Hyperglycemic NOD Research Articles

Reversal of Experimental Autoimmune Diabetes With an sCD39/Anti-CD3 Treatment.

Extracellular (e)ATP, a potent pro-inflammatory molecule, is released by dying/damaged cells at the site of inflammation and it is degraded by the membrane ectonucleotidases CD39 and CD73. In this study, we sought to unveil the role of eATP degradation in autoimmune diabetes, we then assessed the effect of soluble CD39 (sCD39) administration in prevention and reversal studies in NOD mice as well as in mechanistic studies. Our data showed that eATP levels were increased in hyperglycemic NODs as compared to pre-diabetic NODs. CD39 and CD73 were found expressed by both α- and β-cells and by different subsets of T-cells. Importantly, pre-diabetic NOD mice displayed increased frequencies of CD3+CD73+CD39+ cells within their pancreata, pLNs and spleens. The administration of sCD39 into pre-diabetic NODs reduced their eATP levels, abrogated the proliferation of CD4+- and CD8+-autoreactive T-cells and increased the frequency of Tregs; while delaying the onset of T1D. Notably, concomitant administration of sCD39 and anti-CD3 showed a strong synergism in restoring normoglycemia in newly hyperglycemic NOD mice as compared to monotherapy with anti-CD3 or with sCD39. The eATP/CD39 pathway plays an important role in the onset of T1D and its targeting might represent a potential therapeutic strategy in T1D.

Combination treatment of a novel CXCR3 antagonist ACT-777991 with an anti-CD3 antibody synergistically increases persistent remission in experimental models of type 1 diabetes.

Treatment of patients with recent-onset type 1 diabetes with an anti-CD3 antibody leads to the transient stabilization of C-peptide levels in responder patients. Partial efficacy may be explained by the entry of islet-reactive T-cells spared by and/or regenerated after the anti-CD3 therapy. The CXCR3/CXCL10 axis has been proposed as a key player in the infiltration of autoreactive T cells into the pancreatic islets followed by the destruction of β cells. Combining the blockade of this axis using ACT-777991, a novel small-molecule CXCR3 antagonist, with anti-CD3 treatment may prevent further infiltration and β-cell damage and thus, preserve insulin production. The effect of anti-CD3 treatment on circulating T-cell subsets, including CXCR3 expression, in mice was evaluated by flow cytometry. Anti-CD3/ACT-777991 combination treatment was assessed in the virally induced RIP-LCMV-GP and NOD diabetes mouse models. Treatments started at disease onset. The effects on remission rate, blood glucose concentrations, insulitis, and plasma C-peptide were evaluated for the combination treatment and the respective monotherapies. Anti-CD3 treatment induced transient lymphopenia but spared circulating CXCR3+ T cells. Combination therapy in both mouse models synergistically and persistently reduced blood glucose concentrations, resulting in increased disease remission rates compared to each monotherapy. At the study end, mice in disease remission demonstrated reduced insulitis and detectable plasma C-peptide levels. When treatments were initiated in non-severely hyperglycemic NOD mice at diabetes onset, the combination treatment led to persistent disease remission in all mice. These results provide preclinical validation and rationale to investigate the combination of ACT-777991 with anti-CD3 for the treatment of patients with recent-onset diabetes.

ICAM-1 Abundance Is Increased in Pancreatic Islets of Hyperglycemic Female NOD Mice and Is Rapidly Upregulated by NF-κB in Pancreatic β-Cells.

Type 1 diabetes (T1D) is classified as an autoimmune disease where pancreatic β-cells are specifically targeted by cells of the immune system. The molecular mechanisms underlying this process are not completely understood. Herein, we identified that the Icam1 gene and ICAM-1 protein were selectively elevated in female NOD mice relative to male mice, fitting with the sexual dimorphism of diabetes onset in this key mouse model of T1D. In addition, ICAM-1 abundance was greater in hyperglycemic female NOD mice than in age-matched normoglycemic female NOD mice. Moreover, we discovered that the Icam1 gene was rapidly upregulated in response to IL-1β in mouse, rat, and human islets and in 832/13 rat insulinoma cells. This early temporal genetic regulation requires key components of the NF-κB pathway and was associated with rapid recruitment of the p65 transcriptional subunit of NF-κB to corresponding κB elements within the Icam1 gene promoter. In addition, RNA polymerase II recruitment to the Icam1 gene promoter in response to IL-1β was consistent with p65 occupancy at κB elements, histone chemical modifications, and increased mRNA abundance. Thus, we conclude that β-cells undergo rapid genetic reprogramming by IL-1β to enhance expression of the Icam1 gene and that elevations in ICAM-1 are associated with hyperglycemia in NOD mice. These findings are highly relevant to, and highlight the importance of, pancreatic β-cell communication with the immune system. Collectively, these observations reveal a portion of the complex molecular events associated with onset and progression of T1D.

258-LB: ICAM-1 Abundance Is Increased in Pancreatic Islets of Hyperglycemic NOD Mice and Is Rapidly Upregulated by NF-kB in Pancreatic Beta Cells

Type 1 diabetes (T1D) is an autoimmune disease strongly associated with immune cell targeting of pancreatic β-cells, which ultimately produces insulin insufficiency. We identified the Icam1 gene and ICAM-1 protein, which facilitate interaction with immune cells, as elevated in female NOD mice relative to male mice. This observation is consistent with a greater frequency of diabetes onset in female mice in this well-established model of T1D. In addition, ICAM-1 protein abundance was greater in hyperglycemic female NOD compared with age-matched normoglycemic female NOD mice. Upon examining islet β-cell responses to immune cell-derived cytokines, we discovered that the Icam1 gene was rapidly (within 3h) and strongly (> 20-fold) upregulated in mouse, rat, and human islets as well as 832/13 rat insulinoma cells in response to IL-1β (p < 0.01) . Using chromatin immunoprecipitation (ChIP) assays, we identified rapid recruitment (within 30 minutes) of the p65 transcriptional subunit of NF-κB to κB genomic elements within the Icam1 gene promoter (3-fold; p < 0.05 relative to isotype control) . ChIP assays also revealed elevated RNA polymerase II recruitment to the Icam1 gene promoter (within 30 min) in response to IL-1β (7-fold, p < 0.05) . Finally, using single-cell RNA-Sequencing, we discovered that only a few clusters of β-cells expressed Icam1 at very low abundance in the basal (unstimulated) condition, while the majority of β-cell clusters in the IL-1β (3 h) stimulated condition presented with strong Icam1 positive (mRNA expressing) cells. Thus, we conclude that most, if not all, β-cells undergo rapid genetic reprogramming by IL-1β to enhance expression of the Icam1 gene and promote accumulation of the ICAM-1 protein. These findings illustrate the importance of pancreatic β-cell communication with the immune system and reveal inducible events at the β-cell level that are linked with onset and progression of T1D. Disclosure T. Martin: None. S. Burke: None. S. Ghosh: None. M. D. Karlstad: None. J. Collier: None. Funding National Institutes of Health (R03 AI151920-02)

GABAB-Receptor Agonist-Based Immunotherapy for Type 1 Diabetes in NOD Mice.

Some immune system cells express type A and/or type B γ-aminobutyric acid receptors (GABAA-Rs and/or GABAB-Rs). Treatment with GABA, which activates both GABAA-Rs and GABAB-Rs), and/or a GABAA-R-specific agonist inhibits disease progression in mouse models of type 1 diabetes (T1D), multiple sclerosis, rheumatoid arthritis, and COVID-19. Little is known about the clinical potential of specifically modulating GABAB-Rs. Here, we tested lesogaberan, a peripherally restricted GABAB-R agonist, as an interventive therapy in diabetic NOD mice. Lesogaberan treatment temporarily restored normoglycemia in most newly diabetic NOD mice. Combined treatment with a suboptimal dose of lesogaberan and proinsulin/alum immunization in newly diabetic NOD mice or a low-dose anti-CD3 in severely hyperglycemic NOD mice greatly increased T1D remission rates relative to each monotherapy. Mice receiving combined lesogaberan and anti-CD3 displayed improved glucose tolerance and, unlike mice that received anti-CD3 alone, had some islets with many insulin+ cells, suggesting that lesogaberan helped to rapidly inhibit β-cell destruction. Hence, GABAB-R-specific agonists may provide adjunct therapies for T1D. Finally, the analysis of microarray and RNA-Seq databases suggested that the expression of GABAB-Rs and GABAA-Rs, as well as GABA production/secretion-related genes, may be a more common feature of immune cells than currently recognized.

The expression of Tim-1 and Tim-4 molecules in regulatory T cells in type 1 diabetes

The TIM family comprises of eight genes in the mouse, three of which are conserved in humans (TIM-1, TIM-3, and TIM-4). Previous studies have revealed the relationships between Tim3+ Tregs and autoimmune disease. There was little study about the expression of Tim1 and Tim4 surface molecules on Tregs. We evaluated the frequency of the Tim1+Tregs and Tim4+Tregs in type 1 diabetes (T1D) in the present study. A total of 28 patients with T1D and 14 gender-, age-, and ethnically matched healthy volunteers were recruited. PBMCs from these individuals were isolated and analyzed by flow cytometry. Splenocytes from mice were also analyzed by flow cytometry. There is no difference in the frequency of Treg cells in peripheral blood isolated from T1D patients. Tim1 on CD4+CD25+ T cells decreased significantly in PBMC of patients with T1D(1.19 ± 0.17% vs 2.78 ± 0.38%, 95% CI:0.87-2.31, P < 0.0001), while expression of Tim4 on CD4+CD25+ T cells in PBMC was less frequent in patients with T1D than healthy people(3.0 ± 0.39% vs 6.25 ± 1.08%, 95% CI:1.08-5.43, P = 0.0044). The frequencies of CD4+CD25+Tim1+ T cells and CD4+CD25+Tim4+ T cells also decreased in spleen of hyperglycemic NOD mice. There were no significant correlations between CD4+CD25+Tim1+T-cells, CD4+CD25+Tim4+T-cells and any clinical features such as age, HbA1c, Fasting C-peptide, diabetic autoantibodies, disease duration, total cholesterol, LDL, HDL, and TG. It is the first report of the expression of Tim1 and Tim4 molecules on Treg cells in T1D in the present study. We also presented evidence that the frequencies of Tim1+Tregs and Tim4+Tregs decreased significantly in both type 1 diabetic patients and hyperglycemic NOD mice. However, the specific functions of Tim1+Tregs and Tim4+Tregs are still unclear.

Homotaurine Treatment Enhances CD4+ and CD8+ Regulatory T Cell Responses and Synergizes with Low-Dose Anti-CD3 to Enhance Diabetes Remission in Type 1 Diabetic Mice.

Immune cells express γ-aminobutyric acid receptors (GABA-R), and GABA administration can inhibit effector T cell responses in models of autoimmune disease. The pharmacokinetic properties of GABA, however, may be suboptimal for clinical applications. The amino acid homotaurine is a type A GABA-R (GABAA-R) agonist with good pharmacokinetics and appears safe for human consumption. In this study, we show that homotaurine inhibits in vitro T cell proliferation to a similar degree as GABA but at lower concentrations. In vivo, oral homotaurine treatment had a modest ability to reverse hyperglycemia in newly hyperglycemic NOD mice but was ineffective after the onset of severe hyperglycemia. In severely diabetic NOD mice, the combination of homotaurine and low-dose anti-CD3 treatment significantly increased 1) disease remission, 2) the percentages of splenic CD4+ and CD8+ regulatory T cells compared with anti-CD3 alone, and 3) the frequencies of CD4+ and CD8+ regulatory T cells in the pancreatic lymph nodes compared with homotaurine monotherapy. Histological examination of their pancreata provided no evidence of the large-scale GABAA-R agonist–mediated replenishment of islet β-cells that has been reported by others. However, we did observe a few functional islets in mice that received combined therapy. Thus, GABAA-R activation enhanced CD4+ and CD8+ regulatory T cell responses following the depletion of effector T cells, which was associated with the preservation of some functional islets. Finally, we observed that homotaurine treatment enhanced β-cell replication and survival in a human islet xenograft model. Hence, GABAA-R agonists, such as homotaurine, are attractive candidates for testing in combination with other therapeutic agents in type 1 diabetes clinical trials.

Mo1930 - Nature and the Site of Impairment of the Inhibitory Neuromuscular Transmission in the Stomach of Hyperglycemic Nod Mice

Mo1930 - Nature and the Site of Impairment of the Inhibitory Neuromuscular Transmission in the Stomach of Hyperglycemic Nod Mice

Characterization of Diabetogenic CD8+ T Cells: IMMUNE THERAPY WITH METABOLIC BLOCKADE

Type 1 diabetes mellitus is caused by the killing of insulin-producing β cells by CD8+T cells. The disease progression, which is chronic, does not follow a course like responses to conventional antigens such as viruses, but accelerates as glucose tolerance deteriorates. To identify the unique features of the autoimmune effectors that may explain this behavior, we analyzed diabetogenic CD8+ T cells that recognize a peptide from the diabetes antigen IGRP (NRP-V7-reactive) in prediabetic NOD mice and compared them to others that shared their phenotype (CD44(+)CD62L(lo)PD-1(+)CXCR3(+)) but negative for diabetes antigen tetramers and to LCMV (lymphocytic choriomeningitis)-reactive CD8+ T cells. There was an increase in the frequency of the NRP-V7-reactive cells coinciding with the time of glucose intolerance. The T cells persisted in hyperglycemic NOD mice maintained with an insulin pellet despite destruction of β cells. We compared gene expression in the three groups of cells compared with the other two subsets of cells, and the NRP-V7-reactive cells exhibited gene expression of memory precursor effector cells. They had reduced cellular proliferation and were less dependent on oxidative phosphorylation. When prediabetic NOD mice were treated with 2-deoxyglucose to block aerobic glycolysis, there was a reduction in the diabetes antigen versus other cells of similar phenotype and loss of lymphoid cells infiltrating the islets. In addition, treatment of NOD mice with 2-deoxyglucose resulted in improved β cell granularity. These findings identify a link between metabolic disturbances and autoreactive T cells that promotes development of autoimmune diabetes.

Targeting CD44 ameliorates autoimmune diabetes by reducing pancreatic infiltrates and improving Treg efficacy (P5180)

Abstract In type 1 diabetes (T1D), Th1 cells are a major population orchestrating the disease pathology. We showed previously that signals through CD44 are needed for Th1 cell survival and memory development. Therefore, we hypothesized that blocking CD44 could impact progression to overt diabetes. To test this, we treated recently hyperglycemic NOD mice with a short course of anti-CD44 and found a significant delay in the spontaneous development of TID that was associated with a reduction in islet infiltrates. However, previous studies suggested that targeting CD44 might enhance Treg function. To test whether this could contribute to the anti-CD44-mediated effect, we transferred polyclonal in vitro differentiated adaptive Treg cells (aTregs), which we showed can protect ~50% of newly hyperglycemic mice from overt diabetes, together with anti-CD44 treatment. We found a significantly increased the success rate of diabetes prevention. The islet infiltrates in these mice had a higher frequency of CD4+ cells producing IL-10 and IL-2 as well as of endogenous Tregs that displayed enhanced FoxP3 expression. However, anti-CD44 treatment did not alter cytokine profiles or Foxp3 expression of the transferred aTregs. The data therefore suggest that anti-CD44 treatment can alleviate inflammation in the target organ, and can functionally enhance the endogenous Treg population, thereby improving the establishment of long-term control by aTregs, which form a protective memory population.

Transient B-Cell Depletion with Anti-CD20 in Combination with Proinsulin DNA Vaccine or Oral Insulin: Immunologic Effects and Efficacy in NOD Mice

A recent type 1 diabetes (T1D) clinical trial of rituximab (a B cell-depleting anti-CD20 antibody) achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin) with anti-CD3 antibody (a T cell-directed immunomodulator) offers better protection than either entity alone, indicating that novel combination therapies that include a T1D-related autoantigen are possible. To accelerate the identification and development of novel combination therapies that can be advanced into the clinic, we have evaluated the combination of a mouse anti-CD20 antibody with either oral insulin or a proinsulin-expressing DNA vaccine. Anti-CD20 alone, given once or on 4 consecutive days, produced transient B cell depletion but did not prevent or reverse T1D in the NOD mouse. Oral insulin alone (twice weekly for 6 weeks) was also ineffective, while proinsulin DNA (weekly for up to 12 weeks) showed a trend toward modest efficacy. Combination of anti-CD20 with oral insulin was ineffective in reversing diabetes in NOD mice whose glycemia was controlled with SC insulin pellets; these experiments were performed in three independent labs. Combination of anti-CD20 with proinsulin DNA was also ineffective in diabetes reversal, but did show modest efficacy in diabetes prevention (p = 0.04). In the prevention studies, anti-CD20 plus proinsulin resulted in modest increases in Tregs in pancreatic lymph nodes and elevated levels of proinsulin-specific CD4+ T-cells that produced IL-4. Thus, combination therapy with anti-CD20 and either oral insulin or proinsulin does not protect hyperglycemic NOD mice, but the combination with proinsulin offers limited efficacy in T1D prevention, potentially by augmentation of proinsulin-specific IL-4 production.

Inotuzumab Ozogamicin Murine Analog–Mediated B-Cell Depletion Reduces Anti-islet Allo- and Autoimmune Responses

B cells participate in the priming of the allo- and autoimmune responses, and their depletion can thus be advantageous for islet transplantation. Herein, we provide an extensive study of the effect of B-cell depletion in murine models of islet transplantation. Islet transplantation was performed in hyperglycemic B-cell–deficient(μMT) mice, in a purely alloimmune setting (BALB/c into hyperglycemic C57BL/6), in a purely autoimmune setting (NOD.SCID into hyperglycemic NOD), and in a mixed allo-/autoimmune setting (BALB/c into hyperglycemic NOD). Inotuzumab ozogamicin murine analog (anti-CD22 monoclonal antibody conjugated with calicheamicin [anti-CD22/cal]) efficiently depleted B cells in all three models of islet transplantation examined. Islet graft survival was significantly prolonged in B-cell–depleted mice compared with control groups in transplants of islets from BALB/c into C57BL/6 (mean survival time [MST]: 16.5 vs. 12.0 days; P = 0.004), from NOD.SCID into NOD (MST: 23.5 vs. 14.0 days; P = 0.03), and from BALB/c into NOD (MST: 12.0 vs. 5.5 days; P = 0.003). In the BALB/c into B-cell–deficient mice model, islet survival was prolonged as well (MST: μMT = 32.5 vs. WT = 14 days; P = 0.002). Pathology revealed reduced CD3+ cell islet infiltration and confirmed the absence of B cells in treated mice. Mechanistically, effector T cells were reduced in number, concomitant with a peripheral Th2 profile skewing and ex vivo recipient hyporesponsiveness toward donor-derived antigen as well as islet autoantigens. Finally, an anti-CD22/cal and CTLA4-Ig–based combination therapy displayed remarkable prolongation of graft survival in the stringent model of islet transplantation (BALB/c into NOD). Anti-CD22/cal–mediated B-cell depletion promotes the reduction of the anti-islet immune response in various models of islet transplantation.

Synergistic Reversal of Type 1 Diabetes in NOD Mice With Anti-CD3 and Interleukin-1 Blockade

Inflammatory cytokines are involved in autoimmune diabetes: among the most prominent is interleukin (IL)-1β. We postulated that blockade of IL-1β would modulate the effects of anti-CD3 monoclonal antibody (mAb) in treating diabetes in NOD mice. To test this, we treated hyperglycemic NOD mice with F(ab′)2 fragments of anti-CD3 mAb with or without IL-1 receptor antagonist (IL-1RA), or anti–IL-1β mAb. We studied the reversal of diabetes and effects of treatment on the immune system. Mice that received a combination of anti-CD3 mAb with IL-1RA showed a more rapid rate of remission of diabetes than mice treated with anti-CD3 mAb or IL-1RA alone. Combination-treated mice had increased IL-5, IL-4, and interferon (IFN)-γ levels in circulation. There were reduced pathogenic NOD-relevant V7 peptide-V7+ T cells in the pancreatic lymph nodes. Their splenocytes secreted more IL-10, had increased arginase expression in macrophages and dendritic cells, and had delayed adoptive transfer of diabetes. After 1 month, there were increased concentrations of IgG1 isotype antibodies and reduced intrapancreatic expression of IFN-γ, IL-6, and IL-17 despite normal splenocyte cytokine secretion. These studies indicate that the combination of anti-CD3 mAb with IL-1RA is synergistic in reversal of diabetes through a combination of mechanisms. The combination causes persistent remission from islet inflammation.

Reduced Neutrophil Apoptosis in Diabetic Mice during Staphylococcal Infection Leads to Prolonged Tnfα Production and Reduced Neutrophil Clearance

Diabetes is a frequent underlying medical condition among individuals with Staphylococcus aureus infections, and diabetic patients often suffer from chronic inflammation and prolonged infections. Neutrophils are the most abundant inflammatory cells during the early stages of bacterial diseases, and previous studies have reported deficiencies in neutrophil function in diabetic hosts. We challenged age-matched hyperglycemic and normoglycemic NOD mice intraperitoneally with S. aureus and evaluated the fate of neutrophils recruited to the peritoneal cavity. Neutrophils were more abundant in the peritoneal fluids of infected diabetic mice by 48 h after bacterial inoculation, and they showed prolonged viability ex vivo compared to neutrophils from infected nondiabetic mice. These differences correlated with reduced apoptosis of neutrophils from diabetic mice and were dependent upon the presence of S. aureus and a functional neutrophil respiratory burst. Decreased apoptosis correlated with impaired clearance of neutrophils by macrophages both in vitro and in vivo and prolonged production of proinflammatory tumor necrosis factor alpha by neutrophils from diabetic mice. Our results suggest that defects in neutrophil apoptosis may contribute to the chronic inflammation and the inability to clear staphylococcal infections observed in diabetic patients.

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.

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.

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.