Claudy J.-P. Mullon

Treatment of Diabetes by Xenogeneic Islets Without Immunosuppression: Use of a Vascularized Bioartificial Pancreas

Tight glycemic control by intensive insulin therapy effectively delays the onset and slows the progression of diabetic complications but is associated with frequent dose adjustments and a high incidence of hypoglycemia. Successful pancreas transplantation corrects abnormal glucose metabolism but subjects patients to morbidity and mortality associated with chronic immunosuppression. A vascularized artificial pancreas device containing pancreatic islets is designed to provide glycemic control without immunosuppression. We report here that devices seeded with porcine islets implanted into pancreatectomized severely diabetic dogs maintained a marked improvement in glycemic control with reduced exogenous insulin requirements for up to 9 months with improved glucose tolerance and a reduction in glycosylated hemoglobin levels. No immunosuppression was used. Thus, use of a vascularized artificial pancreas containing xenogeneic porcine islets could be an alternative to intensive insulin therapy and pancreatic transplantation in treating diabetic patients before the development of severe diabetic complications.

PRIMARY CULTURES OF RAT HEPATOCYTES IN HOLLOW FIBER CHAMBERS

SummaryHepatocyte culture may represent an alternative to the use of animals to study drug detoxification by the liver. An ideal in vitro system should closely mimic the in vivo environment by providing continuous media perfusion and oxygenation, and should facilitate sampling of cells and culture media. To meet these criteria, a hollow fiber bioreactor seeded with isolated rat hepatocytes was developed and tested by measuring the formation of three products of the oxidative metabolism of diazepam and the glucuronidation of phenolsulfonphthalein (PSP). To compare the performance of conventional monolayer culture to that of the bioreactor system, diazepam metabolism was studied for 45 days in both systems. The oxygen dependency of diazepam metabolism was evaluated by perfusing the bioreactor in an oxygen-rich atmosphere (30%). Total diazepam metabolism was twofold higher in the O2-rich perfused hollow fiber cultures than in the cultures perfused under normal conditions, reflecting an increase in temazepam and oxazepam production. Diazepam detoxification activity was significantly enhanced by oxygen (P≤0.001) over the life of the perfused cultures. PSP metabolism was similar in all three culture systems. By Day 10, diazepam metabolism in the oxygenated bioreactor system was 44% of the in vivo activity of rat hepatocytes. This activity dropped to 30% by Day 25 of culture. These results justify the use of perfused culture systems for in vitro detoxification studies as an alternative to animal use and emphasize the capacity of a culture device perfused under O2-enriched conditions to maintain long-term P450 activity of rat hepatocytes.

Porcine islets for xenotransplantation

Long-term function of isolated porcine islets was investigated in diabetic nude mice. Seven of eight mice that received transplants of porcine islets remained normoglycemic for 1 year with progressive weight gain. Circulating porcine C-peptide was detected throughout the study period. Intravenous glucose tolerance tests showed a rapid glucose clearance rate. Together with our recent finding that porcine islets contained within an immunoexclusion device achieved glycemic control in a totally pancreatectomized dog, these results clearly demonstrate that isolated porcine islets are capable of functioning for prolonged periods in xenogeneic hosts and are suitable for long-term use in an immunoexclusion device in a discordant host.

Improved assessment of isolated islet tissue volume using digital image analysis

Accurate and consistent measurement of tissue volume is critical to performing many types of islet research; however, conventional visual determination of isolated islet yields through a microscope is heavily operator dependent. An improved method of islet volume determination using digital image analysis (DIA) was developed to remove operator bias and automate the islet counting process. A series of 140 porcine islet isolations were used to evaluate the DIA method in three separate stages. In Stage 1 (n = 29 isolations), the conventional and DIA methods were correlated with two other independent islet quantitation methods: insulin extraction, and DNA extraction. It was found that volumes determined by DIA correlated more closely with insulin content and DNA content than did conventionally determined volumes. In Stages 2 and 3 (n = 54 and 57 isolations, respectively), it was shown that an increase in the number of fields analyzed by DIA did not significantly improve the quality of the correlations. Inclusion of very small tissue (<50 microm in diameter), which is ignored in the conventional protocol affected yields by less than 10% and did not significantly improve the correlation with insulin or DNA content. Quantitation of isolated islet tissue volume using DIA has been shown to be rapid, consistent, and objective. In the laboratory, use of this method as the standard for islet volume measurement will allow more meaningful comparison of experimental results between centers. In the clinic, its use will allow more accurate dosing of transplanted tissue.

Novel Delivery of Pancreatic Islet Cells to Treat Insulin-Dependent Diabetes Mellitus

SummaryImmune protective devices containing pancreatic islets are designed to treat insulin-dependent diabetes mellitus by providing glycaemic control without immunosuppression. The immune protection is achieved by separating allogeneic or xenogeneic islets from the host by semipermeable membranes that allow only small molecules such as glucose, insulin and nutrients to pass through. Lymphocytes and immunoglobulins are excluded by the membrane and unable to cause rejection of the islets.Three types of immune protective devices, i.e. microcapsules, diffusion chambers and perfusion devices (vascularised artificial pancreas), have been studied. Microcapsules injected into the abdominal cavity in a large quantity achieved glycaemic control, but required a small amount of immunosuppression to prevent fibrosis around the capsules. A clinical attempt to use microcapsulated human islets in a diabetic patient who has maintained functional kidney allografts has been reported. Intra-abdominal placement of diffusion chambers containing allogeneic islets achieved excellent glycaemic control without immunosuppression in diabetic dogs. However, their use was limited by the eventual breakage of tubular chambers. We have extensively used the vascularised artificial pancreas for treatment of experimental diabetes mellitus. Excellent biocompatibility of the device was evidenced by the extraordinary longevity of the patency of the device in healthy dogs. Long term control of severe diabetes mellitus was achieved in totally pancreatectomised dogs without immunosuppression by devices seeded with allogeneic (canine) and xenogeneic (porcine) islets. The vascularised artificial pancreas could be an excellent alternative to Diabetes Control and Complication Trial (DCCT)-type intensive insulin therapy or pancreatic transplantation by providing tight glycaemic control with minimal exogenous insulin therapy without immunosuppression.

Early Treatment of Diabetes with Porcine Islets in a Bioartificial Pancreas

Successful pancreas transplantation is an effective therapy for insulin-dependent diabetes mellitus (IDDM) but subjects patients to morbidity and mortality associated with chronic immunosuppression. Bioartificial pancreas devices containing pancreatic islets provide glycemic control without immunosuppression by physically separating the islet grafts from immune lymphocytes and immunoglobulins. Because immunosuppression is not required, the bioartificial pancreas may offer early treatment of IDDM prior to the development of debilitating diabetic complications. Use of xenogeneic islets (i.e., porcine islets) in the device also provides a solution to the limited availability of human donor organs. This report provides a brief summary of our experience with vascularized bioartificial pancreas devices containing xenogeneic porcine islets used for treatment of experimental diabetes in dogs and describes our plans for a clinical phase I/II trial of the vascularized bioartificial pancreas in patients with IDDM.

Evaluation of anti-AIDS drugs in conventional mice implanted with a permeable membrane device containing human T cells infected with HIV

We now report the confirmation of the work of Hollingshead et al. (1995) on development of a cell based hollow fiber (HF) system for evaluating potential anti-AIDS drugs in vivo using conventional mice rather than SCID mice. CD4 +, CEM-SS cells infected with HIV/1, strain RF, at a multiplicity of infection of 0.1 were placed into HFs. The fibers were implanted into the peritoneal cavity of outbred Swiss mice. Using this model, the antiviral activity of azidothymidine (AZT) at doses of approximately 150, 75 and 37.5 mg/kg/day was evaluated by administering AZT to the mice in drinking water. Upon fiber removal on day 6, AZT treatment was shown to significantly increase CEM cell viability over the untreated, virus control group and significantly reduced the levels of HIV p24 and HIV RT activity.