Hyperglycemia In NOD Mice Research Articles

A Novel Clinically Relevant Strategy to Abrogate Autoimmunity and Regulate Alloimmunity in NOD Mice

OBJECTIVETo investigate a new clinically relevant immunoregulatory strategy based on treatment with murine Thymoglobulin mATG Genzyme and CTLA4-Ig in NOD mice to prevent allo- and autoimmune activation using a stringent model of islet transplantation and diabetes reversal.RESEARCH DESIGN AND METHODSUsing allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig.RESULTSBALB/c islets transplanted into hyperglycemic NOD mice under prolonged mATG+CTLA4-Ig treatment showed a pronounced delay in allograft rejection compared with untreated mice (mean survival time: 54 vs. 8 days, P < 0.0001). Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment. The striking effect on autoimmunity was confirmed by 100% diabetes reversal in newly hyperglycemic NOD mice and 100% indefinite survival of syngeneic islet transplantation (NOD.SCID into NOD mice).CONCLUSIONSThe capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.

Change you can B(cell)eive in: recent progress confirms a critical role for B cells in type 1 diabetes

Here we review extant recent findings regarding the multiple roles of B cells in type 1 diabetes (T1D) and discuss how autoreactive B cells may become activated by a breach in B cell tolerance, and thereby initiate disease. Finally, we discuss the use of B cell-targeted therapies for treatment of autoimmunity. Anti-CD20-specific depletion of B cells prevents and reverses diabetes in human CD20/non-obese diabetic (NOD) mice. Correspondingly, in nontransgenic NOD mice, B cells are effectively depleted with high dose antimouse CD20 mAbs of varying isotypes, and this also prevents diabetes in more than 60% of the mice when administered early, and significantly delays disease in 15-week-old animals. A separate study revealed that targeting B cells with anti-CD22/cal monoclonal antibody therapy delays diabetes onset in prediabetic NOD mice and restores normoglycemia in new-onset hyperglycemic NOD mice. In humans, a clinical trial of rituximab in new onset type 1 diabetics has yielded promising preliminary findings. B cells are major players in T1D in humans, and clearly essential for disease development in the NOD mouse model of T1D. In this review, we discuss the silencing of autoreactive B cells and how failure of this process may contribute to autoimmunity. Further, we describe the most recent advances in studies of therapeutic effects of B cell depletion in T1D, and provide recent data indicating the diverse functions by which B cells may mediate disease.

A vasoprotective role for free‐radical scavenger therapies in chronically hyperglycemic mice

The presence of a hydrodynamically relevant endothelial‐cell surface glycocalyx, ~ 0.5 µm thick, is well established under normal physiological conditions. This layer is partially degraded by light‐dye treatment and completely degraded following hyaluronidase treatment. Many complications of diabetes are derived from vascular disease, and the glycocalyx is situated as the first line of defense against vascular damage. Therefore, we seek to establish the effects of hyperglycemia on the glycocalyx and the effectiveness of various therapies. We analyzed data obtained in venules of chronically hyperglycemic NOD mice receiving subsistence levels of insulin therapy, half of which were also treated with synthetic catalytic free radical scavengers (SOD). In mice receiving insulin only, we observed that the glycocalyx thickness is degraded to ~ 0.21 µm (n = 10), compared with ~ 0.48 µm (n = 13) in healthy wild type and NOD mice. This degradation is significant (p &lt; 0.05) and is comparable to that seen after free‐radical damage. In mice also treated with SOD, the glycocalyx thickness was ~ 0.39 µm (n = 10); this is not significantly different from the healthy control group. Furthermore, we seek to link glycocalyx degradation to vascular pathology. Preliminary data suggest that chronic enzymatic degradation results in pathological levels of microalbuminuria, an early indicator of kidney dysfunction due to vascular damage.

Suppression of Autoimmune Diabetes by Soluble Galectin-1

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that targets the beta-cells of the pancreas. We investigated the ability of soluble galectin-1 (gal-1), an endogenous lectin that promotes T cell apoptosis, to down-regulate the T cell response that destroys the pancreatic beta-cells. We demonstrated that in nonobese diabetic (NOD) mice, gal-1 therapy reduces significantly the amount of Th1 cells, augments the number of T cells secreting IL-4 or IL-10 specific for islet cell Ag, and causes peripheral deletion of beta-cell-reactive T cells. Administration of gal-1 prevented the onset of hyperglycemia in NOD mice at early and subclinical stages of T1D. Preventive gal-1 therapy shifted the composition of the insulitis into an infiltrate that did not invade the islets and that contained a significantly reduced number of Th1 cells and a higher percentage of CD4(+) T cells with content of IL-4, IL-5, or IL-10. The beneficial effects of gal-1 correlated with the ability of the lectin to trigger apoptosis of the T cell subsets that cause beta-cell damage while sparing naive T cells, Th2 lymphocytes, and regulatory T cells in NOD mice. Importantly, gal-1 reversed beta-cell autoimmunity and hyperglycemia in NOD mice with ongoing T1D. Because gal-1 therapy did not cause major side effects or beta-cell toxicity in NOD mice, the use of gal-1 to control beta-cell autoimmunity represents a novel alternative for treatment of subclinical or ongoing T1D.

Improved Efficacy of a Tolerizing DNA Vaccine for Reversal of Hyperglycemia through Enhancement of Gene Expression and Localization to Intracellular Sites

Insulin is a major target for the autoimmune-mediated destruction of pancreatic beta cells during the pathogenesis of type I diabetes. A plasmid DNA vaccine encoding mouse proinsulin II reduced the incidence of diabetes in a mouse model of type I diabetes when administered to hyperglycemic (therapeutic mode) or normoglycemic (prophylactic mode) NOD mice. Therapeutic administration of proinsulin DNA was accompanied by a rapid decrease in the number of insulin-specific IFN-gamma-producing T cells, whereas prophylactic treatment was accompanied by enhanced IFN-gamma-secreting cells and a decrease in insulin autoantibodies. Adoptive transfer experiments demonstrated that the protection was not mediated by induction of CD25(+)/CD4(+) T regulatory cells. The efficacy of the DNA vaccine was enhanced by increasing the level of expression of the encoded Ag, more frequent dosing, increasing dose level, and localization of the protein product to the intracellular compartment. The efficacy data presented in this study demonstrate that Ag-specific plasmid DNA therapy is a viable strategy for preventing progression of type I diabetes and defines critical parameters of the dosing regime that influences tolerance induction.

Combination therapy with glucagon-like peptide-1 and gastrin restores normoglycemia in diabetic NOD mice.

OBJECTIVE—Glucagon-like peptide-1 (GLP-1) and gastrin promote pancreatic β-cell function, survival, and growth. Here, we investigated whether GLP-1 and gastrin can restore the β-cell mass and reverse hyperglycemia in NOD mice with autoimmune diabetes.RESEARCH DESIGN AND METHODS—Acutely diabetic NOD mice were treated with GLP-1 and gastrin, separately or together, twice daily for 3 weeks. Blood glucose was measured weekly and for a further 5 weeks after treatments, after which pancreatic insulin content and β-cell mass, proliferation, neogenesis, and apoptosis were measured. Insulin autoantibodies were measured, and adoptive transfer of diabetes and syngeneic islet transplant studies were done to evaluate the effects of GLP-1 and gastrin treatment on autoimmunity.RESULTS—Combination therapy with GLP-1 and gastrin, but not with GLP-1 or gastrin alone, restored normoglycemia in diabetic NOD mice. The GLP-1 and gastrin combination increased pancreatic insulin content, β-cell mass, and insulin-positive cells in pancreatic ducts, and β-cell apoptosis was decreased. Insulin autoantibodies were reduced in GLP-1–and gastrin-treated NOD mice, and splenocytes from these mice delayed adoptive transfer of diabetes in NOD-scid mice. Syngeneic islet grafts in GLP-1–and gastrin-treated NOD mice were infiltrated by leukocytes with a shift in cytokine expression from interferon-γ to transforming growth factor-β1, and β-cells were protected from apoptosis.CONCLUSIONS—Combination therapy with GLP-1 and gastrin restores normoglycemia in diabetic NOD mice by increasing the pancreatic β-cell mass and downregulating the autoimmune response.

AAV8-mediated Gene Transfer of Interleukin-4 to Endogenous β-Cells Prevents the Onset of Diabetes in NOD Mice

We have demonstrated the ability to deliver and express genes specifically in beta-cells for at least 6 months, using a murine insulin promoter (mIP) in a double-stranded, self-complementary AAV vector (dsAAV8-mIP). In this study, we evaluated the effects of dsAAV8-mIP-mediated delivery of interleukin 4 (mIL-4) to endogenous beta-cells in nonobese diabetic (NOD) mice. In 4-week-old NOD mice, the extent of gene transfer and expression in endogenous beta-cells after ip delivery of dsAAV8-mIP-enhanced green fluorescent protein (eGFP) was comparable to normal BALB/C mice. Further, after IP delivery of dsAAV8-mIP-IL4, expression of mIL-4 was detected in islets isolated from the treated mice and cultured. AAV8-mIP-mediated gene expression of mIL-4 in endogenous beta- cells of 4- and 8-week-old NOD mice prevented the onset of hyperglycemia in NOD mice and reduced the severity of insulitis. Moreover, expression of mIL-4 also maintained the level of CD4(+)CD25(+)FoxP3(+) cells, and adoptive transfer of splenocytes from nondiabetic dsAAV8-mIP-IL-4 mice to NODscid mice was able to block the diabetes induced by splenocytes co-adoptively transferred from nondiabetic dsAAV-mIP-eGFP mice. Taken together, these results demonstrate that local expression of mIL-4 in islets prevents islet destruction and blocks autoimmunity, partly through regulation of T-cell function.

Using micro‐PIV to assess the role of insulin and free radical scavenger therapies in treating the effects of chronic hyperglycemia on the microvascular glycocalyx of NOD mice

The existence of a hydrodynamically relevant endothelial surface layer (ESL), ~ 0.5 μm thick in microvessels of wild‐type mouse, is well established under normal physiological conditions. Since nearly all chronic complications of diabetes are derived from vascular disease arising from hyperglycemia, and since the ESL is situated as the first line of defense against vascular damage, we seek to establish the effects of hyperglycemia on the ESL. We applied microviscometric analysis to data obtained using micro‐particle image velocimetry in cremaster‐muscle venules (20–60 μm diameter) of chronically hyperglycemic NOD mice. In mice without treatment, we observed that the ESL thickness is degraded to ~ 0.15 μm (n=20), compared with ~ 0.53 μm (n=23) in healthy wild‐type mice. This represents a significant degradation of the ESL (p&lt;0.05), comparable to that seen after free‐radical damage due to light‐dye treatment. In mice treated with insulin therapy (0.3 u/d LinBit implant) the ESL thickness is degraded to ~ 0.18 μm (n=5). This result is not significantly different from the untreated group. In mice treated with synthetic catalytic free radical scavengers (EUK‐207, Proteome Systems, Ltd.) in addition to insulin therapy, the ESL thickness is ~ 0.49 μm (n=5). This is significantly thicker than that found in either of the chronically hyperglycemic groups (p&lt;0.05) and not significantly different from the control group.

ICOS Mediates the Development of Insulin-Dependent Diabetes Mellitus in Nonobese Diabetic Mice

Initiation of diabetes in NOD mice can be mediated by the costimulatory signals received by T cells. The ICOS is found on Ag-experienced T cells where it acts as a potent regulator of T cell responses. To determine the function of ICOS in diabetes, we followed the course of autoimmune disease and examined T cells in ICOS-deficient NOD mice. The presence of ICOS was indispensable for the development of insulitis and hyperglycemia in NOD mice. In T cells, the deletion of ICOS resulted in a decreased production of the Th1 cytokine IFN-gamma, whereas the numbers of regulatory T cells remained unchanged. We conclude that ICOS is critically important for the induction of the autoimmune process that leads to diabetes.

Diabetes induces changes of catecholamines in primary mesangial cells

Diabetes mellitus is a frequent cause of kidney function damage with diabetic nephropathy being predominantly related to glomerular dysfunction. Diabetes is capable of interfering with distinct hormonal systems, as well as catecholamine metabolism. Since mesangial cells, the major constituent of renal glomerulus, constitute a potential site for catecholamine production, the present study was carried out to investigate alterations in catecholamine metabolism in cultured mesangial cells from the nonobese diabetic mouse, a well-established model for type I diabetes. We evaluated mesangial cells from normoglycemic and hyperglycemic nonobese diabetic mice, as well as cells from normoglycemic Swiss mice as control. Mesangial cells from normoglycemic mice presented similar profiles concerning all determinations. However, cells isolated from hyperglycemic animals presented increased dopamine and norepinephrine production/secretion. Among the studied mechanisms, we observed an upregulation of tyrosine hydroxylase expression accompanied by increased tetrahydrobiopterin consumption, the tyrosine hydroxylase enzymatic cofactor. However, this increase in synthetic pathways was followed by decreased monoamine oxidase activity, which corresponds to the major metabolic pathway of catecholamines in mesangial cells. In addition, whole kidney homogenates from diabetic animals also presented increased dopamine and norepinephrine levels when compared to normoglycemic animals. Thus, our results suggest that diabetes alters catecholamine production by interfering with both synthesizing and degrading enzymes, suggesting a possible role of catecholamine in the pathogenesis of acute and chronic renal complications of diabetes mellitus.

262. Suppression of Hyperglycemia in Nod Mice after Inoculation with Recombinant Vaccinia Viruses

262. Suppression of Hyperglycemia in Nod Mice after Inoculation with Recombinant Vaccinia Viruses

ARALAST or ANTI-TNF-a TREATMENT PREVENTS AUTOIMMUNE DESTRUCTION OF ISLET GRAFTS IN NONOBESE DIABETIC (NOD) MICE (131.23)

Abstract The onset hyperglycemia in NOD mice coincides with loss of a critical portion of the islet cell mass. Thus, restoration of a portion of the lost islet cell mass should be sufficient to create euglycemia if a deficit in insulin producing tissue is the sole cause of disease. It is notable that many potent T-cell tolerizing therapies have failed to enable restoration of euglycemia in new onset T1D NOD mice with or without transplantation of islets. Hence, we have searched for other potentially complicating features, e.g. insulin resistance, that may serve as an additional barrier to restoration of euglycemia. We now report that new onset T1D is associated with inflammation induced insulin resistance created by faulty phosphorylation of crucial elements of the insulin signaling pathway (insulin receptor and IRS-1). To test the hypotheses that inflammation, insulin resistance and T1DM disease expression are linked we have tested the effects of select anti-inflammatory agents in T1DM NOD mice. We now report that treatment for 15 days with alpha-1-antitrypsin (AAT) or 20 days with anti-TNF-a (i) permanently restores a euglycemic state in 14 of 16 or 22 of 24 treated new onset diabetic NOD mice (ii) ablated insulin resistance and (iii) produces specific tolerance to syngeneic, not acceptance of allogeneic, islets that is manifest long after cessation of treatment of new onset diabetic NODs while preserving other immune responses. In short, the restoration of euglycemia, self-tolerance, and of the faulty insulin receptors and IRS- 1 phosphorylation patterns can be corrected by treatment with ATT, an acute phase reactant with potent anti-inflammatory activity, and/or anti-TNF-a.

Bromocriptine-induced hyperglycemia in nonobese diabetic mice: kinetics and mechanisms of action.

The effects of bromocriptine (10 mg/kg), known to inhibit prolactin secretion and lower autoimmune processes, were studied on glucose homeostasis in non-fasted non-obese diabetic mice, a spontaneous model of type 1 diabetes. Hyperglycemia was observed 120 and 240 min after i.p. but not s.c. injection. Bromocriptine administration i.p. led to rapid and marked hyperglycemia characterized by sexual dimorphism with males having higher glycemia than females. Bromocriptine induced a rapid but transient decrease in insulinemia in males only and biphasic increases in glucagon levels and a sustained stimulatory effect on circulating corticosterone in both sexes. Bromocriptine-induced hyperglycemia involved D2-dopaminergic receptors, as demonstrated by the inhibitory effect of the D2-dopamine antagonist, metoclopramide (10 mg/kg). Simultaneous injection of bromocriptine and metoclopramide also blocked the rise in blood corticosterone. In conclusion, by inducing hyperglycemia, i.p. bromocriptine administration to prediabetic autoimmune mice may counteract its beneficial anti-immunostimulatory effects.

Glycated Fibroblast Growth Factor-2 Is Quickly Producedin Vitroupon Low-Millimolar Glucose Treatment and Detectedin Vivoin Diabetic Mice

Angiogenesis impairment in hyperglycemic patients represents a leading cause of severe vascular complications of both type-1 and -2 diabetes mellitus (DM). Angiogenesis dysfunction in DM is related to glycemic control; however, molecular mechanisms involved are still unclear. Fibroblast growth factor-2 (FGF-2) is a potent angiogenic factor and, according to previous evidence, may represent a key target of molecular modifications triggered by high-sugar exposure. Therefore, the purpose of this study was to investigate whether short incubation with hyperglycemic levels of glucose affected FGF-2 and whether glucose-modified FGF-2 was detectable in vivo. Biochemical analyses carried out with SDS-PAGE, fluorescence emission, mass-spectrometry, immunoblot, and competitive ELISA experiments demonstrated that human FGF-2 undergoes a rapid and specific glycation upon 12.5-50 mm glucose exposure. In addition, FGF-2 exposed for 30 min to 12.5 mm glucose lost mitogenic and chemotactic activity in a time- and dose-dependent manner. Under similar conditions, binding affinity to FGF receptor 1 was dramatically reduced by 20-fold, as well as FGF receptor 1 and ERK-1/2 phosphorylation, and FGF-2 lost about 45% of angiogenic activity in two different in vivo angiogenic (Matrigel and chorioallantoic-membrane) assays. Such glucose-induced modification was specific, because other angiogenic growth factors, namely platelet-derived growth factor BB and placental-derived growth factor were not significantly or markedly less modified. Finally, for the first time, glycated-FGF-2 was detected in vivo, in tissues from hyperglycemic nonobese diabetic mice, in significantly higher amounts than in normoglycemic mice. In conclusion, hyperglycemic levels of glucose may strongly affect FGF-2 structure and impair its angiogenic features, and endogenous glycated-FGF-2 is present in diabetic mice, indicating a novel pathogenetic mechanism underlying angiogenesis defects in DM.

Preventive and Therapeutic Potential of p38α-Selective Mitogen-Activated Protein Kinase Inhibitor in Nonobese Diabetic Mice with Type 1 Diabetes

Mitogen-activated protein kinases (MAPKs) and heat shock proteins (HSPs) are ubiquitous proteins that function within T cells in both normal and stress-related pathophysiological states, including type 1 diabetes. The nonobese diabetic (NOD) mouse spontaneously develops T cell-mediated autoimmune pancreatic beta cell destruction that is similar to type 1 diabetes in humans. Because p38 MAPKs have been shown to modulate T cell function, we studied the effects of a p38alpha MAPK-selective inhibitor, indole-5-carboxamide (SD-169), on the development and progression of type 1 diabetes in the NOD mouse. In preventive treatment studies, SD-169 significantly reduced p38 and HSP60 expression in T cells of the pancreatic beta islets. Following treatment, the incidence of diabetes as determined by blood glucose levels was significantly lower, and immuno-histochemistry of pancreatic beta islet tissue demonstrated significant reduction in CD5+ T cell infiltration in the SD-169 treatment group as compared with untreated NOD mice. In therapeutic studies using mildly and moderately hyperglycemic NOD mice, SD-169 treatment lowered blood glucose and improved glucose homeostasis. Furthermore, following cessation of SD-169 treatment, NOD mice showed significant arrest of diabetes. In conclusion, we report that this p38alpha-selective inhibitor prevents the development and progression of diabetes in NOD mice by inhibiting T cell infiltration and activation, thereby preserving beta cell mass via inhibition of the p38 MAPK signaling pathway. These results have bearing on current prophylactic and therapeutic protocols using p38alpha-selective inhibitors in the prediabetic period for children at high risk of type 1 diabetes, in the honeymoon period, and for adults with latent autoimmune diabetes.

Combination Therapy With Epidermal Growth Factor and Gastrin Increases β-Cell Mass and Reverses Hyperglycemia in Diabetic NOD Mice

Combination therapy with epidermal growth factor (EGF) and gastrin induces beta-cell regeneration in rodents with chemically induced diabetes. We investigated whether EGF plus gastrin could correct hyperglycemia in NOD mice with autoimmune diabetes. Combined treatment with EGF (1 mug/kg) and gastrin (3 mug/kg) for 2 weeks restored normoglycemia after diabetes onset in NOD mice, whereas EGF or gastrin alone did not. Fasting blood glucose remained normal (3.5-6.5 mmol/l) or mildly elevated (<11 mmol/l) in five of six mice (83%) for 10 weeks after EGF plus gastrin treatment was stopped, whereas all mice treated with vehicle or EGF or gastrin alone became severely hyperglycemic (12-35 mmol/l). Pancreatic beta-cell mass was increased threefold and insulin content was increased eightfold in mice treated with EGF plus gastrin compared with pretreatment values. The correction of hyperglycemia correlated significantly with increases in pancreatic beta-cell mass and insulin content. In addition, splenic cells from mice treated with EGF plus gastrin delayed diabetes induction by adoptive transfer of diabetogenic cells into immunodeficient NOD-scid mice, suggesting the induction of immunoregulatory cells in NOD mice treated with EGF plus gastrin. We conclude that a short course of combined EGF and gastrin therapy increases pancreatic beta-cell mass and reverses hyperglycemia in acutely diabetic NOD mice; the impact of this combined therapy may result from the effects of EGF and gastrin on beta-cells, immune cells, or both.

Receptor for Advanced Glycation End Products and Its Ligands: A Journey from the Complications of Diabetes to Its Pathogenesis

Many studies have suggested that the expression of RAGE (receptor for advanced glycation end products) is upregulated in human tissues susceptible to the long-term complications of diabetes. From the kidneys to the macrovessels of the aorta, RAGE expression is upregulated in a diverse array of cell types, from glomerular epithelial cells (podocytes) to endothelial cells, vascular smooth muscle cells, and inflammatory mononuclear phagocytes and lymphocytes. Although RAGE was first described as a receptor for advanced glycation end products (AGEs), the key finding that RAGE was also a signaling receptor for proinflammatory S100/calgranulins and amphoterin, led to the premise that even in euglycemia, ligand-RAGE interaction propagated inflammatory mechanisms linked to chronic cellular perturbation and tissue injury. Indeed, such considerations suggested that RAGE might even participate in the pathogenesis of type 1 diabetes. Our studies have shown that pharmacological and/or genetic deletion/mutation of the receptor attenuates the development of hyperglycemia in NOD mice; in mice with myriad complications of diabetes, interruption of ligand-RAGE interaction prevents or delays the chronic complications of the disease in both macro- and microvessel structures. Taken together, these findings suggest that RAGE is "at the right place and time" to contribute to the pathogenesis of diabetes and it complications. Studies are in progress to test the premise that antagonism of this interaction is a logical strategy for the prevention and treatment of diabetes.

Accelerated Functional Maturation of Isolated Neonatal Porcine Cell Clusters: In Vitro and In Vivo Results in NOD Mice

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/ differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the beta-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from < 3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were determined. Double fluorescence immunolabeling showed increase in beta-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional beta-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.

155. Oral Delivery of Recombinant Vaccinia Viruses Expressing Adjuvanted Islet Autoantigens Protects NOD Mice from Autoimmune Diabetes

Oral administration of autoantigens can delay or suppress clinical disease in experimental autoimmune disorders. However, repeated feeding of large tolerogen amounts is required over long periods and is only partially effective in animals systemically sensitized to the ingested antigen. The cholera toxin B subunit adjuvant (CTB) when linked to autoantigen epitopes acts as a strong adjuvant to enhance suppression of inflammatory Th1 cell reactivity in both naive and immune animals. Enhanced suppression of inflammatory Th1 cell reactivity was demonstrated in both naive and immune NOD mice following oral inoculation with islet autoantigens coupled to CTB. To stimulate adjuvant-autoantigen fusion protein biosynthesis in the gut mucosae, we evaluated oral inoculation of juvenile NOD mice with recombinant vaccinia virus (rVV) expressing fusion genes encoding CTB linked to the pancreatic islet autoantigens proinsulin (INS) and a 55 kDa peptide from glutamate decarboxylase (GAD55). Oral inoculation with rVV significantly reduced pancreatic insulitis. Hyperglycemia in NOD mice inoculated with CTB::GAD fusion protein was reduced by 40% in comparison with rVV-GAD inoculated mice. Measurement of antibody isotype titers in rVV infected mice suggested activation of autoantigen specific Th2 lymphocytes. These experiments demonstrate the feasibility of vaccinia virus mediated delivery of adjuvanted autoantigens to the mucosae of prediabetic mice for suppression of autoimmune diabetes.

Decreased opioid-induced antinociception but unaltered G-protein activation in the genetic-diabetic NOD mouse

Previous evaluation of antinociceptive action in experimental diabetes has been conducted almost exclusively in chemically induced diabetes mellitus. The purpose of the present study was to evaluate antinociceptive response and G-protein activation by μ-opioid receptor and δ-opioid receptor agonists in the genetic non-obese diabetic (NOD) mouse, a model of type I insulin-dependent diabetes mellitus (IDDM). Tail-flick latency before and after hyperglycemia was unaltered. Hyperglycemic NOD mice were hyporesponsive to intracerebroventricular (i.c.v.) injections of [ d-Ala 2]deltorphin II but not to [ d-Ala 2, N-MePhe 4, Gly-ol 5]enkephalin (DAMGO); however, G-protein activation in pons/medulla assessed by [ 35S]GTPγS binding was not diminished. This suggests that a G-protein defect in signaling cannot account for the hyporesponsiveness of antinociception in this genetic model of IDDM.