g(m1)-ganglioside and Diabetes-Mellitus--Type-1

Cholera toxin and its close relative, Escherichia coli heat-labile enterotoxin, are potent immunogens and mucosal adjuvants. The recent findings that their B subunits can promote tolerance highlights the complexity of their interactions with the immune system. Here, Neil Williams and colleagues review the mechanisms by which these molecules modulate leukocyte populations and seek to explain the paradox.

Topics: Adjuvants, Immunologic; Animals; Arthritis; Autoantigens; Autoimmune Diseases; Bacterial Toxins; Carbohydrate Sequence; Cholera Toxin; Collagen; Diabetes Mellitus, Type 1; Encephalomyelitis, Autoimmune, Experimental; Enterotoxins; Escherichia coli Proteins; G(M1) Ganglioside; Gene Expression Regulation; Hydrogen Bonding; Immune Tolerance; Interferon-gamma; Mice; Mice, Inbred NOD; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Conformation; Rats; Structure-Activity Relationship; Th2 Cells

The destruction of beta cells by the islet infiltrating lymphocytes causes type 1 diabetes. Transgenic mice models expressing interferon (IFN)-beta in beta cells, in the non-obese diabetic (NOD) strain and in a diabetes-free, major histocompatibility complex-matched, homologous strain, the non-obese resistant (NOR) mice, developed accelerated type 1 diabetes after 3 weeks of age. Our aim was to determine if natural killer (NK) cells could affect the acceleration of the disease. We determined the amount of NK cells in the pancreas, spleen and lymph nodes from NOD rat insulin promoter (RIP)-IFN-beta mice. Pancreatic cytokines were assessed by quantitative real-time polymerase chain reaction and protein arrays. To confirm the relevance of NK cells in the acceleration of autoimmune diabetes this subset was depleted with anti-asialo GM1 antibodies. An increase of intrapancreatic NK cells characterized the accelerated onset of diabetes both in NOD and NOR RIP-IFN-beta transgenic models. Cytokines involved in NK function and migration were found to be hyperexpressed in the pancreas from accelerated diabetic mice. Interestingly, the depletion of NK cells in vivo abolished completely the acceleration of diabetes. NK cells connect innate to adaptive immunity and might play a role in autoimmunity. We report here that NK cells are required critically in the pancreas for accelerated diabetes. This model links inflammation to acceleration of beta cell-specific autoimmunity mediated by NK cells.

Topics: Animals; B-Lymphocytes; Cytokines; Diabetes Mellitus, Type 1; Disease Models, Animal; Disease Progression; G(M1) Ganglioside; Interferon-beta; Islets of Langerhans; Killer Cells, Natural; Lymph Nodes; Lymphocyte Subsets; Mice; Mice, Inbred NOD

NOD (non-obese diabetic) mice develop type 1 diabetes mellitus spontaneously and with a strong similarity to the human disease. Differentiation and function of pancreas beta cells are regulated by a variety of hormones and growth factors, including the nerve growth factor (NGF). Gangliosides have multiple immunomodulatory activities with immunosuppressive properties, decreasing lymphoproliferative responses and modulating cytokine production. In the present study, serum, pancreas islets and spleen mononuclear cells from NOD mice treated with monosialic ganglioside GM1 (100 mg/kg/day) and the group control which received saline solution were isolated to investigate the proinflammatory cytokines (IL-1beta, IFN-gamma, IL-12, TNF-alpha), NGF and its high-affinity receptor TrkA, peri-islet Schwann cells components (GFAP, S100-beta) expression and the relationship with diabetes onset and morphological aspects. Our results suggest that GM1 administration to female NOD mice beginning at the 4th week of life is able to reduce the index of inflammatory infiltrate and consequently the expression of diabetes, modulating the expression of proinflammatory cytokines (IL-12, IFN-gamma, TNF-alpha and IL-1beta). Furthermore, GM1 increases GFAP, S-100beta and NGF in pancreas islets, factors involved in beta cell survival.

Topics: Age of Onset; Animals; Cytokines; Diabetes Mellitus, Type 1; Female; G(M1) Ganglioside; Glial Fibrillary Acidic Protein; Humans; Islets of Langerhans; Male; Mice; Mice, Inbred NOD; Nerve Growth Factor; Nerve Growth Factors; Nerve Tissue Proteins; Receptor, trkA; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Schwann Cells; Spleen

Several bacterial and plant enterotoxin B subunit-islet autoantigen fusion proteins were compared for their ability to serve as islet autoantigen carriers and adjuvants for reduction of pancreatic islet inflammation associated with type 1 diabetes. The cholera toxin B subunit (CTB), the heat-labile toxin B subunit from enterotoxigenic Escherichia coli (LTB), the Shigella toxin B subunit (STB), and the plant toxin ricin B subunit (RTB) were genetically linked to the islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The adjuvant-autoantigen gene fusions were transferred to a bacterial expression vector and the corresponding fusion proteins synthesized in E. coli. The purified adjuvant-autoantigen proteins were fed to 5-wk-old nonobese diabetic (NOD) mice once a week for 4 wk. Histological examination of pancreatic islets isolated from inoculated mice showed significant levels of insulitis reduction in comparison with uninoculated mice. The ratio of serum anti-INS and anti-GAD IgG2c to IgG1 antibody isotype titers increased in all ligand-autoantigen inoculated animal groups, suggesting an increase in effector Th2 lymphocytes in B subunit-mediated insulitis suppression. The results of these experiments indicate that bacterial and plant enterotoxin B subunit ligand-autoantigens enhance insulitis reduction in NOD mice. This research prompts further exploration of a multiadjuvant/autoantigen co-delivery strategy that may facilitate type 1 diabetes prevention and suppression in animals and humans.

Topics: alpha-Fetoproteins; Animals; Antibody Formation; Asialoglycoproteins; Autoantigens; Bacterial Toxins; Cholera Toxin; Diabetes Mellitus, Type 1; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Proteins; Female; Fetuins; G(M1) Ganglioside; Glutamate Decarboxylase; Immunoglobulin G; Immunoglobulin Isotypes; Immunotherapy, Active; Islets of Langerhans; Mice; Mice, Inbred NOD; Peptide Fragments; Proinsulin; Protein Subunits; Recombinant Fusion Proteins; Ricin; Shiga Toxin; Trihexosylceramides

Advanced glycation end products (AGEs) are involved in the development of microvascular complications, including alterations of retinal pericyte and renal mesangial cell growth occurring during diabetic retinopathy and diabetic nephropathy, respectively. Because gangliosides are implicated in the regulation of cell proliferation, we hypothesized that AGEs could exert cellular effects in part by modulating ganglioside levels. Results of the present study indicate that AGEs caused an inhibition of both bovine retinal pericyte (BRP) and rat renal mesangial cell (RMC) proliferation, associated with an increase of a-series gangliosides consecutive to GM3 synthase activity increase and GD3 synthase activity inhibition. Similar modifications were also found in the renal cortex of diabetic db/db mice compared with controls. Treatment of BRP and RMC with exogenous a-series gangliosides decreased proliferation and blockade of a-series gangliosides with specific antibodies partially protecting the two cell types from the AGE-induced proliferation decrease. Further, inhibition of GM3 synthase using specific SiRNA partially reversed the AGE effects on mesangial cell proliferation. These results suggest that a-series gangliosides are mediators of the adverse AGE effects on BRP and RMC proliferation. They also raise the hypothesis of common mechanisms involved in the development of diabetic retinopathy and diabetic nephropathy.

Topics: Animals; Cattle; Cell Division; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Diabetic Retinopathy; Disease Models, Animal; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Glomerular Mesangium; Glycation End Products, Advanced; Kidney Cortex; Mice; Microcirculation; Pericytes; Rats; Retinal Vessels; RNA, Small Interfering; Sialyltransferases

The oral administration of disease-specific autoantigens can induce oral immune tolerance and prevent or delay the onset of autoimmune disease symptoms. Here, we describe the construction of an edible vaccine consisting of a fusion protein composed of cholera toxin B subunit (CTB) and insulin that is produced in silkworm larvae at levels of up to 0.3 mg/ml of hemolymph. The silkworm bioreactor produced this fusion protein vaccine as the pentameric CTB-insulin form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and insulin. Non-obese diabetic mice fed hemolymph containing microgram quantities of the CTB-insulin fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of symptoms of clinical diabetes. These results demonstrate that the silkworm bioreactor is a feasible production and delivery system for an oral protein vaccine designed to develop immunological tolerance against T-cell-mediated autoimmune diabetes by regulatory T-cell induction.

Topics: Administration, Oral; Animals; Antibody Formation; Base Sequence; Bombyx; Cholera Toxin; Diabetes Mellitus, Type 1; Female; G(M1) Ganglioside; Insulin; Mice; Mice, Inbred NOD; Molecular Sequence Data; Protein Engineering; Recombinant Fusion Proteins; Vaccines, Edible

Sulfatide (3'sulfogalactosylceramide) is a glycosphingolipid present within the nervous system and in the islets of Langerhans. Anti-sulfatide antibodies have been observed in both pre-diabetic and newly diagnosed type 1 diabetic patients. The aim of this study was to test in vivo, the therapeutic effect of sulfatide on the development of diabetes in the NOD mouse. In four separate experiments diabetogenic splenocytes from newly diabetic NOD mice were injected iv into 7-8 week old irradiated (700R) female NOD mice (4-10 million cells/mouse). Each experiment consisted of four treatment groups to which the mice were randomly divided: 1) sulfatide; 2) galactosylceramide (the precursor to sulfatide without sulfate); 3) GM1, a glycosphingolipid negatively charged as sulfatide but with a different sugar composition; and 4) phosphate buffered saline (PBS). The mice received 100 microg glycosphingolipid iv on the day of cell transfer and 1-3 times thereafter at four day intervals, and were screened for diabetes three times a week the next 52 days. Among all the 35 sulfatide-treated mice 54% became diabetic compared to 93 % of 43 PBS-treated animals (p < 0.00001). Correspondingly, galactosylceramide reduced diabetes incidence to 52% (25 mice, p < 0.00001). On the other hand, 86% of GM1-treated mice (n=28) became diabetic indicating that no effect was obtained by this glycosphingolipid. In two experiments in which less spleen cells were transferred (4-5 mill.) and glycosphingolipids were given 4 times, 35% of the sulfatide-treated animals (n = 17) developed diabetes compared to 85% of PBS-treated mice (n = 20, p < 0.001). A robust proliferative response to sulfatide, but none to GM1, was observed when spleen cells were rechallenged with glycosphingolipid in vitro. Thus, like insulin and GAD, sulfatide is able to prevent diabetes in NOD mice.

Topics: Adoptive Transfer; Animals; Autoantigens; Autoimmunity; Carbohydrate Sequence; Diabetes Mellitus, Type 1; Female; G(M1) Ganglioside; Galactosylceramides; Humans; Islets of Langerhans; Lymphocyte Activation; Mice; Mice, Inbred NOD; Molecular Sequence Data; Spleen; Sulfoglycosphingolipids

Topics: Autoantigens; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Female; G(M1) Ganglioside; Humans; Male; Middle Aged; Motor Neuron Disease

To elucidate the role of natural killer (NK) cells in the pathogenesis of diabetes in the non-obese diabetic (NOD) mouse, we examined whether or not cyclophosphamide-induced diabetes occurs in NOD mice intraperitoneally (i.p.) injected with anti-asialo GM1 antibody. Two weeks after a single intraperitoneal injection of cyclophosphamide, none of the 24 NOD mice which had previously been treated with antiasialo GM1 antibody, 2-3 times per week for either 2 or 3 weeks, had developed indications of diabetes such as glycosuria or a high plasma glucose level. On the other hand, signs of diabetes were found in 10 of 24 control NOD mice injected with normal rabbit Ig instead of anti-asialo GM1 antibody (p less than 0.01). The NK cell activities of spleen cells from anti-asialo GM1 antibody-treated mice were significantly lower than those of control mice (p less than 0.01). Flowcytometry analysis demonstrated that anti-asialo GM1 antibody-positive cells had disappeared from the spleens of anti-asialo GM1 antibody-injected mice but no suppression of CD8+ and CD4+ cells could be demonstrated. These observations suggest that NK cells are involved in the development of diabetes in NOD mice.

Topics: Animals; Antibodies, Anti-Idiotypic; Blood Glucose; Cyclophosphamide; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Female; G(M1) Ganglioside; Islets of Langerhans; Killer Cells, Natural; Mice; Mice, Inbred NOD; Spleen