Orion D. Hegre

Protection of Encapsulated Human Islets Implanted Without Immunosuppression in Patients With Type I or Type II Diabetes and in Nondiabetic Control Subjects

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 β-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.

Central and peripheral localization of somatostatin. Immunoenzyme immunocytochemical studies.

Early in 1973, Brazeau et al. (5), using monolayer cultures of rat pituitary as a bioassay system (34), reported their findings on the chemical structure and physiology of a tetradecapeptide extracted from sheep hypothalami. The new neunohonmone was named somatostatin (SRIF) because it blocked the secretion of growth hormone. Synthetic SRIF proved to be an effective hapten in conjunction with immunogenic molecules, and anti-SRIF antisera were raised in rabbits (3, 7) and guinea pigs (10). Thus, very soon after the initial description of SRIF (5), specific antibodies became available for use in quantitative immunochemical and morphologic cytoendoncninologic studies. Accordingly, Animura et al. (2) and Patel et al. (23) synthesized a tyrosine-containing analogue of SRIF that could be readily iodinated and developed radioimmunoassays (RIA). Subsequent RIA of extracts of brains and peripheral tissues of experimental animals showed that SRIF was not localized exclusively within the hypothalamus. Immunoassayable SRIF is present in significant amounts within extnahypothalamic neural tissue (6, 23, 28) and also within tissues of the gastroentenopancreatic (GEP; see Fujita and Kobayashi (13)) system of endocrine cells (3, 28). Thus SRIF may play a physiologically significant role in these tissues as well as in the hypothalamus. This prospect has become more intriguing in light of numerous reports which have demonstrated that synthetic SRIF, in addition to its inhibitory actions upon adenohypophysial function, blocks pancreatic insulin and glucagon release (see Genich et aL (14), Reichlin et at. (28) and Vale et at. (33)).

Clonal Insulinoma Cell Line That Stably Maintains Correct Glucose Responsiveness

A number of pancreatic β-tumor cell (β 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 β 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 β TC6-F7, was characterized in detail. β 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 β-cells. These results demonstrate that transformed β-cells can maintain a highly differentiated phenotype during prolonged propagation in culture, which has implications for the development of continuous β-cell lines for transplantation therapy of diabetes.

Morphometric Quantitation of the Pancreatic Insulin-, Glucagon-, and Somatostatin-positive Cell Populations in Normal and Alloxan-diabetic Rats

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.

Immunoisolation of adult porcine islets for the treatment of diabetes mellitus. The use of photopolymerizable polyethylene glycol in the conformal coating of mass-isolated porcine islets.

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).

Pancreatic Islet Transplantation in the Rat

Twenty to thirty-five neonatal rat pancreases (2.5 to 4.5 days postpartum) were minced, dissociated in a collagenasetrypsin-EDTA solution, washed, centrifuged, resuspended in Tyrodes solution, and transplanted intraperitoneally to an alloxan-diabetic recipient. All of eight highly inbred moderate-diabetic rats (mean pretransplant blood sugar level and daily urine glucose excretion = 340 mg./100 ml. and 6.0 gm., respectively), showed a reversal of the diabetes within several days following isotransplantation (blood sugar and daily urine glucose excretion levels dropped to less than 150 mg./100 ml. and 0.1 gm., respectively). Two of seven highly inbred severe-diabetic rats (mean pretransplant blood sugar level and daily urine glucose excretion = 440 mg./100 ml. and 8.0 gm., respectively), recovered from their diabetes about two weeks following isotransplantation. In these highly inbred recipients which recovered from their diabetes, normoglycemia has persisted for more than five months. Control animals receiving liver transplants showed no significant changes in their diabetes. Exploration of the peritoneal cavity, after two months of remission, demonstrated isolated implants in association with the liver, spleen, pancreas, abdominal wall, etc. Yascularized grafts consisted primarily of cords of heavily granulated (aldehyde fuchsin positive) beta cells. Acinar cells were not identified. By contrast, the recipient pancreas showed few identifiable beta cells (characteristic of alloxan diabetes), but these were heavily granulated, presumably because of the controlled diabetic state. In some grafts, removed several days following implantation, mitotic figures were seen in cells containing aldehyde fuchsin positve granules. Similar homotransplants were carried out in thirty-seven partially inbred moderate-diabetic recipients; 46 per cent showed a transitory recovery from their diabetes lasting three to thirteen days. However, the pretransplant diabetes was re-established in one to three weeks and this was attributed to the rejection of the transplant. These studies indicate that dissociated neonatal pancreas forms functional islet implants capable of reversing diabetes in alloxanized rats.

Fetal Rat Pancreas: Differentiation of the Acinar Cell Component in Vivo and in Vitro

Fetal rat pancreases, Day 16 through Day 22 postcoitum, were analyzed biochemically for amylase and chymotrypsinogen to assess acinar cell development in vivo. The interval between -eighteen and twenty days proved to be a critical period in the differentiation of the acinar component. Eighty- and thirtyfold increases in, respectively, amylase and chymotrypsinogen concentration were accompanied by a large increase in the acinar cell percentage of the pancreas. Explantation of pancreas either before or after this critical period resulted in a developmental pattern which varied from that observed in vivo. The present organ culture system neither supported the differentiated acinar cell nor favored the further development of new acinar cells. Using the present organ culture system, explants can be produced which are devoid of acinar cells and their exocrine enzymes.

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.

Effect of MtTW15 Mammosomatotropic Tumors on Pancreatic Islet Hormones

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 (2×) 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 (< 70 μm) and more large (> 140 μm) 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.

Isotransplantation of organ-cultured neonatal pancreas: reversal of alloxan diabetes in the rat.

Pancreases from neonatal rats four to 16 days postpartum were grown in organ culture for from two to nine days. Approximately 10-20 explants, each measuring 1 mm.3 (1 mg.), were grown on a single Millipore filter placed at the gas-liquid interface of a medium consisting of 50 per cent horse serum and 50 per cent chick embryo extract. Following organ culture, an estimated 9-20 mg. of cultured islet tissue were dissociated with collagenase and isotransplanted into the peritoneal cavity of alloxan-diabetic recipients. In seven of eight recipients the diabetes was reversed between 11 and 53 days of posttransplantation. Animals receiving 12-16 mg. of cultured islet attained normoglycemia in 11-20 days; animals receiving 9-10 mg. of cultured islet tissue recovered between 45 and 53 days. These animals have remained symptom-free for over six months. Biopsies of grafts taken from the peritoneal cavity following reversal of diabetes contained well-vascularized islets compared primarily of heavily granulated beta cells. Quantitative analysis of host pancreases by the linear scanning method (biopsied at one to two weeks and four to five months following reversal of the diabetes) demonstrated that the total beta-cell mass was 3 per cent and the total insulin content was 6 per cent of the normal values. Little or no evidence of regeneration of host beta cells was observed. These studies show that a period of organ culture prior to isotransplantation does not impair the ability of islet tissue to reverse alloxan diabetes in the rat.