Byoung-Hoon Min

Long-term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets.

Pig islets are an alternative source for islet transplantation to treat type 1 diabetes (T1D), but reproducible curative potential in the pig‐to‐nonhuman primate (NHP) model has not been demonstrated. Here, we report that pig islet grafts survived and maintained normoglycemia for >6 months in four of five consecutive immunosuppressed NHPs. Pig islets were isolated from designated pathogen‐free (DPF) miniature pigs and infused intraportally into streptozotocin‐induced diabetic rhesus monkeys under pretreatment with cobra venom factor (CVF), anti‐thymocyte globulin (ATG) induction and maintenance with anti‐CD154 monoclonal antibody and low‐dose sirolimus. Ex vivo expanded autologous regulatory T cells were adoptively transferred in three recipients. Blood glucose levels were promptly normalized in all five monkeys and normoglycemia (90–110 mg/dL) was maintained for >6 months in four cases, the longest currently up to 603 days. Intravenous glucose tolerance tests during the follow‐up period showed excellent glucose disposal capacity and porcine C‐peptide responses. Adoptive transfer of autologous regulatory T cells was likely to be associated with more stable and durable normoglycemia. Importantly, the recipients showed no serious adverse effects. Taken together, our results confirm the clinical feasibility of pig islet transplantation to treat T1D patients without the need for excessive immunosuppressive therapy.

Failure of transplantation tolerance induction by autologous regulatory T cells in the pig-to-non-human primate islet xenotransplantation model.

Islet allotransplantation is a promising way to treat some type 1 diabetic (T1D) patients with frequent hypoglycemic unawareness, and islet xenotransplantation is emerging to overcome the problem of donor organ shortage. Our recent study showing reproducible long‐term survival of porcine islets in non‐human primates (NHPs) allows us to examine whether autologous regulatory T‐cell (Treg) infusion at peri‐transplantation period would induce transplantation tolerance in xenotransplantation setting.

Porcine antigen-specific IFN-γ ELISpot as a potentially valuable tool for monitoring cellular immune responses in pig-to-non-human primate islet xenotransplantation.

Recent progress in xenotransplantation of porcine islets to non‐human primates (NHPs) gives hope for human clinical trials in the near future. Thus, implementation of an appropriate monitoring method to detect the development of detrimental porcine antigen‐specific cellular immune responses is necessary. The enzyme‐linked immunospot (ELISpot) assay has been widely used to monitor antigen‐specific alloreactive T‐cell responses in humans; however, the utility of porcine islet‐specific ELISpot assay has not yet been thoroughly evaluated for pig‐to‐NHPs intraportal islet xenotransplantation.

Novel culture technique involving an histone deacetylase inhibitor reduces the marginal islet mass to correct streptozotocin-induced diabetes.

Islet transplantation is limited by the difficulties in isolating the pancreatic islets from the cadaveric donor and maintaining them in culture. To increase islet viability and function after isolation, here we present a novel culture technique involving an histone deacetylase inhibitor (HDACi) to rejuvenate the isolated islets. Pancreatic islets were isolated from Sprague-Dawley (SD) rats and one group (FIs; freshly isolated islets) was used after overnight culture and the other group (RIs; rejuvenated islet) was subjected to rejuvenation culture procedure, which is composed of three discrete steps including degranulation, chromatin remodeling, and regranulation. FIs and RIs were compared with regard to intracellular insulin content, glucose-stimulated insulin secretion (GSIS) capacity, gene expression profile, viability and apoptosis rate under oxidative stresses, and the engraftment efficacy in the xenogeneic islet transplantation models. RIs have been shown to have 1.9 ± 0.28- and 1.7 ± 0.31-fold greater intracellular insulin content and GSIS capacity, respectively, than FIs. HDACi increased overall histone acetylation levels, with inducing increased expression of many genes including insulin 1, insulin 2, GLUT2, and Ogg1. This enhanced islet capacity resulted in more resistance against oxidative stresses and increase of the engraftment efficacy shown by reduction of twofold marginal mass of islets in xenogeneic transplantation model. In conclusion, a novel rejuvenating culture technique using HDACi as chromatin remodeling agents improved the function and viability of the freshly isolated islets, contributing to the reduction of islet mass for the control of hyperglycemia in islet transplantation.

Pre-clinical results in pig-to-non-human primate islet xenotransplantation using anti-CD40 antibody (2C10R4)-based immunosuppression

Islet transplantation is an effective therapy for selected patients with type 1 diabetes with labile glycemic control and hypoglycemic unawareness, but donor organs are limited. Islet xenotransplantation using porcine islets will potentially solve this problem. Although successful proof of concept studies using clinically inapplicable anti‐CD154 monoclonal antibody (mAb) in pig‐to‐non‐human primate (NHP) islet xenotransplantation has been demonstrated by several groups worldwide, potentially clinically applicable anti‐CD40 (2C10R4) mAb‐based studies have not been reported.

Induction, management, and complications of streptozotocin-induced diabetes mellitus in rhesus monkeys.

Diabetes mellitus (DM) model using streptozotocin (STZ) which induces chemical ablation of β cell in the pancreas has been widely used for various research purposes in non‐human primates. However, STZ has been known to have a variety of adverse effects such as nephrotoxicity, hepatotoxicity, and even mortality. The purpose of this study is to report DM induction by STZ, toxicity associated with STZ and procedure and complication of exogenous insulin treatment for DM management in rhesus monkeys (Macaca mulatta) that are expected to be transplanted with porcine islets within 2 months.

Tacrolimus-induced asymptomatic thrombotic microangiopathy diagnosed by laboratory tests in pig-to-rhesus corneal xenotransplantation: A case report

Tacrolimus‐associated thrombotic microangiopathy (TA‐TMA) is a rare complication. TA‐TMA is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and organ damage due to thrombus. We report asymptomatic TA‐TMA diagnosed by laboratory tests in pig‐to‐rhesus corneal xenotransplantation. Corneal transplantation had been conducted from a wild‐type SNU miniature pig to a rhesus macaque. The veterinary records were retrospectively reviewed in this case. The immunosuppressive regimen consisted of rituximab, basiliximab, and IVIg as inductive therapies, and steroids with tacrolimus (0.1 mg/kg/day) as maintenance therapies. Although there were no clinical symptoms, increased levels of lactate dehydrogenase, total bilirubin, blood urea nitrogen, and creatinine and decreased levels of hemoglobin and platelet were observed in laboratory tests on Day (D) 61. Systemic TA‐TMA was tentatively diagnosed. Tacrolimus was discontinued starting on D71. Dalteparin, clopidogrel bisulfate (D77‐D99), and IVIg (D72) were administered as a conservative treatment. Abnormal laboratory results were reversed on D99. When low‐dose tacrolimus (0.07 mg/kg/day) was re‐administered on D131 to prevent rejection of the graft, TMA was detected again by laboratory tests on D161, confirming the initial diagnosis. Discontinuation of tacrolimus on D162 and re‐administration of Dalteparin (D161‐D196) corrected the laboratory values on D161. This report shows that in pig‐to‐rhesus corneal xenotransplantation, clinically asymptomatic TMA can be induced by tacrolimus, and the discontinuation of tacrolimus and administration of anticoagulant seems sufficient to correct the laboratory TMA.

Gastrostomy tube placement for long-term oral drug administration in non-human primates.

Non‐human primates (NHPs) are often used as recipients in preclinical transplantation research that in most cases involves administration of various drugs including immunosuppressants. Long‐term oral drug administration, particularly tacrolimus, is challenging in the transplant recipient NHPs. Oral drug administration method using the mixture of drug and fruit juice has been used in NHPs, but this is not always effective in all monkeys. To those monkeys who are poorly compliant, oral drug administration in restraint or administration using gastrostomy tube should be necessary. The aim of this study was to compare the efficacy of between oral drug administration in restraint and administration using gastrostomy tube and to report complications and solutions to overcome the problems related to gastrostomy tube for long‐term oral drug dosing in rhesus monkeys.

A novel method for murine intrahepatic islet transplantation via cecal vein.

Islet transplantation is one of the most beneficial treatment modality to treat type 1 diabetic patients with frequent hypoglycemic unawareness. In clinical setting, human islets are infused via portal vein and are settled in the end-portal venules in the liver. However, mouse islets are transplanted into kidney subcapsule or liver through direct portal vein. These conventional transplantation methods have several drawbacks such as different physiological environments around the transplanted islets in kidney subcapsule from the liver and high mortality rate in direct portal vein approach. In this study, we introduced murine intrahepatic islet transplantation method via cecal vein to have the same surgical operation route in humans as well as guaranteeing low mortality rate after islet transplantation. With this protocol, consistent normoglycemia can be obtained in diabetic mice, while keeping operation-related mortality extremely low. This approach with easier accessibility and low mortality will make murine intrahepatic islet transplantation a useful model for studying immunological mechanisms such as strong innate and adaptive immune responses that occur in human islet transplantation.

Ascites formation accompanied by portal vein thrombosis after porcine islet xenotransplantation via the portal vein in Rhesus macaque (Macaca mulatta)

Pig‐to‐nonhuman primate (NHP) islet transplantation has been widely conducted as a preclinical xenotransplantation model prior to human clinical trial. Portal vein thrombosis is one of the complications associated with islet infusion through the portal vein into the liver. Here, we briefly report severe case of ascites formation accompanied by portal vein thrombi after pig‐to‐NHP islet xenotransplantation in a rhesus monkey. Meticulous prophylactic treatment such as continuous heparin infusion should be implemented to prevent portal vein thrombi in pig‐to‐NHP islet transplantation models.