Acute Allograft Research Articles

Improve in-depth immunological risk assessment to optimize genetic-compatibility and clinical outcomes in child and adolescent recipients of parental donor kidney transplants: protocol for the INCEPTION study

BackgroundParental donor kidney transplantation is the most common treatment option for children and adolescents with kidney failure. Emerging data from observational studies have reported improved short- and medium-term allograft outcomes in recipients of paternal compared to maternal donors. The INCEPTION study aims to identify potential differences in immunological compatibility between maternal and paternal donor kidneys and ascertain how this affects kidney allograft outcomes in children and adolescents with kidney failure.MethodsThis longitudinal observational study will recruit kidney transplant recipients aged ≤18 years who have received a parental donor kidney transplant across 4 countries (Australia, New Zealand, United Kingdom and the Netherlands) between 1990 and 2020. High resolution human leukocyte antigen (HLA) typing of both recipients and corresponding parental donors will be undertaken, to provide an in-depth assessment of immunological compatibility. The primary outcome is a composite of de novo donor-specific anti-HLA antibody (DSA), biopsy-proven acute rejection or allograft loss up to 60-months post-transplantation. Secondary outcomes are de novo DSA, biopsy-proven acute rejection, acute or chronic antibody mediated rejection or Chronic Allograft Damage Index (CADI) score of > 1 on allograft biopsy post-transplant, allograft function, proteinuria and allograft loss. Using principal component analysis and Cox proportional hazards regression modelling, we will determine the associations between defined sets of immunological and clinical parameters that may identify risk stratification for the primary and secondary outcome measures among young people accepting a parental donor kidney for transplantation. This study design will allow us to specifically investigate the relative importance of accepting a maternal compared to paternal donor, for families deciding on the best option for donation.DiscussionThe INCEPTION study findings will explore potentially differential immunological risks of maternal and paternal donor kidneys for transplantation among children and adolescents. Our study will provide the evidence base underpinning the selection of parental donor in order to achieve the best projected long-term kidney transplant and overall health outcomes for children and adolescents, a recognized vulnerable population.Trial registrationThe INCEPTION study has been registered with the Australian New Zealand Clinical Trials Registry, with the trial registration number of ACTRN12620000911998 (14th September 2020).

Respiratory Viruses in Solid Organ Transplant Recipients.

Solid organ transplantation is often lifesaving, but does carry an increased risk of infection. Respiratory viral infections are one of the most prevalent infections, and are a cause of significant morbidity and mortality, especially among lung transplant recipients. There is also data to suggest an association with acute rejection and chronic lung allograft dysfunction in lung transplant recipients. Respiratory viral infections can appear at any time post-transplant and are usually acquired in the community. All respiratory viral infections share similar clinical manifestations and are all currently diagnosed using nucleic acid testing. Influenza has good treatment options and prevention strategies, although these are hampered by resistance to neuraminidase inhibitors and lower vaccine immunogenicity in the transplant population. Other respiratory viruses, unfortunately, have limited treatments and preventive methods. This review summarizes the epidemiology, clinical manifestations, therapies and preventive measures for clinically significant RNA and DNA respiratory viruses, with the exception of SARS-CoV-2. This area is fast evolving and hopefully the coming decades will bring us new antivirals, immunologic treatments and vaccines.

Highest Recorded Serum Creatinine

Serum creatinine is a commonly used laboratory marker to assess kidney function; however, there has not been an established level of serum creatinine to predict mortality. After extensive literature review, we present a case of the highest recorded serum creatinine of 73.8 mg/dL in a 23-year-old male with the history of pediatric deceased donor kidney transplant (DDKT). He initially presented with uremia and signs of acute renal allograft failure after two months of immunosuppressive medication nonadherence, ultimately requiring emergent hemodialysis, which was complicated by new onset seizures. This was the patient's fourth episode of late acute rejection and emphasizes the need for education of immunosuppressant adherence and periodic monitoring of renal function in high-risk patients. Though there is no known creatinine level incompatible with life, this patient appears to have the highest known serum creatinine in a uremic patient on record.

Variable selection for longitudinal zero-inflated power series transition model

ABSTRACT In modeling many longitudinal count clinical studies, the excess of zeros is a common problem. To take into account the extra zeros, the zero-inflated power series (ZIPS) models have been applied. These models assume a latent mixture model consisting of a count component and a degenerated zero component that has a unit point mass at zero. Usually, the current response measurement in a longitudinal sequence is a function of previous outcomes. For example, in a study about acute renal allograft rejection, the number of acute rejection episodes for a patient in current time is a function of this outcome at previous follow-up times. In this paper, we consider a transition model for accounting the dependence of current outcome on the previous outcomes in the presence of excess zeros. New variable selection methods for the ZIPS transition model using least absolute shrinkage and selection operator (LASSO), minimax concave penalty (MCP) and smoothly clipped absolute deviation (SCAD) penalties are proposed. An expectation-maximization (EM) algorithm using the penalized likelihood is applied for both parameters estimations and conducting variable selection. Some simulation studies are performed to investigate the performance of the proposed approach and the approach is applied to analyze a real dataset.

P0302NEUTROPHIL TO LYMPHOCYTE AND PLATELET TO LYMPHOCYTE RATIO IN PATIENTS UNDERGOING KIDNEY TRANSPLANT

Abstract Background and Aims Inflammation plays a central role before and after a kidney transplant recipients (KTR). Despite survival improvement, several factors are associated with poor outcomes after transplantation. In search of a cost effective inflammatory marker neutrophil to lymphocyte ratio (NLR) and the platelet to lymphocyte ratio (PLR) have shown a prognostic value. Therefore, the objective of this study was to determine the association of NLR and PLR and acute kidney allograft dysfunction as markers of inflammatory state in KTR. Method A single center, retrospective study. Our study group included 41 KTR with acute kidney allograft dysfunction from our center at Centro Medico Nacional de Occidente, Jalisco, Mexico. NLR and PLR were collected and evaluated 1 month prior KT, at the time, 6 months and one year after transplantation. Statistical analysis: Data were expressed as the mean ± SD, median and range or frequency, as appropriate. Intergroup comparisons were performed with a chi-squared test for categorical variables and Student’s t test or the Mann-Whitney test for continuous variables. Putative associations between clinical factors, biological factors and mortality were assessed in univariate and multivariate Cox models. Statistical analyses were performed using SPSS v26.0 (IBM Corporation, NY, USA); p < 0.05 was considered statistically significant. Results Mean age was 29.54 ± 7.32 years, and 56% were women. Of those patients, all received living donor kidney transplantation. All patients in the study groups received standardized immunosuppressive regimen consisting of calcineurin inhibitors, mycophenolate mofetil and steroids. Median serum creatinine levels after KT were between 1.11 ± 0.36 mg/dl. The median NLR and PLR levels were significantly higher in the study group, with a median of 3.34 (1.83 –5.14) and 8.65 (343.47 – 360.59), not statistically associated. The best set of predictors in multiple regression analysis of higher levels of NLR were serum albumin (r = -0.432; p = 0.007) and C reactive protein (r = 0.641; p = 0.002). The best set of predictors in multiple regression analysis of higher levels of PLR were hematocrit (r = -0.313; p = 0.055) and serum glucose (r = 0.360; p = 0.026). Conclusion Our data showed that higher values of NLR and PLR are associated with inflammatory markers, leading to the conclusion that this finding must be confirmed with larger, prospective and controlled follow up.

P1678ASSOCIATION OF EPIDERMAL GROWTH FACTOR(EGF) AND EGF RECEPTOR POLYMORPHISMS WITH END STAGE RENAL DISEASE AND ACUTE RENAL ALLOGRAFT REJECTION IN KOREAN POPULATION

Abstract Background and Aims Epidermal growth factor (EGF) has been found to be associated with development and repair mechanisms of several renal diseases. In this study, we hypothesized that single nucleotide polymorphisms (SNPs) of the EGF or its receptor genes might have association with end stage renal disease (ESRD) or acute renal allograft rejection (AR) in Korean patients. Method 347 recipients of the first renal transplants for ESRD including 63 AR patients among them and 289 healthy adults were included in the study. Five EGF gene SNPs (rs11568835, rs11568943, rs2237051, rs11569017, rs3756261) and four EGFR gene SNPs (rs1140475, rs2293347, rs1050171, rs6965469) were analyzed. The genotypes of these SNPs were analyzed using AxiomTM genome-wide human assay. For statistical analysis we used SNPStats and Haploview version 4.2. Multiple logistic regression models (codominant, dominant, recessive and Log-additive) were produced to obtain odds ratio (OR), 95% confidence interval (CI), and p value. Results One SNP (rs11569017) in EGF gene showed significant association with ESRD but not with AR. Another SNP (rs11568835) in EGF gene showed significant association with susceptibility to AR but not with ESRD. One SNP (rs1050171) in EGFR gene showed significant association with susceptibility to AR but not with ESRD. Conclusion We suggest that genetic polymorphisms of EGF and EGFR gene may be associated with the risk of development of ESRD and AR in the Korean population.

P1608CXCL13 IS STRONGLY INDUCED BY RENAL ISCHEMIA REPERFUSION INJURY AND CORRELATES WITH SEVERITY OF RENAL INFLAMMATION

Abstract Background and Aims Ischemia reperfusion injury (IRI) is a strong trigger of inflammation and is tightly linked to renal allograft damage and function. CXCL13 or B lymphocyte chemoattractant (BLC) is a chemokine that controls B cell organization in lymphoid tissues and has recently been described as a biomarker for B cell involvement in acute kidney allograft rejection. Method To show, that CXCL13 is already induced by IRI alone we used renal IRI models in mice with different ischemia times and a kidney transplantation model. We measured serum CXCL13 and characterized cell infiltrates by immunohistochemistry and performed single cell RNA sequencing. Allogenic and isogenic kidney transplantation (KTX) in mice was done to evaluate these findings in the transplant setting. Results Systemic CXCL13 levels increased within hours after surgery in a time dependent manner, both after induction of IRI and KTX. Levels of CXCL13 increased with duration of ischemia time and were higher in allogenic than isogenic transplantation with equal duration of ischemia. CXCR5, the receptor for CXCL13, is mainly expressed on B lymphocytes but also on a subset of T lymphocytes and macrophages. CXCR5 positive infiltrates were detected in IRI kidneys seven days after injury. In kidney allografts interstitial B cell infiltrates were observed as early as seven days and increased further towards three weeks after KTX. Conclusion The current findings indicate that IRI is a strong trigger of CXCL13 expression in the kidney and release into the circulation, which is followed by B lymphocyte infiltration into damaged tissue in ischemic and renal allografts.

P0691LIPOPROTEIN(A) AS A POTENTIAL RISK FACTOR FOR CARDIOVASCULAR (CV) AND THROMBOTIC EVENTS IN CHRONIC KIDNEY DISEASE (CKD) AND TRANSPLANTED PATIENTS

Abstract Background and Aims Several studies suggested an independent association between elevated Lipoprotein (a) [Lp(a)] levels and the incidence of CV events in the normal population. Lp(a) levels start to rise with decreasing glomerular filtration rate. Whether high Lp(a) serum levels may contribute to the pathogenesis of atherosclerosis or thrombosis in CKD and, particularly, in transplanted (Tx) patients is largely unclear. Furthermore, it is still uncertain to which extent very high Lp(a) levels might influence CV events in this population. Aims: 1. To evaluate Lp(a) serum concentrations and lipid profile in CKD, dialysis and Tx patients. 2. To investigate the relationship between Lp(a) serum levels and atherosclerotic cardiovascular disease (ASCVD), including myocardial infarction, coronary revascularization, cerebrovascular accident, carotid endarterectomy, peripheral revascularization, gangrene, or limb amputation. 3. To assess whether Lp(a) may have a role in thrombotic events (failure of vascular access in HD or early renal allograft thrombosis after transplantation) in these patients. Method Serum Lp(a) levels and lipid profile were assessed in 295 patients (M 172, F 123; mean age 57.3, range 19-85 years): 23 with CKD stage I-III, 76 with CKD stage IV-V, 128 on dialysis (68 hemodialysis (HD), 60 peritoneal dialysis) and 68 were kidney Tx recipients (eGFR>30 ml/min). In the latter group, Lp(a) values were assayed before and after transplantation. Lp(a) levels were determined using the Macra® Lp(a) ELISA kit (Trinity Biotech, USA). Values are expressed as median and interquartile range. Logistic regression analysis was used to determine whether Lp(a) is a risk factor for CV and thrombosis disease. The cut-off value was identified according to maximum Youden index by receiver operating characteristic (ROC) curve. Results Increased mean serum levels of LDL-C (93.85 mg/d; normal values=<70 mg/dl) were observed in all groups of patients, while HDL-C and triglycerides serum levels were in the normal range. Serum Lp(a) levels (nv=14-31 mg/dl) were significantly (p<0.001) increased in advanced CKD group (stage IV-V) (45.5, 1.5-216.5 mg/dl) and, mostly, in patients on dialysis treatment (63.2, 2.0-216.6 mg/dl, p=0.001) compared to mild-moderate CKD group (stage I-III) (32, 1.3-149 mg/dl). Lp(a) serum levels correlated significantly with age (r=0.11, p<0.05) and, inversely, with eGFR only in advanced CKD group (r=-0.21 p<0.05). A significant (-56%; p<0.001) decrease of serum Lp(a) was observed after renal Tx, while staying in HD for another year resulted in an increase in serum Lp (a) levels of about 23.7% (p<0.003). No significant relationship was found between Lp(a) values and gender, inflammation (as assessed by serum C-reactive protein levels), or diabetes. By logistic regression analysis, Lp(a) values were found to be a risk factors for ASCVD in the whole population (No events 246 pts: Lp(a) 47.8 mg/dl; Yes events 49 pts: Lp(a) 53.8 mg/dl; p=0.049) and for thrombotic events in HD group (vascular access thrombosis: 4 events) and Tx patients (early acute renal vein thrombosis after Tx: 4 events) (p=0.028). The ROC curve (AUC 0.7829, 95% CI 0.5751-0.9907) allowed us to define the cut-off value for serum Lp(a) (82.45 mg/dl) (sensitivity of 87%; specificity of 73%) as a thrombotic risk factor in HD and Tx patients. Conclusion The study confirms the relevance of Lp(a) as non-traditional CV risk factor in CKD and Tx patients predicting both, the development of ASCVD and thrombosis. Serum Lp(a) levels higher than 82.45 mg/dl are potential thrombotic risk factors for vascular access failure in HD patients or early acute renal allograft thrombosis in Tx patients, suggesting more aggressive strategies to lower Lp(a) serum levels in this patient setting.

Observer variability in the assessment of renal 18F-FDG uptake in kidney transplant recipients

18F-FDG PET/CT imaging may help non-invasively disprove the diagnosis of acute kidney allograft rejection (AR) in kidney transplant recipients (KTR). The present study aims at evaluating the repeatability and reproducibility of the quantification of renal 18F-FDG uptake in KTR. We prospectively performed 18F-FDG PET/CT in 95 adult KTR who underwent surveillance transplant biopsy between 3 to 6 months post transplantation. Images were obtained 180 minutes after injecting 3 MBq 18F-FDG per kg body weight. Mean standard uptake value (SUVmean) of kidney cortex was independently measured by 2 experienced observers in 4 volumes of interest (VOI) distributed in the upper (n = 2) and lower (n = 2) poles. The first observer repeated SUV assessment in the uppermost VOI, blinded to the initial results. Intra-class correlation coefficients (ICC) and Bland-Altman plots were calculated. An ICC of 0.96 with 95%CI of [0.94; 0.97] was calculated for the intra-observer measurements. The ICC for inter-observer reproducibility for each VOI was 0.87 [0.81–0.91], 0.87 [0.81–0.91], 0.85 [0.78–0.89] and 0.83 [0.76–0.88] for the upper to the lower renal poles, respectively. The repeatability and reproducibility of the quantification of kidney allograft 18F-FDG uptake are both consistent, which makes it transferrable to the clinical routine.

Association between epidermal growth factor (EGF) and EGF receptor gene polymorphisms and end-stage renal disease and acute renal allograft rejection in a Korean population

Purpose Epidermal growth factor (EGF) has been found to be associated with the development and repair mechanisms of several renal diseases. In this study, we hypothesized that single nucleotide polymorphisms (SNPs) in EGF or its receptor genes might have an association with end-stage renal disease (ESRD) or acute renal allograft rejection (AR) in a Korean population. Methods Three-hundred and forty seven recipients of the first renal transplants for ESRD, including 63 AR patients along with 289 healthy adults were included in the study. Five EGF gene SNPs (rs11568835, rs11568943, rs2237051, rs11569017, and rs3756261) and four EGFR gene SNPs (rs1140475, rs2293347, rs1050171, and rs6965469) were analyzed. The genotypes of these SNPs were analyzed using the AxiomTM genome-wide human assay. Statistical analysis was performed using SNPStats and Haploview version 4.2 software. Multiple logistic regression models (codominant, dominant, recessive, and Log-additive) were used to estimate the odds ratio (OR), 95% confidence interval (CI), and P value. Results One SNP (rs11569017) in the EGF gene showed significant association with ESRD but not with AR. Another SNP (rs11568835) in the EGF gene showed significant association with susceptibility to AR but not with ESRD. One SNP (rs1050171) in the EGFR gene showed significant association with susceptibility to AR but not with ESRD. Conclusion Our findings suggest that SNPs in the EGF and EGFR gene may be associated with the risk of ESRD and AR development in the Korean population.

P2472Two-dimensional speckle tracking echocardiography in heart transplant patients: five year follow-up of right and left ventricular function

Abstract Background Evolution of left and right ventricular (LV and RV) function after heart transplantation (HT) has not been well described. Our objective was to evaluate the normal evolution of echocardiographic parameters of both ventricles and to explore if there is a link between the decrease of strain values and acute rejection (AR) or coronary allograft vasculopathy (CAV) Methods We followed 29 HT recipients with serial echocardiograms performed between 2011 and 2018, with a median follow-up of 5 years. LV global longitudinal strain (LV GLS) was analyzed by speckle tracking in 12 LV segments in 4 and 2 chamber views, and RV free wall longitudinal strain (RV free Wall LS) was measured in 4 chamber view. Acute rejection was diagnosed by EMB following our HT protocol. We take into consideration only moderate or severe rejection episodes (grade ≥2R).The presence of CAV was studied by coronariography or IVUS one year post-HT. Results As shown in the table below, LVEF was preserved from the begining of the follow up while LV GLS reached the normality in the 6th month, and both remained in normal ranges untill the 5th year. Regarding RV function, TAPSE was impaired in the early post-HT period and increased progressively and reached normality 1 year after HT. RV lateral wall LS rose during follow-up as well, reaching normal values 6 months after HT. Nevertheless, we noticed an impairment in this parameter at 5 years (−20.1±2.7, p=0.001), although it remained within normal ranges compared to guidelines reference parameters. We did not find any correlation between any parameter evaluated and the presence of AR or CAV at five years of follow-up. LV and RV function parameters LVEF LV GLS TAPSE FAC RV free wall LS Basal (14 days) 63.0±7.9 −17.2±3.6 12.1±2.9* 43.7±9.8 −19.3±4.2 3 months 65.0±8.6 −17.7±2.8 14.8±3.4* 45.3±8.2 −22.0±4.6 6 months 65.8±9.6 −18.7±3.4 16.1±3.6 44.6±9.6 −24.6±4.9* 1 year 63.5±8.1 −18.1±2.2 17.1±4.1 44.0±8.1 −26.7±7.1* 2 years 63.8±6.8 −18.3±9.0 19.4±3.7 45.3±7.9 −27.6±6.3* 5 years 64.4±7.3 −18.1±3.3 17.9±3.9 46.6±12.1 −20.1±2.8 P (Anova) 0.85 0.85 <0.001 0.82 <0.001 Conclusion As we show in this series of HT recipients with uneventful postoperative course, all LV and RV function parameters showed normal values 1 year after HT and manteined them during long-term follow-up. The presence of AR or CAV did not have any influence in ventricular function.

P171 Donor specific antibody in simultaneous liver-kidney transplantation: preformed class II HLA antibodies and acute renal and hepatic allograft rejection

P171 Donor specific antibody in simultaneous liver-kidney transplantation: preformed class II HLA antibodies and acute renal and hepatic allograft rejection

P122 Comparison of long-term outcomes between alemtuzumab and rabbit anti-thymocyte globulin for acute kidney allograft rejection

P122 Comparison of long-term outcomes between alemtuzumab and rabbit anti-thymocyte globulin for acute kidney allograft rejection

P213 The association between mononuclear inflammatory cells infiltration and histopathologic changes of acute renal allograft rejection; a local experience and review of literatures

P213 The association between mononuclear inflammatory cells infiltration and histopathologic changes of acute renal allograft rejection; a local experience and review of literatures

Donor-Derived Factors Influence NK Cell Effector Activity in a Model of Antibody-Mediated Chronic Rejection

Purpose Natural killer (NK) cells are key components of the innate immune system that contribute to acute and chronic allograft rejection. Previous studies have shown that donor-specific antibodies (DSA) in concert with NK cells can be sufficient to inflict chronic allograft vasculopathy (CAV) in an Fc dependent manner. However, it has been observed clinically that detectable DSA does not always correlate with demonstrable injury. The modern understanding of NK cell triggering suggests that the transition from quiescence to activation is mediated by a network of activating and inhibitory receptors; it is the integration of the sum of these signals that determines the final NK cell response. Our study aimed to investigate if donor-derived factors can alter the balance of the NK cell effector response. Methods We utilized a murine model of cardiac transplant with C3H/HeJ (C3H, H-2k), BALB/cByJ (BALB/c, H-2d), or C3HxBALB/c F1 (F1) generation donors engrafted into C57Bl/6.rag-/- (B6.rag) recipients. Recipients had no functional T or B cells, but an intact innate immune system. Experimental animals received monoclonal IgG2a Class I DSA (anti-H2k or anti-H2d). Allografts were subsequently recovered and examined after 60 days for the presence of CAV (defined by calculation of the neointimal index). Results Initial studies verified that NK cells alone could not cause significant vascular injury. When C3H mice were used as donors (n=7), administration of Class I DSA resulted in marked neointimal changes consistent with CAV. However, when BALB/c mice were used as donors (n=6), administration of Class I DSA did not result in development of CAV, despite the fact that this donor strain also represented a fully allogeneic mismatch from the recipient. When F1 mice were utilized as donors (allografts expressed both H-2k and H-2d MHC molecules), neither administration of anti-H2k (n=7) nor anti-H2d (n=6) Class I DSA resulted in development of CAV. Conclusion Donor strain determines if the activating signal provided to NK cells by binding to Class I DSA, coupled with the lack of inhibition from “missing self,” is sufficient to result in NK cell triggering with initial results suggesting that certain donor / recipient MHC combinations demonstrate a permissive phenotype towards NK cell quiescence.

Microbiome Dependent Regulatory T Cell Generation Correlates with Acute and Chronic Lung Allograft Rejection in a Murine Model

Purpose Despite the growing experience in lung transplantation a significant number of patients rapidly succumb to chronic rejection in the form of bronchiolitis obliterans (OB). The reasons for such discrepancies are unknown but have been suspected to relate to individual variability in gut and lung microbiome. Methods To study the role of the microbiome in controlling lung immunology we transplanted C57BL/10 lungs to C57BL/6 recipients from either Jackson Lab, Maine or Envigo facilities in Maryland vs Indiana. Histological grading of acute and chronic rejection, in the form of OB was performed on days 7 and 21 respectively with flow cytometric analysis of the lungs in parallel. Results Recipients from the Maine facility rapidly rejected lung grafts with high ISHLT rejection grades (2.8±0.37) by day 7 while Maryland recipients were rejection-free (0.25±0.25). Delayed histologic evaluation on day 21 post-engraftment revealed evidence of OB in 40% of Indiana recipients while 0% in Maine or Maryland mice(Fig.1A). Flow cytometric analysis revealed a high prevalence of CD4+Foxp3+ Tregs in Maryland recipients (29.56±2.32%) but lower in Maine and Indiana mice while T cell IL-17 levels were also different between them. Systemic different TGF-β levels were also observed. Treatment of Maryland recipients with broad-spectrum antibiotics affected TGF-β levels, decreased the Treg proportion (14.24±1.47%, p Conclusion Our data demonstrate that mice from different origins mount different lung allograft-specific microbiome-related immune responses. Furthermore it suggests that microbiome-dependent TGF beta levels may contribute to the generation of protective Tregs and ameliorate both acute and chronic rejection. Such data puts into question the notion that TGF-β contributes to lung fibrosis and opens up therapeutic avenues of microbiome manipulation to augment the TGF-β -Treg axis.

Transcriptome Profiles of the Circulating Extracellular Vesicles in Acute Lung Allograft Rejection

Summary of Objectives The potential of Extracellular Vesicles (EVs) as novel diagnostic, prognostic and predictive biomarkers in transplantation has been noticed as they are known to contain various molecules related to cell-cell communication that vary depending on the EV's origin, such as DNA, RNA and miRNA, and their ability to deliver these molecules to target cells. Given this factor, we hypothesize that circulating EV from patients may contain transcriptomes derived from host immune cells and allograft endothelium, epithelium, and parenchymal cells that regulate the gene expression related to the development of acute rejection and chronic lung allograft dysfunction. Therefore, we will investigate the circulating EVs' transcriptome by next generation sequencing analysis and compare these results with clinical findings and transcriptomics from transbronchial biopsies to discover novel molecular mechanisms and to develop liquid biopsies for acute rejection in a prospective study. Methods We have collected the recipient's and donor's plasma and transbronchial biopsies. The samples are taken at the time of organ procurement from the donor, and at the time of transplantation, 1 week, 1, 3, and 6 months, and 1 year after lung transplantation and every time when there is clinical suspicion of acute rejection. For this preliminary study, four rejection patients proven with the ISHLT grading system and one patient with suspected acute rejection were randomly chosen. All samples from the five patients and their donors were processed to obtain plasma-derived EVs (n=39). EVs were isolated with ExoRNeasy kit (Qiagen) and the whole transcriptome sequencing was performed to obtain at least 15m pair-ended reads by Illumina HiSeq2000. The sequenced data will be processed further with a bioinformatics pipeline for a downstream analysis to observe differential expression patterns, alternative splicing events, and the discovery of biomarkers. Endpoints We were able to validate the morphology and size distribution of EVs isolated from plasma by electron microscopy and nanoparticle tracking analysis. Since the data are currently in the sequencing data analysis pipeline, we expect the results of this preliminary study to be finished by December 2018. If successful, we will confirm the results with a larger cohort.

Cardiac Allograft Vasculopathy is Linked to Impaired Generation of Regulatory M2 Macrophages

Purpose There is mounting evidence for the role of innate immunity in acute cardiac allograft rejection, but this does not necessarily extend to cardiac allograft vasculopathy (CAV) and predictors of CAV remain elusive. We hypothesized that circulating monocytes and their derived macrophage subtypes correlate with CAV. Methods 49 consecutive heart transplant recipients ≥ 2 years post-transplant were enrolled. CAV was assessed on angiogram with IVUS by ISHLT and Stanford classification grading. Peripheral blood mononuclear cells were isolated by density gradient centrifugation, with monocytes stained and measured by flow cytometry for classical (CD14++/CD16−), intermediate (CD14++/CD16+), and nonclassical (CD14lo/CD16++) subtypes. Monocytes were cultured into macrophages, driven by M-CSF, and assayed for polarization markers CD86 and CD206. Results were also compared to 22 non-transplant patients with atherosclerotic coronary artery disease (CAD) and healthy controls (no CAD). Results 22 patients had no significant CAV (Stanford class I-II) while 27 had significant CAV (class III-IV). Other than time since transplant (no CAV 77±13 vs CAV 135±12 months), there were no significant differences, including immunosuppression, between the two groups. There were no differences between monocyte subgroups in isolation, but when combined with derived macrophage polarization, patients with CAV had significantly lower M2 (CD206+) macrophage production (0.19±0.04%) than those without CAV (0.45±0.13%), p Conclusion CAV patients are characterized by reduced ability to generate regulatory M2 macrophages. The circulating monocyte and macrophage subtypes differ from those of native CAD and these cell-based markers could be useful to differentiate high vs. low CAV risk phenotypes.

P041 Renal allograft hyperacute rejection: Reviewing the role of hla and non–hla antibodies

A 35 y/o Caucasian female with bilateral nephrectomies due to Von Hippel-Lindeau disease received a first right kidney transplant from a 14 y/o male deceased donor. At 0.5 day post kidney transplant (POD), ultrasound suggested renal vein thrombosis. Allograft biopsy at the time of thrombectomy showed extensive endothelial injury with diffuse thrombotic angiopathy and microangiopathy. There was moderate microvascular inflammation, though C4d was negative. Overall findings were suggestive of hyperacute rejection. Despite thrombectomy and apheresis, the allograft was not viable and was removed on POD 2. We retrospectively re-analyzed HLA donor specific antibody (DSA) and cross-match (XM) results. Additional retrospective testing included endothelial cell XM using surrogate endothelial cell lines (1.5-fold > negative control as +), non–HLA auto-antibodies (AuAb) against different targets associated with allograft rejection (1.5-fold > negative control as +), and angiotensin II type-I receptor antibody (AT1R-Ab) > 17 U/ml as positive and 10–17 U/ml as borderline positive. Pre-kidney transplant HLA-B7 DSA was 3000 mean fluorescence intensity, but both T/B cell flow cytometry XMs were negative. HLA-B7 DSA reduced to 2100 mean fluorescence intensity at POD 1. Pre- kidney transplant AT1R-Ab was positive, but also declined to borderline positive at POD 1. Both pre and post-kidney transplant endothelial cell XM were consistently positive. Six endothelial targets and 1 chronic kidney disease related AuAbs were positive pre- transplant, but 6 out of 7 decreased to borderline level POD 1 (Table 1). Positive endothelial cell XM combined with weak HLA- B7 DSA and multiple strong direct/indirect endothelial target antigen specificities suggest the possible involvement of AuAbs in severe acute endothelial injury related allograft dysfunction.

GC/MS-based urine metabolomics analysis of renal allograft recipients with acute rejection

BackgroundAcute renal allograft rejection is a common complication after renal transplantation that often leads to chronic rejection and ultimate graft loss. While renal allograft biopsy remains the gold standard for diagnosis of acute rejection, the possibility of biopsy-associated complications cannot be overlooked. The development of noninvasive methods for accurate detection of acute renal allograft rejection is thus of significant clinical importance.MethodsGas chromatography–mass spectrometry (GC/MS) was employed for analysis of urine metabolites in 15 renal allograft recipients with acute rejection and 15 stable renal transplant recipients. Partial least squares (PLS) regression and leave-one-out analyses were performed to ascertain whether the metabolites identified could be exploited to distinguish acute rejection from stable groups as well as their sensitivity and specificity.ResultsOverall, 14 metabolites were significantly altered in the acute rejection group (11 and 3 metabolites displayed higher and lower levels, respectively) relative to the stable transplant group. Data from PLS and leave-one-out analyses revealed that the differential metabolites identified not only distinguished acute rejection from stable transplant recipients but also showed high sensitivity and specificity for diagnosis of renal allograft recipients with acute rejection.ConclusionUrine metabolites identified with GC/MS can effectively distinguish acute rejection from stable transplant recipients, supporting the potential utility of metabolome analysis in non-invasive diagnosis of acute rejection.