Search

Xenotransplantation using porcine donors is rapidly approaching clinical applicability as an alternative therapy for treatment of many end-stage diseases including type 1 diabetes. Porcine neonatal islet cell clusters (NICC) have normalised blood sugar levels for relatively short periods in the preclinical diabetic rhesus model but have met with limited success in the stringent baboon model. Here we report that NICC from genetically modified (GM) pigs deleted for αGal and expressing the human complement regulators CD55 and CD59 can cure diabetes long-term in immunosuppressed baboons, with maximum graft survival exceeding 22 months. Five diabetic baboons were transplanted intraportally with 9,673 – 56,913 islet equivalents (IEQ) per kg recipient weight. Immunosuppression consisted of T cell depletion with an anti-CD2 mAb, tacrolimus for the first 4 months, and maintenance with belatacept and anti-CD154; no anti-inflammatory treatment or cytomegalovirus (CMV) prophylaxis/treatment was given. This protocol was well tolerated, with all recipients maintaining or gaining weight. Recipients became insulin-independent at a mean of 87 ± 43 days post-transplant and remained insulin-independent for 397 ± 174 days. Maximum graft survival was 675 days. Liver biopsies showed functional islets staining for all islet endocrine components, with no evidence of the inflammatory blood-mediated inflammatory reaction (IBMIR) and minimal leukocytic infiltration. The costimulation blockade-based immunosuppressive protocol prevented an anti-pig antibody response in all recipients. In conclusion, we demonstrate that genetic modification of the donor pig enables attenuation of early islet xenograft injury, and in conjunction with judicious immunosuppression provides excellent long-term function and graft survival in the diabetic baboon model. [ABSTRACT FROM AUTHOR]

Machine learning (ML)-workflows enable unprejudiced/robust evaluation of complex datasets. Here, we analyzed over 490,000,000 data points to compare 10 different ML-workflows in a large (N=11,652) training dataset of human pancreatic single-cell (sc-)transcriptomes to identify genes associated with the presence or absence of insulin transcript(s). Prediction accuracy/sensitivity of each ML-workflow was tested in a separate validation dataset (N=2,913). Ensemble ML-workflows, in particular Random Forest ML-algorithm delivered high predictive power (AUC=0.83) and sensitivity (0.98), compared to other algorithms. The transcripts identified through these analyses also demonstrated significant correlation with insulin in bulk RNA-seq data from human islets. The top-10 features, (including IAPP, ADCYAP1, LDHA and SST) common to the three Ensemble ML-workflows were significantly dysregulated in scRNA-seq datasets from Ire-1αβ-/- mice that demonstrate dedifferentiation of pancreatic β-cells in a model of type 1 diabetes (T1D) and in pancreatic single cells from individuals with type 2 Diabetes (T2D). Our findings provide direct comparison of ML-workflows in big data analyses, identify key elements associated with insulin transcription and provide workflows for future analyses. [ABSTRACT FROM AUTHOR]

Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγnull (NSG) mouse model of T-cell-mediated human islet allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin-2 (IL-2) combined with low-dose rapamycin to prolong graft survival. NSG mice that had received renal subcapsular human islet allografts and were transfused with 1 × 107 of human spleen mononuclear cells reconstituted human CD45+ cells that were predominantly CD3+ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3 × 106 IU/m2 or 1 × 106 IU/m2) or rapamycin alone (0.5-1 mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet allograft survival. Graft survival was associated with expansion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) and enhanced transforming growth factor-β production by CD4+ T cells. CD8+ T cells showed reduced interferon-γ production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet allograft rejection by expanding CD4+FOXP3+ Tregs in vivo and suppressing effector cell function and could be the basis of effective tolerance-based regimens. [ABSTRACT FROM AUTHOR]

Normothermic machine perfusion (NMP) is an emerging modality for kidney preservation prior to transplantation. NMP may allow directed pharmacomodulation of renal ischemia-reperfusion injury (IRI) without the need for systemic donor/recipient therapies. Three proven anti-IRI agents not in widespread clinical use, CD47-blocking antibody (αCD47Ab), soluble complement receptor 1 (sCR1), and recombinant thrombomodulin (rTM), were compared in a murine model of kidney IRI. The most effective agent was then utilized in a custom NMP circuit for the treatment of isolated porcine kidneys, ascertaining the impact of the drug on perfusion and IRI-related parameters. αCD47Ab conferred the greatest protection against IRI in mice after 24 hours. αCD47Ab was therefore chosen as the candidate agent for addition to the NMP circuit. CD47 receptor binding was demonstrated by immunofluorescence. Renal perfusion/flow improved with CD47 blockade, with a corresponding reduction in oxidative stress and histologic damage compared to untreated NMP kidneys. Tubular and glomerular functional parameters were not significantly impacted by αCD47Ab treatment during NMP. In a murine renal IRI model, αCD47Ab was confirmed as a superior anti-IRI agent compared to therapies targeting other pathways. NMP enabled effective, direct delivery of this drug to porcine kidneys, although further efficacy needs to be proven in the transplantation setting. [ABSTRACT FROM AUTHOR]

Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Immunophenotyping of whole peripheral blood (WPB) by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. Healthy individuals and patients with type 1 diabetes (T1D) enrolled in islet transplantation were recruited and WPB was collected. 46 fluorochrome-conjugated mouse-anti-human antibodies were used (43 of 46 antibodies were titrated). BD cytometer setup and tracking beads were used to characterize and adjust for cytometer performance. Antibody cocktails were pre-mixed <60 minutes before staining. Multicolour panels were designed based on fluorochrome brightness, antigen density, co-expression, and fluorochrome spillover into non-primary detectors in each panel on a 5 laser flow cytometer. WPB sample staining used 50–300 μl WPB for each panel and was performed within 2 hours of blood sample collection. Samples were acquired on a BD-LSRFortessa. The operating procedures, including specimen collection, antibody cocktails, staining protocol, flow-cytometer setup and data analysis, were standardized. The staining index of 43 antibodies and the spillover spreading matrix for each panel was calculated. The final concentrations for the 46 antibodies used was determined for staining of WPB samples. Absolute cell-count and 7 leukocyte profiling panels consisting of subsets and/or status of granulocytes, monocytes, dendritic, B, NK, and T cells including regulatory T cells (Tregs) and NKT were designed and established on a 5 laser BD-LSR Fortessa. 13 T1D patients, including 4 islet transplant recipients and 8 healthy controls, were evaluated. The ability to reproducibly measure immune subsets and immune-profiles of islet transplant patients up to 18 months post transplantation has been established as a tool to measure immune cell reconstitution after transplantation. [ABSTRACT FROM AUTHOR]

Background: The ongoing supply‐demand gap with respect to donor kidneys for transplantation necessitates the increased use of higher kidney donor profile index and/or donation after circulatory death (DCD) kidneys. Machine perfusion (MP) preservation has become increasingly popular as a means to preserve such organs. Human data regarding normothermic kidney MP (NMP) is in its infancy, and such a system has not been established in the Australasian clinical setting. Methods: Modified cardio‐pulmonary bypass technology was utilized to develop a viable NMP kidney perfusion system using a porcine DCD model. System development and optimization occurred in two stages, with system components added in each experiment to identify optimal perfusion conditions. Results: Device functionality was demonstrated by the successful perfusion of and urine production by, eight porcine kidneys. Urine production diminished in the presence of colloid in the perfusate. Pressure‐controlled (compared with flow‐controlled) perfusion is preferable as a safe perfusion pressure range can be maintained. More physiologic perfusion conditions are achieved if oxygenation is provided by an oxygen/carbon dioxide mixture compared to 100% oxygen. Conclusion: A viable and reproducible NMP system was established and tested in porcine kidneys, which was able to simulate graft function extra‐corporeally. Further work is required to identify the most optimal perfusion conditions. Prior to its utilization in clinical transplantation, the system should be tested in non‐transplanted human kidneys. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cold ischemia and hence travel time can adversely affect outcomes of islet isolation. The aim of this study was to compare the isolation and transplant outcomes of donor pancreata according to the distance from islet isolation facility. Principally, those within a 50 km radius of the centre were compared with those from regional areas within the state and those from interstate donors within Australia. Organ donors were categorised according to distance from National Pancreas Transplant Unit Westmead (NPTU). Donor characteristics were analysed statistically against islet isolation outcomes. These were age, BMI, cause and mechanism of death, days in ICU, gender, inotrope and steroid use, cold ischemia time (CIT) and retrieval surgical team. Between March 2007 and December 2020, 297 islet isolations were performed at our centre. A total of 149 donor pancreata were local area, and 148 non-local regions. Mean distance from the isolation facility was 780.05 km. Mean pancreas CIT was 401.07 ± 137.71 min and was significantly different between local and non-local groups (297.2 vs. 487.5 min, p < 0.01). Mean age of donors was 45.22 years, mean BMI was 28.82, sex ratio was 48:52 F:M and mean time in ICU was 3.07 days. There was no significant difference between local and non-local for these characteristics. The mean CIT resulting in islet transplantation was 297.1 ± 91.5 min and longest CIT resulting in transplantation was 676 min. There was no significant difference in islet isolation outcomes between local and non-local donors for characteristics other than CIT. There was also no significant effect of distance from the isolation facility on positive islet transplant outcomes (C-peptide > 0.2 at 1 month post-transplant). Conclusions: Distance from the isolation centre did not impact on isolation or transplant outcomes supporting the ongoing nationwide use of shipping pancreata for islet isolation and transplantation. [ABSTRACT FROM AUTHOR]