Purpose: The use of rodent models for diabetes, particularly with pancreatic islet transplantation, has been prevalent in various preclinical trials. The purpose of this study is to establish a diabetes mellitus (DM) model in Sprague Dawley (SD) rats using alloxan evaluated by assessing alloxan dosage, the induction rate of diabetes, and glucose stability through insulin treatment. Methods: Over the course of 13 experimental rounds, diabetes was induced in 86 SD rats using alloxan at concentrations of 200 mg/kg (16 rats) or 150 mg/kg (70 rats). Various parameters, including diabetes induction rates, average insulin doses, extent of weight loss, and adverse effects such as diabetic ketoacidosis (DKA), were measured. Results: The administration of 200 mg/kg of alloxan in rats resulted in severe diabetes induction, leading to DKA in three individuals, despite daily insulin glargine administration, DKA prevention was unsuccessful. The stability of alloxan decreases over time, especially when refrigeration is compromised during weighing. In the group treated with 150 mg/kg of alloxan, the diabetes induction rate was 83%. The average insulin dose was 2.21 units/kg/day. In contrast, the group treated with 200 mg/kg of alloxan exhibited a diabetes induction rate of 81% with a statistically significant higher average insulin requirement at 7.58 units/kg/day compared to 150 mg/kg of alloxan. Conclusion: Inducing diabetes in rats with 150 mg/kg of alloxan is considered more suitable for creating a diabetes model for xenogeneic islet transplantation compared to using 200 mg/kg of alloxan. This is due to fewer complications related to DKA or hyperglycemia and reduced need for exogenous insulin treatment.

Background: Pancreatic islet transplantation is currently proven as a promising treatment for type 1 diabetes patients with labile glycemic control and severe hypoglycemia unawareness. Upon islet transplantation, revascularization is essential for proper functioning of the transplanted islets. As IL‐6 is important for endothelial cell survival and systemic inflammation related to xenograft, the effect of IL‐6 receptor antagonist, tocilizumab, on revascularization of the transplanted islets was examined in pig to non‐human primate islet xenotransplantation model. Also, the endothelial cell origin in a new vessel of the transplanted pig islets was determined.Methods: Pig islets were isolated from designated pathogen‐free (DPF) SNU miniature pigs and transplanted via portal vein into five streptozotocin‐induced diabetic monkeys. One group (n = 2, basal group) was treated with anti‐thymoglobulin (ATG), anti‐CD40 antibody (2C10R4), sirolimus, and tacrolimus, and the other group was additionally given tocilizumab on top of basal immunosuppression (n = 3, Tocilizumab group). To confirm IL‐6 blocking effect, C‐reactive protein (CRP) levels and serum IL‐6 concentration were measured. Scheduled biopsy of the margin of the posterior segment right lobe inferior of the liver was performed at 3 weeks after transplantation to assess the degree of revascularization of the transplanted islets. Immunohistochemical staining using anti‐insulin, anti‐CD31 antibodies, and lectin IB4 was conducted to find the origin of endothelial cells in the islet graft.Results: CRP significantly increased at 1~2 days after transplantation in Basal group, but not in Tocilizumab group, and higher serum IL‐6 concentration was measured in latter group, showing the biological potency of tocilizumab. In Basal group, well‐developed endothelial cells were observed on the peri‐ and intraislet area, whereas the number of CD31⁺ cells in the intraislet space was significantly reduced in Tocilizumab group. Finally, new endothelial cells in the pig islet graft were positive for CD31, but not for lectin IB4, suggesting that they are originated from the recipient monkey.Conclusions: Our results demonstrated that tocilizumab can delay revascularization of the transplanted islet, although this effect had no significant correlation to the overall islet graft survival. In the pig to NHP islet xenotransplantation model, the endothelial cells from recipient monkey form new blood vessels in and around pig islets.

Porcine corneas may be good substitutes for human corneas in donor shortage. Therefore, we evaluated the efficacy and safety of an anti‐CD40 antibody‐based regimen compared to an anti‐CD20 antibody‐based regimen on the survival of full‐thickness corneas in pig‐to‐rhesus xenotransplantation. Thirteen Chinese rhesuses underwent full‐thickness corneal xenotransplantation. Six were administered anti‐CD40 antibody, and the others were administered anti‐CD20 antibody, basiliximab, and tacrolimus. Graft survival and changes in lymphocyte, donor‐specific and anti‐αGal antibody, and aqueous complement levels were evaluated. Treatment with the anti‐CD40 antibody (>511, >422, >273, >203, >196, 41 days) and anti‐CD20 antibody (>470, 297, >260, >210, >184, 134, >97 days) resulted in long‐term survival of grafts. In the anti‐CD20 group, the number of activated B cells was significantly lower than that in the anti‐CD40 group, and the level of aqueous complements at 6 months was significantly higher than the preoperative level. There were no differences in the levels of T cells or donor‐specific and anti‐αGal antibodies between the two groups. In the anti‐CD20 group, three primates suffered from adverse reactions. In conclusion, both the anti‐CD40 antibody‐ and the anti‐CD20 antibody‐based protocols were effective for the long‐term survival of full‐thickness corneal xenografts, but the anti‐CD40 antibody‐based treatment had fewer adverse effects.

Background: Safety concerns exist for corneal recipients under immunosuppression. We report long‐term safety results of porcine corneal xenotransplantation under immunosuppression in nonhuman primates.Methods: Systemic monitoring data from 49 Chinese rhesus macaques that received pig corneal transplant between 2009 and 2018 were retrospectively reviewed. The recipients were divided into 4 groups depending on the systemic immunosuppressants used: (a) conventional steroid group; costimulation blockade groups ([b] anti‐CD154 antibody, [c] anti‐CD40 antibody); and (d) commercially available immunosuppressants (anti‐CD20 antibody, tacrolimus, basiliximab) group. We compared results of general condition monitoring; hematologic, biochemical, and electrolyte tests; and Rhesus Cytomegalovirus infection monitoring.Results: All recipients recovered from early weight loss. White blood cell counts significantly decreased at 6 months in the steroid and anti‐CD154 groups. Abnormal liver and kidney function and electrolyte imbalance were not observed in all groups. The mean value of Rhesus Cytomegalovirus DNA copies was consistently lower than 200 copies/mL, and antibody titers did not change over time in all groups. Tacrolimus‐associated thrombotic microangiopathy was developed in one case, which resolved after discontinuation of tacrolimus. In 2017, a simian varicella virus outbreak led to clinical signs in 5 that received immunosuppressive therapies, of which 3 died.Conclusion: Costimulatory blockade‐based and anti‐CD20 antibody/tacrolimus‐based immunosuppressive therapies seem to be comparably safe with steroid therapy in nonhuman primates receiving corneal xenotransplantation, as they did not reactivate Rhesus Cytomegalovirus and maintained manageable systemic status. Although reactivation is rare, antiviral prophylaxis for simian varicella virus should be considered in immunocompromised hosts.