Prospective observational study to validate a next-generation sequencing blood RNA signature to predict early kidney transplant rejection.

The objective of this study was to validate the performance of Tutivia, a peripheral blood gene expression signature, in predicting early acute rejection (AR) post-kidney transplant. Recipients of living or deceased donor kidney transplants were enrolled in a nonrandomized, prospective, global, and observational study (NCT04727788). The main outcome was validation of the area under the curve (AUC) of Tutivia vs serum creatinine at biopsy alone, or Tutivia + serum creatinine at biopsy. Of the 151 kidney transplant recipients, the mean cohort age was 53 years old, and 64% were male. There were 71% (107/151) surveillance/protocol biopsies and 29% (44/151) for-cause biopsies, with a 31% (47/151) overall rejection rate. Tutivia (AUC 0.69 [95% CI: 0.59-0.77]) and AUC of Tutivia + creatinine at biopsy (0.68 [95% CI: 0.59-0.77]) were greater than the AUC of creatinine at biopsy alone (0.51.4 [95% CI: 0.43-0.60]). Applying a model cut-off of 50 (scale 0-100) generated a high- and low-risk category for AR with a negative predictive value of 0.79 (95% CI: 0.71-0.86), a positive predictive value of 0.60 (95% CI: 0.45-0.74), and an odds ratio of 5.74 (95% CI: 2.63-12.54). Tutivia represents a validated noninvasive approach for clinicians to accurately predict early AR, beyond the current standard of care.

Single nuclei transcriptomics delineates complex immune and kidney cell interactions contributing to kidney allograft fibrosis.

Chronic allograft dysfunction (CAD), characterized histologically by interstitial fibrosis and tubular atrophy, is the major cause of kidney allograft loss. Here, using single nuclei RNA sequencing and transcriptome analysis, we identified the origin, functional heterogeneity, and regulation of fibrosis-forming cells in kidney allografts with CAD. A robust technique was used to isolate individual nuclei from kidney allograft biopsies and successfully profiled 23,980 nuclei from five kidney transplant recipients with CAD and 17,913 nuclei from three patients with normal allograft function. Our analysis revealed two distinct states of fibrosis in CAD; low and high extracellular matrix (ECM) with distinct kidney cell subclusters, immune cell types, and transcriptional profiles. Imaging mass cytometry analysis confirmed increased ECM deposition at the protein level. Proximal tubular cells transitioned to an injured mixed tubular (MT1) phenotype comprised of activated fibroblasts and myofibroblast markers, generated provisional ECM which recruited inflammatory cells, and served as the main driver of fibrosis. MT1 cells in the high ECM state achieved replicative repair evidenced by dedifferentiation and nephrogenic transcriptional signatures. MT1 in the low ECM state showed decreased apoptosis, decreased cycling tubular cells, and severe metabolic dysfunction, limiting the potential for repair. Activated B, T and plasma cells were increased in the high ECM state, while macrophage subtypes were increased in the low ECM state. Intercellular communication between kidney parenchymal cells and donor-derived macrophages, detected several years post-transplantation, played a key role in injury propagation. Thus, our study identified novel molecular targets for interventions aimed to ameliorate or prevent allograft fibrogenesis in kidney transplant recipients.

Single-cell RNA sequencing reveals peripheral blood mononuclear immune cell landscape associated with operational tolerance in a kidney transplant recipient.

Operational tolerance (OT) after kidney transplantation is defined as stable graft acceptance without the need for immunosuppression therapy. However, it is not clear which cellular and molecular pathways are driving tolerance in these patients. In this first-of-its-kind pilot study, we assessed the immune landscape associated with OT using single-cell analyses. Peripheral mononuclear cells from a kidney transplant recipient with OT (Tol), 2 healthy individuals (HC), and a kidney transplant recipient with normal kidney function on standard-of-care immunosuppression (SOC) were evaluated. The immune landscape of the Tol was drastically different from that of SOC and emerged closer to the profile of HC. TCL1A+ naive B cells and LSGAL1+ regulatory T cells (Tregs) were in higher proportions in Tol. We were unable to identify the Treg subcluster in SOC. The ligand-receptor analysis in HC and Tol identified interactions between B cells, and Tregs that enhance the proliferation and suppressive function of Tregs. SOC reported the highest proportion of activated B cells with more cells in the G2M phase. Our single-cell RNA sequencing study identified the mediators of tolerance; however, it emphasizes the requirement of similar investigations on a larger cohort to reaffirm the role of immune cells in tolerance.

Immune landscape of the kidney allograft in response to rejection.

Preventing kidney graft dysfunction and rejection is a critical step in addressing the nationwide organ shortage and improving patient outcomes. While kidney transplants (KT) are performed more frequently, the overall number of patients on the waitlist consistently exceeds organ availability. Despite improved short-term outcomes in KT, comparable progress in long-term allograft survival has not been achieved. Major cause of graft loss at 5 years post-KT is chronic allograft dysfunction (CAD) characterized by interstitial fibrosis and tubular atrophy (IFTA). Accordingly, proactive prevention of CAD requires a comprehensive understanding of the immune mechanisms associated with either further dysfunction or impaired repair. Allograft rejection is primed by innate immune cells and carried out by adaptive immune cells. The rejection process is primarily facilitated by antibody-mediated rejection (ABMR) and T cell-mediated rejection (TCMR). It is essential to better elucidate the actions of individual immune cell subclasses (e.g. B memory, Tregs, Macrophage type 1 and 2) throughout the rejection process, rather than limiting our understanding to broad classes of immune cells. Embracing multi-omic approaches may be the solution in acknowledging these intricacies and decoding these enigmatic pathways. A transition alongside advancing technology will better allow organ biology to find its place in this era of precision and personalized medicine.

European Society for Organ Transplantation Consensus Statement on Biomarkers in Liver Transplantation.

Currently, one-year survival following liver transplantation (LT) exceeds 90% in large international registries, and LT is considered definitive treatment for patients with end-stage liver disease and liver cancer. Recurrence of disease, including hepatocellular carcinoma (HCC), significantly hampers post-LT outcomes. An optimal approach to immunosuppression (IS), including safe weaning, may benefit patients by mitigating the effect on recurrent diseases, as well as reducing adverse events associated with over-/under-IS, including chronic kidney disease (CKD). Prediction of these outcome measures-disease recurrence, CKD, and immune status-has long been based on relatively inaccurate clinical models. To address the utility of new biomarkers in predicting these outcomes in the post-LT setting, the European Society of Organ Transplantation (ESOT) and International Liver Transplant Society (ILTS) convened a working group of experts to review literature pertaining to primary disease recurrence, development of CKD, and safe weaning of IS. Summaries of evidence were presented to the group of panelists and juries to develop guidelines, which were discussed and voted in-person at the Consensus Conference in Prague November 2022. The consensus findings and recommendations of the Liver Working Group on new biomarkers in LT, clinical applicability, and future needs are presented in this article.

Pretransplant kidney transcriptome captures intrinsic donor organ quality and predicts 24-month outcomes.

With the development of novel prognostic tools derived from omics technologies, transplant medicine is entering the era of precision medicine. Currently, there are no established predictive biomarkers for posttransplant kidney function. A total of 270 deceased donor pretransplant kidney biopsies were collected and posttransplant function was prospectively monitored. This study first assessed the utility of pretransplant gene expression profiles in predicting 24-month outcomes in a training set (n = 174). Nearly 600 differentially expressed genes were associated with 24-month graft function. Grafts that progressed to low function at 24 months exhibited upregulated immune responses and downregulated metabolic processes at pretransplantation. Using penalized logistic regression modeling, a 55 gene model area under the receiver operating curve (AUROC) for 24-month graft function was 0.994. Gene expression for a subset of candidate genes was then measured in an independent set of pretransplant biopsies (n = 96) using quantitative polymerase chain reaction. The AUROC when using 13 genes with three donor characteristics (age, race, body mass index) was 0.821. Subsequently, a risk score was calculated using this combination for each patient in the validation cohort, demonstrating the translational feasibility of using gene markers as prognostic tools. These findings support the potential of pretransplant transcriptomic biomarkers as novel instruments for improving posttransplant outcome predictions and associated management.

When is it safe for the liver donor to be discharged home and prevent unnecessary re-hospitalizations? - A systematic review of the literature and expert panel recommendations.

BACKGROUND: Few data are available on discharge criteria after living liver donation (LLD). OBJECTIVES: To identify the features for fit for discharge checklist after LLD to prevent unnecessary re-hospitalizations and to provide international expert recommendations. DATA SOURCES: Ovid MEDLINE, Embase, Scopus, Google Scholar, and Cochrane Central. METHODS: Systematic review following PRISMA guidelines and recommendations using the GRADE approach derived from an international expert panel. The critical outcomes included were complications rates and liver function (defined by elevated bilirubin and INR) (CRD42021260725). RESULTS: Total 57/1710 studies were included in qualitative analysis and 28/57 on the final analysis. No randomized controlled trials were identified. The complications rate was reported in 20/28 studies and ranged from 7.8% to 71.2%. Post hepatectomy liver function was reported in 13 studies. The Quality of Evidence (QoE) was Low and Very-Low for complications rate and liver function test, respectively. CONCLUSIONS: Monitoring and prevention of donor complications should be crucial in decision making of discharge. Pain and diet control, removal of all drains and catheters, deep venous thrombosis prophylaxis, and use routine imaging (CT scan or liver ultrasound) before discharge should be included as fit for discharge checklist (QoE; Low | GRADE of recommendation; Strong). Transient Impaired liver function (defined by elevated bilirubin and INR), a prognostic marker of outcome after liver resection, usually occurs after donor right hepatectomy and should be monitored. Improving trends for bilirubin and INR value should be observed by day 5 post hepatectomy and be included in the fit for discharge checklist. (QoE; Very-Low | GRADE; Strong).

Comparison of Biliary Complications Rates After Brain Death, Donation After Circulatory Death, and Living-Donor Liver Transplantation: A Single-Center Cohort Study.

Donation-after-circulatory-death (DCD), donation-after-brain-death (DBD), and living-donation (LD) are the three possible options for liver transplantation (LT), each with unique benefits and complication rates. We aimed to compare DCD-, DBD-, and LD-LT-specific graft survival and biliary complications (BC). We collected data on 138 DCD-, 3,027 DBD- and 318 LD-LTs adult recipients from a single center and analyzed patient/graft survival. BC (leak and anastomotic/non-anastomotic stricture (AS/NAS)) were analyzed in a subset of 414 patients. One-/five-year graft survival were 88.6%/70.0% for DCD-LT, 92.6%/79.9% for DBD-LT, and, 91.7%/82.9% for LD-LT. DCD-LTs had a 1.7-/1.3-fold adjusted risk of losing their graft compared to DBD-LT and LD-LT, respectively (p < 0.010/0.403). Bile leaks were present in 10.1% (DCD-LTs), 7.2% (DBD-LTs), and 36.2% (LD-LTs) (ORs, DBD/LD vs. DCD: 0.7/4.2, p = 0.402/<0.001). AS developed in 28.3% DCD-LTs, 18.1% DBD-LTs, and 43.5% LD-LTs (ORs, DBD/LD vs. DCD: 0.5/1.8, p = 0.018/0.006). NAS was present in 15.2% DCD-LTs, 1.4% DBDs-LT, and 4.3% LD-LTs (ORs, DBD/LD vs. DCD: 0.1/0.3, p = 0.001/0.005). LTs w/o BC had better liver graft survival compared to any other groups with BC. DCD-LT and LD-LT had excellent graft survival despite significantly higher BC rates compared to DBD-LT. DCD-LT represents a valid alternative whose importance should increase further with machine/perfusion systems.

Recent progress and remaining hurdles toward clinical xenotransplantation.

BACKGROUND: Xenotransplantation has made tremendous progress over the last decade. METHODS: We discuss kidney and heart xenotransplantation, which are nearing initial clinical trials. RESULTS: Life sustaining genetically modified kidney xenografts can now last for approximately 500 days and orthotopic heart xenografts for 200 days in non-human primates. Anti-swine specific antibody screening, preemptive desensitization protocols, complement inhibition and targeted immunosuppression are currently being adapted to xenotransplantation with the hope to achieve better control of antibody-mediated rejection (AMR) and improve xenograft longevity. These newest advances could probably facilitate future clinical trials, a significant step for the medical community, given that dialysis remains difficult for many patients and can have prohibitive costs. Performing a successful pig-to-human clinical kidney xenograft, that could last for more than a year after transplant, seems feasible but it still has significant potential hurdles to overcome. The risk/benefit balance is progressively reaching an acceptable equilibrium for future human recipients, e.g. those with a life expectancy inferior to two years. The ultimate question at this stage would be to determine if a "proof of concept" in humans is desirable, or whether further experimental/pre-clinical advances are still needed to demonstrate longer xenograft survival in non-human primates. CONCLUSION: In this review, we discuss the most recent advances in kidney and heart xenotransplantation, with a focus on the prevention and treatment of AMR and on the recipient's selection, two aspects that will likely be the major points of discussion in the first pig organ xenotransplantation clinical trials.

An Integrated Transcriptomic Approach to Identify Molecular Markers of Calcineurin Inhibitor Nephrotoxicity in Pediatric Kidney Transplant Recipients.

Calcineurin inhibitors are highly efficacious immunosuppressive agents used in pediatric kidney transplantation. However, calcineurin inhibitor nephrotoxicity (CNIT) has been associated with the development of chronic renal allograft dysfunction and decreased graft survival. This study evaluated 37 formalin-fixed paraffin-embedded biopsies from pediatric kidney transplant recipients using gene expression profiling. Normal allograft samples (n = 12) served as negative controls and were compared to biopsies exhibiting CNIT (n = 11). The remaining samples served as positive controls to validate CNIT marker specificity and were characterized by other common causes of graft failure such as acute rejection (n = 7) and interstitial fibrosis/tubular atrophy (n = 7). MiRNA profiles served as the platform for data integration. Oxidative phosphorylation and mitochondrial dysfunction were the top molecular pathways associated with overexpressed genes in CNIT samples. Decreased ATP synthesis was identified as a significant biological function in CNIT, while key toxicology pathways included NRF2-mediated oxidative stress response and increased permeability transition of mitochondria. An integrative analysis demonstrated a panel of 13 significant miRNAs and their 33 CNIT-specific gene targets involved with mitochondrial activity and function. We also identified a candidate panel of miRNAs/genes, which may serve as future molecular markers for CNIT diagnosis as well as potential therapeutic targets.

Integrative Analyses of Circulating Small RNAs and Kidney Graft Transcriptome in Transplant Glomerulopathy.

Transplant glomerulopathy develops through multiple mechanisms, including donor-specific antibodies, T cells and innate immunity. This study investigates circulating small RNA profiles in serum samples of kidney transplant recipients with biopsy-proven transplant glomerulopathy. Among total small RNA population, miRNAs were the most abundant species in the serum of kidney transplant patients. In addition, fragments arising from mature tRNA and rRNA were detected. Most of the tRNA fragments were generated from 5' ends of mature tRNA and mainly from two parental tRNAs: tRNA-Gly and tRNA-Glu. Moreover, transplant patients with transplant glomerulopathy displayed a novel tRNA fragments signature. Gene expression analysis from allograft tissues demonstrated changes in canonical pathways related to immune activation such as iCos-iCosL signaling pathway in T helper cells, Th1 and Th2 activation pathway, and dendritic cell maturation. mRNA targets of down-regulated miRNAs such as miR-1224-5p, miR-4508, miR-320, miR-378a from serum were globally upregulated in tissue. Integration of serum miRNA profiles with tissue gene expression showed that changes in serum miRNAs support the role of T-cell mediated mechanisms in ongoing allograft injury.

Rapamycin Alternatively Modifies Mitochondrial Dynamics in Dendritic Cells to Reduce Kidney Ischemic Reperfusion Injury.

Dendritic cells (DCs) are unique immune cells that can link innate and adaptive immune responses and Immunometabolism greatly impacts their phenotype. Rapamycin is a macrolide compound that has immunosuppressant functions and is used to prevent graft loss in kidney transplantation. The current study evaluated the therapeutic potential of ex-vivo rapamycin treated DCs to protect kidneys in a mouse model of acute kidney injury (AKI). For the rapamycin single (S) treatment (Rapa-S-DC), Veh-DCs were treated with rapamycin (10 ng/mL) for 1 h before LPS. In contrast, rapamycin multiple (M) treatment (Rapa-M-DC) were exposed to 3 treatments over 7 days. Only multiple ex-vivo rapamycin treatments of DCs induced a persistent reprogramming of mitochondrial metabolism. These DCs had 18-fold more mitochondria, had almost 4-fold higher oxygen consumption rates, and produced more ATP compared to Veh-DCs (Veh treated control DCs). Pathway analysis showed IL10 signaling as a major contributing pathway to the altered immunophenotype after Rapamycin treatment compared to vehicle with significantly lower cytokines Tnfa, Il1b, and Il6, while regulators of mitochondrial content Pgc1a, Tfam, and Ho1 remained elevated. Critically, adoptive transfer of rapamycin-treated DCs to WT recipients 24 h before bilateral kidney ischemia significantly protected the kidneys from injury with a significant 3-fold improvement in kidney function. Last, the infusion of DCs containing higher mitochondria numbers (treated ex-vivo with healthy isolated mitochondria (10 µg/mL) one day before) also partially protected the kidneys from IRI. These studies demonstrate that pre-emptive infusion of ex-vivo reprogrammed DCs that have higher mitochondria content has therapeutic capacity to induce an anti-inflammatory regulatory phenotype to protect kidneys from injury.

Epigenetic modifications and the development of kidney graft fibrosis.

PURPOSE OF REVIEW: To outline recent discoveries in epigenetic regulatory mechanisms that have potential implications in the development of renal fibrosis following kidney transplantation. RECENT FINDINGS: The characterization of renal fibrosis following kidney transplantation has shown TGFß/Smad signaling to play a major role in the progression to chronic allograft dysfunction. The onset of unregulated proinflammatory pathways are only exacerbated by the decline in regulatory mechanisms lost with progressive patient age and comorbidities such as hypertension and diabetes. However, significant developments in the recognition of epigenetic regulatory markers upstream of aberrant TGFß-signaling has significant clinical potential to provide therapeutic targets for the treatment of renal fibrosis. In addition, discoveries in extracellular vesicles and the characterization of their cargo has laid new framework for the potential to evaluate patient outcomes independent of invasive biopsies. SUMMARY: The current review summarizes the main findings in epigenetic machinery specific to the development of renal fibrosis and highlights therapeutic options that have significant potential to translate into clinical practice.

Applications of CRISPR technologies in transplantation.

In transplantation, the ever-increasing number of an organ's demand and long-term graft dysfunction constitute some of the major problems. Therefore, alternative solutions to increase the quantity and quality of the organ supply for transplantation are desired. On this subject, revolutionary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology holds enormous potential for the scientific community with its expanding toolbox. In this minireview, we summarize the history and mechanism of CRISPR/Cas9 systems and explore its potential applications in cellular- and organ-level transplantation. The last part of this review includes future opportunities as well as the challenges in the transplantation field.

Living donor program crisis management plans: Current landscape and talking point recommendations.

Although minimized by expert evaluation, operative technique, and postoperative care, the extremely low risk of perioperative mortality following living kidney or liver donation will never be eliminated. Furthermore, anticipation of poor donor outcome may simultaneously be a source of anxiety for physicians and programs and also be a circumstance for which they are unprepared. We conducted a national survey of US transplant surgeons to understand experiences with and systemic preparedness for the event of a living donor death. Respondents represented 87 unique transplant programs (71 kidney and 16 liver donor programs). Perioperative deaths were rare, as expected. Although most respondents (N = 57, 64% of total respondents; 88% of liver programs) reported being moderately to extremely concerned about a future living donor death at their institution, only 30 (33% of total program respondents) had a written plan available in the case of such an event; 63% of programs would find guidance and recommendations useful. To help address this gap, the American Society of Transplantation Live Donor Community of Practice (AST LDCOP) developed Living Donor Crisis Management Plan Talking Points suitable to guide crisis plan development at transplant programs.

Predicting harmful alcohol relapse after liver transplant: The HALT score.

BACKGROUND: Alcohol-associated liver disease (AALD) is a rapidly growing indication for liver transplantation (LT). We aimed to examine various clinical, demographic, and behavioral factors to predict post-LT alcohol relapse and graft survival. METHODS: Retrospective analysis was performed on 241 LT recipients with AALD as either a primary or secondary indication for LT (2006-2015). RESULTS: Patients with <6 months of alcohol abstinence had significantly increased cumulative incidence for alcohol relapse compared to those with >6 months of abstinence (P = .0041, Log-Rank). We identified four variables to predict harmful alcohol relapse post-LT: age at LT, non-alcohol-related criminal history, pre-LT abstinence period (Ref >6 months of alcohol abstinence), and drinks per day (Ref <10 drinks/day). Area under the curve (AUC) for the final model was 0.79 (95% CI: 0.68-0.91). Our multivariable model was evaluated with internal cross-validation; random sampling of the study subjects 100 times yielded a median C statistic of 75 (±SD 0.097) and accuracy of 91 (±SD 0.026). The four-variable model served to form the harmful alcohol use post-LT (HALT) score. Graft survival remained significantly lower in patients with <6 months of pre-LT alcohol abstinence and those with blue-collar jobs. CONCLUSION: The HALT score identifies LT candidates with AALD at significant risk for alcohol relapse, potentially guiding transplant centers for pre- and post-LT interventions for improved patient outcomes.

Transplantation of kidneys from hepatitis C-infected donors to hepatitis C-negative recipients: Single center experience.

Our aim was to evaluate the safety of transplanting kidneys from HCV-infected donors in HCV-uninfected recipients. Data collected from 53 recipients in a single center, observational study included donor and recipient characteristics, liver and kidney graft function, new infections and de novo donor-specific antibodies and renal histology. Treatment with a direct-acting antiviral regimen was initiated when HCV RNA was detected. The mean ± SD age of recipients was 53 ± 11 years, 34% were female, 19% and 79% of recipients were white and African American, respectively. The median and interquartile range (IQR) time between transplant and treatment initiation was 76 (IQR: 68-88) days. All 53 recipients became viremic (genotype: 1a [N = 34], 1b [N = 1], 2 [N = 3], and 3 [N = 15]). The majority (81%) of recipients did not experience clinically significant increases (>3 times higher than upper limit of the normal value) in aminotransferase levels and their HCV RNA levels were in the 5 to 6 log range. One patient developed fibrosing cholestatic hepatitis with complete resolution. All recipients completed antiviral treatment and 100% were HCV RNA-negative and achieved 12-week sustained virologic response. The estimated GFRs at end of treatment and 12-week posttreatment were 67 ± 21 mL/min/1.73 m2 and 67 ± 17 mL/min/1.73 m2 , respectively. Four recipients developed acute rejection. Kidney transplantation from HCV-infected donors to HCV-negative recipients should be considered in all eligible patients.

Adenosine 2A Receptor Activation Attenuates Ischemia Reperfusion Injury During Extracorporeal Cardiopulmonary Resuscitation.

OBJECTIVE: We tested the hypothesis that systemic administration of an A2AR agonist will reduce multiorgan IRI in a porcine model of ECPR. SUMMARY BACKGROUND DATA: Advances in ECPR have decreased mortality after cardiac arrest; however, subsequent IRI contributes to late multisystem organ failure. Attenuation of IRI has been reported with the use of an A2AR agonist. METHODS: Adult swine underwent 20 minutes of circulatory arrest, induced by ventricular fibrillation, followed by 6 hours of reperfusion with ECPR. Animals were randomized to vehicle control, low-dose A2AR agonist, or high-dose A2AR agonist. A perfusion specialist using a goal-directed resuscitation protocol managed all the animals during the reperfusion period. Hourly blood, urine, and tissue samples were collected. Biochemical and microarray analyses were performed to identify differential inflammatory markers and gene expression between groups. RESULTS: Both the treatment groups demonstrated significantly higher percent reduction from peak lactate after reperfusion compared with vehicle controls. Control animals required significantly more fluid, epinephrine, and higher final pump flow while having lower urine output than both the treatment groups. The treatment groups had lower urine NGAL, an early marker of kidney injury (P = 0.01), lower plasma aspartate aminotransferase, and reduced rate of troponin rise (P = 0.01). Pro-inflammatory cytokines were lower while anti-inflammatory cytokines were significantly higher in the treatment groups. CONCLUSIONS: Using a novel and clinically relevant porcine model of circulatory arrest and ECPR, we demonstrated that a selective A2AR agonist significantly attenuated systemic IRI and warrants clinical investigation.

Intragraft Molecular Pathways Associated with Tolerance Induction in Renal Transplantation.

The modern immunosuppression regimen has greatly improved short-term allograft outcomes but not long-term allograft survival. Complications associated with immunosuppression, specifically nephrotoxicity and infection risk, significantly affect graft and patient survival. Inducing and understanding pathways underlying clinical tolerance after transplantation are, therefore, necessary. We previously showed full donor chimerism and immunosuppression withdrawal in highly mismatched allograft recipients using a bioengineered stem cell product (FCRx). Here, we evaluated the gene expression and microRNA expression profiles in renal biopsy samples from tolerance-induced FCRx recipients, paired donor organs before implant, and subjects under standard immunosuppression (SIS) without rejection and with acute rejection. Unlike allograft samples showing acute rejection, samples from FCRx recipients did not show upregulation of T cell- and B cell-mediated rejection pathways. Gene expression pathways differed slightly between FCRx samples and the paired preimplantation donor organ samples, but most of the functional gene networks overlapped. Notably, compared with SIS samples, FCRx samples showed upregulation of genes involved in pathways, like B cell receptor signaling. Additionally, prediction analysis showed inhibition of proinflammatory regulators and activation of anti-inflammatory pathways in FCRx samples. Furthermore, integrative analyses (microRNA and gene expression profiling from the same biopsy sample) identified the induction of regulators with demonstrated roles in the downregulation of inflammatory pathways and maintenance of tissue homeostasis in tolerance-induced FCRx samples compared with SIS samples. This pilot study highlights the utility of molecular intragraft evaluation of pathways related to FCRx-induced tolerance and the use of integrative analyses for identifying upstream regulators of the affected downstream molecular pathways.

Geographic variation in liver transplantation persists despite implementation of Share35.

AIM: Geographic disparities persist in the USA despite locoregional organ sharing policies. The impact of national organ sharing policies on waiting-list mortality on a regional basis remains unknown. METHODS: Data on all adult liver transplants between 1 February 2002 and 31 March 2015 were obtained from the United Network for Organ Sharing/Organ and Transplantation Network. Multivariable Cox proportional hazards models were constructed in a time-to-event analysis to estimate waiting-list mortality for the pre- and post-Share35 eras. RESULTS: In the analyzed time period, 134 247 patients were listed for transplantation and 54 510 received organs (42.8%). Listing volume increased following the introduction of the Share35 organ sharing policy (15 976 candidates pre- vs. 18 375 post) without significant regional changes as did the number of transplants (7210 pre- vs. 8224 post). Waiting-list mortality improved from 12.2% to 8.1% (P < 0.001). Adjusted waiting-list mortality ratios remained geographically disparate. Region 10 and region 11 had lower hazard ratios (HR) but still had increased mortality (1.46, 95% confidence interval [CI] 1.34-1.60, P < 0.001; and HR 1.49, 95% CI 1.37-1.62, P < 0.001, respectively). Regions 3 and 6 had increased HR with persistently elevated waiting-list mortality (1.79, 95% CI 1.66-1.93, P < 0.001; and HR 1.29, 95% CI 1.16-1.45, P < 0.001, respectively). Model for End-state Liver Disease (MELD) exception continued to propagate a survival benefit (HR 0.65, 95% CI 0.63-0.68, P < 0.001). CONCLUSIONS: Although overall waiting-list mortality has decreased, geographic disparities persist, but appear reduced despite broader sharing policies enacted by Share35. The advantage afforded by MELD exception, while still present, was diminished by Share35 as organs are being shifted to MELD >35 candidates. The disparities highlighted by our findings imply a need to review current allocation policies to best balance local, regional, and national transplant environments.