Balanced crystalloid solution versus saline in deceased donor kidney transplantation (BEST-Fluids): a pragmatic, double-blind, randomised, controlled trial.

BACKGROUND: Delayed graft function (DGF) is a major adverse complication of deceased donor kidney transplantation. Intravenous fluids are routinely given to patients receiving a transplant to maintain intravascular volume and optimise graft function. Saline (0·9% sodium chloride) is widely used but might increase the risk of DGF due to its high chloride content. We aimed to test our hypothesis that using a balanced low-chloride crystalloid solution (Plasma-Lyte 148) instead of saline would reduce the incidence of DGF. METHODS: BEST-Fluids was a pragmatic, registry-embedded, multicentre, double-blind, randomised, controlled trial at 16 hospitals in Australia and New Zealand. Adults and children of any age receiving a deceased donor kidney transplant were eligible; those receiving a multi-organ transplant or weighing less than 20 kg were excluded. Participants were randomly assigned (1:1) using an adaptive minimisation algorithm to intravenous balanced crystalloid solution (Plasma-Lyte 148) or saline during surgery and up until 48 h after transplantation. Trial fluids were supplied in identical bags and clinicians determined the fluid volume, rate, and time of discontinuation. The primary outcome was DGF, defined as receiving dialysis within 7 days after transplantation. All participants who consented and received a transplant were included in the intention-to-treat analysis of the primary outcome. Safety was analysed in all randomly assigned eligible participants who commenced surgery and received trial fluids, whether or not they received a transplant. This study is registered with Australian New Zealand Clinical Trials Registry, (ACTRN12617000358347), and ClinicalTrials.gov (NCT03829488). FINDINGS: Between Jan 26, 2018, and Aug 10, 2020, 808 participants were randomly assigned to balanced crystalloid (n=404) or saline (n=404) and received a transplant (512 [63%] were male and 296 [37%] were female). One participant in the saline group withdrew before 7 days and was excluded, leaving 404 participants in the balanced crystalloid group and 403 in the saline group that were included in the primary analysis. DGF occurred in 121 (30%) of 404 participants in the balanced crystalloid group versus 160 (40%) of 403 in the saline group (adjusted relative risk 0·74 [95% CI 0·66 to 0·84; p<0·0001]; adjusted risk difference 10·1% [95% CI 3·5 to 16·6]). In the safety analysis, numbers of investigator-reported serious adverse events were similar in both groups, being reported in three (<1%) of 406 participants in the balanced crystalloid group versus five (1%) of 409 participants in the saline group (adjusted risk difference -0·5%, 95% CI -1·8 to 0·9; p=0·48). INTERPRETATION: Among patients receiving a deceased donor kidney transplant, intravenous fluid therapy with balanced crystalloid solution reduced the incidence of DGF compared with saline. Balanced crystalloid solution should be the standard-of-care intravenous fluid used in deceased donor kidney transplantation. FUNDING: Medical Research Future Fund and National Health and Medical Research Council (Australia), Health Research Council (New Zealand), Royal Australasian College of Physicians, and Baxter.

The impact of the cytoplasmic ubiquitin ligase TNFAIP3 gene variation on transcription factor NF-κB activation in acute kidney injury.

Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3+ T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI.

Deep learning identified pathological abnormalities predictive of graft loss in kidney transplant biopsies.

Interstitial fibrosis, tubular atrophy, and inflammation are major contributors to kidney allograft failure. Here we sought an objective, quantitative pathological assessment of these lesions to improve predictive utility and constructed a deep-learning-based pipeline recognizing normal vs. abnormal kidney tissue compartments and mononuclear leukocyte infiltrates. Periodic acid- Schiff stained slides of transplant biopsies (60 training and 33 testing) were used to quantify pathological lesions specific for interstitium, tubules and mononuclear leukocyte infiltration. The pipeline was applied to the whole slide images from 789 transplant biopsies (478 baseline [pre-implantation] and 311 post-transplant 12-month protocol biopsies) in two independent cohorts (GoCAR: 404 patients, AUSCAD: 212 patients) of transplant recipients to correlate composite lesion features with graft loss. Our model accurately recognized kidney tissue compartments and mononuclear leukocytes. The digital features significantly correlated with revised Banff 2007 scores but were more sensitive to subtle pathological changes below the thresholds in the Banff scores. The Interstitial and Tubular Abnormality Score (ITAS) in baseline samples was highly predictive of one-year graft loss, while a Composite Damage Score in 12-month post-transplant protocol biopsies predicted later graft loss. ITASs and Composite Damage Scores outperformed Banff scores or clinical predictors with superior graft loss prediction accuracy. High/intermediate risk groups stratified by ITASs or Composite Damage Scores also demonstrated significantly higher incidence of estimated glomerular filtration rate decline and subsequent graft damage. Thus, our deep-learning approach accurately detected and quantified pathological lesions from baseline or post-transplant biopsies and demonstrated superior ability for prediction of post-transplant graft loss with potential application as a prevention, risk stratification or monitoring tool.

Post-transplant cyclophosphamide limits reactive donor T cells and delays the development of graft-versus-host disease in a humanized mouse model.

Graft-versus-host disease (GVHD) is a major complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) that develops when donor T cells in the graft become reactive against the host. Post-transplant cyclophosphamide (PTCy) is increasingly used in mismatched allo-HSCT, but how PTCy impacts donor T cells and reduces GVHD is unclear. This study aimed to determine the effect of PTCy on reactive human donor T cells and GVHD development in a preclinical humanized mouse model. Immunodeficient NOD-scid-IL2Rγnull mice were injected intraperitoneally (i.p.) with 20 × 106 human peripheral blood mononuclear cells stained with carboxyfluorescein succinimidyl ester (CFSE) (day 0). Mice were subsequently injected (i.p.) with PTCy (33 mg kg-1 ) (PTCy-mice) or saline (saline-mice) (days 3 and 4). Mice were assessed for T-cell depletion on day 6 and monitored for GVHD for up to 10 weeks. Flow cytometric analysis of livers at day 6 revealed lower proportions of reactive (CFSElow ) human (h) CD3+ T cells in PTCy-mice compared with saline-mice. Over 10 weeks, PTCy-mice showed reduced weight loss and clinical GVHD, with prolonged survival and reduced histological liver GVHD compared with saline-mice. PTCy-mice also demonstrated increased splenic hCD4+ :hCD8+ T-cell ratios and reduced splenic Tregs (hCD4+  hCD25+  hCD127lo ) compared with saline-mice. This study demonstrates that PTCy reduces GVHD in a preclinical humanized mouse model. This corresponded to depletion of reactive human donor T cells, but fewer human Tregs.

Strategic plan for integrated care of patients with kidney failure.

There is a huge gap between the number of patients worldwide requiring versus those actually receiving safe, sustainable, and equitable care for kidney failure. To address this, the International Society of Nephrology coordinated the development of a Strategic Plan for Integrated Care of Patients with Kidney Failure. Implementation of the plan will require engagement of the whole kidney community over the next 5-10 years.

Relative survival and quality of life benefits of pancreas-kidney transplantation, deceased kidney transplantation and dialysis in type 1 diabetes mellitus-a probabilistic simulation model.

For patients with type 1 diabetes mellitus who progress to the point of requiring renal replacement therapy, the relative benefits of simultaneous pancreas and kidney transplantation (SPK) and deceased donor kidney transplantation across different age categories compared to dialysis are uncertain. Using Australian and New Zealand registry data from 2006 to 2016, a probabilistic Markov model (n = 10 000) was built comparing patient survival between SPK and deceased donor kidney transplantation with dialysis. Compared to dialysis, the average life years saved (LYS) and quality-adjusted life years (QALY) for SPK and deceased donor kidney transplantation were 5.48 [95% CI 5.47, 5.49] LYS and 6.48 [6.47, 6.49] QALY, and 3.38 [3.36, 3.40] LYS and 2.46 [2.45, 2.47] QALY, respectively. For recipients aged 50 years or younger, receiving a deceased donor kidney, the average incremental gains compared to dialysis were 4.13 [4.10, 4.16] LYS and 2.99 [2.97, 3.01] QALY, and for recipients older than 50 years, 3.05 [3.02, 3.08] LYS and 2.25 [2.23, 2.27] QALY. Compared to dialysis, SPK transplantation incurs the greatest benefits in LYS and QALY for patients with type 1 diabetes requiring renal replacement therapy. Patients older than 50 years still experience survival benefits from deceased donor kidney transplantation compared to dialysis.

A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection.

BACKGROUND: In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies. METHODS: We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort. RESULTS: Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss. CONCLUSIONS: Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients.

Increasing access to integrated ESKD care as part of universal health coverage.

The global nephrology community recognizes the need for a cohesive strategy to address the growing problem of end-stage kidney disease (ESKD). In March 2018, the International Society of Nephrology hosted a summit on integrated ESKD care, including 92 individuals from around the globe with diverse expertise and professional backgrounds. The attendees were from 41 countries, including 16 participants from 11 low- and lower-middle-income countries. The purpose was to develop a strategic plan to improve worldwide access to integrated ESKD care, by identifying and prioritizing key activities across 8 themes: (i) estimates of ESKD burden and treatment coverage, (ii) advocacy, (iii) education and training/workforce, (iv) financing/funding models, (v) ethics, (vi) dialysis, (vii) transplantation, and (viii) conservative care. Action plans with prioritized lists of goals, activities, and key deliverables, and an overarching performance framework were developed for each theme. Examples of these key deliverables include improved data availability, integration of core registry measures and analysis to inform development of health care policy; a framework for advocacy; improved and continued stakeholder engagement; improved workforce training; equitable, efficient, and cost-effective funding models; greater understanding and greater application of ethical principles in practice and policy; definition and application of standards for safe and sustainable dialysis treatment and a set of measurable quality parameters; and integration of dialysis, transplantation, and comprehensive conservative care as ESKD treatment options within the context of overall health priorities. Intended users of the action plans include clinicians, patients and their families, scientists, industry partners, government decision makers, and advocacy organizations. Implementation of this integrated and comprehensive plan is intended to improve quality and access to care and thereby reduce serious health-related suffering of adults and children affected by ESKD worldwide.

SHROOM3-FYN Interaction Regulates Nephrin Phosphorylation and Affects Albuminuria in Allografts.

BACKGROUND: We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. METHODS: We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli. RESULTS: Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3-binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration. CONCLUSIONS: We demonstrate a novel mechanism that may explain SHROOM3's dichotomous associations in glomerular versus nonglomerular compartments in CKD.

The causes, significance and consequences of inflammatory fibrosis in kidney transplantation: The Banff i-IFTA lesion.

Inflammation within areas of interstitial fibrosis and tubular atrophy (i-IFTA) is associated with adverse outcomes in kidney transplantation. We evaluated i-IFTA in 429 indication- and 2052 protocol-driven biopsy samples from a longitudinal cohort of 362 kidney-pancreas recipients to determine its prevalence, time course, and relationships with T cell-mediated rejection (TCMR), immunosuppression, and outcome. Sequential histology demonstrated that i-IFTA was preceded by cellular interstitial inflammation and followed by IF/TA. The prevalence and intensity of i-IFTA increased with developing chronic fibrosis and correlated with inflammation, tubulitis, and immunosuppression era (P < .001). Tacrolimus era-based immunosuppression was associated with reduced histologic inflammation in unscarred and scarred i-IFTA compartments, ameliorated progression of IF, and increased conversion to inactive IF/TA (compared with cyclosporine era, P < .001). Prior acute (including borderline) TCMR and subclinical TCMR were followed by greater 1-year i-IFTA, remaining predictive by multivariate analysis and independent of humoral markers. One-year i-IFTA was associated with accelerated IF/TA, arterial fibrointimal hyperplasia, and chronic glomerulopathy and with reduced renal function (P < .001 versus no i-IFTA). In summary, i-IFTA is the histologic consequence of active T cell-mediated alloimmunity, representing the interface between inflammation and tubular injury with fibrotic healing. Uncontrolled i-IFTA is associated with adverse structural and functional outcomes.

Transplantation sites for human and murine islets.

AIMS/HYPOTHESIS: Beta cell replacement is a potential cure for type 1 diabetes. In humans, islet transplants are currently infused into the liver via the portal vein, although this site has disadvantages. Here, we investigated alternative transplantation sites for human and murine islets in recipient mice, comparing the portal vein with quadriceps muscle and kidney, liver and spleen capsules. METHODS: Murine islets were isolated from C57BL6/J mice and transplanted into syngeneic recipients. Human islets were isolated and transplanted into either severe combined immunodeficiency (SCID) or recombination-activating gene 1 (RAG-1) immunodeficient recipient mice. All recipient mice were 8-12 weeks of age and had been rendered diabetic (defined as blood glucose concentrations ≥20 mmol/l on two consecutive days before transplantation) by alloxan tetrahydrate treatment. Islets were transplanted into five different sites (portal vein, quadriceps muscle, kidney, liver and spleen capsules). Blood glucose concentrations were monitored twice weekly until mice were killed. Dose-response studies were also performed to determine the minimum number of islets required to cure diabetes ('cure' is defined for this study as random fed blood glucose of <15 mmol/l). RESULTS: For transplantation of murine islets into the different sites, the kidney yielded 100% success, followed by muscle (70%), portal vein (60%), spleen capsule (29%) and liver capsule (0%). For human islets, transplantation into the kidney cured diabetes in 75-80% of recipient mice. Transplantation into muscle and portal vein had intermediate success (both 29% at 2000 islet equivalents), while transplantation into liver and spleen capsule failed (0%). With increased islet mass, success rates for muscle grafts improved to 52-56%. CONCLUSIONS/INTERPRETATION: For both human and murine islets, equivalent or superior glucose lowering results were obtained for transplantation into skeletal muscle, compared with the portal vein. Unfortunately, kidney grafts are not feasible in human recipients. Skeletal muscle offers easier access and greater potential for protocol biopsies. This study suggests that human trials of muscle as a transplant site may be warranted.

Transplantation sites for porcine islets.

AIMS/HYPOTHESIS: Xenotransplantation has great potential to provide beta cell replacement and thereby provide a cure for large numbers of people with type 1 diabetes. Crucial to the success of xenotransplantation is establishment of the most viable sites for transplantation. METHODS: We compared porcine islet tissue transplanted into kidney, liver and spleen in pig recipients as assessed by blood glucose levels and IVGTT. RESULTS: Kidney was the superior site for porcine islet tissue transplantation, followed by liver then spleen. This was demonstrated by IVGTTs showing significant difference between the peak glucose levels: 22.8 ± 2.9 mmol/l for kidney compared with 26.8 ± 1.3 mmol/l for spleen and 24.7 ± 1.7 mmol/l for liver. CONCLUSIONS/INTERPRETATION: Kidney grafts are not as feasible in humans and liver results were relatively poorer than spleen. For islet transplantation to be viable and successful in the longer term, there remains a need for future investigation of alternative sites.

Biopsy transcriptome expression profiling to identify kidney transplants at risk of chronic injury: a multicentre, prospective study.

BACKGROUND: Chronic injury in kidney transplants remains a major cause of allograft loss. The aim of this study was to identify a gene set capable of predicting renal allografts at risk of progressive injury due to fibrosis. METHODS: This Genomics of Chronic Allograft Rejection (GoCAR) study is a prospective, multicentre study. We prospectively collected biopsies from renal allograft recipients (n=204) with stable renal function 3 months after transplantation. We used microarray analysis to investigate gene expression in 159 of these tissue samples. We aimed to identify genes that correlated with the Chronic Allograft Damage Index (CADI) score at 12 months, but not fibrosis at the time of the biopsy. We applied a penalised regression model in combination with permutation-based approach to derive an optimal gene set to predict allograft fibrosis. The GoCAR study is registered with ClinicalTrials.gov, number NCT00611702. FINDINGS: We identified a set of 13 genes that was independently predictive for the development of fibrosis at 1 year (ie, CADI-12 ≥2). The gene set had high predictive capacity (area under the curve [AUC] 0·967), which was superior to that of baseline clinical variables (AUC 0·706) and clinical and pathological variables (AUC 0·806). Furthermore routine pathological variables were unable to identify which histologically normal allografts would progress to fibrosis (AUC 0·754), whereas the predictive gene set accurately discriminated between transplants at high and low risk of progression (AUC 0·916). The 13 genes also accurately predicted early allograft loss (AUC 0·842 at 2 years and 0·844 at 3 years). We validated the predictive value of this gene set in an independent cohort from the GoCAR study (n=45, AUC 0·866) and two independent, publically available expression datasets (n=282, AUC 0·831 and n=24, AUC 0·972). INTERPRETATION: Our results suggest that this set of 13 genes could be used to identify kidney transplant recipients at risk of allograft loss before the development of irreversible damage, thus allowing therapy to be modified to prevent progression to fibrosis. FUNDING: National Institutes of Health.

Ex vivo-expanded baboon CD39 + regulatory T cells prevent rejection of porcine islet xenografts in NOD-SCID IL-2rγ-/- mice reconstituted with baboon peripheral blood mononuclear cells.

BACKGROUND: A high immunosuppressive burden is required for long-term islet xenograft survival in non-human primates even using genetically modified donor pigs. AIMS: We aimed to investigate the capacity of baboon regulatory T cells (Treg) to suppress islet xenograft rejection, thereby developing a potential immunoregulatory or tolerance therapy that could be evaluated in NHP models of xenotransplantation. MATERIALS & METHODS: Baboon Treg expanded with stimulation by porcine peripheral blood mononuclear cells (PBMC) were characterized by cell phenotyping and suppressive activity assays in vitro. Their function in vivo was evaluated in neonatal porcine islet cell clusters (NICC) transplanted NOD-SCID IL-2rγ-/- (NSG) mice receiving baboon PBMC alone or with expanded autologous Treg. RESULTS: The majority of expanded Treg coexpressed Foxp3 and CD39 and were highly suppressive of the baboon anti-pig xenogeneic T cell response in vitro. Reconstitution of mice with baboon PBMC alone resulted in NICC xenograft rejection within 35 days. Cotransfer with baboon PBMC and Treg prolonged islet xenograft survival beyond 100 days, correlating with Treg engraftment, intragraft CD39 and Foxp3 gene expression, and reduced graft infiltrating effector T cells and reduced interferon-γ production. DISCUSSION & CONCLUSION: Our data supports the capacity of ex vivo expanded CD39+ baboon Treg to suppress islet xenograft rejection in primatized mice, suggesting it has potential as an adjunctive immunotherapy in preclinical NHP models of xenotransplantation.

Determinants of regional differences in rates of overeducation in Europe.

This paper examines the factors determining variations in spatial rates of overeducation. A quantile regression model has been implemented on a sample of region-yearly data drawn from the EU Survey on Income and Living Conditions (EU-SILC) and several institutional and macroeconomic features captured from other data-sets. Potential determinants of overeducation rates include factors such as labour market risk, financial aid to university students, excess labour demand and institutional factors. We find significant effects both for labour market structural imbalances and institutional factors. The research supports the findings of micro based studies which have found that overeducation is consistent with an assignment interpretation of the labour market.

Regulatory T cells in kidney disease and transplantation.

Regulatory T cells (Tregs) have been shown to be important in maintaining immune homeostasis and preventing autoimmune disease, including autoimmune kidney disease. It is also likely that they play a role in limiting kidney transplant rejection and potentially in promoting transplant tolerance. Although other subsets of Tregs exist, the most potent and well-defined Tregs are the Foxp3 expressing CD4(+) Tregs derived from the thymus or generated peripherally. These CD4(+)Foxp3(+) Tregs limit autoimmune renal disease in animal models, especially chronic kidney disease, and kidney transplantation. Furthermore, other subsets of Tregs, including CD8 Tregs, may play a role in immunosuppression in kidney disease. The development and protective mechanisms of Tregs in kidney disease and kidney transplantation involve multiple mechanisms of suppression. Here we review the development and function of CD4(+)Foxp3(+) Tregs. We discuss the specific application of Tregs as a therapeutic strategy to prevent kidney disease and to limit kidney transplant rejection and detail clinical trials in this area of transplantation.

Human islet cells are killed by BID-independent mechanisms in response to FAS ligand.

Cell death via FAS/CD95 can occur either by activation of caspases alone (extrinsic) or by activation of mitochondrial death signalling (intrinsic) depending on the cell type. The BH3-only protein BID is activated in the BCL-2-regulated or mitochondrial apoptosis pathway and acts as a switch between the extrinsic and intrinsic cell death pathways. We have previously demonstrated that islets from BID-deficient mice are protected from FAS ligand-mediated apoptosis in vitro. However, it is not yet known if BID plays a similar role in human beta cell death. We therefore aimed to test the role of BID in human islet cell apoptosis immediately after isolation from human cadaver donors, as well as after de-differentiation in vitro. Freshly isolated human islets or 10-12 day cultured human islet cells exhibited BID transcript knockdown after BID siRNA transfection, however they were not protected from FAS ligand-mediated cell death in vitro as determined by DNA fragmentation analysis using flow cytometry. On the other hand, the same cells transfected with siRNA for FAS-associated via death domain (FADD), a molecule in the extrinsic cell death pathway upstream of BID, showed significant reduction in cell death. De-differentiated islets (human islet-derived progenitor cells) also demonstrated similar results with no difference in cell death after BID knockdown as compared to scramble siRNA transfections. Our results indicate that BID-independent pathways are responsible for FAS-dependent human islet cell death. These results are different from those observed in mouse islets and therefore demonstrate potentially alternate pathways of FAS ligand-induced cell death in human and mouse islet cells.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 6: patient selection for pilot clinical trials of islet xenotransplantation.

Patients in whom type 1 diabetes is complicated by impaired awareness of hypoglycemia and recurrent episodes of severe hypoglycemia are candidates for islet or pancreas transplantation if severe hypoglycemia persists after completion of a structured stepped care approach or a formalized medical optimization run-in period that provides access to hypoglycemia-specific education including behavioral therapies, insulin analogs, and diabetes technologies under the close supervision of a specialist hypoglycemia service. Patients with type 1 diabetes and end-stage renal failure who cannot meet clinically appropriate glycemic goals or continue to experience severe hypoglycemia after completion of a formalized medical optimization program under the guidance of an expert diabetes care team are candidates for islet or pancreas transplantation either simultaneously with or after a previous kidney transplant. Similarly, patients with type 2 diabetes and problematic hypoglycemia or renal failure who meet these criteria are considered candidates for islet replacement. Likewise, patients with pancreatectomy-induced diabetes in whom an islet autograft was not available or deemed inappropriate are candidates for islet or pancreas transplantation if extreme glycemic lability persists despite best medical therapy. To justify participation of these transplant candidates in early-phase trials of porcine islet cell products, lack of timely access to islet or pancreas allotransplantation due to allosensitization, high islet dose requirements, or other factors, or alternatively, a more favorable benefit-risk determination associated with the xenoislet than the alloislet or allopancreas transplant must be demonstrated. Additionally, in non-uremic xenoislet recipients, the risks associated with diabetes must be perceived to be more serious than the risks associated with the xenoislet product and the rejection prophylaxis, and in xenoislet recipients with renal failure, the xenoislet product and immunosuppression must not impact negatively on renal transplant outcomes. The most appropriate patient group for islet xenotransplantation trials will be defined by the specific characteristics of each investigational xenoislet product and related technologies applied for preventing rejection. Selecting recipients who are more likely to experience prolonged benefits associated with the islet xenograft will help these patients comply with lifelong monitoring and other public health measures.

Long-term cultured neonatal islet cell clusters demonstrate better outcomes for reversal of diabetes: in vivo and molecular profiles.

BACKGROUND: Porcine neonatal islet-like cell clusters (NICC) are being considered as a source of ß-cell replacement. However, the lag time to full function due to hormonal immaturity remains a problem. This study aimed to determine whether time in culture was important for NICC function in vivo. METHODS: Neonatal islet-like cell clusters were isolated from piglets aged between 1 and 3 days, and cultured for up to 27 days post-isolation. Each week, NICC number, viability, and function were determined. RESULTS: Neonatal islet-like cell clusters cultured for 12, 19, and 27 days achieved normal blood glucose levels at 46 days (85% of animals), 32 days (100% of animals), and 35 days (81% of animals), respectively. By comparison, standard 6-day culture took a mean of 63 days to achieve normoglycemia in 35% of animals. Longer time in culture resulted in a significant loss of islet equivalent over time. However, insulin gene expression levels were significantly higher at days 12, 19, 27 compared to day 6. Glucagon gene expression was highest at day 12, and significantly higher than day 6 at all time points. Bcl-2 gene expression increased over time, and tissue factor (TF) gene expression was highest on day 6 and then decreased over the remaining time points. CONCLUSION: Culture of NICC for 12 days provides the best balance in vivo functional outcome for transplantation, shown by better reversal of diabetes, and higher levels of gene expression for insulin, glucagon and Bcl-2 and lower levels of TF expression with acceptable NICC number loss in terms of time and expense.

Clonidine inhibits anti-non-Gal IgM xenoantibody elicited in multiple pig-to-primate models.

BACKGROUND: Survival of vascularized xenografts is dependent on pre-emptive inhibition of the xenoantibody response against galactosyltransferase knockout (GTKO) porcine organs. Our analysis in multiple GTKO pig-to-primate models of xenotransplantation has demonstrated that the anti-non-gal-α-1,3-gal (anti-non-Gal) xenoantibody response displays limited structural diversity. This allowed our group to identify an experimental compound which selectively inhibited induced anti-non-Gal IgM xenoantibodies. However, because this compound had an unknown safety profile, we extended this line of research to include screening small molecules with known safety profiles allowing rapid advancement to large animal models. METHODS: The NIH clinical collections of small molecules were screened by ELISA for their ability to inhibit xenoantibody binding to GTKO pig endothelial cells. Serum collected from non-immunosuppressed rhesus monkeys at day 14 post-injection with GTKO pig endothelial cells was utilized as a source of elicited xenoantibody for initial screening. Virtual small molecule screening based on xenoantibody structure was used to assess the likelihood that the identified small molecules bound xenoantibody directly. As a proxy for selectivity, ELISAs against tetanus toxoid and the natural antigens laminin, thyroglobulin, and single-stranded DNA (ssDNA) were utilized to assess the ability of the identified reagents to inhibit additional antibody responses. The identified inhibitory small molecules were further tested for their ability to inhibit xenoantibody elicited in multiple settings, including rhesus monkeys pre-treated with an anti-non-Gal selective anti-idiotypic antibody, non-immunosuppressed rhesus monkeys immunized with wild-type fetal pig isletlike cell clusters, and non-immunosuppressed baboons transplanted with GTKO multiple transgenic pig kidneys. RESULTS: Four clinically relevant small molecules inhibited anti-non-Gal IgM binding to GTKO pig endothelial cells in vitro. Three of these drugs displayed a limited region of structural similarity suggesting they may inhibit xenoantibody by a similar mechanism. One of these, the anti-hypertensive agent clonidine, displayed only minimal inhibition of antibodies elicited by vaccination against tetanus toxoid or pre-existing natural antibodies against laminin, thyroglobulin, or ssDNA. Furthermore, clonidine inhibited elicited anti-non-Gal IgM from all animals that demonstrated a xenoantibody response in each experimental setting. CONCLUSIONS: Clinically relevant small molecule drugs with known safety profiles can inhibit xenoantibody elicited against non-Gal antigens in diverse experimental xenotransplantation settings. These molecules are ready to be tested in large animal models. However, it will first be necessary to optimize the timing and dosing required to inhibit xenoantibodies in vivo.