RORγt inverse agonist TF-S14 inhibits Th17 cytokines and prolongs skin allograft survival in sensitized mice.

Chronic antibody mediated rejection (AMR) is the major cause of solid organ graft rejection. Th17 contributes to AMR through the secretion of IL17A, IL21 and IL22. These cytokines promote neutrophilic infiltration, B cell proliferation and donor specific antibodies (DSAs) production. In the current study we investigated the role of Th17 in transplant sensitization. Additionally, we investigated the therapeutic potential of novel inverse agonists of the retinoic acid receptor-related orphan receptor gamma t (RORγt) in the treatment of skin allograft rejection in sensitized mice. Our results show that RORγt inverse agonists reduce cytokine production in human Th17 cells in vitro. In mice, we demonstrate that the RORγt inverse agonist TF-S14 reduces Th17 signature cytokines in vitro and in vivo and leads to blocking neutrophilic infiltration to skin allografts, inhibition of the B-cell differentiation, and the reduction of de novo IgG3 DSAs production. Finally, we show that TF-S14 prolongs the survival of a total mismatch grafts in sensitized mice. In conclusion, RORγt inverse agonists offer a therapeutic intervention through a novel mechanism to treat rejection in highly sensitized patients.

Discovery, Synthesis, and In Vitro Characterization of 2,3 Derivatives of 4,5,6,7-Tetrahydro-Benzothiophene as Potent Modulators of Retinoic Acid Receptor-Related Orphan Receptor γt.

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor that is expressed in a variety of tissues and is a potential drug target for the treatment of inflammatory and auto-immune diseases, metabolic diseases, and resistant cancer types. We herein report the discovery of 2,3 derivatives of 4,5,6,7-tetrahydro-benzothiophene modulators of RORγt. We also report the solubility in acidic/neutral pH, mouse/human/dog/rat microsomal stability, Caco-2, and MDR1-MDCKII permeabilities of a set of these derivatives. For this group of modulators, inverse agonism by steric clashes and push-pull mechanisms induce greater instability to protein conformation compared to agonist lock hydration. Independent of the two mechanisms, we observed a basal modulatory activity of the tested 2,3 derivatives of 4,5,6,7-tetrahydro-benzothiophene toward RORγt due to the interactions with the Cys320-Glu326 and Arg364-Phe377 hydrophilic regions. The drug discovery approach reported in the current study can be employed to discover modulators of nuclear receptors and other globular protein targets.

Outcomes of kidney transplant recipients who underwent pre-transplant bariatric surgery for severe obesity: a long-term follow-up study.

BACKGROUND: Kidney transplantation (KT) is the preferred therapy for end-stage renal disease (ESRD). While a major cause for ESRD, obesity is also a key obstacle to candidacy for KT. Bariatric surgery, particularly sleeve gastrectomy (SG), is increasingly used to improve access to KT in patients with obesity, but the literature especially on outcomes post-KT remains lacking. We aimed to provide a long-term follow-up analysis of efficacy and outcomes of a previously described cohort of patients with obesity, who had SG as a means for access to KT. METHODS: This is a single-center retrospective follow-up study of 32 patients with advanced chronic kidney disease or ESRD, who were referred and underwent SG between 2013 and 2018 as an access strategy to KT. The primary outcome was successful KT. Ninety-day outcomes, long-term graft function, and changes in weight and obesity-related comorbidities after KT were assessed. Descriptive statistics are presented as count (percentage) or median (interquartile range). RESULTS: At baseline, 18 (56%) were male with a median age and BMI of 51 (11) years and 42.3 (5.2) kg/m2, respectively. Median follow-up time post-SG was 53 (58) months. At last follow-up, 23 (72%) patients received KT. Median time to KT was 16 (20) months and BMI was 34.0 (5.1) kg/m2 at time of transplant. At KT, 13 (57%) and 20 (87%) had diabetes and hypertension, respectively. Median follow-up post-KT was 16 (47) months. There was one graft loss requiring return to dialysis. At 5-year post-KT, median serum creatinine was 136 (66) µmol/l. At last follow-up post-KT, median BMI remained at 33.7 (7.6) kg/m2. Among patients with diabetes and hypertension, 7/13 (54%) and 5/20 (25%) had either improvement or remission of their comorbidities, respectively. CONCLUSION: SG is an effective strategy to improve access to KT in patients with severe obesity. Transplant recipients also continue to benefit from sustained weight loss and improved related comorbidities that may positively impact their graft function after KT.

TFNR2 in Ischemia-Reperfusion Injury, Rejection, and Tolerance in Transplantation.

Tumor necrosis factor receptor 2 (TNFR2) has been shown to play a crucial role in CD4+ T regulatory cells (CD4+Tregs) expansion and suppressive function. Increasing evidence has also demonstrated its role in a variety of immune regulatory cell subtypes such as CD8+ T regulatory cells (CD8+ Tregs), B regulatory cells (Bregs), and myeloid-derived suppressor cells (MDSCs). In solid organ transplantation, regulatory immune cells have been associated with decreased ischemia-reperfusion injury (IRI), improved graft survival, and improved overall outcomes. However, despite TNFR2 being studied in the context of autoimmune diseases, cancer, and hematopoietic stem cell transplantation, there remains paucity of data in the context of solid organ transplantation and islet cell transplantation. Interestingly, TNFR2 signaling has found a clinical application in islet transplantation which could guide its wider use. This article reviews the current literature on TNFR2 expression in immune modulatory cells as well as IRI, cell, and solid organ transplantation. Our results highlighted the positive impact of TNFR2 signaling especially in kidney and islet transplantation. However, further investigation of TNFR2 in all types of solid organ transplantation are required as well as dedicated studies on its therapeutic use during induction therapy or treatment of rejection.

Extracellular Vesicles From Kidney Allografts Express miR-218-5p and Alter Th17/Treg Ratios.

Delayed graft function (DGF) in kidney transplantation is associated with ischemic injury and carries long term functional and immunological risks. Extracellular vesicles (EV) released from allografts may signal a degree of ischemic stress, and are thought to play an important role in the development of anti-donor immunity. Here, we show that kidney perfusate-derived extracellular vesicles (KP-EV) express donor-specific human leukocyte antigen. KP-EV from kidneys that experience DGF increase the T-helper 17 (Th17) to T-regulatory (Treg) ratio in third party peripheral blood mononuclear cells to a greater degree than those from kidneys with immediate function. We report miR-218-5p upregulation in KP-EV of kidney transplant recipients with DGF. Levels of miR-218-5p in KP-EV inversely correlated with recipient eGFR at multiple time points following transplantation. Additionally, the degree of increase in Th17/Treg ratio by KP-EV positively correlated with miR-218-5p expression in KP-EV samples. Taken together, these data provide evidence that KP-EV may contribute to modulating immune responses in transplant recipients. This could lead to novel intervention strategies to inhibit DGF in order to improve graft function and survival.

Kidney Transplantation in Times of Covid-19: Decision Analysis in the Canadian Context.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic impacted transplant programs across Canada. OBJECTIVE: We evaluated the implications of delays in transplantation among Canadian end-stage kidney disease (ESKD) patients to allow pretransplant vaccination. DESIGN: We used a Markov microsimulation model and ESKD patient perspective to study the effectiveness (quality-adjusted life years [QALY]) of living (LD) or deceased donor (DD) kidney transplantation followed by 2-dose SARS-CoV-2 vaccine versus delay in LD ("Delay LD") or refusal of DD offer ("Delay DD") to receive 2-dose SARS-CoV-2 vaccine pretransplant. SETTING: Canadian dialysis and transplant centers. PATIENTS: We simulated a 10 000-waitlisted ESKD patient cohort, which was predictively modeled for a lifetime horizon in monthly cycles. MEASUREMENTS: Inputs on patient and graft survival estimates by patient, LD or DD characteristics, were extracted from the Treatment of End-Stage Organ Failure in Canada, Canadian Organ Replacement Register, 2009 to 2018. In addition, a literature review provided inputs on quality of life, SARS-CoV-2 transmissibility, new variants of concern, mortality risk, and antibody responses to 2-dose SARS-CoV-2 mRNA vaccines. METHODS: We conducted base case, scenario, and sensitivity analyses to illustrate the impact of patient, donor, vaccine, and pandemic characteristics on the preferred strategy. RESULTS: In the average waitlisted Canadian patient, receiving 2-dose SARS-CoV-2 vaccine post-transplant provided an effectiveness of 22.32 (95% confidence interval: 22.00-22.7) for LD and 19.34 (19.02-19.67) QALYs for DD. Delaying transplants for 6 months to allow 2-dose SARS-CoV-2 vaccine before LD and DD transplant yielded effectiveness of 22.83 (21.51-23.14) and 20.65 (20.33-20.96) QALYs, respectively. Scenario analysis suggested a benefit to short delays in DD transplants to receive 2-dose SARS-CoV-2 vaccine in waitlisted patients ≥55 years. Two-way sensitivity analysis suggested decreased effectiveness of the strategy prioritizing 2-dose SARS-CoV-2 vaccine prior to DD transplant the longer the delay and the higher the Kidney Donor Risk Index of the eventual DD transplant. When assessing the impact of SARS-CoV-2 variants of concern (infection rates ≥10-fold and associated mortality ≥3-fold vs base case), we found short delays to allow 2-dose SARS-CoV-2 vaccine administration pretransplant to be preferable. LIMITATIONS: Risks associated with nosocomial exposure of LDs were not considered. There was uncertainty regarding input parameters related to SARS-CoV-2 infection, new variants, and COVID-19 severity in ESKD patients. Given rollout of population-level SARS-CoV-2 vaccination, we assumed a linear decrease in infection rates over 1 year. Proportions of patients mounting an antibody response to 2-dose SARS-CoV-2 mRNA vaccines were considered in lieu of data on vaccine efficacy in dialysis and following transplantation. Non-age-stratified annual mortality rates were used for waitlisted candidates. CONCLUSIONS: Our analyses suggest that short delays allowing pretransplant vaccination offered comparable to greater effectiveness than pursuing transplantation without delay, proposing transplant candidates should be prioritized to receive at least 2 doses of SARS-CoV-2 vaccine. Our scenario and sensitivity analyses suggest that caution must be exercised when declining DD offers in patients offered low risk DD and who are likely to incur significant delays in access to transplantation. While population-level herd immunity may decrease infection risk in transplant patients, more data are required on vaccine efficacy against SARS-CoV-2 and variants of concern in ESKD, and how efficacy may be modified by a third vaccine dose, maintenance immunosuppression and timing of induction and rejection therapies.

An in vitro Perfused Macroencapsulation Device to Study Hemocompatibility and Survival of Islet-Like Cell Clusters.

Transplantation of hydrogel-encapsulated pancreatic islets is a promising long-term treatment for type 1 diabetes that restores blood glucose regulation while providing graft immunoprotection. Most human-scale islet encapsulation devices that rely solely on diffusion fail to provide sufficient surface area to meet islet oxygen demands. Perfused macroencapsulation devices use blood flow to mitigate oxygen limitations but increase the complexity of blood-device interactions. Here we describe a human-scale in vitro perfusion system to study hemocompatibility and performance of islet-like cell clusters (ILCs) in alginate hydrogel. A cylindrical perfusion device was designed for multi-day culture without leakage, contamination, or flow occlusion. Rat blood perfusion was assessed for prothrombin time and international normalized ratio and demonstrated no significant change in clotting time. Ex vivo perfusion performed with rats showed patency of the device for over 100 min using Doppler ultrasound imaging. PET-CT imaging of the device successfully visualized metabolically active mouse insulinoma 6 ILCs. ILCs cultured for 7 days under static conditions exhibited abnormal morphology and increased activated caspase-3 staining when compared with the perfused device. These findings reinforce the need for convective transport in macroencapsulation strategies and offer a robust and versatile in vitro system to better inform preclinical design.

Relative decrease in hemoglobin and outcomes in patients undergoing kidney transplantation surgery: A retrospective cohort study.

BACKGROUND: Recent surgical literature suggests that a relative decrease in hemoglobin (ΔHb) is predictive of adverse outcomes regardless of the absolute level. We aimed to examine the association between perioperative ΔHb and kidney transplantation (KT) outcomes. METHODS: This was a retrospective cohort study of transplant recipients, where ΔHb = [Hb0- Hb1Hb0]x 100 (Hb0 = hemoglobin pre-KT and Hb1 = lowest hemoglobin 24-h post-KT). The main outcome of interest was immediate graft function (IGF). RESULTS: Of the 899 eligible patients, 38% experienced IGF, and ΔHb was associated with 36% lower odds of IGF. Also, ΔHb was associated with higher all-cause graft failure and longer length of stay but not death-censored graft failure or mortality. ΔHb ≥30% was the threshold beyond which the odds of IGF were significantly lower even if Hb1 was ≥7 g/dL. CONCLUSION: ΔHb is associated with inferior outcomes independent of Hb1; whether it can be used to guide transfusion practices should be explored.

The impact of COVID-19 on the Canadian Kidney Paired Donation program: an opportunity for universal implementation of kidney shipping.

SUMMARY: "Never let a good crisis go to waste." - Sir Winston Churchill.The value of Canada's Kidney Paired Donation program to the population cannot be overstated. Its greatest challenge as a national program, however, is the geographic separation of recipient and matched donor. Representatives from every transplant program in the country have been working toward increased use of kidney shipping in order to diminish the disincentive of donor travel. With transplantation program and travel restrictions in place to minimize the risk of coronavirus disease 2019 (COVID-19), the time to make a full transition from donor travel to the shipment of donor kidneys has clearly arrived.

Extracellular Vesicles in Type 1 Diabetes: Messengers and Regulators.

PURPOSE OF REVIEW: Theories about the pathogenesis of type 1 diabetes (T1D) refer to the potential of primary islet inflammatory signaling as a trigger for the loss of self-tolerance leading to disease onset. Emerging evidence suggests that extracellular vesicles (EV) may represent the missing link between inflammation and autoimmunity. Here, we review the evidence for a role of EV in the pathogenesis of T1D, as well as discuss their potential value in the clinical sphere, as biomarkers and therapeutic agents. RECENT FINDINGS: EV derived from ß cells are enriched in diabetogenic autoantigens and miRNAs that are selectively sorted and packaged. These EV play a pivotal role in antigen presentation and cell to cell communication leading to activation of autoimmune responses. Furthermore, recent evidence suggests the potential of EV as novel tools in clinical diagnostics and therapeutic interventions. In-depth analysis of EV cargo using modern multi-parametric technologies may be useful in enhancing our understanding of EV-mediated immune mechanisms and in identifying robust biomarkers and therapeutic strategies for T1D.

Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling.

BACKGROUND: All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS. METHODS: Human pancreata were preserved by SCS or by persufflation in combination with SCS. Islets were subsequently isolated, and preparations in each group matched for SCS or total preservation time were compared using dynamic glucose-stimulated insulin secretion as a measure of ß-cell function and RNA sequencing to elucidate transcriptomic changes. RESULTS: Persufflated pancreata had reduced SCS time, which resulted in islets with higher glucose-stimulated insulin secretion compared to islets from SCS only pancreata. RNA sequencing of islets from persufflated pancreata identified reduced inflammatory and greater metabolic gene expression, consistent with expectations of reducing cold ischemic exposure. Portions of these transcriptional responses were not associated with time spent in SCS and were attributable to pancreatic reoxygenation. Furthermore, persufflation extended the total preservation time by 50% without any detectable decline in islet function or viability. CONCLUSIONS: These data demonstrate that pancreas preservation by persufflation rather than SCS before islet isolation reduces inflammatory responses and promotes metabolic pathways in human islets, which results in improved ß cell function.

Immune Response to Extracellular Vesicles From Human Islets of Langerhans in Patients With Type 1 Diabetes.

The autoimmune response that characterizes type 1 diabetes (T1D) has no clear cause. Extracellular vesicles (EVs) play an important role in triggering the immune response in other contexts. Here, we propose a model by which EVs isolated from human islets stimulate proinflammatory immune responses and lead to peripheral blood mononuclear cell (PBMC) activation. We show that human islet EVs are internalized by monocytes and B cells and lead to an increase in T-helper 1, 2, and 17 cytokine expression, as well as T and B cell proliferation. Importantly, we demonstrate memory T and B cell activation by EVs selectively in PBMCs of patients with T1D. Additionally, human islet EVs induce an increase in antibodies against glutamic acid decarboxylase 65 (GAD65) in T1D PBMCs. Furthermore, pretreatment of T1D PBMCs with ibrutinib, an inhibitor of Bruton tyrosine kinase, dampens EV-induced memory B cell activation and GAD65 antibody production. Collectively, our findings indicate a role for human islet EVs in mediating activation of B and T cells and GAD65 autoantibody production.

Kidney paired donation and the unique challenges of kidney shipment in Canada.

SUMMARY: Kidney paired donation (KPD) programs are an effort to bridge the disparity between kidney supply and demand. These programs combine several incompatible donor-recipient pairs in a national paired exchange database, thereby increasing the number of compatible matches. But KPD programs face unique challenges, particularly the large distances that often separate donors and recipients. Here we discuss key factors to consider when transitioning from a donor travelling model to a kidney shipment model in the Canadian context.

Renal resistance thresholds during hypothermic machine perfusion and transplantation outcomes - a retrospective cohort study.

Renal resistance (RR), of allografts undergoing hypothermic machine perfusion (HMP), is considered a measure of organ quality. We conducted a retrospective cohort study of adult deceased donor kidney transplant (KT) recipients whose grafts underwent HMP. Our aim was to evaluate whether RR is predictive of death-censored graft failure (DCGF). Of 274 KT eligible for analysis, 59% were from expanded criteria donor. RR was modeled as a categorical variable, using a previously identified terminal threshold of 0.4, and 0.2 mmHg/ml/min (median in our cohort). Hazard ratios (HR) of DCGF were 3.23 [95% confidence interval (CI): 1.12-9.34, P = 0.03] and 2.67 [95% CI: 1.14-6.31, P = 0.02] in univariable models, and 2.67 [95% CI: 0.91-7.86, P = 0.07] and 2.42 [95% CI: 1.02-5.72, P = 0.04] in multivariable models, when RR threshold was 0.4 and 0.2, respectively. Increasing risk of DCGF was observed when RR over the course of HMP was modeled using mixed linear regression models: HR of 1.31 [95% CI: 1.07-1.59, P < 0.01] and 1.25 [95% CI: 1.00-1.55, P = 0.05], in univariable and multivariable models, respectively. This suggests that RR during HMP is a predictor of long-term KT outcomes. Prospective studies are needed to assess the survival benefit of patients receiving KT with higher RR in comparison with staying wait-listed.

Machine perfusion and long-term kidney transplant recipient outcomes across allograft risk strata.

Background: The use of machine perfusion (MP) in kidney transplantation lowers delayed graft function (DGF) and improves 1-year graft survival in some, but not all, grafts. These associations have not been explored in grafts stratified by the Kidney Donor Profile index (KDPI). Methods: We analyzed 78 207 deceased-donor recipients using the Scientific Registry of Transplant Recipients data from 2006 to 2013. The cohort was stratified using the standard criteria donor/expanded criteria donor (ECD)/donation after cardiac death (DCD)/donation after brain death (DBD) classification and the KDPI scores. In each subgroup, MP use was compared with cold storage. Results: The overall DGF rate was 25.4% and MP use was associated with significantly lower DGF in all but the ECD-DCD donor subgroup. Using the donor source classification, the use of MP did not decrease death-censored graft failure (DCGF), except in the ECD-DCD subgroup from 0 to 1 year {adjusted hazard ratio [aHR] 0.56 [95% confidence interval (CI) 0.32-0.98]}. In the ECD-DBD subgroup, higher DCGF from 1 to 5 years was noted [aHR 1.15 (95% CI 1.01-1.31)]. Also, MP did not lower all-cause graft failure except in the ECD-DCD subgroup from 0 to 1 year [aHR = 0.59 (95% CI 0.38-0.91)]. Using the KDPI classification, MP did not lower DCGF or all-cause graft failure, but in the ≤70 subgroup, higher DCGF [aHR 1.16 (95% CI 1.05-1.27)] and higher all-cause graft failure [aHR 1.10 (95% CI 1.02-1.18)] was noted. Lastly, MP was not associated with mortality in any subgroup. Conclusions: Overall, MP did not lower DCGF. Neither classification better risk-stratified kidneys that have superior graft survival with MP. We question their widespread use in all allografts as an ideal approach to organ preservation.

Presence of diabetes autoantigens in extracellular vesicles derived from human islets.

Beta-cell (ß-cell) injury is the hallmark of autoimmune diabetes. However, the mechanisms by which autoreactive responses are generated in susceptible individuals are not well understood. Extracellular vesicles (EV) are produced by mammalian cells under normal and stressed physiological states. They are an important part of cellular communication, and may serve a role in antigen processing and presentation. We hypothesized that isolated human islets in culture produce EV that contain diabetes autoantigens (DAA) from these otherwise normal, non-diabetic donors. Here we report the caspase-independent production of EV by human islets in culture, and the characterization of DAA glutamic acid decarboxylase 65 (GAD65) and zinc transporter 8 (ZnT8), as well as the ß-cell resident glucose transporter 2 (Glut2), present within the EV.