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1.  Prevention of Cyclophosphamide-induced Accelerated Diabetes in the NOD Mouse by Nicotinamide or a Soy Protein-based Infant Formula 
Spontaneous diabetes in the NOD mouse can be prevented by nicotinamide or by an infant formula diet in which the protein source is replaced with casein hydrolysate (Pregestimil) or soy protein (Prosobee). NOD mice maintained on the standard diet (chow and water) and given cyclophosphamide (Cy) at day 95 develop accelerated and synchronised diabetes within 14 days. Here, we compared the ability of oral nicotinamide or Prosobee, either given alone or concurrently, from weaning, in preventing diabetes in the Cy model. The resulting insulitis and the expression of intra-islet inducible nitric oxide synthase (iNOS) were examined at days 0, 4, 7, 11 and 14 following Cy administration. Intra-islet CD4 and CD8 cells and macrophages were also enumerated at day 11. In mice given the standard diet and injected with Cy at day 95 (group 5), diabetes developed in 7/11 mice, 14 days later. Mice exposed to oral nicotinamide (group 2), Prosobee (group 3) or both (group 4), did not develop the disease during this period and until a further 30 days (p = 0.03). In mice exposed to the standard diet and without Cy treatment (group 1) the insulitis scores increased slowly until day 11 and then declined slightly at day 14 whereas mice exposed to the same diet but given Cy at day 95, showed a sharp decline at day 4 followed by a rapid increase between day 7–14. However, in mice given either nicotinamide, Prosobee or both, the insulitis scores at most time-points were generally lower than in Cy-teated animals on the standard diet. In the latter group, CD4 and CD8 cells and macrophages were also higher at day 11 than all other 4 groups (CD4: p < 0.05; CD8: p < 0.05; macrophages: p < 0.0001). The number of iNOS labelled cells increased progressively in mice on the standard diet and given Cy and were significantly higher at days 4, 7 and 11 than in the 3 dietary groups. Thus, oral nicotinamide or Prosobee, either alone or together, prevents Cy induced diabetes in the NOD mouse. The protective diets suppress Cyinduced intra-islet immune cell influx and iNOS expression.
PMCID: PMC2477741  PMID: 11467420
2.  Expression of genetically determined diabetes and insulitis in the nonobese diabetic (NOD) mouse at the level of bone marrow-derived cells. Transfer of diabetes and insulitis to nondiabetic (NOD X B10) F1 mice with bone marrow cells from NOD mice 
The Journal of Experimental Medicine  1988;167(6):1801-1810.
The development of autoimmune diabetes in the nonobese diabetic (NOD) mouse is controlled by at least three recessive loci, including one linked to the MHC. To determine whether any of these genetic loci exert their effects via the immune system, radiation bone marrow chimeras were constructed in which (NOD X B10)F1-irradiated recipients were reconstituted with NOD bone marrow cells. Unmanipulated (NOD X B10)F1 mice, or irradiated F1 mice reconstituted with F1 or B10 bone marrow, did not display insulitis or diabetes. In contrast, insulitis was observed in a majority of the NOD----F1 chimeras and diabetes developed in 21% of the mice. These data demonstrate that expression of the diabetic phenotype in the NOD mouse is dependent on NOD-derived hematopoietic stem cells. Diabetogenic genes in the NOD mouse do not appear to function at the level of the insulin-producing beta cells since NOD----F1 chimeras not only developed insulitis and diabetes but also rejected beta cells within pancreas transplants from newborn B10 mice. These data suggest that the beta cells of the NOD mouse do not express a unique antigenic determinant that is the target of the autoimmune response.
PMCID: PMC2189678  PMID: 3290380
3.  Pancreatic Lymph Nodes Are Required for Priming of β Cell Reactive T Cells in NOD Mice 
Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that results from the destruction of insulin secreting β cells by diabetogenic T cells. The time and location of the encounter of autoantigen(s) by naive autoreactive T cells in normal NOD mice are still elusive. To address these issues, we analyzed diabetes development in mice whose spleen or pancreatic lymph nodes (panLNs) had been removed. Excision of panLNs (panLNx) at 3 wk protected mice against insulin autoantibodies (IAAs), insulitis, and diabetes development almost completely, but had no effect when performed at 10 wk. The protection afforded by panLNx at weaning was not due to modifications of the immune system, the absence of autoreactive T cells, or the increase in the potency of regulatory T cells. That panLNs are dispensable during adult life was confirmed by the capacity of 10-wk-old panLNx irradiated recipients to develop diabetes upon transfer of diabetogenic T cells. In contrast, splenectomy had no effect at any age. Partial excision of mesenteric LN at 3 wk did not prevent accelerated diabetes by cyclophosphamide as panLNx did. Thus, in normal NOD mice, autoreactive T cell initial priming occurs in LNs draining the target organ of the disease from 3 wk of age.
PMCID: PMC2193939  PMID: 12163565
insulin-dependent diabetes mellitus; lymph node excision; splenectomy; T cell activation; autoimmunity
4.  Requirement of Fas for the Development of Autoimmune Diabetes in Nonobese Diabetic Mice  
Insulin-dependent diabetes mellitus (IDDM) is assumed to be a T cell–mediated autoimmune disease. To investigate the role of Fas-mediated cytotoxicity in pancreatic β cell destruction, we established nonobese diabetic (NOD)-lymphoproliferation (lpr)/lpr mice lacking Fas. Out of three genotypes, female NOD-+/+ and NOD-+/lpr developed spontaneous diabetes by the age of 10 mo with the incidence of 68 and 62%, respectively. In contrast, NOD-lpr/lpr did not develop diabetes or insulitis. To further explore the role of Fas, adoptive transfer experiments were performed. When splenocytes were transferred from diabetic NOD, male NOD-+/+ and NOD-+/lpr developed diabetes with the incidence of 89 and 83%, respectively, whereas NOD-lpr/lpr did not show glycosuria by 12 wk after transfer. Severe mononuclear cell infiltration was revealed in islets of NOD-+/+ and NOD-+/lpr, whereas islet morphology remained intact in NOD-lpr/lpr. These results suggest that Fas-mediated cytotoxicity is required to initiate β cell autoimmunity in NOD mice. Fas–Fas ligand system might be critical for autoimmune β cell destruction leading to IDDM.
PMCID: PMC2199040  PMID: 9254659
5.  Restoration of impaired intestinal barrier function by the hydrolysed casein diet contributes to the prevention of type 1 diabetes in the diabetes-prone BioBreeding rat 
Diabetologia  2010;53(12):2621-2628.
Impaired intestinal barrier function is observed in type 1 diabetes patients and animal models of the disease. Exposure to diabetogenic antigens from the intestinal milieu due to a compromised intestinal barrier is considered essential for induction of the autoimmune process leading to type 1 diabetes. Since a hydrolysed casein (HC) diet prevents autoimmune diabetes onset in diabetes-prone (DP)-BioBreeding (BB) rats, we studied the role of the HC diet on intestinal barrier function and, therefore, prevention of autoimmune diabetes onset in this animal model.
DP-BB rats were fed the HC diet from weaning onwards and monitored for autoimmune diabetes development. Intestinal permeability was assessed in vivo by lactulose–mannitol test and ex vivo by measuring transepithelial electrical resistance (TEER). Levels of serum zonulin, a physiological tight junction modulator, were measured by ELISA. Ileal mRNA expression of Myo9b, Cldn1, Cldn2 and Ocln (which encode the tight junction-related proteins myosin IXb, claudin-1, claudin-2 and occludin) and Il-10, Tgf-ß (also known as Il10 and Tgfb, respectively, which encode regulatory cytokines) was analysed by quantitative PCR.
The HC diet reduced autoimmune diabetes by 50% in DP-BB rats. In DP-BB rats, prediabetic gut permeability negatively correlated with the moment of autoimmune diabetes onset. The improved intestinal barrier function that was induced by HC diet in DP-BB rats was visualised by decreasing lactulose:mannitol ratio, decreasing serum zonulin levels and increasing ileal TEER. The HC diet modified ileal mRNA expression of Myo9b, and Cldn1 and Cldn2, but left Ocln expression unaltered.
Improved intestinal barrier function might be an important intermediate in the prevention of autoimmune diabetes by the HC diet in DP-BB rats. Effects on tight junctions, ileal cytokines and zonulin production might be important mechanisms for this effect.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-010-1903-9) contains supplementary material, which is available to authorised users.
PMCID: PMC2974912  PMID: 20853098
BB rat; Cytokines; Hydrolysed casein diet; Intestinal barrier; Tight junctions; Type 1 diabetes
6.  Rotavirus Infection Accelerates Type 1 Diabetes in Mice with Established Insulitis▿  
Journal of Virology  2008;82(13):6139-6149.
Infection modulates type 1 diabetes, a common autoimmune disease characterized by the destruction of insulin-producing islet β cells in the pancreas. Childhood rotavirus infections have been associated with exacerbations in islet autoimmunity. Nonobese diabetic (NOD) mice develop lymphocytic islet infiltration (insulitis) and then clinical diabetes, whereas NOD8.3 TCR mice, transgenic for a T-cell receptor (TCR) specific for an important islet autoantigen, show more rapid diabetes onset. Oral infection of infant NOD mice with the monkey rotavirus strain RRV delays diabetes development. Here, the effect of RRV infection on diabetes development once insulitis is established was determined. NOD and NOD8.3 TCR mice were inoculated with RRV aged ≥12 and 5 weeks, respectively. Diabetes onset was significantly accelerated in both models (P < 0.024), although RRV infection was asymptomatic and confined to the intestine. The degree of diabetes acceleration was related to the serum antibody titer to RRV. RRV-infected NOD mice showed a possible trend toward increased insulitis development. Infected males showed increased CD8+ T-cell proportions in islets. Levels of β-cell major histocompatibility complex class I expression and islet tumor necrosis factor alpha mRNA were elevated in at least one model. NOD mouse exposure to mouse rotavirus in a natural experiment also accelerated diabetes. Thus, rotavirus infection after β-cell autoimmunity is established affects insulitis and exacerbates diabetes. A possible mechanism involves increased exposure of β cells to immune recognition and activation of autoreactive T cells by proinflammatory cytokines. The timing of infection relative to mouse age and degree of insulitis determines whether diabetes onset is delayed, unaltered, or accelerated.
PMCID: PMC2447104  PMID: 18417562
7.  Autoimmune syndromes in major histocompatibility complex (MHC) congenic strains of nonobese diabetic (NOD) mice. The NOD MHC is dominant for insulitis and cyclophosphamide-induced diabetes 
The development of autoimmune diabetes in the nonobese diabetic (NOD) mouse is controlled by multiple genes. At least one diabetogenic gene is linked to the major histocompatibility complex (MHC) of the NOD and is most likely represented by the two genes encoding the alpha and beta chains of the unique NOD class II molecule. Three other diabetogenic loci have recently been identified in the NOD mouse and are located on chromosomes 1, 3, and 11. In addition to the autoimmune diabetes which is caused by destruction of the insulin-producing beta cells in the pancreas, other manifestations of autoimmunity are seen in the NOD mouse. These include mononuclear cell inflammation of the submandibular and lacrimal glands, as well as the presence of circulating autoantibodies. To determine the effect of the non-MHC diabetogenic genes on the development of autoimmunity, we constructed the NOD.B10-H- 2b (NOD.H-2b) strain, which possesses the non-MHC diabetogenic genes from the NOD mouse, but derives its MHC from the C57BL/10 (B10) strain. The NOD.H-2b strain does not develop insulitis, cyclophosphamide- induced diabetes, or spontaneous diabetes. It does, however, develop extensive lymphocytic infiltrates in the pancreas and the submandibular glands that are primarily composed of Thy 1.2+ T cells and B220+ B cells. In addition, autoantibodies are present in NOD.H-2b mice which recognize the "polar antigen" on the insulin-secreting rat tumor line RINm38. These observations demonstrate that the non-MHC genes in the NOD strain, in the absence of the NOD MHC, significantly contribute to the development of autoimmunity. The contribution of a single dose of the NOD MHC to autoimmunity was assessed with a (NOD x NOD.H-2b)F1 cross. Although only approximately 3% of F1 females developed spontaneous diabetes, approximately 50% of both female and male F1 mice developed insulitis, and 25% of females and 17% of males became diabetic after treatment with cyclophosphamide. These data demonstrate that the MHC-linked diabetogenic genes of the NOD mouse are dominant with decreasing levels of penetrance for the following phenotypes: insulitis greater than cyclophosphamide-induced diabetes greater than spontaneous diabetes.
PMCID: PMC2119272  PMID: 1613467
8.  Pancreatic expression of interleukin-4 abrogates insulitis and autoimmune diabetes in nonobese diabetic (NOD) mice 
The Journal of Experimental Medicine  1996;184(3):1093-1099.
Diabetes in nonobese diabetic (NOD) mice is a T cell-dependent autoimmune disease. The destructive activities of autoreactive T cells have been shown to be tightly regulated by effector molecules. In particular, T helper (Th) 1 cytokines have been linked to diabetes pathogenesis, whereas Th2 cytokines and the cells that release them have been postulated to be protective from disease. To test this hypothesis, we generated transgenic NOD mice that express interleukin (IL) 4 in their pancreatic beta cells under the control of the human insulin promoter. We found that transgenic NOD-IL-4 mice, both females and males, were completely protected from insulitis and diabetes. Induction of functional tolerance to islet antigens in these mice was indicated by their inability to reject syngeneic pancreatic islets and the failure of diabetogenic spleen cells to induce diabetes in transgenic NOD-IL-4 recipients. Interestingly, however, islet expression of IL-4 was incapable of preventing islet rejection in overtly diabetic NOD recipient mice. These results demonstrate that the Th2 cytokine IL-4 can prevent the development of autoimmunity and destructive autoreactivity in the NOD mouse. Its ability to regulate the disease process in the periphery also indicates that autoimmune diabetes in NOD mice is not a systemic disease, and it can be modulated from the islet compartment.
PMCID: PMC2192796  PMID: 9064326
9.  Abrogation of autoimmune diabetes in nonobese diabetic mice and protection against effector lymphocytes by transgenic paracrine TGF-beta1. 
Journal of Clinical Investigation  1998;102(3):499-506.
Paracrine effect of transforming growth factor-beta1 (TGF-beta1) on autoimmune insulitis and diabetes was studied by transgenic production of the active form of porcine TGF-beta1 (pTGF-beta1) in pancreatic islet (islet) alpha cells in nonobese diabetic (NOD) mice under the control of rat glucagon promoter (RGP) (NOD-RGP-TGF-beta1). None of 27 NOD-RGP-TGF- beta1 mice developed diabetes by 45 wk of age, in contrast to 40 and 71% in male and female nontransgenic mice, respectively. None of the NOD-RGP-TGF-beta1 mice developed diabetes after cyclophosphamide (CY) administration. Adoptive transfer of splenocytes of NOD-RGP-TGF-beta1 mice to neonatal NOD mice did not transfer diabetes after CY administration. Adoptive transfer of three types of diabetogenic lymphocytes to NOD-RGP-TGF-beta1 and nontransgenic mice after CY administration led to the lower incidence of diabetes in NOD-RGP-TGF-beta1 mice versus that in nontransgenic mice: 29 vs. 77% for diabetogenic splenocytes, 25 vs. 75% for islet beta cell-specific Th1 clone cells, and 0 vs. 50% for islet beta cell-specific CD8(+) clone cells, respectively. Based on these, it is concluded that autoimmune diabetes in NOD mice is not a systemic disease and it can be completely prevented by the paracrine TGF-beta1 in the islet compartment through protection against CD4(+) and CD8(+) effector lymphocytes.
PMCID: PMC508910  PMID: 9691086
10.  T cell-mediated inhibition of the transfer of autoimmune diabetes in NOD mice 
The Journal of Experimental Medicine  1989;169(5):1669-1680.
The nonobese diabetic (NOD) mouse has recently been introduced as a model for insulin-dependent diabetes mellitus. The role of regulatory T cells in the development of antipancreatic autoimmunity in this model remains unclear. To evaluate the presence of suppressive phenomena, we used disease transfer by spleen cells from diabetic NOD mice into preirradiated adult recipients as a model for accelerated disease. Suppressor phenomena were detected by testing the protection afforded by lymphoid cells from nondiabetic NOD mice against diabetes transfer in irradiated recipients. Transfer of diabetes was delayed by reconstituting recipients with spleen cells from nondiabetic NOD donors. The greatest protection against diabetes transfer was conferred by spleen cells from 8-wk-old nondiabetic female NOD mice. Depletion experiments showed that the protection was dependent on CD4+ cells. Protection was also detected within thymic cells from nondiabetic NOD mice and protection conferred by spleen cells was abrogated by thymectomy of nondiabetic female, but not male, NOD donors at 3 wk of age. These findings indicate that suppressive CD4+ T cells that are dependent on the presence of the thymus may delay the onset of diabetes in female diabetes-prone NOD mice.
PMCID: PMC2189316  PMID: 2523954
11.  Immune Depletion With Cellular Mobilization Imparts Immunoregulation and Reverses Autoimmune Diabetes in Nonobese Diabetic Mice 
Diabetes  2009;58(10):2277-2284.
The autoimmune destruction of β-cells in type 1 diabetes results in a loss of insulin production and glucose homeostasis. As such, an immense interest exists for the development of therapies capable of attenuating this destructive process through restoration of proper immune recognition. Therefore, we investigated the ability of the immune-depleting agent antithymocyte globulin (ATG), as well as the mobilization agent granulocyte colony–stimulating factor (GCSF), to reverse overt hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes.
Effects of each therapy were tested in pre-diabetic and diabetic female NOD mice using measurements of glycemia, regulatory T-cell (CD4+CD25+Foxp3+) frequency, insulitis, and/or β-cell area.
Here, we show that combination therapy of murine ATG and GCSF was remarkably effective at reversing new-onset diabetes in NOD mice and more efficacious than either agent alone. This combination also afforded durable reversal from disease (>180 days postonset) in animals having pronounced hyperglycemia (i.e., up to 500 mg/dl). Additionally, glucose control improved over time in mice subject to remission from type 1 diabetes. Mechanistically, this combination therapy resulted in both immunological (increases in CD4-to-CD8 ratios and splenic regulatory T-cell frequencies) and physiological (increase in the pancreatic β-cell area, attenuation of pancreatic inflammation) benefits.
In addition to lending further credence to the notion that combination therapies can enhance efficacy in addressing autoimmune disease, these studies also support the concept for utilizing agents designed for other clinical applications as a means to expedite efforts involving therapeutic translation.
PMCID: PMC2750219  PMID: 19628781
12.  B lymphocytes are essential for the initiation of T cell-mediated autoimmune diabetes: analysis of a new "speed congenic" stock of NOD.Ig mu null mice 
The Journal of Experimental Medicine  1996;184(5):2049-2053.
The T lymphocytes mediating autoimmune destruction of pancreatic beta cells in the nonobese diabetic (NOD) mouse model of insulin-dependent diabetes mellitus (IDDM) may be generated due to functional defects in hematopoietically derived antigen-presenting cells (APC). However, it has not been clear which particular subpopulations of APC (B lymphocytes, macrophages, and dendritic cells) contribute to the development and activation of diabetogenic T cells in NOD mice. In the current study we utilized a functionally inactivated immunoglobulin (Ig) mu allele (Ig mu null) to generate a "speed congenic" stock of B lymphocyte-deficient NOD mice that are fixed for linkage markers delineating previously identified diabetes susceptibility (Idd) genes. These B lymphocyte NOD.Ig mu null mice had normal numbers of T cells but were free of overt IDDM and insulitis resistant, while the frequency of disease in the B lymphocyte intact segregants was equivalent to that of standard NOD mice in our colony. Thus, B lymphocytes play a heretofore unrecognized role that is essential for the initial development and/or activation of beta cell autoreactive T cells in NOD mice.
PMCID: PMC2192892  PMID: 8920894
13.  Pancreatic islet beta cells drive T cell-immune responses in the nonobese diabetic mouse model 
The Journal of Experimental Medicine  1995;181(5):1635-1642.
The role of autoantigens and that of target organs in which tissue lesions develop remains elusive in most spontaneous models of autoimmune diseases. Whether the presence of target autoantigens is required for the recruitment of autoreactive lymphocytes is unknown in most cases. To evaluate the importance of islet cells in the development of autoimmunity in the nonobese diabetic (NOD) mouse, we generated beta cell-deprived mice by injecting a high dose of alloxan, a toxic agent specific for beta cells. In contrast with spleen cells from 6-mo-old naive NOD mice which transfer diabetes in irradiated 8-mo- old male recipients, spleen cells from age-matched NOD mice which received a single injection of alloxan at 3 wk of age did not transfer diabetes. With the exception of the ability to transfer diabetes, beta cell-deprived NOD mice showed maintained immune competence. Furthermore, sialitis developed with the expected intensity and prevalence in beta cell-deprived mice. Already committed "diabetogenic" spleen cells collected from spontaneously diabetic mice also showed a reduced capacity to transfer diabetes after their removal from the diabetic mice and transient "parking" in beta cell-deprived mice. Taken together, our data bring evidence that involvement of autoreactive T cells detected by the capacity to transfer diabetes requires the presence of target beta cells.
PMCID: PMC2192008  PMID: 7722443
14.  Effect of tumor necrosis factor alpha on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process 
The Journal of Experimental Medicine  1994;180(3):995-1004.
Tumor necrosis factor (TNF) alpha is a cytokine that has potent immune regulatory functions. To assess the potential role of this cytokine in the early development of autoimmunity, we investigated the effect of TNF on the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice, a spontaneous murine model for autoimmune, insulin-dependent type I diabetes. Treatment of newborn female NOD mice with TNF every other day for 3 wk, led to an earlier onset of disease (10 versus 15 wk of age in control mice) and 100% incidence before 20 wk of age (compared to 45% at 20 wk of age in control phosphate-buffered saline treated female mice). In contrast, administration of an anti-TNF monoclonal antibody, TN3.19.12, resulted in complete prevention of IDDM. In vitro proliferation assays demonstrated that mice treated with TNF developed an increased T cell response to a panel of beta cell autoantigens, whereas anti-TNF treatment resulted in unresponsiveness to the autoantigens. In addition, autoantibody responses to the panel of beta cell antigens paralleled the T cell responses. The effects mediated by TNF appear to be highly age dependent. Treatment of animals either from birth or from 2 wk of age had a similar effect. However, if treatment was initiated at 4 wk of age, TNF delayed disease onset. These data suggest that TNF has a critical role in the early development of autoimmunity towards beta-islet cells.
PMCID: PMC2191653  PMID: 8064245
15.  Diabetes Acceleration or Prevention by a Coxsackievirus B4 Infection: Critical Requirements for both Interleukin-4 and Gamma Interferon 
Journal of Virology  2005;79(2):1045-1052.
Type 1 diabetes acceleration in nonobese diabetic (NOD) mice through coxsackievirus B4 (CVB4) infection requires a preexisting critical mass of autoreactive T cells in pancreatic islets, and in the absence of this insulitic threshold, CVB4 infection leads to long-term disease protection. To understand this acceleration and protection process, we challenged 8- and 12-week-old NOD mice containing a disruption in interleukin-4 (IL-4) or gamma interferon (IFN-γ) genes (NOD IL-4−/− and NOD IFN-γ−/−, respectively) with a diabetogenic, pancreatropic Edwards strain of CVB4. The elimination of IL-4 did not alter the rate of insulitis or diabetes development in NOD mice, while the elimination of IFN-γ delayed these events several weeks. CVB4 infection in 8-week-old mice only significantly accelerated the onset of diabetes in a subset of standard, but not IL-4- or IFN-γ-deficient, NOD mice. Long-term diabetes protection was established in standard NOD mice as well as in the NOD IFN-γ−/− mice that did not rapidly develop disease following CVB4 infection at 8 weeks of age. When mice were infected at 12 weeks of age, the onset of diabetes was accelerated in NOD IL-4−/− mice, while neither acceleration nor long-term protection was elicited in NOD IFN-γ−/− mice. No differences were observed in the kinetics of CVB4 clearance in pancreases from NOD, NOD IL-4−/−, and NOD IFN-γ−/− mice. Collectively, these results suggest that at the insulitis threshold at which CVB4 infection can first accelerate the onset of diabetes in NOD mice, IL-4 as well as IFN-γ contributes to this pathogenic process. The protective mechanism against diabetes elicited in NOD mice infected with CVB4 prior to the development of a critical threshold level of insulitis requires neither IL-4 nor IFN-γ.
PMCID: PMC538592  PMID: 15613333
16.  HLA-A2–Matched Peripheral Blood Mononuclear Cells From Type 1 Diabetic Patients, but Not Nondiabetic Donors, Transfer Insulitis to NOD-scid/γcnull/HLA-A2 Transgenic Mice Concurrent With the Expansion of Islet-Specific CD8+ T cells 
Diabetes  2011;60(6):1726-1733.
Type 1 diabetes is an autoimmune disease characterized by the destruction of insulin-producing β-cells. NOD mice provide a useful tool for understanding disease pathogenesis and progression. Although much has been learned from studies with NOD mice, increased understanding of human type 1 diabetes can be gained by evaluating the pathogenic potential of human diabetogenic effector cells in vivo. Therefore, our objective in this study was to develop a small-animal model using human effector cells to study type 1 diabetes.
We adoptively transferred HLA-A2–matched peripheral blood mononuclear cells (PBMCs) from type 1 diabetic patients and nondiabetic control subjects into transgenic NOD-scid/γcnull/HLA-A*0201 (NOD-scid/γcnull/A2) mice. At various times after adoptive transfer, we determined the ability of these mice to support the survival and proliferation of the human lymphoid cells. Human lymphocytes were isolated and assessed from the blood, spleen, pancreatic lymph node and islets of NOD-scid/γcnull/A2 mice after transfer.
Human T and B cells proliferate and survive for at least 6 weeks and were recovered from the blood, spleen, draining pancreatic lymph node, and most importantly, islets of NOD-scid/γcnull/A2 mice. Lymphocytes from type 1 diabetic patients preferentially infiltrate the islets of NOD-scid/γcnull/A2 mice. In contrast, PBMCs from nondiabetic HLA-A2–matched donors showed significantly less islet infiltration. Moreover, in mice that received PBMCs from type 1 diabetic patients, we identified epitope-specific CD8+ T cells among the islet infiltrates.
We show that insulitis is transferred to NOD-scid/γcnull/A2 mice that received HLA-A2–matched PBMCs from type 1 diabetic patients. In addition, many of the infiltrating CD8+ T cells are epitope-specific and produce interferon-γ after in vitro peptide stimulation. This indicates that NOD-scid/γcnull/A2 mice transferred with HLA-A2–matched PBMCs from type 1 diabetic patients may serve as a useful tool for studying epitope-specific T-cell–mediated responses in patients with type 1 diabetes.
PMCID: PMC3114397  PMID: 21521873
17.  Genetic control of diabetes and insulitis in the nonobese diabetic (NOD) mouse 
The Journal of Experimental Medicine  1987;165(6):1639-1654.
Genetic analysis of the development of diabetes and insulitis has been performed in the nonobese diabetic (NOD) mouse strain, a model of insulin-dependent (type I) diabetes mellitus. (NOD X C57BL/10)F1, F2, and (F1 X NOD) first-, second-, and third-backcross generations were studied. The data obtained were consistent with the hypothesis that diabetes is controlled by at least three functionally recessive diabetogenic genes, or gene complexes, one of which is linked to the MHC of the NOD. In contrast, pancreatic inflammation leading to insulitis was found to be controlled by a single incompletely dominant gene. One of the two diabetogenic loci that is not linked to the MHC appears to be essential for the development of severe insulitis. This diabetogenic gene may be identical to the gene that controls the initiation of the autoimmune response that progresses to insulitis. Although this gene appears to be functionally recessive in its control of diabetes, it is incompletely dominant in its control of insulitis. The MHC-linked diabetogenic gene, although not required for the development of insulitis, apparently influences the progression of the autoimmune response since NOD MHC homozygotes in the backcross generations displayed the highest incidence and most severe cases of insulitis. Interestingly, we have found two MHC heterozygous backcross females that have become diabetic, suggesting that either the MHC- linked diabetogenic gene is not strictly recessive or that a recombination event has occurred between the diabetogenic gene and the K or I-A regions of the MHC. The third diabetogenic locus appears to influence the progression of severe insulitis to overt diabetes. In animals homozygous at this locus, diabetes may result from a decreased ability to develop a protective suppressor response to the autoimmune process.
PMCID: PMC2188363  PMID: 3585250
18.  Androgen treatment prevents diabetes in nonobese diabetic mice 
The Journal of Experimental Medicine  1992;175(5):1409-1412.
The nonobese diabetic (NOD) mouse strain provides a model system for human autoimmune diabetes. This disease model is extensively used not only to examine the etiology and pathogenesis of diabetes, but also as a means to evaluate therapies. In NOD mice, the disease progresses from insulitis to islet destruction and clinical diabetes in a high percentage of female mice. In this study, androgen therapy, begun after the onset of insulitis, was found to prevent islet destruction and diabetes without eliminating the islet inflammation in female NOD mice. However, diabetes can be adoptively transferred into such hormone- treated recipients. The prevention of disease onset by androgen is likely due to the hormonal alteration of the development or function of the immune cells necessary for islet destruction.
PMCID: PMC2119211  PMID: 1569406
19.  Effect of Insulin on ACE2 Activity and Kidney Function in the Non-Obese Diabetic Mouse 
PLoS ONE  2014;9(1):e84683.
We studied the non-obese diabetic (NOD) mice model because it develops autoimmune diabetes that resembles human type 1 diabetes. In diabetic mice, urinary albumin excretion (UAE) was ten-fold increased at an “early stage” of diabetes, and twenty-fold increased at a “later stage” (21 and 40 days, respectively after diabetes diagnosis) as compared to non-obese resistant controls. In NOD Diabetic mice, glomerular enlargement, increased glomerular filtration rate (GFR) and increased blood pressure were observed in the early stage. In the late stage, NOD Diabetic mice developed mesangial expansion and reduced podocyte number. Circulating and urine ACE2 activity were markedly increased both, early and late in Diabetic mice. Insulin administration prevented albuminuria, markedly reduced GFR, blood pressure, and glomerular enlargement in the early stage; and prevented mesangial expansion and the reduced podocyte number in the late stage of diabetes. The increase in serum and urine ACE2 activity was normalized by insulin administration at the early and late stages of diabetes in Diabetic mice. We conclude that the Diabetic mice develops features of early kidney disease, including albuminuria and a marked increase in GFR. ACE2 activity is increased starting at an early stage in both serum and urine. Moreover, these alterations can be completely prevented by the chronic administration of insulin.
PMCID: PMC3882249  PMID: 24400109
20.  Development of Spontaneous Autoimmune Peripheral Polyneuropathy in B7-2–Deficient Nod Mice 
An increasing number of studies have documented the central role of T cell costimulation in autoimmunity. Here we show that the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain, deficient in B7-2 costimulation, is protected from diabetes but develops a spontaneous autoimmune peripheral polyneuropathy. All the female and one third of the male mice exhibited limb paralysis with histologic and electrophysiologic evidence of severe demyelination in the peripheral nerves beginning at 20 wk of age. No central nervous system lesions were apparent. The peripheral nerve tissue was infiltrated with dendritic cells, CD4+, and CD8+ T cells. Finally, CD4+ T cells isolated from affected animals induced the disease in NOD.SCID mice. Thus, the B7-2–deficient NOD mouse constitutes the first model of a spontaneous autoimmune disease of the peripheral nervous system, which has many similarities to the human disease, chronic inflammatory demyelinating polyneuropathy (CIDP). This model demonstrates that NOD mice have “cryptic” autoimmune defects that can polarize toward the nervous tissue after the selective disruption of CD28/B7-2 costimulatory pathway.
PMCID: PMC2195945  PMID: 11535635
autoimmunity; peripheral neuropathy; B7-2 costimulation; animal model; NOD mice
21.  The Programmed Death-1 (PD-1) Pathway Regulates Autoimmune Diabetes in Nonobese Diabetic (NOD) Mice 
Programmed death-1 (PD-1) receptor, an inhibitory costimulatory molecule found on activated T cells, has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in the development of autoimmune diabetes. PD-1 or PD-L1 but not PD-L2 blockade rapidly precipitated diabetes in prediabetic female nonobese diabetic (NOD) mice regardless of age (from 1 to 10-wk-old), although it was most pronounced in the older mice. By contrast, cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) blockade induced disease only in neonates. Male NOD mice also developed diabetes after PD-1–PD-L1 pathway blockade, but NOR mice, congenic to NOD but resistant to the development of diabetes, did not. Insulitis scores were significantly higher and frequency of interferon γ–producing GAD-reactive splenocytes was increased after PD-1–PD-L1 pathway blockade compared with controls. Interestingly, PD-L1 but not PD-L2 was found to be expressed on inflamed islets of NOD mice. These data demonstrate a central role for PD-1–PD-L1 interaction in the regulation of induction and progression of autoimmune diabetes in the NOD mouse and provide the rationale to develop new therapies to target this costimulatory pathway in this disease.
PMCID: PMC2196083  PMID: 12847137
diabetes mellitus, insulin-dependent; mice, inbred NOD; autoimmunity; self-tolerance; programmed cell death protein 1
22.  T cell populations in the pancreatic lymph node naturally and consistently expand and contract in NOD mice as disease progresses 
Molecular immunology  2012;52(1):9-18.
Nonobese diabetic (NOD) mice develop spontaneous autoimmune Type 1 diabetes (T1D) that results from the destruction of insulin secreting β cells by diabetogenic T cells. The activation of autoreactive T cells occurs in the pancreatic lymph nodes (PLN) from where effector T cells migrate to the pancreas. This study was designed to explore whether T cell populations in the NOD PLN expand in a predictable and reproducible way during disease progression. Complementary determining region (CDR) 3 length spectratype analysis of 19 TCR Vβ families was used to identify the relative frequency of T populations in PLN of 4 and 10 week old NOD mice and mice at T1D onset. Significant and highly reproducible changes in specific T cell populations were detected in 14 of Vβ families tested at all stages of disease. However, of these, the CDR3 spectratype of only four Vβ families was significantly more perturbed at T1D onset than in 10 week old mice. Intriguingly, when diabetes was induced in 10 week old mice with cyclophosphamide (CYP) the same four Vβ families, Vβ5.1, Vβ9, Vβ10, and Vβ15, were again significantly more perturbed than in the untreated non-diabetic age matched mice. Taken together the data show that while T cell responses in PLN of NOD mice are heterogeneous, they are ordered and consistent throughout disease development. The finding that within this heterogeneous response four Vβ families are significantly more perturbed in diabetic mice, whether spontaneous or induced, strongly suggests their selection as part of the disease process.
PMCID: PMC3774157  PMID: 22580347
Autoimmune disease; TCR; CDR3; Diabetes; Pancreatic lymph nodes; Perturbation
23.  Low Incidence of Spontaneous Type 1 Diabetes in Non-Obese Diabetic Mice Raised on Gluten-Free Diets Is Associated with Changes in the Intestinal Microbiome 
PLoS ONE  2013;8(11):e78687.
Human and animal studies strongly suggest that dietary gluten could play a causal role in the etiopathogenesis of type 1 diabetes (T1D). However, the mechanisms have not been elucidated. Recent reports indicate that the intestinal microbiome has a major influence on the incidence of T1D. Since diet is known to shape the composition of the intestinal microbiome, we investigated using non-obese diabetic (NOD) mice whether changes in the intestinal microbiome could be attributed to the pro- and anti-diabetogenic effects of gluten-containing and gluten-free diets, respectively. NOD mice were raised on gluten-containing chows (GCC) or gluten-free chows (GFC). The incidence of diabetes was determined by monitoring blood glucose levels biweekly using a glucometer. Intestinal microbiome composition was analyzed by sequencing 16S rRNA amplicons derived from fecal samples. First of all, GCC-fed NOD mice had the expected high incidence of hyperglycemia whereas NOD mice fed with a GFC had significantly reduced incidence of hyperglycemia. Secondly, when the fecal microbiomes were compared, Bifidobacterium, Tannerella, and Barnesiella species were increased (p = 0.03, 0.02, and 0.02, respectively) in the microbiome of GCC mice, where as Akkermansia species was increased (p = 0.02) in the intestinal microbiomes of NOD mice fed GFC. Thirdly, both of the gluten-free chows that were evaluated, either egg white based (EW-GFC) or casein based (C-GFC), significantly reduced the incidence of hyperglycemia. Interestingly, the gut microbiome from EW-GFC mice was similar to C-GFC mice. Finally, adding back gluten to the gluten-free diet reversed its anti-diabetogenic effect, reduced Akkermansia species and increased Bifidobacterium, Tannerella, and Barnesiella suggesting that the presence of gluten is directly responsible for the pro-diabetogenic effects of diets and it determines the gut microflora. Our novel study thus suggests that dietary gluten could modulate the incidence of T1D by changing the gut microbiome.
PMCID: PMC3827256  PMID: 24236037
24.  A Defect in Tryptophan Catabolism Impairs Tolerance in Nonobese Diabetic Mice 
The predisposition of nonobese diabetic (NOD) mice to develop autoimmunity reflects deficiencies in both peripheral and central tolerance. Several defects have been described in these mice, among which aberrant antigen-presenting cell function and peroxynitrite formation. Prediabetes and diabetes in NOD mice have been targeted with different outcomes by a variety of immunotherapies, including interferon (IFN)-γ. This cytokine may be instrumental in specific forms of tolerance by virtue of its ability to activate immunosuppressive tryptophan catabolism. Here, we provide evidence that IFN-γ fails to induce tolerizing properties in dendritic cells from highly susceptible female mice early in prediabetes. This effect is associated with impaired tryptophan catabolism, is related to transient blockade of the Stat1 pathway of intracellular signaling by IFN-γ, and is caused by peroxynitrite production. However, the use of a peroxynitrite inhibitor can rescue tryptophan catabolism and tolerance in those mice. This is the first report of an experimental autoimmune disease in which defective tolerance is causally linked to impaired tryptophan catabolism.
PMCID: PMC2196078  PMID: 12835483
autoimmunity; indoleamine 2,3-dioxygenase; dendritic cells; tolerance; NOD mice
25.  T Helper 2 (Th2) T Cells Induce Acute Pancreatitis and Diabetes in Immune-compromised Nonobese Diabetic (NOD) Mice 
Autoimmune diabetes is caused by the CD4+, T helper 1 (Th1) cell-mediated apoptosis of insulin-producing β cells. We have previously shown that Th2 T cells bearing the same T cell receptor (TCR) as the diabetogenic Th1 T cells invade islets in neonatal nonobese diabetic (NOD) mice but fail to cause disease. Moreover, when mixed in excess and cotransferred with Th1 T cells, Th2 T cells could not protect NOD neonates from Th1-mediated diabetes. We have now found, to our great surprise, the same Th2 T cells that produced a harmless insulitis in neonatal NOD mice produced intense and generalized pancreatitis and insulitis associated with islet cell necrosis, abscess formation, and subsequent diabetes when transferred into immunocompromised NOD.scid mice. These lesions resembled allergic inflamation and contained a large eosinophilic infiltrate. Moreover, the Th2-mediated destruction of islet cells was mediated by local interleukin-10 (IL-10) production but not by IL-4. These findings indicate that under certain conditions Th2 T cells may not produce a benign or protective insulitis but rather acute pathology and disease. Additionally, these results lead us to question the feasibility of Th2-based therapy in type I diabetes, especially in immunosuppressed recipients of islet cell transplants.
PMCID: PMC2198973  PMID: 9221759

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