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The usefulness of interfacial photopolymerization of poly(ethylene glycol) (PEG) diacrylate at a variety of concentrations and molecular weights to form hydrogel membranes for encapsulating porcine islets of Langerhans was investigated. The results from this study show in vitro and in vivo function of PEG-encapsulated porcine islets and the ability of PEG membranes to prevent immune rejection in a discordant xenograft model. Encapsulated islets demonstrated an average viability of 85% during the first week after encapsulation, slightly but significantly lower than unencapsulated controls. Encapsulated porcine islets were shown to be glucose responsive using static glucose stimulation and perifusion assays. Higher rates of insulin release were observed for porcine islets encapsulated in lower concentrations of PEG diacrylate (10-13%), not significantly reduced relative to unencapsulated controls, than were observed in islets encapsulated in higher concentrations (25%) of PEG diacrylate. Perifusion results showed biphasic insulin release from encapsulated islets in response to glucose stimulation. Streptozotocin-induced diabetic athymic mice maintained normoglycemia for up to 110 days after the implantation of 5,000-8,000 encapsulated porcine islet equivalents into the peritoneal cavity. Normoglycemia was also confirmed in these animals using glucose tolerance tests. PEG diacrylate-encapsulated porcine islets were shown to be viable and contain insulin after 30 days in the peritoneal cavity of Sprague-Dawley rats, a discordant xenograft model. From these studies, we conclude that PEG diacrylate encapsulation of porcine islets by interfacial photopolymerization shows promise for use as a method of xenoprotection toward a bioartificial endocrine pancreas.

A method has been defined to interfacially photopolymerize poly(ethylene glycol) diacrylates (PEG diacrylates) to form a crosslinked hydrogel membrane upon the surfaces of porcine islets of Langerhans to serve as an immune barrier for allo- and xenotransplantation. A sensitivity study of six key parameters in the interfacial photopolymerization process was performed to aid in determination of the optimal encapsulation conditions, leading to the most uniform hydrogel membranes and viable islets. The key parameters included the concentrations of the components of the initiation scheme, namely eosin Y, triethanolamine, and 1-vinyl 2-pyrrolidinone. Other parameters investigated included the duration and flux of laser irradiation and the PEG diacrylate molecular weight. Each parameter was doubled and halved from the standard conditions used in the encapsulation process while holding all the remaining parameters at the standard conditions. The effects of changing each parameter on islet viability, encapsulation efficiency, and gel thickness were quantified. Islet viability was sensitive to the duration of laser illumination, viability significantly increasing as the duration was reduced. Encapsulation efficiency was sensitive to the concentrations of eosin Y, triethanolamine, and 1-vinyl 2-pyrrolidinone, to the laser flux, and to the PEG diacrylate molecular weight. Increasing the concentration of eosin Y significantly improved the encapsulation efficiency, while decreasing the concentration of 1-vinyl 2-pyrrolidinone and increasing the concentration of triethanolamine had the greatest effects in significantly reducing the encapsulation efficiency. Gel thickness was sensitive to the concentrations of triethanolamine and 1-vinyl 2-pyrrolidinone, to the duration of laser illumination, and to the PEG diacrylate molecular weight. Increasing the PEG diacrylate molecular weight significantly increased the gel thickness, while decreasing the concentration of 1-vinyl 2-pyrrolidinone and increasing the concentration of triethanolamine had the greatest effects in significantly reducing the gel thickness. From this sensitivity study, conditions were determined to encapsulate porcine islets, resulting in greater than 90% islet viability and greater than 90% encapsulation efficiency., (Copyright 1998 John Wiley & Sons, Inc.)

Functional porcine islets, free of known pathogens, can serve as a source of insulin producing cells for the treatment of experimentally induced insulin dependent Diabetes Mellitus. Porcine islets can be conformally coated (microencapsulated) with a covalently linked, stable permselective membrane while maintaining islet viability and function. The PEG conformal coating is immunoprotective in a discordant xenograft animal model (porcine islets to rat).

A number of pancreatic beta-tumor cell (beta TC) lines have been derived from insulinomas arising in transgenic mice expressing the SV40 T antigen gene under control of the insulin promoter. Some of these lines secrete insulin in response to physiological glucose concentrations. However, this phenotype is unstable. After propagation in culture, these nonclonal lines become responsive to subphysiological glucose levels and/or manifest reduced insulin release. Here we report the use of soft-agar cloning to isolate single-cell clones from a beta TC line, which give rise to sublines that maintain correct glucose responsiveness and high insulin production and secretion for > 55 passages (over a year) in culture. One of these clonal lines, denoted beta TC6-F7, was characterized in detail. beta TC6-F7 cells expressed high glucokinase and low hexokinase activity, similarly to normal islets. In addition, they expressed mRNA for the GLUT2 glucose transporter isotype and no detectable GLUT1 mRNA, as is characteristic of normal beta-cells. These results demonstrate that transformed beta-cells can maintain a highly differentiated phenotype during prolonged propagation in culture, which has implications for the development of continuous beta-cell lines for transplantation therapy of diabetes.

Human islets were macroencapsulated in permselective hollow fiber membrane devices and successfully allotransplanted subcutaneously with > 90% viability after 2 weeks in situ. Recipients were patients with type I or type II diabetes and normal control subjects; none was immunosuppressed. Between 150 and 200 islet equivalents were implanted in each of the nine patients. No adverse patient complications were observed. Biocompatibility of devices was excellent. Insulin-positive beta-cells were confirmed in encapsulated islets recovered from the implanted devices in all patient populations including the type I diabetic patients. Glucose-stimulated insulin release could be demonstrated in vitro from recovered islets. These data demonstrate that macroencapsulated human islets can survive at the subcutaneous site and that permselective membranes can be designed to protect against both allogeneic immune responses as well as the autoimmune component of type I diabetes.

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.

Dendritic cells (DCs) are potent antigen-presenting cells for a variety of immune responses; however, their mechanism of action has not been established. It is known that DCs can cluster with one another and with other cell types during in vitro immune responses, and clustering may be essential for the activation of resting lymphocytes. In this study, ultrastructural examination of clusters that form during extended culture of enriched rat splenic DCs (approximately 70% DCs) is reported. DCs were readily distinguished from other cell types, which included lymphocytes and macrophages. DCs displayed characteristic veils and/or dendritic processes that intertwined with processes of other cells within the cluster, or extended from the cluster periphery. Occasional DCs contained large vacuoles lined with small vesicles. A paramount feature of DCs is their constitutive expression of high levels of surface major histocompatibility complex class II antigens. The surface distribution of class II antigens on clustering DCs was examined using 10 nm immunogold labeling techniques and high-resolution scanning electron microscopy. DCs were readily distinguished by morphologic criteria, and examination of various surface membrane regions revealed a differential distribution of class II antigens. Gold label was frequently distributed in linear arrays and clusters, suggesting a cytoskeletal role in the recycling/redistribution of Class II antigens. These morphologic findings further an understanding of basic DC biology and their mechanism of action as antigen-presenting cells.

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 grafts from 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 micrograms, 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 micrograms of islet tissue that increased in mass to 1790, 3270, and 4107 micrograms, 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal rat islets derived by nonenzymic (in vitro) isolation procedures from the Fischer-344 (F-344, Rt1lv1) strain have been shown to be freely transplantable across complete MHC barriers without the use of any form of immunosuppression. However, islets obtained by in vitro isolation from some donor strains, such as the ACI, retain a degree of immunogenicity upon transplantation. This study examined the immunogenic nature of these neonatal ACI islets, and correlated this with the presence of a residual population of MHC class II-positive antigen presenting cells within the islets. In addition, further reduction of immunogenicity of isolated neonatal ACI islets by extension of the culture period was examined. Islets were obtained from neonatal ACI donors by culture-isolation and placed in secondary culture for either 2 days (10 day total culture period, standard culture) or 42 days (50 day total culture period, extended culture). Standard-culture ACI islets were rejected 33% to 100% of the time in five different recipient strains (depending on recipient strain). Compared with control grafts of fresh pancreatic fragments, rejection rates were significantly lower in two strain combinations (ACI to BUF, P = 0.044 and ACI to F-344, P = 0.028), and for all recipient strains combined (P less than 0.001). Extended-culture ACI islets survived significantly longer in all five recipient strains tested (ACI to BN, P = 0.024; ACI to BUF, P = 0.005; ACI to LEW, P = 0.004; ACI to F-344, P = 0.008 and ACI to WF, P = 0.024) and for all strains combined (P less than 0.001). No MHC class II-positive cells were detected upon examination of sectioned neonatal ACI islets using peroxidase immunocytochemistry (OX-6 antigen) in either the standard-culture or extended-culture groups. These results suggest that the increased survival of grafts of extended culture ACI islets is due to a mechanism other than the elimination of class II-positive APCs from the islets. Although possible down-regulation of MHC class II expression on residual APCs during the culture process may make identification of these cells difficult, alternative mechanisms such as the down-regulation of MHC class I antigen, resulting in the reduction of available donor target, or the existence of an accessory cell that is not constitutively MHC class II-positive, may be responsible for this increased graft survival.

The goal of islet transplantation in human diabetes is to maintain the islet grafts in the recipients without the use of immunosuppression. One approach is to encapsulate the donor islets in permselective membranes. Hollow fibers fabricated from an acrylic copolymer were used to encapsulate small numbers of rat islets that were immobilized in an alginate hydrogel for transplantation in diabetic mice. The fibers were biocompatible, prevented rejection, and maintained normoglycemia when transplanted intraperitoneally; hyperglycemia returned when the fibers were removed at 60 days. Normoglycemia was also maintained by subcutaneous implants that had an appropriately constructed outer surface on the fibers.

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 10(5)-cell dosage; 10(5) dendritic cells induced graft rejection in five of six rats (1 rat required 2 injections), whereas 10(5) SACs failed to induce rejection in four of four rats (P less than 0.10, chi 2 test). Challenge stimuli consisting of less than or equal to 10(5) SACs or less than or equal to 10(4) 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 10(3) 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.

Culture-derived neonatal Fischer-344 (Rtllvl) rat islets have reduced immunogenicity and have been shown to be fully transplantable into Wistar Furth (Rtlu) recipients. In studies designed to test the MHC-restriction of the autoimmune disease process in the BB/Wor rat, MHC-matched (Wistar Furth) and MHC-mismatched (Fischer-344) neonatal islets were isolated by a nonenzymatic tissue culture procedure and transplanted to the renal subcapsular site of BB/Wor rats before the onset of disease. All MHC-mismatched and most MHC-matched grafts survived intact. In this study we report the growth of MHC-matched and mismatched islet tissue at the graft site. Grafts in diabetes resistant animals averaged 60 +/- 26 micrograms, a comparable mass to that which was transplanted. Grafts in diabetic animals were significantly increased in size, averaging 176 +/- 156 micrograms (P = 0.01). Analysis of nuclear to cytoplasmic volume ratios indicates that the increase in mass was primarily due to islet hyperplasia rather than hypertrophy. Factors contributing to the growth of islet tissue in situ are discussed.

Neonatal Fischer-344 (FSH) islets isolated by a nonenzymatic method have been shown to survive indefinitely in Wistar/Furth (WF) recipients. We have applied this islet isolation method to six different donor strains and transplanted the resulting islets across 20 different strain combinations. We report the variable results obtained, with FSH being the most consistently successful donor strain and ACI being the best recipient strain. Based on the complete success of culture-derived FSH (Rt1lv1) and BN (Rt1n) transplants to WF (Rt1u) recipients, we used this system to test the MHC-restriction of autoimmune beta cell destruction in the BB/Wor rat (Rt1u). Culture-derived FSH and/or WF islets were transplanted to pre-diabetic BB/Wor recipients. Animals were sacrificed at the onset of disease or up to 33 days after disease onset. No immune response developed in FSH and WF grafts in non-diabetic animals. In diabetic animals, all FSH grafts and 12/14 WF grafts survived intact, although some grafts exhibited mild-moderate infiltration which was more pronounced in MHC-matched grafts. In two animals which developed severe hyperglycemia and ketosis, the FSH grafts were found to be intact while the WF grafts were destroyed by disease recurrence. In addition, FSH and BN islets were transplanted to diabetic BB/Wor recipients. Three of four FSH islet recipients and two of four BN islet recipients were reversed of their disease. Neither these nor the non-reversed animals showed signs of disease recurrence in the MHC-mismatched grafts. Therefore, in these studies, MHC-mismatched cultured grafts survived well and were capable of reversing the diabetic syndrome in spontaneously diabetic BB/Wor recipients.

The effects of hypersecretion of growth hormone and prolactin on islet endocrine cells have been studied by radioimmunoassays, immunocytochemistry, and morphometry in randomized samples of pancreata from MtTW15 mammosomatotropic tumor-bearing and control rats. The randomized sampling procedure, validated by immunoassays, allowed evaluation of both hormone content (immunoassay) and endocrine cell population (immunocytochemistry) on samples derived from the same origin. Hyperinsulinemia (2x) and non-fasting hypoglycemia in 10-wk-tumor rats were normalized 3 wk after tumor removal. Pancreatic weight was doubled, but proportional to body weight increases. Islet/pancreas ratio was constant (1.29 +/- 0.05%) and the same in tumor, tumor-removed, and control animals, but average islet dimensions were increased by 30% and average area doubled in tumor animals. Frequency analysis showed fewer small (less than 70 micrometers) and more large (greater than 140 micrometers) islets in tumor animals, but no change in average islet shape shown by average axis ratios of 1.4 in all groups. Pancreatic content of insulin and glucagon was doubled, while that of somatostatin was constant. These changes were not completely reversed in tumor-removed animals. Similarly, a significant doubling in islet-derived mass was mainly due to a doubling of the B-cell mass as the average proportion of endocrine cells per islet shifted from 66%, 26%, and 18% to 81%, 18%, and 3% for B-, A-, and D-cells of control and tumor-bearing rats, respectively. Immunocytochemically detectable insulin was found in duct cells of tumor animals, but not controls. Whether such cells represent a functional reserve remains to be determined.

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.

Long term reversal of alloxan diabetes has been accomplished by intraperitoneal isotransplantation of enzymatically dispersed neonatal pancreas. In contrast, allotransplanted recipients showed only a transient recovery from the alloxan diabetes followed by a return to the diabetic state at the time of the homograft rejection. These data strongly suggest that the reversal of the diabetic state was a consequence of the transplanted islets. This conclusion is further supported by quantitative analysis of biopsied pancreases from successfully reversed recipients which reveals only 3% of the normal beta cell mass. By comparison, recovery of transplanted islets composed primarily of aldehyde fuchsin positive beta cells was routinely accomplished in these recipients. Utilization of the more specific unlabeled immunoperoxidase method has revealed that some of the transplanted islets are composed of cells positive for glucagon and somatostatin, as well as insulin. Other recovered transplanted islets (generally smaller in size) are composed primarily of one cell type or the other. The presence of insulin, glucagon, somatostatin, and delete pancreatic polypeptide positive cells in the islets of normal rat pancreas has been confirmed. In addition, cells reacting positively for these hormones have been observed in the alloxan diabetic rat pancreatic islets and in islets from reversed recipients. The time required for the disappearance of glycosuria and hyperglycemia (usually occurring from one to eleven weeks posttransplantation) appeared to be related to the amount and age of the donor islet tissue transplanted. Fetal islet tissue was more effective on a per milligram basis in reversing the diabetic state. In addition, while reversal was obtained by transplantation of as little as 5 mg of neonatal islet tissue, relatively large amounts (20 mg) were required before successfully reversed recipients responded normally to glucose tolerance test. By comparison, a similar reversal of diabetes with normal response to glucose load was attained by transplanting only 3 mg of fetal islet tissue. Quantitative morphological evidence of large increases in absolute islet mass, obtained in fetal transplants at the renal subcapsular site suggests that the superiority of fetal islet donor tissue may by in its high growth potential. No adverse effects of an in vitro organ culture period, prior to transplantation, were observed with regard to the ability of neonatal tissue to reverse the diabetic state or for fetal islet tissue to continue to survive at the renal subcapsular site. Likewise, no advantage in regard to amelioration of the homograft rejection response was observed in cultured islet tissue; allotransplants of which were rejected at the kidney site.

Proislets, derived from fetal mouse pancreata by collagenase digestion and subsequent organ culture, can be frozen to -196 degrees C and stored in a viable condition before successful syngeneic transplantation. Cryopreserved proislets are relatively undifferentiated morphologically, but continue to differentiate into mature islets after transplantation.

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.

When highly purified neonatal rat islet tissue, derived after 10 days in vitro, was allografted, it was found to be nonimmunogenic or weakly immunogenic. In contrast, nonislet pancreatic components, derived from the same culture system, transplanted with highly purified islet tissue resulted in rejection in 88% of cases. Extension of the culture period did not result in reduced immunogenicity of the nonislet material. Immunostaining of islet or nonislet tissue from the culture system failed to demonstrate major histocompatibility complex (MHC) class II positive cells in the islet tissue, whereas the presence of MHC class II staining cells in the nonislet components was clearly demonstrable. These results demonstrate that the islet tissue obtained by culture isolation is free of cells capable of stimulating an allogeneic immune response and are consistent with the hypothesis that the absence of MHC class II positive antigen-presenting cells reduces the immunogenicity of the tissue and enhances the survival of allogeneic grafts.

Purified epithelial-cell monolayers were generated in vitro from explants of fetal rat pancreas. The extent of the development of the epithelial monolayer, as determined by planimetric analysis, was enhanced by the application of two methodological procedures: (a) preincubation of fetal pancreas in situ at 27 degrees C for 5 hr prior to dissection and explantation; and (b) incubation of the explants in medium containing a high concentration (50% to 70%) of fetal bovine serum. By utilizing such culture conditions, sheets of contiguous epithelial cells, with little or no peripheral fibroblastic contamination, were maintained for 9 days. Whereas the majority of cells within the monolayer had morphological characteristics of pancreatic ductal cells, endocrine cells were identified by the specific immunocytochemical localization of insulin and glucagon. In addition, insulin could be detected in the incubation medium throughout the course of experiment. The simplicity of this preparation offers some advantages over other techniques including reduced chance of contamination and reduced cellular damage or death. It provides a model for future studies directed toward developing individual cell strains derived from pancreatic epithelial cells.

Foetal rat pancreas (20-day postcoitum) was grown in organ culture using a natural media (serum and chick embryo extract). The media was supplemented with several adrenal and gonadal steroids; precursors, glucocorticoids, mineralocorticoids, estrogens, androgens and progesterone. The effects of the steroids on the pancreatic explants were quantitated biochemically by analysis of amylase and insulin in the incubated culture media and in the explanted pancreatic tissue, and morphologically by quantitative morphometric analysis. corticosterone, hydrocortisone, aldosterone and dexamethasone fully preserved the acinar cell component. The effect was concentration-dependent. Deoxycorticosterone and cortexolone had intermediate effects even at the highest concentration. Gonadal steroids had no effect on the acinar or islet component in this culture system. Some of the steroids inhibited the selective islet growth seen in the control explants as well as inhibiting insulin secretion. The relationship between these data and other work in this area is summarised. In addition, the possible implications of these data relating to normal in vivo pancreatic development are discussed.

The purpose of this study was to determine the in vitro effect of ovine PRL (oPRL) on the dynamics of insulin secretion and dye coupling among islet B cells. The effect of oPRL (2 micrograms/ml) on insulin secretion was time dependent and reached a maximum on day 4 when there was a 2.4-fold increase in insulin secretion from cultured neonatal rat islets (n = 6, P less than 0.001). When islets cultured in the presence of oPRL for 4 days were perifused, 300 mg/dl glucose stimulation resulted in insulin release of 131 +/- 20 microU/ml.100 micrograms islet tissue as compared to control islets 94 +/- 20 microU/ml.100 micrograms islet tissue (n = 7, P less than 0.02). Stimulation of the islets with a linear 30-250 mg/dl glucose gradient resulted in a threshold for glucose-stimulated insulin secretion of 73 +/- 6 mg/dl glucose for the oPRL treated islets (n = 7) as compared to a threshold of 123 +/- 6 mg/dl glucose for control islets (n = 7, P less than 0.001). Mean islet volume was unchanged after 4 days of oPRL treatment but was 34% greater after 8 days (n = 6, P less than 0.001). Dye coupling among central islet B cells was also increased after in vitro treatment with oPRL for 4 days. The mean projected area of dye spread was 2-fold greater in the oPRL treated islets (n = 33) in comparison to the control islets (n = 33, P less than 0.05). These results indicate that in vitro lactogen treatment, in the form of oPRL, alters insulin secretory behavior and B cell junctional communication and supports our hypothesis that lactogen, insulin secretion, and junctional communication among B cells are related.

A method of perfusion organ culture is described in which explants cultured at the air-medium interface are bathed by a continuous flow of nutrient medium. Morphological studies on the fetal rat lung indicate that explant development in this system is comparable to that obtained using standard organ-culture dishes. Medium supply is easily manipulated and continuous sampling of the effluent stream is possible without disturbing the immediate explant environment. The basic design facilitates secretory-response studies on cultured organ explants as demonstrated by a study of glucose-stimulated insulin release by the neonatal rat pancreas.

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 beta-cell population of the endogenous pancreas. Four manifestly hyperglycemic 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.

Eight 18-days-postcoitum fetal pancreases were transplanted to isogenic alloxan-diabetic male rats. Some recipients were treated with insulin for seven days immediately after transplantation. Eight animals in both the insulin-treated group and control group were killed 15days after transplantation for morphologic and hormonal studies of the transplanted tissue. Using the morphometric technique of linear scanning, the insulin, glucagon, and somatostatin immunocytochemically positive, cell masses of the fetal pancreatic implants were quantitated. The beta cell mass of the implants from the control animals increased roughly eightfold from the time of transplant; insulin treatment resulted in a further two- to threefold increase. The insulin content of the implants increased more than did the beta cell mass, resulting in the fivefold increase in insulin per beta cell. The alpha cell and delta cell masses did not change during the transplant site, the mass of functional beta cells, and the cell-to-cell content of the implanted tissue. These results are discussed in relation to previous quantitative studies of pancreatic islet cell growth. The relationships of the transplant site, the mass of functional beta cell, and the cell-to-cell interaction within the islet to the maintenance of glucose homeostasis are also discussed.

Eight 18-316 fetal pancreases were transplanted to syngeneic alloxan diabetic male rats. Some of the recipients were treated with insulin for a 7-day period immediately after transplant. By previously published clinical criteria, three groups of recipients could be identified after reversal of diabetes by the transplanted tissue: insulin-treated rapid reversal; insulin-treated slow reversal; and control (not treated with insulin). Five animals in each group were sacrificed after glucose tolerance testing for morphologic and hormonal analysis of the transplanted tissue. The insulin-,glucagon-, and somatostatin-positive islet cell masses of the fetal pancreatic implants were quantitated. There was a correlation between the beta cell mass of the implants and the glucose tolerance exhibited by the host animals. The rapid response insulin-treated recipients had significantly greater implant beta cell mass and insulin content compared with the other groups. There was no difference in implant alpha cell mass among the groups, but the insulin-treated implants had a significantly greater glucagon content. The delta cell mass of insulin-treated rapid response was less than that of the other two groups. The results are discussed in relation to previously reported morphometric analysis 15 days after transplantation. The relationships of transplanted beta cell mass, beta cell differentiation, transplant site, and cell-to-cell interactions within the transplanted islet to the control of glucose homeostasis are also discussed.

When pancreases from fetal rats were transplanted beneath the kidney capsule of isologous normal adult recipients, continued growth and differentiation of the endocrine portion of the pancreas occurred. While limited amounts of acinar tissues were identifiable in the early transplant period (7 days), such cells were absent in long term transplants (14 and 21 days). In contrast, while few definitive islet beta cells were present at the time of transplantation, following 21 days at the kidney site large circumscribed islets comprised of heavily granulated beta cells in association with duct epithelial cells predominated. Mitotic figures were seen in both these cell populations. Total islet mass had increased over 20-fold during the transplantation period. Similar results were observed if fetal pancreases were grown in organ culture for ten days prior to transplantation. Continued islet and duct cell growth, as evidenced by mitotic figures and an increase in absolute islet cell mass was obtained in such cultured explants when transplanted to either isogenic or allogenic recipients. These observations support the hypothesis that fetal pancreas may be the best source of donor material for transplantation to diabetic recipients, in part due to the continued growth and differentiation of the islet tissue during the transplantation period.

Fetal rat pancreas (ages 16 to 22 days postcoitum) and neonatal pancreas (4 days postnatal) were grown in organ culture for four days. The medium consisted of chick embryo extract and rooster serum either with or without the addition of corticosterone (3 X 10(-5) M). Acinar cell differentiation was assessed using quantitative light microscopic linear scanning of tissue sections and enzymatic analysis of amylase in the culture media and in the explants. In the younger fetal tissue of 16 and 18 days postcoitum exocrine differentiation continued in vitro. The effect of corticosterone was an enhancement of the degree of in vitro differentiation. Even with corticosterone, however, the degree of differentiation in vitro was less than that observed during a similar period in vivo. In differentiated pancreas (20- and 22-day fetal neonatal) the acinar pancreas degenerated under control conditions and a selective growth of the endocrine pancreas was observed. The addition of corticosterone to the media resulted in a maintenance of the differentiated state of the acini except in 22-day fetal tissue in which the acini were not preserved. The differences between these results and the work of other investigators and the possible in vivo role of adrenocorticosteroids in exocrine pancreatic differentiation is discussed.

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.

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 degrees 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 well-granulated 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.

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.

Early in gestation, fetal rat kidneys are less immunogenic than in later fetal stages, and also less immunogenic than early-gestation fetal hepatic tissue. The purpose of this study was to test this observation in an allogeneic model, and to explore the basis of the decreased immunogenicity of early-gestation fetal kidneys. Kidneys were harvested from Fischer (FSH) fetal rats on the 15th, 17th, 18th, and 19th gestational day (GD), and then grafted under the kidney capsule of incompatible Wistar/Furth (W/F) adult male rats. Fetal hepatic tissue was harvested from 15th-GD FSH fetal rats, and then grafted under the kidney capsule of W/F adult male rats. Each recipient received either one kidney or a 3-mm piece of liver. Graft biopsies were obtained 10, 20, 30, and 40 days posttransplantation, and evaluated histologically. The severity of rejection was divided into three grades according to the degree of mononuclear infiltration and percentage of original fetal structures preserved. All fetal hepatic grafts were completely rejected (grade III) by the tenth posttransplantation day. In contrast, the degree of rejection of the kidneys was age-dependent. Fifteenth-GD kidneys showed a minimal or moderate degree of rejection (grade I or II) at 10, 20, and 30 days; however, 18th- and 19-GD kidneys were rejected (grade III) by the tenth post-transplantation day. To explore the basis for the decreased immunogenicity of 15th-GD kidneys, 20 adult W/F rats were divided into two groups. Each animal in the first group received one FSH 15th-GD kidney implanted under the kidney capsule.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of insulinopenia, hyperglycemia, glycosuria, and polyuria associated with severe alloxan diabetes in the rat was accomplished by syngeneic transplantation of whole late-gestation fetal rat pancreata. Intravenous glucose tolerance test (GTT) revealed an improved yet still abnormal glucose and insulin response in reversed recipients reconstituted with as few as two pancreata from fetal donors. Eight fetal donors were sufficient to return glucose and insulin response following GTT to normal. Seventy to eighty percent fewer donors were required when the pancreata were transplanted in their entirely as opposed to transplantation of pancreata subjected to prior enzymatic and mechanical dissociation. The facility and simplicity of the whole fetal pancreas implantation technique makes it an appealing model for further study of islet growth and differentiation at the transplant site and of its effect on the metabolic state of the recipient.

Foetal rat pancreatic explants (20-day postcoitum) were grown in organ culture on medium enriched with serum and embryo extract containing various concentrations of corticosterone. Normal pancreatic exocrine morphology was preserved. In addition, media amylase concentration remained high and media insulin was suppressed. This is in sharp contrast to explants incubated on control medium without addition of steroid in which a rapid dissappearance of the acinar component and a selective proliferation of the islet cells was noted. The magnitude of these effects was related to the concentration of the steroid. Corticosterone was effective in preserving pancreatic acinar cells through 8-10 days in vitro. Removal of high levels of corticosterone from the incubation medium after 4 days of culture resulted in a decrease in media amylase and a fall in explant acinar cell mass. The media insulin returned to control levels during the following 4 days of culture. Addition of corticosterone to the media following 4 days of control culture resulted in no increase in media amylase and no statistically significant differences in explant acinar cell mass. Media insulin was decreased from control levels following the additional 4 days of incubation. However, corticosterone, even at a concentration of 10.0 mug/ml, was not effective in depressing insulin secretion as was foetal adrenal co-culture. It is proposed that adrenal corticosteroids are responsible for the maintenance of differentiated acinar cells previously observed in pancreatic adrenal co-culture. This suggests that corticosteroids may play an important role in in vivo pancreatic morphogenesis and cytodifferentiation. In addition, adrenal corticosteroids directly inhibit insulin release from the explant beta cells in vitro.

The pancreatic insulin-, glucagon-, and somatostatin-positive cell populations were quantitated in normal and alloxan-diabetic rats. The method of quantitation (linear scanning) allowed an estimation of absolute changes in these cell populations through 14 months of diabetes. The changes in cell masses were correlated with changes in plasma and pancreatic immunoreactive insulin and glucagon. A marked reduction in the insulin-positive beta cells was demonstrated within seven days after alloxan treatment. No significant change in the glucagon-positive alpha cell population was noted in the diabetic rats when compared with normoglycemic controls. A statistically significant increase in the pancreatic somatostatin-positive delta cell population was demonstrable only after 14 months of alloxan diabetes. The results would suggest that the hyperglucagonemia of insulin-deficient diabetes is not a consequence of an increased pancreatic alpha cell population. In addition, since the increase in the pancreatic delta cell mass was found only late in the course of alloxan diabetes in the rat, the increase in delta cells is probably not of significance in the pathophysiology of diabetes in this experimental model.