William L. Chick

Somatostatinoma: a somatostatin-containing tumor of the endocrine pancreas.

We studied the pancreatic and enteric hormone profile of a 46-year-old woman who had hyperglycemia and a pancreatic tumor. Before operation, there was no evidence of overproduction of glucagon or insulin. The tumors ultrastructure had a distinctive endocrine morphology, resembling D cells. Prompted by the recent demonstration of somatostatin in D cells of pancreatic islets, we analyzed the tumor and found a large quantity of immunoreactive somatostatin (301 ng per milligram of tissue). Insulin, glucagon, gastrin, vasoactive intestinal polypeptide and human pancreatic polypeptide were present in only trace quantities. The tumor cells were cultured in monolayers, which remained viable up to 51 days and released somatostatin into the culture medium. In seven insulinomas and two glucagonomas, we found the somatostatin content either much lower (less than 0.6 ng per milligram of tissue) or undetectable. After complete resection of the tumor, our patient became euglycemic and has remained so for the past 20 months.

Cell Encapsulation Technology and Therapeutics

Encapsulated cells offer enormous potential for the treatment of human disease. This work includes detailed descriptions of chemical properties of encapsulation materials, purification, biocompatibility issues and experimental protocols.

Beta cell culture on synthetic capillaries: an artificial endocrine pancreas

Beta cells from neonatal rats were cultured on bundles of artificial capillaries perfused with tissue culture medium. Cells continued to release insulin and remained responsive to changes in glucose concentration. The quantity of insulin released was similar to that of conventional flask cultures.

XENOTRANSPLANTATTON OF PORCINE AND BOVINE ISLETS WITHOUT IMMUNOSUPPRESSION USING UNCOATED ALGINATE MICROSPHERES

Uncoated spherical hydrogel microspheres (calcium alginate, nominal M(r) exclusion of > 600 kD) 800-900 microns in diameter were employed to prevent immune rejection of discordant islet xenografts isolated from pigs and cows. The islets were immobilized in the microspheres and injected into the peritoneum of 14 nonimmunosuppressed streptozotocin (STZ)-induced diabetic C57BL/6J mice. Four recipients received islet grafts from bovine calves, and 10 received islet grafts from pigs. In the control group of 15 diabetic mice implanted with nonencapsulated islets, 6 received i.p. porcine islets and 5 received i.p. bovine islets, whereas remaining 4 received porcine islets under the kidney capsule. Plasma glucose concentrations in recipients of the alginate-encapsulated islets promptly dropped from a preimplantation value of 498 +/- 47 (mean +/- SEM) to 142 +/- 6 (bovine) and 178 +/- 7 mg/dl (porcine) during the first wk. All the animals sustained these levels for at least 1 mo. Two mice implanted with bovine islets subsequently reverted to diabetes (plasma glucose > 250 mg/dl) at 43 days postimplantation. The remaining grafts maintained function for > 10 wk. In contrast, nonencapsulated islets failed to function, or sustained euglycemia for < 4 days. Mice receiving encapsulated islets showed a 23-38% gain in body weight during the first mo after implantation, compared with < 1% (P < 0.002) and 32% (P = 0.84) for the untreated diabetic (n = 6) and normal control (n = 6) groups. Immunohistochemical staining of long-term grafts (> 10 wk) revealed viable islets, with well-granulated alpha, beta, and delta cells; the external surfaces of the microreactors were free of fibrotic overgrowth and exhibited only occasional host cell adherence. Uptake studies with IgG and thyroglobulin (M(r) of 669 kD) suggest that the microreactors were permeable to molecules with a molecular weight of up to > 600 kD (including the various proteins of the complement system, M(r) of 24-570 kD). Spheres implanted in the peritoneum after only 1 wk stained positive for both IgG and for the C3 component of complement. These findings suggest that prolonged survival of discordant xenografts of porcine and bovine islets in the STZ diabetic mouse model can be achieved with uncoated alginate microspheres that are permeable to IgG and complement. The question of whether similar results can be achieved with uncoated alginate microspheres in higher animals remains to be fully determined.

Islet Transplantation With Immunoisolation

Immunoisolation is a potentially important approach to transplanting islets without need for immunosuppressive drugs. Immunoisolation systems have been conceived in which the transplanted tissue Is separated from the immune system of the host by an artificial barrier. These systems offer a solution to the problem of human islet procurement by permitting use of Islets Isolated from animal pancreases. The devices used are referred to as biohybrid artificial organs because they combine synthetic, selectively permeable membranes that block immune rejection with living transplants. Three major types of biohybrid pancreas devices have been studied. These include devices anastomosed to the vascular system as AV shunts, diffusion chambers, and microcapsules. Results In diabetic rodents and dogs indicate that biohybrid pancreas devices significantly improve glucose homeostasis and can function for more than a year. Recent progress made with this approach is discussed, and some of the remaining problems that must be resolved to bring this technology to clinical reality are addressed.

Transplantation of encapsulated cells and tissues

fue body by a selectively permeable membrane.1-3 The membrane allows fue free exchange of nutrients, oxygen, and biotherapeutic substances between fue blood or plasma and fue encapsulated cells, whereas high molecular weight substances such as immunocytes, antibodies, and other transplant rejection effector mechanisms are excluded. These systems mar algO modulate fue bidirectional diffusion of antigens, cytokines, and other immunologic moieties on fue basis of fue chemical characteristics of fue membrane and matrix support. Encapsulated cell. technology offers a solution to fue problem of donar organ supply, not only by allowing fue transplantation of human cells and tissues without immunosuppression but algO by permitting use of tissues isolated from animals. The ability to cross species lines has fue potential to expand dramatically fue number of patients and fue scope of diseases that can be treated successfully with transplantation.

Beta cell replication in rat pancreatic monolayer cultures. Effects of glucose, tolbutamide, glucocorticoid, growth hormone and glucagon.

Rat pancreatic monolayer cultures were utilized to study the effects of glucose, tolbutamide, dexamethasone phosphate, bovine growth hormone and porcine glucagon on beta cell replication. Cultures were established in medium 199 (glucose concentration 1 mg. per milliliter) containing 10 per cent fetal bovine serum. They were subsequently incubated for two consecutive two day intervals in this same control medium or in media containing the test agents. Cultures were then labeled with 3-H-thymidine and reincubated in control medium for two days to permit beta cell regranulation, thus facilitating staining. Replication was estimated by determining the frequency of labeling of aldehydethionin positive (AT+) cells in stained radioautographs. Both glucose (3 mg. per milliliter medium) and tolbutamide (100 μ. per milliliter) caused a three- to fourfoldincrease in labeling compared to controls (14 labeled AT+ cells per 500 AT+ cells). In -contrast, dexamethasone (10 μ. per milliliter) produced a two-thirds decrease in labeling. Growth hormone (10 μ. per milliliter) and glucagon (10 μ per milliliter) produced no statistically significant effects. Alterations in labeling correlated with changes in the quantity of insulin released into the medium during each of the two day incubation intervals. Both glucose and tolbutamide increased insulin release; dexamethasone decreased insulin release, while growth hormone and glucagon produced no significant effects. These data suggest that agents which stimulate insulin release can trigger beta cell replication in vitro. In addition, since growth hormone, glucocorticoid and glucagon stimulate beta cell replication in vivo but not in vitro, this action in vivo may be mediated by effects on other tissues rather than by direct action on the beta cell.

Treatment of Severely Diabetic Pancreatectomized Dogs Using a Diffusion-Based Hybrid Pancreas

Long-term survival of dog islet allografts implanted in diabetic pancreatectomized dogs was achieved by islet encapsulation inside cylindrical chambers fabricated from permselective acrylic membranes (nominal Mr exclusion of 50,000–80,000). Dog islets were isolated from the pancreases of outbred mongrel dogs by collagenase digestion. Chambers containing mean ± SE 316 ± 63K islet equivalents (mean islet volume, 558 ± 111 mm3, purity 90–95%) were peritoneally implanted into six totally pancreatectomized dogs. The dogs were monitored for glycemic control by fasting and postprandial blood glucose determinations, and responses to both intravenous glucose (intravenous glucose tolerance test 0.5 g/kg) and oral glucose (oral glucose tolerance test 1 g/kg). All of the dogs required appreciably lower dosages of exogenous insulin therapy for control of fasting blood glucose levels, with the mean daily insulin dose dropping from 38 ± 7 to 5 ± 1 U/day during the 1st wk. Three recipients required no insulin for >82, >68, and 51 days. Intravenous glucose tolerance test K values (decline in glucose levels, %%min) at 1 and 2 mo postimplantation were 2.7 ± 0.4 and 2.0 ± 0.5, respectively compared with 3.5 ± 0.5 before pancreatectomy. The glucose values during oral glucose tolerance tests at 2 wk, although returning to < 125 mg/dl (< 7.0 mM) by 2 h, exceeded the normal range, with peak values of 174 to 202 mg/dl (9.7 to 11.3 mM). These preliminary results are encouraging, and represent an important step in determining the feasibility of using this type of diffusion-based hybrid artificial pancreas as treatment for diabetes mellitus in humans.

Transplantation of islets using microencapsulation: studies in diabetic rodents and dogs

Studies involving the transplantation of human islets in Type I diabetics have been of significant value both in documenting the potential importance of islet transplantation as a therapeutic modality, and in defining some of the problems which must be overcome before this approach can be used in large numbers of patients. The currently limited supply of adult human pancreatic glands, and the fact that chronic immunosuppression is required to successfully transplant islets into patients, indicate that techniques must be further developed and refined for allo- and xenografting of isolated islets from human and animal sources to diabetic patients. An increasing body of evidence using microencapsulation techniques strongly suggests that this will be achieved during the next few years. Data from our laboratory in rodents and dogs indicate that these systems can function for extended periods of time. In one study, insulin independence was achieved in spontaneously diabetic dogs by islet microencapsulation inside uncoated alginate gel spheres (Mr exclusion >600 kD). No synthetic materials or membrane coatings were employed in this study. Spheres containing canine islets were implanted into the peritoneum of 4 diabetic dogs. The animals received low-dose CsA (levels below readable limits by HPLC at 3 weeks). Implantation of these spheres completely supplanted exogenous insulin therapy in the dogs for 60 to >175 days. Blood glucose concentration averaged 122±4 mg/dl for these animals during the first 2 months. The glycosylated hemoglobin (HbAIC) levels during this period dropped from 6.7±0.5% to 4.2±0.2% (P<0.001). IVGTT K-values at 1 and 2 months postimplantation were 1.6±0.1 (P<0.002) and 1.9±0.1 (P<0.001), respectively compared with 0.71±0.3 before implantation. In a second group of studies, bovine islets were immobilized inside a new type of selectively permeable ”microreactor” (Mr exclusion <150 kD) and implanted into the peritoneum of 33 STZ-induced diabetic rats without any immunosuppression. Diabetes was promptly reversed, and normoglycemia maintained for periods of several weeks to months. Immunohistochemical staining of microreactors recovered from these animals revealed well-granulated β-cells consistent with functionally active insulin synthesis and secretion. To test further the secretory function of the islets, some of the explanted microreactors were incubated in media containing either basal or stimulatory concentrations of glucose. The islets responded with an approximately 3- to 5-fold average increase above basal insulin secretion. These results are encouraging, and may have important implications in assessing the potential role of these microencapsulation systems as therapy for human insulin-dependent diabetes.

Xenogeneic humoral responses to islets transplanted in biohybrid diffusion chambers

It has been hypothesized that chronic antigen leakage from the hybrid artificial pancreas could stimulate a host humoral response. Such antibodies could be induced by antigens shed from the islet cell surface, or by proteins secreted by live cells or liberated after cell death. To determine if this humoral response occurs, porcine (n = 15) or canine (n = 7) islets were seeded (2-5 x 10(4) equivalent islet number, density 30 islets/mm3) into diffusion chambers fabricated from permselective acrylic membranes (nominal M(r) exclusion of 80,000). The chambers were implanted intraperitoneally into streptozotocin-induced diabetic rats. Sera were collected at various intervals (0-12 weeks) and tested against isolated canine and porcine islets, for tissue specificity and interspecies cross-reactivity by fluorescence immunocytochemistry. No immunofluorescence (or only weak background staining) was obtained when islets were exposed to horse sera, or to sera obtained before to xenodevice implantation. Within 2-6 weeks, however, the postimplantation sera showed strong immunoreactivity. The antibodies were found to be reactive to multiple tissues, and to possess little or no interspecies cross-reactivity. The appearance of these xenoantibodies coincided with the appearance of circulating soluble immune complexes. However, none of the respiratory, cutaneous, or gastrointestinal manifestations that are characteristic of an anaphylactic reaction, or of the diseases of immediate-type hypersensitivity, were observed, even after intraperitoneal injection of additional naked islet tissue. Renal glomeruli did not stain for IgG or C3 in islet recipients. These results suggest that islet cell antigens crossed the membrane and stimulated antibody formation in the host, although they did not appear to cause renal or immune complex disease during the course of this study.