Allograft Injury Research Articles

Baseline Lung Allograft Dysfunction After Bilateral Lung Transplantation Is Associated With an Increased Risk of Death: Results From a Multicenter Cohort Study.

A prior single-center, retrospective cohort study identified baseline lung allograft dysfunction (BLAD) as a risk factor for death in bilateral lung transplant recipients. In this multicenter prospective cohort study, we test the association of BLAD with death in bilateral lung transplant recipients, identify clinical risk factors for BLAD, and assess its association with allograft injury on the molecular level. This multicenter, prospective cohort study included 173 bilateral lung transplant recipients that underwent serial pulmonary function testing and plasma collection for donor-derived cell-free DNA at prespecified time points. BLAD was defined as failure to achieve ≥80% predicted for both forced expiratory volume in 1 s and forced vital capacity after lung transplant, on 2 consecutive measurements at least 3 mo apart. BLAD was associated with increased risk of death (hazard ratio, 1.97; 95% confidence interval [CI], 1.05-3.69; P = 0.03) but not chronic lung allograft dysfunction alone (hazard ratio, 1.60; 95% CI, 0.87-2.95; P = 0.13). Recipient obesity (odds ratio, 1.69; 95% CI, 1.15-2.80; P = 0.04) and donor age (odds ratio, 1.03; 95% CI, 1.02-1.05; P = 0.004) increased the risk of developing BLAD. Patients with BLAD did not demonstrate higher log10(donor-derived cell-free DNA) levels compared with no BLAD (slope [SE]: -0.0095 [0.0007] versus -0.0109 [0.0007]; P = 0.15). BLAD is associated with an increased risk of death following lung transplantation, representing an important posttransplant outcome with valuable prognostic significance; however, early allograft specific injury on the molecular level does not increase the risk of BLAD, supporting further mechanistic insight into disease pathophysiology.

Immunosuppression with cyclosporine versus tacrolimus shows distinctive nephrotoxicity profiles within renal compartments.

Calcineurin inhibitors (CNIs) are the backbone for immunosuppression after solid organ transplantation. Although successful in preventing kidney transplant rejection, their nephrotoxic side effects contribute to allograft injury. Renal parenchymal lesions occur for cyclosporine A (CsA) as well as for the currently favored tacrolimus (Tac). We aimed to study whether chronic CsA and Tac exposures, before reaching irreversible nephrotoxic damage, affect renal compartments differentially and whether related pathogenic mechanisms can be identified. CsA and Tac were administered chronically in wild type Wistar rats using osmotic minipumps over 4 weeks. Functional parameters were controlled. Electron microscopy, confocal, and 3D-structured illumination microscopy were used for histopathology. Clinical translatability was tested in human renal biopsies. Standard biochemical, RNA-seq, and proteomic technologies were applied to identify implicated molecular pathways. Both drugs caused significant albeit differential damage in vasculature and nephron. The glomerular filtration barrier was more affected by Tac than by CsA, showing prominent deteriorations in endothelium and podocytes along with impaired VEGF/VEGFR2 signaling and podocyte-specific gene expression. By contrast, proximal tubule epithelia were more severely affected by CsA than by Tac, revealing lysosomal dysfunction, enhanced apoptosis, impaired proteostasis and oxidative stress. Lesion characteristics were confirmed in human renal biopsies. We conclude that pathogenetic alterations in the renal compartments are specific for either treatment. Considering translation to the clinical setting, CNI choice should reflect individual risk factors for renal vasculature and tubular epithelia. As a step in this direction, we share protein signatures identified from multiomics with potential pathognomonic relevance.

Innate and adaptive effector immune drivers of cytomegalovirus disease in lung transplantation: a double-edged sword.

Up to 90% of the global population has been infected with cytomegalovirus (CMV), a herpesvirus that remains latent for the lifetime of the host and drives immune dysregulation. CMV is a critical risk factor for poor outcomes after solid organ transplant, though lung transplant recipients (LTR) carry the highest risk of CMV infection, and CMV-associated comorbidities compared to recipients of other solid organ transplants. Despite potent antivirals, CMV remains a significant driver of chronic lung allograft dysfunction (CLAD), re-transplantation, and death. Moreover, the extended utilization of CMV antiviral prophylaxis is not without adverse effects, often necessitating treatment discontinuation. Thus, there is a critical need to understand the immune response to CMV after lung transplantation. This review identifies key elements of each arm of the CMV immune response and highlights implications for lung allograft tolerance and injury. Specific attention is paid to cellular subsets of adaptive and innate immune cells that are important in the lung during CMV infection and reactivation. The concept of heterologous immune responses is reviewed in depth, including how they form and how they may drive tissue- and allograft-specific immunity. Other important objectives of this review are to detail the emerging role of NK cells in CMV-related outcomes, in addition to discussing perturbations in CMV immune function stemming from pre-existing lung disease. Finally, this review identifies potential mechanisms whereby CMV-directed treatments may alter the cellular immune response within the allograft.

Cell-free DNA for the detection of kidney allograft rejection

Donor-derived cell-free DNA (dd-cfDNA) is an emerging noninvasive biomarker that has the potential to detect allograft injury. The capacity of dd-cfDNA to detect kidney allograft rejection and its added clinical value beyond standard of care patient monitoring is unclear. We enrolled 2,882 kidney allograft recipients from 14 transplantation centers in Europe and the United States in an observational population-based study. The primary analysis included 1,134 patients. Donor-derived cell-free DNA levels strongly correlated with allograft rejection, including antibody-mediated rejection (P < 0.0001), T cell-mediated rejection (P < 0.0001) and mixed rejection (P < 0.0001). In multivariable analysis, circulating dd-cfDNA was significantly associated with allograft rejection (odds ratio 2.275; 95% confidence interval (CI) 1.902–2.739; P < 0.0001) independently of standard of care patient monitoring parameters. The inclusion of dd-cfDNA to a standard of care prediction model showed improved discrimination (area under the curve 0.777 (95% CI 0.741–0.811) to 0.821 (95% CI 0.784–0.852); P = 0.0011) and calibration. These results were confirmed in the external validation cohorts (n = 1,748) including a cohort of African American patients (n = 439). Finally, dd-cfDNA showed high predictive value to detect subclinical rejection in stable patients. Our study provides insights on the potential value of assessing dd-cfDNA, in addition to standard of care monitoring, to improve the detection of allograft rejection. ClinicalTrials.gov registration: NCT05995379.

#1658 Association of blood dd-cfDNA levels with Banff scores and histopathological lesions in kidney allograft biopsies: results from an observational trial

Abstract Background and Aims Donor-derived cell-free DNA (dd-cfDNA) is a novel blood biomarker of kidney allograft injury. While primarily studied to discriminate rejection from no rejection, knowledge about its dynamics in other graft pathologies is scarce and merits further investigation. Previous research has highlighted its superior diagnostic performance in detecting ABMR compared to TCMR and borderline changes, however, recent studies have suggested that its increase is not limited to damage due to alloimmune injury. Therefore, we aimed to assess the association between dd-cfDNA release and diverse histopathological patterns as well as its correlation with Banff lesion scores in renal allograft biopsies. Method For this analysis, we identified all dd-cfDNA-paired allograft biopsies from a prospective observational trial of kidney transplant recipients undergoing an indication biopsy with deterioration in graft function, persistence of anti-HLA donor-specific antibodies (DSA), or proteinuria. Dd-cfDNA was collected at the time of allograft biopsy and measured using droplet-digital PCR. Both relative and absolute quantification of dd-cfDNA, with cutoffs of 0.5% and 50cp/ml, respectively, were available for clinical interpretation. All biopsy specimens were examined by a central pathologist blinded to the dd-cfDNA results. Histopathological diagnoses were assigned according to the Banff 2019 classification. Results In total, we examined 125 dd-cfDNA paired biopsies from 110 patients performed at our center between April 2020 and October 2023. Absolute levels of dd-cfDNA were significantly higher in mixed rejection (median 156 cp/mL, IQR 138-176), antibody-mediated rejection (ABMR) (median 63cp/mL, IQR 48-81), DSA-negative microvascular inflammation (DSAnegMVI) (median 74 cp/mL, IQR 35-134), and BK-virus associated nephropathy (BKVAN) (median 168cp/mL, IQR 54-311) in comparison to glomerulonephritis (median 12cp/mL, IQR 8-19; p &amp;lt; 0.001, &amp;lt;0.001, 0.02, 0.01, respectively) as well as in comparison CNI-toxicity (median 10, IQR 6-15; p &amp;lt;0.001, &amp;lt;0.001, 0.004, 0.003, respectively). Additionally, patients with mixed rejection, ABMR, and BKVAN had higher absolute dd-cfDNA levels than patients with IFTA (median 13, IQR 8-16; p 0.004, 0.004, and 0.04, respectively), as well as normal histology (median 7, IQR 7-11, p 0.004, 0.008, 0.03, respectively). Finally, absolute dd-cfDNA levels were higher in patients with mixed rejection than in those with UTI (median 14, IQR 12-16, p = 0.02) (Fig. 1). In the univariable analysis, absolute dd-cfDNA levels were significantly correlated with Banff lesion scores for glomerulitis (g), peritubular capillaritis (ptc), and tubulitis (t). In the multivariable analysis, ptc and t were independently correlated with absolute dd-cfDNA levels. Conclusion Increased donor-derived cell-free DNA is not specific for rejection, but also occurs in alternative injury patterns showing microvascular inflammation and severe tubulointerstitial inflammation, such as DSAnegMVI and BKVAN (BK-virus associated nephropathy). Interestingly, dd-cfDNA levels remain low in the setting of de novo or recurrent glomerulonephritis and CNI-toxicity, which are important differential diagnoses in patients with clinical indication biopsy.

Immune monitoring in pediatric kidney transplant.

Long-term outcomes in pediatric kidney transplantation remain suboptimal, largely related to chronic rejection. Creatinine is a late marker of renal injury, and more sensitive, early markers of allograft injury are an active area of current research. This is an educational review summarizing existing strategies for monitoring for rejection in kidney transplant recipients. We summarize supporting currently available clinical tests, including surveillance biopsy, donor specific antibodies, and donor-derived cell free DNA, as well as the potential limitations of these studies. In addition, we review the current avenues of active research, including transcriptomics, proteomics, metabolomics, and torque tenovirus levels. Advancing the use of noninvasive immune monitoring will depend on well-designed multicenter trials that include patients with stable graft function, include biopsy results on all patients, and can demonstrate both association with a patient-relevant clinical endpoint such as graft survival or change in glomerular filtration rate and a potential timepoint for intervention.

Transcriptional Profiling Underscores the Role of Preprocurement Allograft Metabolism and Innate Immune Status on Outcomes in Human Liver Transplantation.

The adverse effects of ischemia-reperfusion injury (IRI) remain a principal barrier to a successful outcome after lifesaving orthotopic liver transplantation (OLT). Gene expression during different phases of IRI is dynamic and modified by individual exposures, making it attractive for identifying potential therapeutic targets for improving the number of suitable organs for transplantation and patient outcomes. However, data remain limited on the functional landscape of gene expression during liver graft IRI, spanning procurement to reperfusion and recovery. Therefore, we sought to characterize transcriptomic profiles of IRI during multiple phases in human OLT. We conducted clinical data analyses, histologic evaluation, and RNA sequencing of 17 consecutive human primary OLT. We performed liver allograft biopsies at 4 time points: baseline (B, before donor cross-clamp), at the end of cold ischemia (CI), during early reperfusion (ER, after revascularization), and during late reperfusion (LR). Data were generated and then recipients grouped by post-OLT outcomes categories: immediate allograft function (IAF; n = 11) versus early allograft dysfunction (EAD; n = 6) groups. We observed that CI (vs B) modified a transcriptomic landscape enriched for a metabolic and immune process. Expression levels of hallmark inflammatory response genes were higher transitioning from CI to ER and decreased from ER to LR. IAF group predominantly showed higher bile and fatty acid metabolism activity during LR compared with EAD group, while EAD group maintained more immunomodulatory activities. Throughout all time points, EAD specimens exhibited decreased metabolic activity in both bile and fatty acid pathways. We report transcriptomic profiles of human liver allograft IRI from prepreservation in the donor to posttransplantation in the recipient. Immunomodulatory and metabolic landscapes across ER and LR phases were different between IAF and EAD allografts. Our study also highlights marker genes for these biological processes that we plan to explore as novel therapeutic targets or surrogate markers for severe allograft injury in clinical OLT.

Sequential administration of anti-complement component C5 eculizumab and type-2 anti-CD20 obinutuzumab for the treatment of early antibody-mediated rejection after kidney transplantation: A proof of concept

Kidney transplant (KT) candidates with donor-specific antibodies (DSA) exhibit exceedingly high antibody-mediated rejection (ABMR) and allograft loss rates. Currently, treatment of ABMR remains an unmet clinical need. We report the use of the anti-C5 eculizumab and the type-2 anti-CD20 obinutuzumab in two patients with early ABMR. Eculizumab (900 mg IV) led to complete inhibition of the terminal complement cascade (unremarkable AP50 and CH50 activity) and prompt stoppage of complement-dependent antibody-mediated allograft injury (clearance of intra-graft C4d and C5b-9 deposition). Despite complement inhibition, obinutuzumab (1000 mg IV) determined full and long-lasting peripheral B-cell depletion, with significant reduction in all DSA. Graft function improved, remaining stable up to three years of follow-up. No signs of active ABMR and rebound DSA were detected. Obinutuzumab B-cell depletion and inhibition of DSA production were not affected by complement blockage. Further studies are needed to confirm the potential benefit of obinutuzumab in association with complement inhibitors.

Extreme elevations of donor-derived cell-free DNA increases the risk of chronic lung allograft dysfunction and death, even without clinical manifestations of disease

Lung transplant recipients are traditionally monitored with pulmonary function testing (PFT) and lung biopsy to detect post-transplant complications and guide treatment. Plasma donor-derived cell free DNA (dd-cfDNA) is a novel molecular approach of assessing allograft injury, including subclinical allograft dysfunction. The aim of this study was to determine if episodes of extreme molecular injury (EMI) in lung transplant recipients increases the risk of chronic lung allograft dysfunction (CLAD) or death. This multicenter prospective cohort study included 238 lung transplant recipients. Serial plasma samples were collected for dd-cfDNA measurement by shotgun sequencing. EMI was defined as a dd-cfDNA above the third quartile of levels observed for acute rejection (dd-cfDNA level of ≥5% occurring after 45 days post-transplant). EMI was categorized as Secondary if associated with co-existing acute rejection, infection or PFT decline; or Primary if not associated with these conditions. EMI developed in 16% of patients at a median 343.5 (IQR: 177.3-535.5) days post-transplant. Over 50% of EMI episodes were classified as Primary. EMI was associated with an increased risk of severe CLAD or death (HR: 2.78, 95% CI: 1.26-6.22,p = 0.012). The risk remained consistent for the Primary EMI subgroup (HR: 2.34, 95% CI 1.18-4.85, p=0.015). Time to first EMI episode was a significant predictor of the likelihood of developing CLAD or death (AUC=0.856, 95% CI=0.805-0.908, p<0.001). Episodes of EMI in lung transplant recipients are often isolated and may not be detectable with traditional clinical monitoring approaches. EMI is associated with an increased risk of severe CLAD or death, independent of concomitant transplant complications.

Multi-centre analytical performance verification of an IVD assay to quantify donor-derived cell-free DNA in solid organ transplant recipients.

Donor-derived cell-free DNA (dd-cfDNA) has been widely studied as biomarker for non-invasive allograft rejection monitoring. Earlier rejection detection enables more prompt diagnosis and intervention, ultimately improving patient treatment and outcomes. This multi-centre study aims to verify analytical performance of a next-generation sequencing-based dd-cfDNA assay at end-user environments. Three independent laboratories received the same experimental design and 16 blinded samples to perform cfDNA extraction and the dd-cfDNA assay workflow. dd-cfDNA results were compared between sites and against manufacturer validation to evaluate concordance, reproducibility, repeatability and verify analytical performance. A total of 247 sample libraries were generated across 18 runs, with completion time of <24 h. A 96.0% first pass rate highlighted minimal failures. Overall observed versus expected dd-cfDNA results demonstrated good concordance and a strong positive correlation with linear least squares regression r2 = 0.9989, and high repeatability and reproducibility within and between sites, respectively (p > 0.05). Manufacturer validation established limit of blank 0.18%, limit of detection 0.23% and limit of quantification 0.23%, and results from independent sites verified those limits. Parallel analyses illustrated no significant difference (p = 0.951) between dd-cfDNA results with or without recipient genotype. The dd-cfDNA assay evaluated here has been verified as a reliable method for efficient, reproducible dd-cfDNA quantification in plasma from solid organ transplant recipients without requiring genotyping. Implementation of onsite dd-cfDNA testing at clinical laboratories could facilitate earlier detection of allograft injury, bearing great potential for patient care.

Donor-derived cell-free DNA, gene expression profile, and acute rejection in donation after circulatory death (DCD) heart transplant recipients

BackgroundAcute rejection (AR) is a leading cause of early morbidity and mortality in heart transplant (HTx) recipients. There is limited data in donor after circulatory death (DCD) HTx recipients where ischemia reperfusion injury may contribute to development of AR and may be detected early with non-invasive biomarkers such as donor-derived cell-free DNA (dd-cfDNA) and gene expression profile (GEP). The goal of this study is to compare dd-cfDNA, GEP and rejection outcomes in DCD and donor after brain death (DBD) HTx recipients. MethodsThis single-center, retrospective study included DCD and DBD HTx recipients from January 2020 to September 2022. Patients were excluded from the study if dd-cfDNA and GEP were not available. The CareDx HeartCare platform was used to obtain dd-cfDNA (AlloSure-Heart) and GEP (AlloMap) with the highest values for each patient recorded at 6 and 12 months. The mean values for these non-invasive markers were compared between DCD and DBD groups. Patients were followed for clinical outcomes, including treated AR, coronary allograft vasculopathy (CAV) and death, through September 2023. ResultsA total of 156 HTx patients were included with 50 DCD and 106 DBD recipients. Baseline characteristics were similar between DCD and DBD recipients including mean age (58.5 years vs 56.9 years, p = 0.48), male sex (82% vs 76%, p = 0.56) and Caucasian race (46% vs 43%, p = 0.78). There were no significant differences in mean AlloSure-Heart at 6 and 12 months between DCD and DBD recipients. AlloMap was significantly lower at 6 months (p = 0.04), but not at 12 months between DCD and DBD recipients. With respect to clinical outcomes at 1 year, there were no significant differences in treated AR (22% vs 14%, p = 0.32), ISHLT grade ≥ 2 CAV (0 vs 4%, p = 0.44) or mortality (2% vs 0%, p = 0.69) between DCD and DBD recipients. ConclusionsIn this single center study, there were no significant differences in mean dd-cfDNA and treated AR at 1 year between DCD and DBD HTx recipients. There was a significant increase in mean GEP at 6 months, but not at 12 months in the DBD cohort. Overall, there does not appear to be clinically significant cardiac allograft injury related to DCD as measured by non-invasive markers. Further work is needed to confirm these findings.

Epithelial cell states associated with kidney and allograft injury.

The kidney epithelium, with its intricate arrangement of highly specialized cell types, constitutes the functional core of the organ. Loss of kidney epithelium is linked to the loss of functional nephrons and a subsequent decline in kidney function. In kidney transplantation, epithelial injury signatures observed during post-transplantation surveillance are strong predictors of adverse kidney allograft outcomes. However, epithelial injury is currently neither monitored clinically nor addressed therapeutically after kidney transplantation. Several factors can contribute to allograft epithelial injury, including allograft rejection, drug toxicity, recurrent infections and postrenal obstruction. The injury mechanisms that underlie allograft injury overlap partially with those associated with acute kidney injury (AKI) and chronic kidney disease (CKD) in the native kidney. Studies using advanced transcriptomic analyses of single cells from kidney or urine have identified a role for kidney injury-induced epithelial cell states in exacerbating and sustaining damage in AKI and CKD. These epithelial cell states and their associated expression signatures are also observed in transplanted kidney allografts, suggesting that the identification and characterization of transcriptomic epithelial cell states in kidney allografts may have potential clinical implications for diagnosis and therapy.

The CaMK Family Differentially Promotes Necroptosis and Mouse Cardiac Graft Injury and Rejection.

Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.

FOXP3 full length splice variant is associated with kidney allograft tolerance.

Progressive decline of allograft function leads to premature graft loss. Forkhead box P3 (FOXP3), a characteristic gene of T-regulatory cells, is known to be essential for auto-antigen tolerance. We assessed the hypothesis that low FOXP3 mRNA splice variant levels in peripheral blood cells early after transplantation are associated with progressive allograft injury. Blood samples were prospectively collected from 333 incident kidney transplant recipients on the first and 29th postoperative day. We used quantitative polymerase chain reaction to determine transcripts of 3 isotypes of FOXP3 splice variants, including pre-mature FOXP3 and full length FOXP3 (FOXP3fl). We investigated the association between FOXP3 splice variant levels and the declines in estimated glomerular filtration rate (eGFR) of more than 5ml/min/1.73m2 within the first-year post-transplant using logistic regression. We observed lower FOXP3fl levels in recipients with declining eGFR (N = 132) than in recipients with stable eGFR (N = 201), (logarithmic value -4.13 [IQR -4.50 to -3.84] vs -4.00 [4.32 to -3.74], p=0.02). In ad hoc analysis pre-transplant FOXP3fl levels were similar in both groups. The association between FOXP3fl and declining eGFR was confirmed by multivariable analysis adjusted for potential confounding factors (Odds Ratio 0.51, 95% confidence interval 0.28 to 0.91: p=0.02). When stratifying FOXP3fl levels into quartiles, recipients with lower day1 FOXP3fl had the highest rate of declining eGFR (p=0.04). Low FOXP3fl splice variant levels at the first postoperative day in kidney transplant recipients were associated with severe decline of eGFR, a well-known surrogate for hard endpoints.

Novel non-invasive method for urine mapping: Deep-learning-enabled SERS spectroscopy for the rapid differential detection of kidney allograft injury

The kidney allograft has been under continuous attack from diverse injuries since the very beginning of organ procurement, leading to a gradual decline in function, chronic fibrosis, and allograft loss. It is vital to routinely and precisely monitor the risk of injuries after renal transplantation, which is difficult to achieve because the traditional laboratory tests lack sensitivity and specificity, and graft biopsies are invasive with the risk of many complications and time-consuming. Herein, a novel method for the diagnosis of graft injury is demonstrated, using deep learning-assisted surface-enhanced Raman spectroscopy (SERS) of the urine analysis. Specifically, we developed a hybrid SERS substrate composed of gold and silver with high sensitivity to the urine composition under test, eliminating the need for labels, which makes measurements easy to perform and meanwhile results in extremely abundant and complex Raman vibrational bands. Deep learning algorithms were then developed to improve the interpretation of the SERS spectral fingerprints. The deep learning model was trained with SERS signals of urine samples of recipients with different injury types including delayed graft function (DGF), calcineurin-inhibitor toxicity (CNIT), T cell-mediated rejection (TCMR), antibody-mediated rejection (AMR), and BK virus nephropathy (BKVN), which explored the features of these types and achieved the injury differentiation with an overall accuracy of 93.03%. The results highlight the potential of combining label-free SERS spectroscopy with deep learning as a method for liquid biopsy of kidney allograft injuries, which can provide great potential to diagnose and evaluate allograft injuries, and thus extend the life of kidney allografts.

(925) - Donor-Derived Cell-Free DNA for Early Detection of Rejection and Allograft Injury in Lung Transplant Recipients

(925) - Donor-Derived Cell-Free DNA for Early Detection of Rejection and Allograft Injury in Lung Transplant Recipients

CX3CR1 deficiency promotes resolution of hepatic ischemia-reperfusion injury by regulating homeostatic function of liver infiltrating macrophages

Hepatic ischemia-reperfusion injury(HIRI) remains to be an unsolved risk factor that contributes to organ failure after liver surgery. Our clinical retrospective study showed that lower donor liver CX3-C chemokine receptor-1(CX3CR1) mRNA expression level were correlated with upregulated pro-resolved macrophage receptor MERTK, as well as promoted restoration efficiency of allograft injury in liver transplant. To further characterize roles of CX3CR1 in regulating resolution of HIRI, we employed murine liver partial warm ischemia-reperfusion model by Wt &Cx3cr1−/− mice and the reperfusion time was prolonged from 6 h to 4–7 days. Kupffer cells(KCs) were depleted by clodronate liposome(CL) in advance to focus on infiltrating macrophages, and repopulation kinetics were determined by FACS, IF and RNA-Seq. CX3CR1 antagonist AZD8797 was injected i.p. to interrogate potential pharmacological therapeutic strategies. In vitro primary bone marrow macrophages(BMMs) culture by LXR agonist DMHCA, as well as molecular and functional studies, were undertaken to dissect roles of CX3CR1 in modulating macrophages cytobiological development and resolutive functions. We observed that deficiency or pharmacological inhibition of CX3CR1 facilitated HIRI resolution via promoted macrophages migration in CCR1/CCR5 manner, as well as enhanced MerTK-mediated efferocytosis. Our study demonstrated the critical roles of CX3CR1 in progression of HIRI and identified it as a potential therapeutic target in clinical liver transplantation.

Concurrent abnormal non-acid reflux is associated with additional chronic rejection risk in lung transplant patients with increased acid exposure.

Acid reflux has been associated with allograft injury and rejection in lung transplant patients; however, the pathogenic role of non-acid reflux remains debated. We aimed to evaluate the impact of concurrent abnormal non-acid reflux with acid reflux on chronic rejection in lung transplant patients with acid reflux. This was a retrospective cohort study of lung transplant recipients who underwent pre-transplant combined impedance-pH study off acid suppression. Only subjects with acid exposure >4% were included. Non-acid reflux (pH > 4) episodes >27 was considered abnormal per prior normative studies. Chronic rejection was defined as chronic lung allograft dysfunction (CLAD) per International Society for Heart and Lung Transplantation criteria. Time-to-event analyses were performed using Cox proportional hazards and Kaplan-Maier methods, with censoring at death, anti-reflux surgery, or last follow-up. In total, 68 subjects (28 abnormal/40 normal non-acid reflux) met inclusion criteria for the study. Baseline demographic/clinical characteristics were similar between groups. Among this cohort of patients with increased acid exposure, subjects with concurrent abnormal non-acid reflux had significantly higher risk of CLAD than those without on Kaplan-Meier analysis (log-ranked P = 0.0269). On Cox multivariable regression analysis controlling for body mass index, age at transplantation, and proton pump inhibitor use, concurrent abnormal non-acid reflux remained independently predictive of increased CLAD risk (hazard ratio 2.31, confidence interval: 1.03-5.19, P = 0.04). Presence of concurrent abnormal non-acid reflux in lung transplant subjects with increased acid exposure is associated with additional risk of chronic rejection. Non-acid reflux may also contribute to pathogenicity in lung allograft injury/rejection, supporting a potential role for impedance-based testing in this population.

Sex-specific patterns of donor-derived cell-free DNA in heart transplant rejection: An analysis from the Genomic Research Alliance for Transplantation (GRAfT)

IntroductionNon-invasive methods for surveillance of acute rejection are increasingly used in heart transplantation (HT), including donor-derived cell-free DNA (dd-cfDNA). As other cardiac biomarkers differ by sex, we hypothesized that there may be sex-specific differences in performance of dd-cfDNA for detection of acute rejection. The purpose of the current study was to examine patterns of dd-cfDNA seen in quiescence and acute rejection in males and female transplant recipients. MethodsPatients enrolled in the Genomic Research Alliance for Transplantation (GRAfT) who were ≥18 years at the time of HT were included. Rejection was defined by endomyocardial biopsy with ISHLT acute cellular rejection (ACR) grade ≥2R and/or antibody-mediated rejection ≥ pAMR 1. dd-cfDNA was quantitated using shotgun sequencing. Median dd-cfDNA levels were compared between sexes during quiescence and rejection. The performance of dd-cfDNA by sex was assessed using AUROC. Allograft injury was defined as ddcfDNA ≥0.25%. Results151 unique patients (49 female, 32%) were included in the analysis with 1119 available dd-cfDNA measurements. Baseline characteristics including demographics and comorbidities were not significantly different between sexes. During quiescence, there were no significant sex differences in median dd-cfDNA level (0.04% [IQR 0.00, 0.16] in females vs 0.03% [IQR 0.00, 0.12] in males, p = 0.22). There were no significant sex differences in median dd-cfDNA for ACR (0.33% [0.21, 0.36] in females vs 0.32% [0.21, 1.10] in males, p = 0.57). Overall, median dd-cfDNA levels were higher in AMR than ACR but did not significantly differ by sex (0.50% [IQR 0.18, 0.82] in females vs 0.63% [IQR 0.32, 1.95] in males, p = 0.51). Elevated dd-cfDNA detected ACR/AMR with an AUROC of 0.83 in females and 0.89 in males, p-value for comparison = 0.16. DiscussionThere were no significant sex differences in dd-cfDNA levels during quiescence and rejection. Performance characteristics were similar, suggesting that similar diagnostic thresholds can be used in men and women for rejection surveillance.

The effect of primary renal disease upon outcomes after renal transplant.

This study investigated whether nature of primary renal disease affects clinical outcomes after renal transplantation at a single center in the United Kingdom. This was a retrospective cohort study of 961 renal transplant recipientsfollowed up at a large renal center from 2000 to 2020. Separation of diseases responsible for end-stage kidney disease included glomerulonephritis, diabetic kidney disease, hypertensive nephropathy, autosomal dominant polycystic kidney disease, unknown cause, other causes and chronic pyelonephritis. Outcome data included graft loss, cardiovascular events, malignancy, post-transplant diabetes mellitus and death, analyzed according to primary disease type. The mean age at transplantation was 47.3 years. During a mean follow-up of 7.6 years, 18% of the overall cohort died corresponding to an annualised mortality rate of 2.3%. Death with a functioning graft occurredat a rate of 2.1% per annum, with the highest incidence observed in in patients with diabetic kidney disease (4.1%/year). Post-transplant cardiovascular events occurred in 21% of recipients(2.8% per year), again highest in recipients with diabetic kidney disease (5.1%/year) and hypertensive nephropathy (4.5%/year). Post-transplant diabetes mellitus manifested in 19% of the cohort at an annualized rate of2.1% while cancer incidence stood at 9% with an annualized rate of 1.1% . Graft loss occurred in 6.8% of recipients at the rate of1.2% per year with chronic allograft injury, acute rejection and recurrent glomerulonephritis being the predominant causative factors. Median + IQR dialysis-free survival of the whole cohort was 16.2 (9.9 ->20) years, being shortest for diabetic kidney disease (11.0 years) and greatest for autosomal dominant polycystic kidney disease (18.2 years) .The collective mean decline in eGFR over time was -1.14ml/min/year. Recipients with Pre-transplant diabetic kidney disease exhibited the fastest rate of decline(-2.1ml/min/year) a statistically significant difference in comparison to the other native kidney diseases with Autosomal dominant polycystic kidney disease exhibiting the lowest rate of decline(-0.05ml/min/year) CONCLUSION: Primary renal disease can influence the outcome after renal transplantation, with patients with prior diabetic kidney disease having the poorest outcome in terms of dialysis-free survival and loss of transplant function. Autosomal polycystic kidney disease, other cause and unknown cause had the best outcomes compared to other primary renal disease groups.