Giuseppe Basta

Long-Term Metabolic and Immunological Follow-Up of Nonimmunosuppressed Patients With Type 1 Diabetes Treated With Microencapsulated Islet Allografts Four cases

OBJECTIVE To assess long-term metabolic and immunological follow-up of microencapsulated human islet allografts in nonimmunosuppressed patients with type 1 diabetes (T1DM). RESEARCH DESIGN AND METHODS Four nonimmunosuppressed patients, with long-standing T1DM, received intraperitoneal transplant (TX) of microencapsulated human islets. Anti-major histocompatibility complex (MHC) class I–II, GAD65, and islet cell antibodies were measured before and long term after TX. RESULTS All patients turned positive for serum C-peptide response, both in basal and after stimulation, throughout 3 years of posttransplant follow-up. Daily mean blood glucose, as well as HbA1c levels, significantly improved after TX, with daily exogenous insulin consumption declining in all cases and being discontinued, just transiently, only in patient 4. Anti-MHC class I–II and GAD65 antibodies all tested negative at 3 years after TX. CONCLUSIONS The grafts did not elicit any immune response, even in the cases where more than one preparation was transplanted, as a unique finding, compatible with encapsulation-driven “bioinvisibility” of the grafted islets. This result had never been achieved with the recipient’s general immunosuppression.

Transplantation of Pancreatic Islets Contained in Minimal Volume Microcapsules in Diabetic High Mammalians

ABSTRACT: To minimize technical problems relating to excessive size (600–800μ in diameter) of standard alginate microcapsules (CSM) for pancreatic islet graft immunoisolation, we have developed two novel minimal volume, chemically identical, capsule prototypes (MVC): 1) coherent microcapsules (CM), and 2) medium‐size microcapsules (300–400μ, MSM). CM, which envelop each individual islet within a thin alginate hydrogel cast, are prepared by emulsification, whereas MSM are made by atomizing the islet‐alginate suspension through a special microdroplet generator. Upon graft into diabetic rodents, CM have shown to immunoprotect both allo‐ and xenogeneic nondiscordant islets, and restored normoglycemia. In higher mammals, at sub‐therapeutic doses, CM fully immunoprotected islet allografts (pig→pig), but only temporarily xenografts (dog→pig). We then used MSM to immunoisolate canine islet allografts in the peritoneal cavity of dogs with spontaneous insulin‐dependent diabetes. Of three grafted dogs, two showed full remission of hyperglycemia with insulin withdrawal. MSM could represent an intermediate solution between CSM and CM for peritoneal immunoisolated islet transplants.

Studies on overnight insulin requirements and metabolic clearance rate of insulin in normal and diabetic man: relevance to the pathogenesis of the dawn phenomenon

SummaryIn order to assess whether the metabolic clearance of insulin changes overnight, 11 patients with Type 1 (insulin-dependent) diabetes and low insulin antibody titre, and 6 nondiabetic subjects were studied. In these studies insulin was always infused by a Harvard pump. Initially, the nocturnal insulin requirements were assessed in the diabetic patients by an overnight feedback insulin infusion to maintain euglycaemia. The insulin requirements decreased continuously after midnight to a nadir of 0.115 ± 0.014 mU · kg−1 · min−1 at 04.30 hours, but after 05.00 hours the insulin requirements increased nearly 40 percent to a maximum of 0.16 ±0.012 mU · kg−1 · min−1 at 07.00 hours. To assess whether plasma insulin clearance changes overnight, the diabetic patients were studied on two different occasions, from 22.00–02.30 hours and from 04.00–08.30 hours. During each of these two studies insulin was infused in sequential steps of 90 min each at the rate of 0.13, 0.40 and 0.20 mU · kg−1 · min−1. Despite changes in plasma free insulin concentration, the metabolic clearance of insulin in the interval 22.00–02.30 hours (12.6±0.17 ml · kg−1 · min−1) was no different from that of the interval 04.00–08.30 hours (12.5 ± 0.19 ml · kg−1 · min−1). The nondiabetic subjects were studied on two different occasions to assess whether the metabolic clearance of insulin changes overnight. Somatostatin (0.25 mg/h) and insulin (0.3 mU kg−1 · min−1) were infused from 22.00–02.30 hours on one occasion, and from 04.00–08.30 hours on the other. The metabolic clearance of plasma free insulin in the interval 22.00–02.30 hours was no different from that of the interval 04.00–08.30 hours (12.6 ± 0.20 vs 12.9 ± 0.25 ml · kg−1 · min−1 nor was it different from that of the diabetic patients.It is concluded that, first, the metabolic clearance rate of insulin does not change overnight either in diabetic or in nondiabetic subjects; second, that it is independent of plasma insulin concentration; and third, that its value is comparable in nondiabetic subjects and in diabetic patients with a low titre of insulin antibodies. Thus, changes in insulin sensitivity rather than changes in insulin clearance are implicated in the pathogenesis of the dawn phenomenon.

A method for the massive separation of highly purified, adult porcine islets of langerhans

A method for the massive and reproducible isolation of highly purified, adult porcine islets of Langerhans is described. The successful combination of donor animal-strain selection with original procedures for pancreas retrieval and enzymatic digestion permitted us to separate uniquely massive concentrations of pure porcine islets with no need for mechanical disruption of the pancreatic tissue. Following our procedure, porcine islets, which fully retain viability and function, can be harvested easily and rapidly. Xenotransplantation of such islets, immunoprotected within algin/polyaminoacidic microcapsules, was associated with complete reversal of hyperglycemia in rodents with either spontaneous or streptozotocin-induced diabetes mellitus.

Grafts of microencapsulated pancreatic islet cells for the therapy of diabetes mellitus in non-immunosuppressed animals.

Pancreatic‐islet‐cell transplantation may reverse hyperglycaemia in diabetic recipients that undertake general pharmacological immunosuppression. A major challenge that remains is the need to avoid immunosuppression associated with the use of allogeneic or heterologous islet cells. In the present study we demonstrate the use of microencapsulation of cells using artificial biocompatible and permselective membranes prepared with alginic acid derivatives and polyamino acids. While characterization of the microcapsule constituent polymers continues to progress, other technical issues such as definition of the immunobarrier capacity, biocompatibility, size, shape and graft site have come into sharper focus. Assessment of microcapsules properties, in order to establish possible guidelines for fabrication of reproducible membranes, and results from both in vitro functional testing, and in vivo encapsulated‐islet‐transplant outcome in several animal models of diabetes are reported.

Multifunctional microcapsules for pancreatic islet cell entrapment: design, preparation and in vitro characterization.

Great advances in cell transplantation have been made, including the recent, remarkable success in pancreatic islet transplantation for the treatment of type 1 diabetes mellitus. Unfortunately, the transplanted cells are very susceptible to oxidative stress that cause severe damage to either allo- or xenogeneic islets upon graft in diabetic patients. Consequently, the transplanted islet functional life span is significantly shortened. The aim of this study was to examine the possible effects of antioxidants on in vitro cultured adult rat islets, and to evaluate the effects of a prolonged-release formulation, in form of cellulose acetate (CA) microspheres, on Vitamin D(3) activity. Isolated rat islets, both free and entrapped in microspheres were treated with Vitamin D(3). The effects of the vitamin were studied at 3, 6 and 9 days of in vitro cell culture. According to insulin secretory patterns, treatment with Vitamin D(3) of both free and CA entrapped microspheres, increased the insulin output as compared to untreated controls. Such positive effects were confirmed under islet static incubation with glucose at day 6. These results suggest that pancreatic islets can be advantageously treated with anti-oxidising vitamins before implantation, and speculatively, with the help of special delivery systems, throughout the islet cell life span, in the post-transplant time period.

Immunoisolation of Pancreatic Islet Grafts with No Recipient’s Immunosuppression: Actual and Future Perspectives

In spite of steady and remarkable progress, islet transplantation in patients with type 1 diabetes mellitus (T1DM) continues to face two major bottlenecks: inadequate availability of human pancreatic donors and necessity to totally immunosuppress the graft recipients lifelong. Microencapsulation of the islet grafts within highly biocompatible and selective permeable biomembranes could obviate use of the immunosuppressants, while potentially offering the opportunity to use a wide array of insulin-producing cells, in active development, including xenogeneic pig islets. Although macrodevices and microcapsules, which essentially differ by size/configuration, and both serve for immunoisolation devices, have been used for many years with initial human applications, new products on development in both areas might open new perspectives for more focused use in patients with T1DM. Physical-chemical properties and material engineering of these devices are critically reviewed to assess where we actually stand and where the future expansion of these technologies may go.

Cellular Support Systems for Alginate Microcapsules Containing Islets, as Composite Bioartificial Pancreas

Abstract: To improve the functional performance of microencapsulated islets, we examined the effects of putative cellular support systems, consisting of rat purified Sertoli cells (SC) and astrocytes (AA), on coenveloped allogeneic islets. Coincubation of islets with SC but not AA, resulted in significant stimulation of β cell mitogenesis, coupled with a significant increase in in vitro glucose‐stimulated insulin release. Preliminarily, the xenotransplantation of coencapsulated rat islets and homologous SC significantly prolonged remission of hyperglycemia in diabetic mice.

Improved function of rat islets upon co-microencapsulation with Sertoli's cells in alginate/poly-L-ornithine

The purpose of this study was to assess whether Sertolis cells would improve functional performance of homologous pancreatic islets within microcapsules. Purified rat Sertolis cells were co-enveloped with islets in microcapsules that had been fabricated with alginic acid and poly-L-ornithine Confocal laser microscopy was used to determine any mitogenic effects of Sertolis cells on islets ß-cells. Insulin secretion from islets, with or without Sertolis cells, was examined and grafts of Sertolis cells with islets in microcapsules into diabetic mice were carried out. Co-incubation of Sertolis cells with islets resulted in a significant increase in the islet ß-cell mitotic rate, which was coupled with significantly higher insulin release under glucose stimulation, as compared to controls. Grafts of co-microencapsulated Sertolis cells with islets resulted in prolongation of the achieved normoglycemia in the animals receiving Sertolis cells with islets as compared to controls that received islets only. Sertolis cells do promote mitogenic activities upon in vitro co-incubation with islets, whose in vitro functional and in vivo post-transplant consequences were evident. Sertolis cells could, therefore, be comicroencapsulated with islets for transplantation in diabetic recipients.

Alginate/Polyaminoacidic Coherent Microcapsules for Pancreatic Islet Graft Immunoisolation in Diabetic Recipients

Pancreatic islet cell transplantation could result in restoration of normoglycemia, thereby allowing for withdrawal of exogenous insulin treatment, in patients with insulin-dependent diabetes mellitus (IDDM). However, the invariable requirement for general pharmacological immunosuppression, in order to prevent islet graftdirected immune destruction, strictly limits progress of this approach into clinical trials. Moreover, owing to immunosuppression-related restrictions, only patients with IDDM, who also require transplant of another major organ (e.g., liver, kidney) are usually enrolled in combined liveror kidney-islet graft trials. Unfortunately this procedure, if ethically correct, invariably cuts off the majority of IDDM-patients, who while not requiring solid organ transplantation, could potentially benefit from this strategy. Finally, the restricted availability of cadaveric human donor pancreata represents, an additional, significant limiting factor.