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.

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.

Successful Islet Outcomes Using Australia-Wide Donors: A National Centre Experience.

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.

Culture of Transplant Perfusate Using BACTEC Technology and Antibiotic Prophylaxis Influences Wound Complications Within a Kidney Transplant and SPK Transplant Cohort.

PURPOSE: Routine screening for microbial contamination in organ recovery perfusion transport solution (ORPTS) is by microbiological culture without broth enrichment. Our aim was to examine the clinical utility of broth enrichment of perfusion solution, through use of BACTEC (Becton Dickinson) blood culture media, in preventing wound complications for transplant recipients in comparison with culture without enrichment. METHODS: We prospectively collected samples of ORPTS of 395 kidney (n = 250) or simultaneous pancreas-kidney (SPK, n = 145) donors over a 7-year period. Results of culture with and without broth enrichment (n = 285) using BACTEC blood culture media were examined to compare the sensitivity of BACTEC with non-BACTEC methods. We then conducted a paired analysis of 110 recipients with both BACTEC and non-BACTEC culture organ perfusion media. We examined the rates of wound infection and whether the use of targeted antimicrobials reduced infections in the BACTEC group and recipients with both types of cultures. RESULTS: Of 395 patients with cultures of ORPTS, first, the results of 79 cultures performed using BACTEC media only were compared with 206 non-BACTEC cultures (n = 285). Second, 110 cultures were performed using both methods. For the first part of the study, BACTEC media detected significantly greater microbial growth than non-BACTEC methods (n = 79, 64.6% vs n = 206, 14.6%; P < .001). In the 110 patients with both BACTEC (52.3%) and non-BACTEC cultures (9.9%), there was significantly higher sensitivity of the BACTEC method (P < .001); 68.2% of these patients had antimicrobial cover in the days immediately following transplant sufficient to cover the cultured organism. In the patients with appropriate antimicrobial cover, the rate of recipient wound infection was significantly reduced (P = .003). CONCLUSIONS: Routine screening of ORPTS with BACTEC broth enrichment should always be employed. When paired with antimicrobial prophylaxis, it has the potential to significantly reduce the risk of recipient wound infection.