Graft Injury Research Articles

Donor-derived cell-free DNA versus left ventricular global longitudinal strain in noninvasive detection of heart allograft injury

Abstract Introduction Invasive endomyocardial biopsy (EMB) is still considered the gold standard method for monitoring heart transplant (HTx) rejection. However, it is associated with potentially serious complications, and its histopathologic interpretation has high interobserver variability. Thus, noninvasive methods for surveillance of the transplanted organ remains of great interest. The noninvasive donor-derived cell-free DNA is a marker of graft injury which can indicate acute rejection, even before the histopathological diagnosis of rejection detected by EMB. The assay has a high negative predictive value for acute rejection. Left ventricular global longitudinal strain (LV GLS) is an emerging marker of subclinical myocardial injury which plays a crucial role in the evaluation of several cardiac diseases; however, its role in HTx injury has so far been poorly investigated. Purpose We aimed to investigate the efficacy of a local laboratory-run dd-cfDNA assay-based heart transplant rejection surveillance programme, and the prognostic and discriminatory performance of LV GLS for subsequent heart allograft injury. Methods HTx recipients without a history of allograft rejection requiring intensification of immunosuppressive therapy were transitioned from our EMB-based surveillance protocol to dd-cfDNA-based rejection surveillance. We assessed the percentage of dd-cfDNA to total cell-free DNA. Injury cut-off was defined by a dd-cfDNA level ≥0.20%, while the severe injury threshold was at ≥0.35%. LV ejection fraction was calculated using 2D Simpson’s method. To derive LV GLS, we analyzed apical 4-chamber, 2-chamber, and long-axis images by 2D speckle tracking echocardiography. Results A total of 244 dd-cfDNA samples were analyzed from 46 patients in fifteen runs performed monthly between October 2022 and December 2023. The dd-cfDNA fraction remained below the injury cut-off in most patients (81%). There were no missed rejection episodes. 88% of routine EMBs that would have otherwise been performed were safely avoided. Eighteen for-cause EMBs were performed based on dd-cfDNA levels suggesting injury. One EMB verified moderate cellular rejection (ISHLT grade 2R) that required steroid pulse therapy. Two EMBs proved mild cellular rejection, one EMB confirmed mild antibody-mediated rejection, while one EMB proved mixed rejection. Thirteen EMBs found no rejection. Mean LVEF was preserved (57.6%±7.6%). Mean LV GLS was -15.1%±2.5% in the whole cohort, while -15.6%±4.7% in recipients with HTx rejection on indication EMB. There was no significant correlation between dd-cfDNA and LV GLS (n=159). Conclusion The noninvasive dd-cfDNA assay is effective in ruling out acute rejection and safely decreases the necessity of invasive surveillance EMBs after HTx. LV GLS is less sensitive in comparison with the dd-cfDNA assay for detection of myocardial injury in the early stages of graft rejection in HTx recipients at low risk for rejection.

Increased thrombin generation in kidney transplant recipients with donor-specific antibodies directed against human leukocyte antigens

IntroductionThe development of de novo anti-HLA donor specific antibodies (DSAs) is associated with poor outcomes in kidney transplant recipients. It is surmised that an interaction between DSAs and the graft endothelium cause tissue injury, however, the exact underlying pathomechanism and optimal management of patients with DSAs remain undetermined.AimsWe hypothesized that in kidney transplant recipients the presence of DSAs induce hemostasis alterations, including hypercoagulability, as assessed by the thrombin generation assay (TGA). Patients and methods. In this observational cohort study, 27 kidney transplant recipients with DSAs (DSA+ group) and 16 without DSAs (DSA– group) were enrolled. Venous blood samples were obtained, and besides routine laboratory tests, von Willebrand factor antigen (VWF), FVIII activity, soluble E selectin (sEsel), soluble P selectin (sPsel), TGA, clot lysis assay (CLA), complement levels (C3, C4) were measured. To correlate results with potential changes in DSA status over time, patients were followed and reassessed 6 ± 1.5 months later.ResultsVWF and sPsel did not differ between groups, but both parameters were increased in the majority of patients. Endogenous thrombin potential (ETP) was significantly higher in the DSA+ group as compared to DSA– patients (median:1666; IQR:1438-2012 vs. 1230; IQR:1097-1659 nM*min, p=0.0019). Follow-up measurements indicated that the observed hemostasis alterations were not transient. CLA parameters, C3 and C4 did not differ between DSA+ and DSA– groups. The extent of anti-HLA II DSA positivity correlated positively with ETP, while tacrolimus levels negatively correlated with ETP and VWF/FVIII levels.ConclusionsIn patients with anti-HLA class II DSAs, thrombin generation was significantly increased as compared to DSA– kidney transplant recipients, suggesting that the presence of antibodies is associated with hypercoagulability. Tacrolimus levels were negatively associated with TGA parameters. Hypercoagulability, associated with the presence of DSAs, may potentially contribute to the pathomechanism of antibody-mediated graft injury, warranting future prospective studies.

FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

Fibroblast growth factor 21 (FGF21) is essential for modulating hepatic homeostasis, but the impact of FGF21 on liver graft injury remains uncertain. Here, we show that high FGF21 levels in liver graft and serum are associated with improved graft function and survival in liver transplantation (LT) recipients. FGF21 deficiency aggravates early graft injury and activates arachidonic acid metabolism and regional inflammation in male mouse models of hepatic ischemia/reperfusion (I/R) injury and orthotopic LT. Mechanistically, FGF21 deficiency results in abnormal activation of the arachidonate 15-lipoxygenase (ALOX15)/15-hydroxy eicosatetraenoic acid (15-HETE) pathway, which triggers a cascade of innate immunity-dominated pro-inflammatory responses in grafts. Notably, the modulating role of FGF21/ALOX15/15-HETE pathway is more significant in steatotic livers. In contrast, pharmacological administration of recombinant FGF21 effectively protects against hepatic I/R injury. Overall, our study reveals the regulatory mechanism of FGF21 and offers insights into its potential clinical application in early liver graft injury after LT.

B-234 Evaluating the diagnostic utility of dd-cf-dna in renal allograft surveillance: a single-center perspective

Abstract Background Donor-derived cell-free DNA (dd-cf-DNA) tests offer a non-invasive alternative to biopsy for allograft surveillance in solid organ transplantation. Unlike biopsies, dd-cf-DNA tests are not subject to interpretation bias and have the potential to detect subclinical rejection, which can lead to graft dysfunction and poor outcomes. At our institution, dd-cf-DNA testing is currently offered as a send out test and we sought to evaluate its efficacy in monitoring our post-transplant patients. Methods We retrospectively examined electronic health records of 50 renal transplant patients with dd-cf-DNA (using AlloSure and Prospera assays) between January 1st, 2019, and December 31st, 2022 to assess diagnostic utility of dd-cf-DNA relative to donor-specific antibodies (DSA) and biopsy (all predictors of graft function). Statistical analysis included correlation testing with t-tests. Results Overall, dd-cf-DNA levels correlated with DSA levels (p = 0.0002) and patients with both HLA-class I and II DSA had significantly higher dd-cf-DNA levels (p< 0.05). The Prospera dd-cf-DNA assay appeared to be a better predictor of DSA levels (p = 0.04) than the Allosure dd-cf-DNA assay. Though not statistically significant, there was trend towards an increase in dd-cf-DNA in patient with biopsy-proven graft injury and rejection vs no rejection. Interestingly, dd-cf-DNA levels were significantly elevated in patients with biopsy-proven antibody mediated rejection (ABMR). Because dd-cfDNA levels have been reported to be impacted by sensitizing events including prior transplant, we evaluated and observed levels of dd-cf-DNA to be higher in patients with a history of more than one graft vs single graft recipients (p< 0.0001) and this observation was prominent in the Allosure assay. Conclusions Our findings suggest that dd-cf-DNA testing holds promise as a valuable tool for graft surveillance and early detection of ABMR. The lack of correlation with biopsy-confirmed rejections may be attributed to the dd-cf-DNA assays’ ability to detect rejection events earlier in the disease progression process compared to biopsy. Time course analysis will be beneficial. Additionally, the observed differences between AlloSure and Prospera emphasize the significance of thoroughly evaluating and selecting assays, particularly in multi-transplant recipients. We highlight that our initial analysis is limited due to a small sample size. However, we aim to assess 200 patients by the conclusion of this study.

Clinical Examples of the Additive Value of Absolute Quantification of Cell-Free DNA After Heart Transplantation.

Cell-free DNA (cfDNA) is used as a biomarker after transplantation to detect graft injury, relying on the donor fraction (DF). We have established a PCR-based approach allowing us to separately quantify absolute values of dd-cfDNA and recipient-derived cfDNA (rd-cfDNA). We aimed to present typical clinical scenarios after heart transplantation (HTx) to illustrate the advantages of absolute cfDNA values over DF. We used the cfDNA results of our cohort (509 samples of 52 patients followed during the first year after HTx) as background and determined the trajectories of cfDNA in specific clinical situations. We profiled an uncomplicated clinical course, viral and bacterial infections, acute and chronic rejection, and false-negative and false-positive rejections in six patients (five adults, one child). There was a substantial discrepancy between relative (DF) and absolute cfDNA-levels in several clinical situations. Rd- and dd-cfDNA were independently elevated during episodes of rejection and infection and were better suited to depict treatment response than DF alone. Absolute quantification of cfDNA may offer clinically relevant information additive to DF in various situations after HTx and could be helpful for more accurate monitoring of diagnosis and treatment of rejection.

Application of graft-derived cell-free DNA for solid organ transplantation.

Monitoring the status of grafts and the occurrence of postoperative complications, such as rejection, is crucial for ensuring the success and long-term survival of organ transplants. Traditional histopathological examination, though effective, is an invasive procedure and poses risks of complications, making frequent use impractical. In recent years, graft-derived cell-free DNA (gd-cfDNA) has emerged as a promising non-invasive biomarker. It not only provides early warnings of rejection and other types of graft injury but also offers important information about the effectiveness of immunosuppressive therapy and prognosis. gd-cfDNA shows potential in the monitoring of organ transplants. The early, real-time information on graft injury provided by gd-cfDNA facilitates timely individualized treatment and improves patient outcomes. However, the progress of research on gd-cfDNA varies across different organs. Therefore, this article will comprehensively review the application and findings of gd-cfDNA in monitoring various solid organs, discussing the advantages, limitations, and some future research directions to aid in its clinical application.

Urinary CXCL-10, a prognostic biomarker for kidney graft injuries: a systematic review and meta-analysis

The challenges of long-term graft survival and the side effects of current immunosuppressive therapies in kidney transplantation highlight the need for improved drugs with fewer adverse effects. Biomarkers play a crucial role in quickly detecting post-transplant complications, with new biomarkers showing promise for ongoing monitoring of disease and potentially reducing the need for unnecessary invasive biopsies. The chemokines such as C-X-C motif chemokine ligand 10 (CXCL10), are particularly promising protein biomarkers for acute renal rejection, with urine samples being a desirable source for biomarkers. The aim of this review is to analyze the literature on the potential role of urinary CXCL10 protein in predicting kidney graft injuries. The results of this study demonstrate that evaluating urinary CXCL10 levels is more successful in identifying post-transplant injuries compared to assessing the CXCL10/Cr ratio.

Assessment of liver graft quality during hypothermic oxygenated perfusion: the first international validation study

Background and AimWhile it is currently assumed that liver assessment is only possible during normothermic machine perfusion (NMP), there is uncertainty regarding a reliable and quick prediction of graft injury during ex situ hypothermic oxygenated perfusion (HOPE). We therefore intended to test, in an international liver transplant cohort, recently described mitochondrial injury biomarkers measured during HOPE before liver transplantation. Study designPerfusate samples of human livers from 10 centers in 7 countries with HOPE-experience were analyzed for released mitochondrial compounds, i.e. flavin mononucleotide (FMN), NADH, purine derivates and inflammatory markers. Perfusate FMN was correlated with graft loss due to primary non-function or symptomatic non-anastomotic biliary strictures (NAS), and kidney failure, as well as liver injury after transplantation. Livers deemed unsuitable for transplantation served as negative control. ResultsWe collected 473 perfusate samples of human DCD (n=315) and DBD livers (n=158). Fluorometric assessment of FMN in perfusate was validated by mass spectrometry (R=0.7011,p<0.0001). Graft loss due to primary non-function or cholangiopathy was predicted by perfusate FMN values (c-statistic mass spectrometry 0.8418 (95%CI 0.7466-0.9370,p<0.0001), c-statistic fluorometry 0.7733 (95%CI 0.7006-0.8461,p<0.0001). Perfusate FMN values were also significantly correlated with symptomatic NAS and kidney failure, and superior in prediction of graft loss when compared to conventional scores derived from donor and recipient parameters, such as the donor risk index and the balance of risk score. Mitochondrial FMN values in liver tissues of non-utilized livers were low, and inversely correlated to high perfusate FMN values and purine metabolite release. ConclusionsThis first international study validates the predictive value of the mitochondrial co-factor FMN, released from complex I during HOPE, and may therefore contribute to a better risk stratification of injured livers before implantation. Impact and ImplicationsAnalysis of 473 perfusates, collected from 10 international centers during hypothermic oxygenated perfusion (HOPE), revealed that mitochondria derived flavin mononucleotide (FMN) values in perfusate is predictive for graft loss, cholangiopathy, and kidney failure after liver transplantation. This result is of high clinical relevance, as recognition of graft quality is urgently needed to improve the safe utilization of marginal livers. Ex-situ machine perfusion approaches, such as HOPE, are therefore likely to increase the number of useable liver grafts.

Donor-derived cell-free DNA is a valuable monitoring tool after single lung transplantation: Multicenter analysis

Donor-derived cell-free DNA (dd-cfDNA) is a nonspecific plasma biomarker for tissue injury which has been validated for monitoring of acute rejection (AR) after lung transplantation (LT). However, no studies to date have focused specifically on single lung transplantation (SLT). Herein we report the performance of dd-cfDNA in detecting AR in SLT from six academic centers who implemented this biomarker surveillance in their standard of practice (SOP). Dd-cfDNA test results were corrected for SLT by an algorithm in the CLIA-laboratory to permit comparison against the same 1.0% threshold used in double-lung transplant. Investigators reviewed patient SOP EMR clinical data to assign test results into cohorts based on clinical allograft health status. To avoid ambiguity in interpretation, samples drawn after a prior acute rejection or infection event or without histopathologic confirmation of acute rejection, were excluded from further analysis. Diagnostic cohorts included: Acute Rejection (AR, N=25 samples), healthy (STABLE, N=137), allograft-related Infection (INFXN, N=41), Chronic Lung Allograft Dysfunction (CLAD, N=7), and “OTHER” (N=12) types of graft injury. The study included a total of 257 dd-cfDNA results from 103 SLT patients while one patient was excluded due to active cancer. Samples were drawn a median of 233 days (IQR: 96-489) after SLT. Analysis of laterality for SLT (R vs L) and median dd-cfDNA fraction in AR and STABLE cohort was not statistically different. The median dd-cfDNA fraction was elevated with AR (1.8%) and INFXN (1.1%) vs STABLE (0.46%; p<0.0001). dd-cfDNA with CLAD was also significantly higher than STABLE cohort (p=0.0155). The Area Under Receiver Operator Characteristics (AUROC) Curve was 0.850 (95% CI: 0.72 - 0.95, p<0.0001) for AR vs STABLE cohort. Applying the dd-cfDNA threshold ≥ 1.0% for AR, yielded a Sensitivity= 77.8%, Specificity= 84.6%, Positive (PPV)= 38.31%, and Negative Predictive Value (NPV)= 96.83%. These multi-center data, incorporating real-world experiences, support the clinical validity and utility of dd-cfDNA monitoring of SLT recipients. Additional studies of the impact of biomarker surveillance on clinically meaningful outcomes should be forthcoming from robust, prospective, clinical trials already in progress.

Analysis of Perioperative Factors Leading to Postoperative Pulmonary Complications, Graft Injury and Increased Postoperative Mortality in Lung Transplantation

ObjectivesPostoperative complications such as postoperative pulmonary complications (PPC) and other organ complications are associated with increased morbidity and mortality after successful lung transplantation and have a detrimental effect on patient recovery. The aim of this study was to investigate perioperative risk factors for in-hospital mortality and postoperative complications with a focus on PPC and graft injury in patients undergoing lung transplantation DesignSingle-center retrospective cohort study of 173 patients undergoing lung transplantation SettingUniversity Hospital, Medical Center Freiburg Main ResultsIn the stepwise multivariate regression analysis, donor age >60 years (OR 1.85, 95% CI 1.27-2.81), intraoperative ECMO (OR 2.4, 95% CI 1.7-3.3), transfusion of > 4 red blood cell concentrates (OR 3.1, 95% CI 1.82-5.1), mean pulmonary artery pressure > 30 mmHg at the end of surgery (OR 3.5, 95% CI 2-6.3), the occurrence of postoperative graft injury (OR 4.1, 95% CI 2.8-5.9), PPCs (OR 2.1, 95% CI 1.7-2.6), sepsis (OR 4.5, 95% CI 2.8-7.3) and acute renal failure KDIGO 3 (OR 4.3, 95% CI 2.4-7.7) were associated with increased in hospital mortality, while COPD patients had a lower in-hospital mortality (OR 1.6, 95% CI 1.4-1.9). The frequency and number of PPC correlated with postoperative mortality. ConclusionClinical management and risk stratification focusing on the underlying, identified factors could help improve patients’ outcome.

Dexmedetomidine and argon in combination against ferroptosis through tackling TXNIP-mediated oxidative stress in DCD porcine livers

Graft availability from donation after circulatory death (DCD) is significantly limited by ischaemia reperfusion (IR) injury. Effective strategies to mitigate IR injury in DCD grafts are essential to improve graft quality and expand the donor pool. In this study, liver grafts from DCD pigs were preserved in the University of Wisconsin (UW) solution saturated with 0.1 nM dexmedetomidine (Dex) and various concentrations of noble gases Argon (Ar) and/or Xenon (Xe) at 4 °C for 24 or 72 h. The combined 50% Ar and Dex provided maximum protection to liver grafts by reducing morphological damage, apoptosis, necroptosis, ferroptosis, hepatocyte glycogen depletion, reticulin framework collapse, iron deposition, and oxidative stress. In vitro, human liver Hep G2 cells were preserved in the UW solution saturated with 0.1 nM Dex and 50% Ar in combination at 4 °C for 24 h, followed by recovery in medium at 37 °C for up to 48 h to mimic clinical IR injury. This treatment significantly increased the expression of anti-oxidative stress proteins by promoting the translocation of thioredoxin-interacting protein (TXNIP) to mitochondria, thereby inhibiting ferroptosis, increasing plasma membrane integrity, and maintaining cell viability.In summary, The combination of 0.1 nM Dex and 50% Ar may be a promising strategy to reduce ferroptosis and other form cell death, and preserve liver grafts.

Enhancing Liver Transplant Outcomes through Liver Precooling to Mitigate Inflammatory Response and Protect Mitochondrial Function.

Transplanted organs experience several episodes of ischemia and ischemia-reperfusion. The graft injury resulting from ischemia-reperfusion (IRI) remains a significant obstacle to the successful survival of transplanted grafts. Temperature significantly influences cellular metabolic rates because biochemical reactions are highly sensitive to temperature changes. Consequently, lowering the temperature could reduce the degradative reactions triggered by ischemia. In mitigating IRI in liver grafts, the potential protective effect of localized hypothermia on the liver prior to blood flow obstruction has yet to be explored. In this study, we applied local hypothermia to mouse donor livers for a specific duration before stopping blood flow to liver lobes, a procedure called "liver precooling". Mouse donor liver temperature in control groups was controlled at 37 °C. Subsequently, the liver donors were preserved in cold University of Wisconsin solution for various durations followed by orthotopic liver transplantation. Liver graft injury, function and inflammation were assessed at 1 and 2 days post-transplantation. Liver precooling exhibited a significant improvement in graft function, revealing more than a 47% decrease in plasma aspartate transaminase (AST) and alanine aminotransferase (ALT) levels, coupled with a remarkable reduction of approximately 50% in liver graft histological damage compared to the control group. The protective effects of liver precooling were associated with the preservation of mitochondrial function, a substantial reduction in hepatocyte cell death, and a significantly attenuated inflammatory response. Taken together, reducing the cellular metabolism and enzymatic activity to a minimum level before ischemia protects against IRI during transplantation.

Serum from patients with cirrhosis undergoing liver transplantation induces permeability in human pulmonary microvascular endothelial cells ex vivo.

Patients with cirrhosis undergoing liver transplantation frequently exhibit systemic inflammation, coagulation derangements, and edema, indicating endothelial dysfunction. This syndrome may worsen after ischemia-reperfusion injury of the liver graft, coincident with organ dysfunction that worsens patient outcomes. Little is known about changes in endothelial permeability during liver transplantation. We hypothesized that sera from these patients would increase permeability in cultured human endothelial cells ex vivo. Adults with cirrhosis presenting for liver transplantation provided consent for blood collection during surgery. Sera were prepared at five time points spanning the entire operation. The barrier function of human pulmonary microvascular endothelial cells in culture was assessed by transendothelial resistance measured using the ECIS ZΘ system. Confluent cells from two different endothelial cell donors were stimulated with human serum from liver transplant patients. Pooled serum from healthy men and purified inflammatory agonists served as controls. The permeability response to serum was quantified as the area under the normalized resistance curve. Responses were compared between time points and analyzed for associations with clinical characteristics of liver transplant patients and their grafts. Liver transplant sera from all time points during surgery-induced permeability in both endothelial cell lines. The magnitude of permeability change was heterogeneous between patients, and there were differences in the effects of sera on the two endothelial cell lines. In one of the cell lines, the severity of liver disease was associated with greater permeability at the start of surgery. In the same cell line, serum collected 15 min after liver reperfusion induced significantly more permeability as compared to that collected at the start of surgery. Early postreperfusion sera from patients undergoing living donor transplants induced more permeability than sera from deceased donor transplants. Sera from two exemplary cases of patients on preoperative dialysis, and one patient with an unexpectedly long warm ischemia time of the liver graft, induced exaggerated and prolonged endothelial permeability. Serum from patients with cirrhosis undergoing liver transplantation induces permeability of cultured human pulmonary microvascular endothelial cells. Increased endothelial permeability during liver transplantation may contribute to organ injury and present a target for future therapeutics.

Role of Donor-derived Cell-free DNA In Predicting Short-term Allograft Health In Liver Transplant Recipients

BackgroundPredicting allograft dysfunction prior to clinical or biochemical evidence remains one of the challenges in transplantation, and a preclinical detection and early management of its cause allows for improved post-transplant outcomes. Donor derived cell-free DNA (ddcfDNA) has been proposed as an important biomarker of allograft injury and has shown to predict dysfunction prior to any biochemical derangements. We aimed to investigate the diagnostic performance of ddcfDNA in detecting and differentiating the causes of early pre-biochemical detection of graft injury and in predicting the short-term outcomes of graft health using a patented protocol and proprietary set of single nucleotide polymorphisms. MethodsBlood samples were collected on defined postoperative days (1-, 3-, 7- and at 3-months) and were analysed through relatively economical patented protocol (TrunomeTM). Biopsy, biochemical tests, and clinical criteria were analysed between various subgroups. ResultOf a total 50 patients, percentage ddcfDNA (%ddcfDNA) levels were significantly elevated in the rejection group (n=8) as compared to the non-rejection group (n= 42; median elevation 12.8% vs 4.3% respectively) with a significant correlation (r = 0.92, p<0.0001). AUC-ROC analysis revealed that the %ddcfDNA levels can predict graft health more precisely when compared to the conventional liver function tests (AUC for %ddcfDNA- 0.86; p<0.001; AUC for AST-0.65, p=0.08; AUC for ALT- 0.75, p<0.01). Moreover, %ddcfDNA levels (with a threshold >10.2%) on post-operative day 7 accurately predicted short-term (3-months) health status of the graft with 93.33% sensitivity, 94.44% specificity, 87.50% positive predictive value, 97.14% negative predictive value and 94.12% accuracy. ConclusionA single time point ddcfDNA on postoperative day 7 accurately predicts graft health and improves risk stratification in the short-term.

Oxidized ATP Suppresses B Lymphocyte Activity to Attenuate Antibody-mediated Rejection of Kidney Allografts in Mice

Background. Antibody-mediated rejection (AMR) is a major cause of renal allograft dysfunction and loss. Targeting B cells and/or donor-specific antibody removal using plasma exchange and anti-CD20 antibodies are increasingly used in clinical practice, but the efficacy remains limited. Recent studies suggest that targeting purinergic P2X7 receptor/ATP axis can have profound immune regulatory effects in transplant models, but the mechanisms involved remain incompletely defined. Methods. Purified B cells were isolated from the spleen of Balb/C mice and cultured with oxidized ATP at different concentrations. Proliferation and differentiation of B cells were examined. Effects of oxidized ATP were examined in a presensitized animal model where kidney allograft rejection mimics aspects of clinical AMR. Histopathology was assessed at the time of rejection or on day 5 after kidney transplantation. Infiltrating immune cells in renal allografts were detected by flow cytometry. Results. Oxidized ATP inhibited B-cell activation and proliferation in vitro, significantly attenuated histological signs of graft injury and prolonged kidney allograft survival. Mechanistically, oxidized ATP inhibited antibody secretion by activated B cells in response to lipopolysaccharide stimulation and markedly suppressed the production of donor-specific antibody in kidney allograft recipients. Oxidized ATP also reduced graft infiltration by other inflammatory cells. Conclusions. These findings provide evidence for the involvement of the purinergic P2X7 receptor pathway in AMR and suggest that targeting this pathways may have important clinical implications.

Rejection Surveillance After Heart Transplantation: Is Paired Noninvasive Testing the New Gold Standard?

Rejection surveillance after heart transplantation has traditionally relied on numerous endomyocardial biopsies, most of which occur during the first posttransplant year. With the introduction of gene expression profiling and, more recently, donor-derived cell-free DNA, a great proportion of surveillance is being performed noninvasively with both tests. Although patients have welcomed the use of paired testing because of the decreased risk and inconvenience, interpretation of both tests can sometimes be challenging, particularly when the test results are discordant. Growing evidence from both single-center experiences and large national databases has given insights that have allowed the field to operationalize dual testing and provide physicians with algorithms to approach paired testing. The increased use of noninvasive testing has also begun to challenge the role of biopsy as the gold standard for graft monitoring, not only for rejection but over the life of the heart transplant. In a growing number of circumstances, cell-free DNA not only may be a better means of assessing rejection but could also redefine how clinicians approach the diagnosis and even treatment of graft injury. As the heart transplant community garners more experience and generates more data, the current paradigms of heart transplant surveillance will continue to be challenged.

Donor-specific graft injury in solid organ transplantation: potential mechanisms and therapeutic strategies.

Donor-specific graft injury in solid organ transplantation: potential mechanisms and therapeutic strategies.

1506-P: Genomic Monitoring of Islet Graft Injury in Type 1 Diabetic Patients after Islet Transplantation

1506-P: Genomic Monitoring of Islet Graft Injury in Type 1 Diabetic Patients after Islet Transplantation

Outcomes of Anterior Cruciate Ligament Reconstruction in Patients with Associated Anterolateral Ligament Injury.

Objective To evaluate if there is a significant difference in the outcomes of isolated anterior cruciate ligament (ACL) reconstruction in patients with or without associated anterolateral ligament (ALL) injury. Methods We conducted a retrospective cross-sectional study through the analysis of medical records and the application of the questionnaires of the Lysholm Knee Scoring Scale and the International Knee Documentation Committee (IKDC) Subjective Knee Form to patients undergoing isolated ACL reconstruction. Results The 52 participants included were divided into two groups: 19 with associated ALL injury and 33 with no associated ALL injury. None of the patients with associated ALL injury suffered an ACL rerupture, and 21.1% presented injuries to other knee structures after surgery. Among the patients with no associated injury, 6.1% suffered ACL rerupture, and 18.2% presented injuries to other structures after surgery ( p = 0.544). Return to activities at the same level as that of the preoperative period occurred in 60% of the patients with associated ALL injury and in 72% of those with no associated injury ( p = 0.309). The mean score on the Lysholm Knee Scoring Scale was of 81.6 points in patients with associated ALL injury, and of 90.1 in those with no associated injury ( p = 0.032). The mean score on the IKDC Subjective Knee Form was of 70.3 points in patients with associated ALL injury and of 76.7 in those with no associated injury ( p = 0.112). Conclusion There was no statistically significant difference regarding graft injuries or new injuries to other structures, satisfaction with the operated knee, or the score on the IKDC Subjective Knee Form. Return to activity was similar in the groups with and without associated ALL injuries. The scores on the Lysholm Knee Scoring Scale were better, with a statistically significant difference in the group with no associated ALL injuries.

Early Injury Landscape in Vein Harvest by Single-Cell and Spatial Transcriptomics.

Vein graft failure following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. Although previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on vein graft failure. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing and spatial transcriptomics analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-carotid vein bypass implantation in a canine model (n=4). Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P<0.05) involved in the activation of endothelial cells (ECs), fibroblasts, and vascular smooth muscle cells, namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and ECM (extracellular matrix) remodeling throughout the vein wall. Subsequent single-nuclei RNA-sequencing analysis supported these findings and further unveiled distinct EC and fibroblast subpopulations with significant upregulation (P<0.05) of markers related to endothelial injury response and cellular activation of ECs, fibroblasts, and vascular smooth muscle cells. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN, FBN1, and VEGFC, in addition to novel genes of interest, such as GLIS3 and EPHA3. These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the spatial transcriptomics and single-nuclei RNA-sequencing data sets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and fibroblasts were notably enriched in the intima and media of distended veins. Finally, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal-transitioning ECs, protomyofibroblasts, and vascular smooth muscle cells in upregulating signaling pathways associated with cellular proliferation (MDK [midkine], PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor]), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.