Yi Vee Chew

Characterizing the Mechanistic Pathways of the Instant Blood-Mediated Inflammatory Reaction in Xenogeneic Neonatal Islet Cell Transplantation

Introduction The instant blood-mediated inflammatory reaction (IBMIR) causes major loss of islets after transplantation and consequently represents the initial barrier to survival of porcine neonatal islet cell clusters (NICC) after xenotransplantation. Methods This study used novel assays designed to characterize the various immunologic components responsible for xenogeneic IBMIR to identify initiators and investigate processes of IBMIR-associated coagulation, complement activation and neutrophil infiltration. The IBMIR was induced in vitro by exposing NICC to platelet-poor or platelet-rich human plasma or isolated neutrophils. Results We found that xenogeneic IBMIR was characterized by rapid, platelet-independent thrombin generation, with addition of platelets both accelerating and exacerbating this response. Platelet-independent complement activation was observed as early as 30 minutes after NICC exposure to plasma. However, membrane attack complex formation was not observed in NICC histopathology sections until after 60 minutes. We demonstrated for the first time that NICC-mediated complement activation was necessary for neutrophil activation in the xenogeneic IBMIR setting. Finally, using the Seahorse extracellular flux analyzer, we identified substantial loss of islet function (up to 40%) after IBMIR with surviving NICC showing evidence of mitochondrial damage. Conclusions This study used novel assays to describe multiple key pathways by which xenogeneic IBMIR causes islet destruction, allowing further refinement of future interventions aimed at resolving the issue of IBMIR in xenotransplantation.

Clinical Islet Isolation

The overarching success of islet transplantation relies on the success in the laboratory to isolate the islets. This chapter focuses on the processes of human islet cell isolation and the ways to optimally provide islet cells for transplantation. The major improvements in regards to the choice of enzyme type, way the digested pancreas tissue is handled to best separate islets from the acinar and surrounding tissues, the various methods of purification of the islets, their subsequent culture and quality assurance to improve outcomes to culminate in safe and effective islet transplantation will be discussed. After decades of improvements, islet cell isolation and transplantation now clearly offer a safe, effective and feasible therapeutic treatment option for an increasing number of patients suffering from type 1 diabetes specifically for those with severe hypoglycaemic unawareness.

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

A high immunosuppressive burden is required for long‐term islet xenograft survival in non‐human primates even using genetically modified donor pigs.

Outcomes for Islet Transplantation in Donation After Circulatory Death compared with Donation after Brain Death in Australia

Introduction Islet cell transplantation has become a clinically accepted transplant technique providing long-term insulin independence, restoring normoglycaemia and treating T1D patient’s severe hypoglycaemic unawareness. Unfortunately, the significant lack of organ donors results in patients remaining on the waitlist for years. Donation after Circulatory Death (DCD) donors have been reported by other groups as having up to 60% of selected DCD islet preparations proceed to transplant with similar outcomes to donation after brain death (DBD) organs. We review our isolation and transplantation outcomes from DCD donors and compare these to DBD donors over the same time period in the Australian program. Materials and Methods Islet donor pancreata were compared from the Australian National Islet Transplant program with direct comparison of organ donor, islet isolation variables and transplantation outcomes. Donor variables included; sex, age, weight, BMI, cause of death, CIT, WIT, pancreas weight. Isolation outcomes included; pre- and post-purification islet yield, post-culture yield, purity and viability, and transplantation outcomes such as islet number transplanted and abrogation of severe hypoglycaemic unawareness. Results and Discussion A total of 27 DCD and 73 DBD islet donor pancreata were compared with no significant differences seen in donor characteristics between DCD and DBD. However, upon evaluation of the isolation outcomes it showed that post-purification yield (IEQ) was significantly lower from the DCD group (146,518±28,971) compared to the DBD group (256,986±17,652; P=0.001). Post-purification yield per gram of pancreas was also lower from the DCD group (2,154±504 vs. 2,681±372 IEQ/g); and was significant (P<0.0001). Post-culture yields in terms of total IEQ and IEQ per gram of pancreas were more than four times lower from DCD pancreata (37,634±27,786 IEQ and 455±305 IEQ/g) compared to DBD pancreata (234,860±18,132 IEQ and 2,280±199 IEQ/g), which reached extreme significance (P<0.0001). The quality and functionality of DCD and DBD islets were also significantly different in terms of the viability (%) – P=0.017 (higher in DBD than DCD), purity (%) – P=0.001 (higher in DBD than DCD), stimulation index, and beta cell viability index outcomes were not different. The proportion of DCD islets transplanted (1/27) was significantly lower than DBD (29/73) going to transplant (OR, 0.1093; 95% CI; P=0.001). Conclusion In the Australian setting with vast distances to ship pancreata we have had poorer outcomes from DCD pancreata for islet isolation and have thus far not yielded outcomes comparable to those from our DBD donors. Earlier intervention, the use of ante mortem heparin and faster logistics in transport may not only improve the DCD organs for transplantation but also help alleviate donor shortages allowing treatment of those with T1DM and severe hypoglycaemic unawareness.

Long-Term Function of Genetically Modified Porcine Neonatal Islet Xenografts in Baboons

Introduction In xenotransplantation the stringent pig-to-baboon islet xenograft preclinical model is a difficult one with the maximum period of normoglycemia reported to only 1 day. Previously we have shown that neonatal islet cell clusters (NICC) from GTKO/CD55-CD59-HT pigs prevent both hyper acute rejection (HAR) and the instant blood mediated inflammatory response (IBMIR) in immunosuppressed baboons. However, the maturation and function of the NICC were not examined in detail in the initial study, because (i) the recipients were not diabetic, and (ii) the standard clinical-based immunosuppression used was insufficient to prevent biopsy-proven rejection within 1 month. In the current study, therefore, we used streptozotocin-induced diabetic recipients and changed to a costimulation blockade-based immunosuppressive regimen. Aims To achieve long-term normoglycemia in diabetic baboons transplanted with neonatal pig islets, and to investigate the effect of ceasing immunosuppression. Materials and Methods: Five diabetic baboons received intraportal infusion of NICC (10,000-50,000 IEQ/kg) from 1-5 day old GTKO/CD55-CD59-HT piglets. From day -3 recipients were treated with anti-CD2 induction and subsequently maintenance with oral tacrolimus, anti-CD154 and belatacept, which were progressively ceased. Graft survival and function was followed by daily blood sugar levels (BSL), IVGTT, OGTT and immunohistochemical analysis of liver biopsies taken at various time points over the study period. Results and Discussion None of the baboons exhibited signs of thrombosis associated with IBMIR, with no change to platelet counts, vWF, fibrinogen or D-dimer levels from baseline. Recipients developed normal fasting BSL and had normal IVGTT and OGTT, with porcine insulin and C-peptide secreted in response to glucose bolus stimulus. All animals have become normoglycaemic off all exogenous insulin. Liver biopsies revealed strong positive staining for insulin, glucagon and somatostatin in xenografts. One recipient receiving 50,000 IEQ/kg was insulin-independent for >7 months, including 7 weeks after the last drug (belatacept) was ceased. A second recipient receiving 10,000 IEQ/kg took longer to achieve insulin independence, but remained insulin independent >18 months, including 6-months off all immunosuppression. The fourth and fifth animals are being followed out past 6-months and 2-months post transplant. Conclusion We have confirmed that GTKO/CD55-CD59-HT neonatal islets are protected from IBMIR in the baboon model. More importantly, we have demonstrated for the first time long-term survival and function of porcine islets in baboons. The costimulation blockade-based immunosuppression permitted maturation of the islets such that the dose required to achieve normoglycemia (10,000 IEQ/kg) was lower than that reported in any NHP model. Remarkably, this recipient remained normoglycemic for 6 months after immunosuppression was ceased. Bristol-Myers Squibb Company for donation of Belatacept,. Keith Reimann at NIH Nonhuman Primate Reagent Resource for supply of Anti CD154 antibody.

Standardized Whole-Blood Immunophenotyping Panels on Flow Cytometry for Transplant Recipients and Clinical Trials

Introduction Immunophenotyping of whole blood by flow cytometry is a reliable, fast, and easy method used to obtain a large amount of information on the effects and outcomes of different treatments in transplantation on immune phenotype with minimal impact on the patient. Aim: 1) To establish whole-blood immunophenotyping panels for transplant recipients, 2) To develop a concise method involving the standardisation of reagents, sample handling, instrument setup and data analysis. Methods Absolute cell count (TruCount) and seven leukocyte-profiling panels containing 8-10 marker-antigens (46 of which were unique within the panels) consisting of subsets and/or status of granulocytes, monocytes, DCs, B, NK,, and T cells including Tregs and NKT were used to monitor the immune profiles of paediatric kidney transplant and adult islet cell transplant recipients. Whole-blood samples were stained and acquired on a BD-LSRFortessa and Flowjo was used for data analysis. BD™ Cytometer Setup and Tracking beads monitored cytometer performance. Sample staining was performed within 2 hours of blood sample collection. The accuracy and variability of these panels were determined and 100-300 &mgr;l whole-blood was used for each panel. Results: The 46 antibodies were titrated and the staining index (SI) was calculated (CD45BUV395 in Fig.1A) for panel optimisation. Application settings on a BD LSRFortessa, measurement of the spillover spreading matrix (SSM) for each panel [SSM in Panel 3 (Tab.1)], and optimal antibody quantity for the panel-cocktail were established. Auto-analysis templates and gating strategies (Panel 1 in Fig.1B) to target subsets of immune-cells were set up and blood sample collection, preparation, antibody cocktails, and staining protocols were standardised. 9 paediatric kidney transplant patients, 4 islet transplant patients (which are to be followed until two years post third islet transplant), 13 T1D patients and 8 control samples have been evaluated to date. The ability to identify consistent immune subsets across all panels over 6 months (Fig.1C from TruCount Panel) was achieved, and was used to longitudinally track the proportions of cell populations in transplant patients (Fig.1D Panel 1). Conclusion We standardised immune panels and procedures for absolute cell numbers and multiple subsets of immune cells for monitoring a range of individuals including healthy controls, paediatric kidney recipients, T1D, and islet transplant recipients. We have demonstrated that immunophenotyping by flow cytometry is a reliable, consistent, and fast technique that allows the detection of changes in absolute cell numbers of leukocyte subsets from any recipients’ whole blood. This immunophenotyping by flow cytometry is a non-invasive technique which requires less than 1.5ml of whole blood and may be a useful tool for monitoring changes in the immune profile of individuals in a range of clinical trials. Birgit Sawitzki. Mathias Streitz. Figure. No caption available. Table. No title available.

Extra-corporeal normothermic machine perfusion of the porcine kidney: working towards future utilization in Australasia: Normothermic machine perfusion system development

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.