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... more
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.
Aims/hypothesis Recent observations suggest the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural... more
Aims/hypothesis Recent observations suggest the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of probiotic bacteria can modulate local and systemic immune responses; consequently, we investigated the effects of oral administration of the probiotic compound VSL#3 on the occurrence of diabetes in non-obese diabetic (NOD) mice. Methods VSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. A control group received PBS. Whole blood glucose was measured twice a week. IFN-γ and IL-10 production/expression was evaluated by ELISA in culture supernatants of mononuclear cells isolated from Peyer’s patches and the spleen, and by real-time PCR in the pancreas. Insulitis was characterised by immunohistochemistry and histomorphometric studies. Results Early oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a decreased rate of beta cell destruction. Prevention was associated with an increased production of IL-10 from Peyer’s patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive islet-infiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice. Conclusions/interpretation Orally administered VSL#3 prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis severity. Our results provide a sound rationale for future clinical trials of the primary prevention of type 1 diabetes by oral VSL#3 administration.
Parenteral and oral administration of autoantigens can induce immune tolerance in autoimmune diseases. Prophylactic therapy based on oral administration of human autoantigens is not, however, feasible when sufficient quantities of... more
Parenteral and oral administration of autoantigens can induce immune tolerance in autoimmune diseases. Prophylactic therapy based on oral administration of human autoantigens is not, however, feasible when sufficient quantities of candidate autoantigens are not available. Transgenic plants that express high levels of recombinant proteins would allow large quantities of autoantigens to be produced at relatively low costs. In addition, transgenic food would provide a simple and direct method of delivering autoantigens. The production and the characterization of transgenic tobacco and carrot plants expressing human GAD65, a major autoantigen in human insulin-dependent diabetes mellitus (IDDM), is reported. Immunogold labeling and electron microscopy of transgenic tobacco tissue shows the selective targeting of human GAD65 to chloroplast tylacoids and mitochondria. In planta expressed GAD65 has a correct immunoreactivity with IDDM-associated autoantibodies and retains enzymatic activity, a finding that suggests a correct protein folding. In transgenic tobacco and carrot the expression levels of human GAD65 varies between 0.01% and 0.04% of total soluble proteins. Transgenic edible plant organs are now available to study the feasibility of inducing immune tolerance in IDDM animals by oral administration of GAD65.
Abstract: The presence of islet cell autoantibodies in adult diabetic subjects who do not require insulin treatment for at least six months after the initial clinical diagnosis identifies the so-called latent autoimmune diabetes in the... more
Abstract: The presence of islet cell autoantibodies in adult diabetic subjects who do not require insulin treatment for at least six months after the initial clinical diagnosis identifies the so-called latent autoimmune diabetes in the adult (LADA). Glutamic acid decarboxylase autoantibodies (GAD65Ab) are the best immune marker to identify LADA patients, while other islet autoantibodies, such as IA-2 autoantibodies, have a very low diagnostic sensitivity. Islet cell antibodies, as detected by indirect immunofluorescence, may improve the diagnostic specificity of the immune analysis when detected in GAD65Ab-positive patients. Although the majority of LADA patients progresses towards insulin dependency within a few years after the diagnosis, this form of diabetes is heterogeneous. The presence of high titers of GAD65Ab and/or GAD65Ab directed towards COOH-terminal epitopes of the autoantigen (GAD65-CAb) identifies a subgroup of LADA patients with clinical characteristics similar to those of typical type 1 diabetes and at very high risk of progression toward insulin dependency. On the other hand, low titers of GAD65Ab, or the exclusive presence of GAD65Ab directed to middle epitopes of the autoantigen, characterizes LADA patients with clinical characteristics almost indistinguishable from those of GAD65Ab-negative type 2 diabetic patients. LADA subjects, especially in the presence of high titers of GAD65Ab and/or GAD65-CAb, have an increased risk for other organ-specific autoimmune diseases such as thyroid autoimmune diseases or autoimmune Addison's. LADA should be considered a major component of the autoimmune polyendocrine syndromes, and screening for other autoimmune diseases should be performed in all LADA patients.
To circumvent pancreatic islet graft-directed immune destruction we enveloped porcine islets within highly biocompatible and selectively permeable algin/polyaminoacid microcapsules. These special microspheres were deposited between the... more
To circumvent pancreatic islet graft-directed immune destruction we enveloped porcine islets within highly biocompatible and selectively permeable algin/polyaminoacid microcapsules. These special microspheres were deposited between the inner (permeable) and the outer (impermeable) layers of an artificial, coaxial vascular prosthesis, directly anastomized to blood vessels. Five dogs with spontaneous, insulin-dependent diabetes received microencapsulated porcine islets in arterio-vein iliac prosthesis by-passes. One showed complete and the remainder partial, sustained reversal of hyperglycemia. Microencapsulation may be a potential solution to immunological problems related to islet transplantation.
