THREE MONTH IMMUNE PROTECTION OF MICROENCAPSULATED PORCINE ISLETS TRANSPLANTED IN PRIMATES WITHOUT IMMUNOSUPPRESSION: PRELIMINARY RESULTS

Abstract

P1072 Aims: Survival of xenogeneic pig pancreatic islets into primates has been casuistically reported. Although the major hurdle for pig islets graft remains the control of primary non-function, acute vascular and cellular rejection, there is no uniform regimen which is able to overcome all immunological problems. The aim of this study was to assess the ability of alginate micro-encapsulation to protect islets in the gold standard discordant pig to primate model. Methods: Pancreases were obtained from adult (>2 years) Landrace pigs. All pig islets isolations were performed with Liberase PI following a static digestion method. Pig islets encapsulation with alginate solution (1% w/v), was carried out by vibrating nozzle and cross-linked with calcium ions (100mM). The mean size of capsules was 600-800μm and contained an average of three pig islets. After microencapsulation, pig islets (15,000 islets equivalent/kg) were transplanted under the capsule of both kidneys of non-diabetic Cynomolgus (n=2) and streptozotocin-induced diabetic primates (n=2). Porcine C-peptide and anti-pig IgM and IgG antibodies were assessed in serum prior and up to 90 days after transplantation by radioimmunoassay and flow cytometry, respectively. As controls, two animals received either non-encapsulated pig islets or empty capsules. Primate kidneys were removed after 30, 60 and 90 days post-transplantation, fixed in Bouin solution and immunostained for insulin. Viability of islets was assessed by Trypan blue and dithyzone staining. Results: The first non-diabetic animal underwent a total pancreatectomy 30 days after graft and serum porcine C-peptide was detected at 0.121ng/ml thereby demonstrating that islets could still produce C-peptide even without immunosuppression. This animal unfortunately died three days after surgery for unknown reasons. The two kidneys of the second primate were removed 60 and 90 days after graft. The alginate capsules were intact and a majority of islets were positive for dithyzone coloration and insulin immunostaining as well as negative by trypan blue. To induce diabetes in the third animal, high dose of STZ (130mg/kg) was used. After encapsulated pig islet graft, the non-fasting blood glucose decreased from 600 to 214 mg/dl day 2 after transplantation and the porcine C-peptide level went from 0 to 3.6 ng/ml. Unfortunately, this animal died on post-graft day 3 due toxic STZ side-effects (liver steatosis and acute tubular necrosis (creatinine 9mg/dl)). In the fourth primate, diabetes was induced with a lower STZ dose (55mg/kg). The kidneys were removed 30 and 60 days after graft. The alginate capsules were intact and insulin and dithyzone positive cells could be evidenced. One additional animal received empty capsules and the kidney removed 146 days after transplantation demonstrated the integrity of the capsules. The animal which received non-encapsulated pig islets demonstrated 10 days after the graft a complete destruction of the cells, a cellular infiltration and major fibrosis. In all these animals, but one which received empty capsules, there was a strong anti-porcine IgM and IgG antibody response thereby demonstrating the xenogeneic sensitization. Conclusions: A calcium-alginate microencapsulation is able to protect, for at least 90 days, pig islets again a strong discordant humoral immune response without any immunosuppression. Functional beta-cells were present after one month in two animals since swine C-peptide could be detected. This method must be confirmed in additional diabetic primates.