Janet R. Serie

Nonenzymic in vitro isolation of perinatal islets of Langerhans

SummaryWe 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 μg 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° 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.

Islet Transplantation in Spontaneously Diabetic BB/Wor Rats

We investigated the effectiveness of islet transplantation as therapy in an animal model of spontaneous type I (insulin-dependent) diabetes mellitus. Grafting MHC-matched and -mismatched islets with the spontaneously diabetic BB rat as a model has been previously reported to result in recurrence of the disease in the grafted tissue. When transplanted with nonimmunogenic islets isolated by nonenzymatic culture, we found that MHC-matched grafts proved to be susceptible to disease recurrence when allowed to remain in situ until ketosis developed in the host. Conversely, the MHC-mismatched grafts did not succumb to the disease process despite the destruction of the β-cell population of the endogenous pancreas. Four manifestly hyperglycemie BB/Wor rats received sufficient islet mass by allotransplantation to reverse this state. All four animals had ameliorated conditions, and three of the four were restored to a normoglycemic state. Recurrence of diabetes in the BB rat was not observed.

Comparative Analysis of Potency of Splenic Dendritic and Adherent Cells (Macrophages) as Alloantigen Presenters In Vivo

Dendritic cells and macrophages have been attributed with stimulatory capacity for in vivo and in vitro immune responses. However, the relative contribution of each of these cell types has long been in dispute. Therefore, the differential ability of dendritic cells and macrophages (splenic adherent cells [SACs]) to stimulate pancreatic islet allograft rejection in reversed alloxan-induced diabetic rats was examined. Rats bearing established allografts were challenged with various dosages of donor-strain dendritic cells or SACs, and graft rejection was assessed by analysis of plasma glucose levels and/or histological criteria. Marked differences in the ability to stimulate allograft rejection were observed at the 105-cell dosage; 105 dendritic cells induced graft rejection in five of six rats (1 rat required 2 injections), whereas 105 SACs failed to induce rejection in four of four rats (P < 0.10, χ2 test). Challenge stimuli consisting of ≤ 105 SACs or ≤104 dendritic cells failed to induce graft rejection. These findings indicate that dendritic cells are potent stimulator cells for in vivo immune responses. Previous studies indicated that as few as 103 dendritic cells initiate allograft rejection in nondiabetic recipients. That more dendritic cells were required to stimulate rejection in reversed diabetic recipients compared with nondiabetic recipients suggests that other factors, such as the diabetic state and the production of a tolerant status achieved by larger amounts of grafted tissue, may influence graft survival.

Growth of Neonatal Islet Transplants in the Spontaneously Diabetic BB/Wor Rat

We have previously shown that culture-isolated neonatal islets are able to survive both rejection and the recurrence of autoimmunity in the spontaneously diabetic BB/Wor rat. In trials designed to demonstrate the MHC restriction of the autoimmune response in this model, we discovered that neonatal islet graftsfrom diabetic BB rats appeared larger than grafts from nondiabetic controls. This study was undertaken to quantify the mass difference seen in this original study and to determine the characteristics of graft growth in more highly controlled trials. Grafts from diabetic animals in the original study were significantly larger than those from nondiabetic animals (81 ± 36 vs. 238 ± 216 μg, P = 0.01). These findings were supported by results from a second series of experiments, in which the mean growth index of grafts from diabetic animals was 7.25 ± 4.91, whereas that from nondiabetic animals was 2.5 ± 1.15 (P = 0.011). Three animals in this study were reversed of hyperglycemia: two had normal and one had a subdiabetic ip GTTs. These three rats received 97, 317, and 408 ng of islet tissue that increased in mass to 1790, 3270, and 4107 fig, respectively. Nuclear/total cell area percentages were the same in diabetic and nondiabetic grafts (P = 0.76), suggesting that the increase in mass was attributable primarily to proliferation rather than hypertrophy. Limited studies that use BrDU incorporation support this conclusion. High glucose levels have been shown to stimulate β-cell replication in fetal, neonatal, and adult islets and may be the stimulus for enhanced graft growth in this model. This study shows that small, immunomodulated neonatal grafts placed in the diabetic environment of the spontaneously diabetic BB/Wor rat can increase in mass >10-fold and can ameliorate the symptoms of diabetes.

The successful allotransplantation of neonatal rat islets across multiple combined major and minor histocompatibility barriers

Cultured neonatal rat islets were transplanted across six strain combinations into nonimmunosuppressed allogeneic recipients. Islets were isolated nonenzymatically by an in vitro method and were cultured at 37°C in 5% CO2 in air for 10 days prior to transplant. Transplants to nondiabetic recipients across four allogeneic barriers resulted in morphologically intact and wellgranulated islet tissue present at the graft site in 54 of 55 cases for periods lasting as long as 445 days (mean day of sacrifice was 163). In trials using diabetic recipients, ACIs receiving WF islets (n = 3) and outbred Holtzmans receiving Holtzman islets (n = 3) were reversed and did not return to the hyperglycemic state for experimental periods of up to 430 days.

Long-term survival and strain-specific tolerance induction in rat-to-mouse neonatal islet xenografts

These studies were designed to determine (1) if culture-isolated, neonatal rat islets are capable of inducing xenogeneic tolerance in mice and (2) whether this tolerance is species- or strain-specific. We attempted to induce xenogeneic tolerance by transplanting culture-isolated neonatal FSH islets to 26 diabetic C57B1/6 recipients. These animals received one injection of ALS at the time of transplant. Fifteen (58%) animals remained reversed by xenotransplant for > 173 days. To assess the development of strain or species-specific tolerance, 14 of the animals bearing long-term surviving FSH grafts were divided into 3 treatment groups. Animals in group 1 were nephrectomized to remove the initial graft and then retransplanted with uncultured, adult FSH islets; animals in group 2 were retransplanted with uncultured, adult FSH islets without nephrectomy; and group 3 animals were nephrectomized and retransplanted with uncultured, adult third-party islets (WF). In naive controls, adult FSH islets were rejected in 9 +/- 2 days. The MST for adult FSH grafts transplanted to nephrectomized recipients was 104 +/- 54 days, with 4 out of 5 (80%) surviving until sacrifice 90-171 days posttransplant. The MST for FSH grafts transplanted to nonnephrectomized recipients was 120 +/- 70 days with 3 out of 4 (75%) surviving until sacrifice 143-154 days posttransplant. Thus, it appears that the initial neonatal FSH transplant induced the development of immune tolerance to highly immunogenic FSH islet tissue. In contrast, the MST for third-party adult WF grafts was 27 +/- 13 days compared with an MST of 36 +/- 24 days in naive controls. Thus, it appears that the xenogeneic tolerance induced by neonatal FSH islets was strain rather than species-specific. Factors such as the close evolutionary relationship between rats and mice, the neonatal condition of the initial graft, and its relative lack of donor APCs are included in a discussion of possible mechanisms of tolerance induction.