated closely with acute rejection during the first 3 months posttransplant. Biological evaluation indicated that the differentially expressed genes encompassed processes related to immune response, signal transduction, and cytoskeletal reorganization. Preliminary evidence indicates that gene expression in the peripheral blood may yield a relevant measure for the occurrence of BCAR and offer a potential tool for immunologic monitoring. These results now require confirmation in a larger cohort.