Modification of allograft immunogenicity in perinatal islets isolated and purified in vitro.

Perinatal rat islets of Langerhans, isolated and cultured in vitro, were examined following long-term allotransplantation across a major histocompatibility barrier in nonimmunosuppressed recipients. Islets were isolated to varying degrees of purity without the use of collagenase digestion. Newborn bovine serum was a component of the incubation medium and the atmosphere during culture was air: 5% CO2. Islets transplanted without rigorous purification were fully rejected by 14 days posttransplantation. However, if islets were maintained in subculture, permitting their subsequent meticulous purification, no evidence of rejection was observed after 45 days at the kidney subcapsular site. Grafts consisted of morphologically intact islets. The three major endocrine cell types of the islet were identified by immunocytochemical localization of insulin, glucagon, and somatostatin. These results demonstrate that perinatal islets can exhibit altered immunogenicity, as evidenced by prolonged allograft survival, when isolated and purified by the nonenzymic in vitro method.

Spermidine cytotoxicity in vitro: effect of serum and oxygen tension.

Plasma amine oxidase activities (benzylamine oxidase and spermine oxidase) were determined in the sera of a number of species of various ages. Benzylamine oxidase (BZO) activity, measured spectrophotometrically, was present in bovine, equine, and ovine species examined. Generally its activity in serum increased with the age of the animal. Spermine oxidase activity (SPO) was estimated by a bioassay of in vitro toxicity and did not necessarily correlate with BZO. Cytotoxicity in the presence of spermidine was found only in the sera of the ruminant species examined. Serum activity tended to rise with animal age; however, great variability was found in perinatal bovine sera. The 50% lethal dose (LD50) of spermidine in the presence of 5% serum and 4 X 10(4) NS1 cells/ml was in the micromolar range. Aminoguanidine, a known inhibitor of SPO, could prevent the cytotoxic effects of exogenously added spermidine in vitro. In contrast, raising the ambient oxygen tension in the incubation environment to 95% lowered the LD50 dose of spermidine required for cytotoxicity. The results suggest that a cell line of hematogenous origin is susceptible to the cytotoxic effects of the products of oxidative deamination of spermidine by SPO, an enzyme present in perinatal bovine sera, and that these cytotoxic effects are potentiated in the presence of an oxygen-enriched environment in vitro.

Nonenzymic in vitro isolation of perinatal islets of Langerhans.

We have developed a method to circumvent the use of exogenous proteolytic enzymes in the isolation of islets of Langerhans from the perinatal rodent pancreas. Advantage is taken of the propensity of fibroblastlike cells to attach and migrate on polystyrene at low-serum concentrations (5%). In contrast, at this serum level, rat islet epithelial cells tend not to adhere to the substrate. At 3 d of culture, islets are visible at the edges of the explants. With further fibroblast outgrowth the majority of islets are freefloating by 7 d. Simple agitation of the medium and centrifugation yields approximately 50 micrograms of islet tissue per perinatal pancreas. Further purification of the islets can be obtained by subculture. Rat islets can be maintained in this manner for several months in Medium F12 supplemented with 25% horse serum in an atmosphere of 5% CO2 and air at 37 degrees C. Hormone content of the islet tissue remains constant during prolonged subculture and such islets continue to exhibit appropriate insulin and glucagon responses to glucose and theophylline. The morphological integrity of the endocrine cells within the cultured islets was confirmed by immunocytochemistry and ultrastructural study. Nonendocrine cells are not identifiable within the long-term cultured islets.

Prolongation of culture-isolated neonatal islet xenografts without immunosuppression.

Xenogeneic transplantation of 300-400 neonatal rat islets (Fischer 344) to the kidney subcapsular site of streptozotocin-induced nonimmunosuppressed diabetic adult mice (C57BL/6ByJ) resulted in a return to normoglycemia in 87% of the recipients. Of the 13 successfully reversed recipients, 5 exhibited graft rejection (hyperglycemia of +250 mg/dl) within 2 weeks posttransplantation, and 2 mice had rejected their rat islets by 3 weeks. The 6 remaining recipients exhibited significantly prolonged survival of the cultured islets: 1 remained reversed until 4 weeks posttransplantation, 2 remained normoglycemic for 5 weeks, in 3 diabetes remained reversed for more than 7 weeks--in one of these animals the disease was reversed for 17 weeks. Transplanted islets were isolated from neonatal rat pancreas during a period in culture that varied from 8 to 17 days. Although morphological integrity of the endocrine cells was confirmed by ultrastructural study, nonendocrine cells were not identifiable within the islets after 8 days of culture. Xenografted islets examined morphologically prior to obtaining physiological evidence of rejection were associated with extensive peripheral lymphocytic accumulation. Modification of islet immunogenicity leading to prolonged xenograft survival may reflect the degree to which the in vitro environment permits the differential survival of endocrine cells while purging the islet of cells initiating the immune response.