Female NOD Mice Research Articles

PGC nanocarrier-formulated native GLP-1 prevents diabetes in NOD mice (42.3)

Abstract Formulation of peptides in a nanocarrier made up of protected-graft-copolymer (PGC) can greatly enhance pharmacokinetics and biodistribution of the peptides in vivo. Glucagon-like peptide 1 (GLP-1) is β-cell protective and decreases the severity symptoms of diabetes in Type 2 and streptozotocin models. We hypothesized that PGC-formulated GLP-1 can prevent Type 1 diabetes in NOD mice. 4-5 week old female NOD mice [n=25] were treated for eight weeks with 3mg/kg PGC-formulated GLP-1 administered s.c. either once or three times per week. After the treatment period, the mice were monitored for signs of diabetes until they reached 36 weeks of age. A group of NOD mice treated with anti-CD3 antibodies (0.5mg/kg i.p. once at treatment week 0 and once at treatment week 1) was used as a positive control for diabetes prevention. At 36 weeks of age, 48% of the mice treated with saline had developed diabetes. Anti-CD3 treatment prevented 50% of the diabetes cases in the mice with only 24% of the mice in this group having developed diabetes at 36 weeks of age. Once a week GLP-1 treatment (3mg/kg s.c.) was as effective in preventing diabetes as anti-CD3 treatment with only 28% of mice being diabetic at 36 weeks of age. Surprisingly, there was no difference between the three times per week GLP-1 treatment group (3mg/kg s.c.) and the saline control: 54.1% of the mice treated three times per week became diabetic, which was not statistically significantly different form the 48% diabetes incidence in the saline group.

Islet lymphocyte subsets in male and female NOD mice are qualitatively similar but quantitatively distinct

Islet lymphocyte subsets in male and female NOD mice are qualitatively similar but quantitatively distinct

T-Cell Promiscuity in Autoimmune Diabetes

OBJECTIVE—It is well established that the primary mediators of β-cell destruction in type 1 diabetes are T-cells. Nevertheless, the molecular basis for recognition of β-cell–specific epitopes by pathogenic T-cells remains ill defined; we seek to further explore this issue.RESEARCH DESIGN AND METHODS—To determine the properties of β-cell–specific T-cell receptors (TCRs), we characterized the fine specificity, functional and relative binding avidity/affinity, and diabetogenicity of a panel of GAD65-specific CD4+ T-cell clones established from unimmunized 4- and 14-week-old NOD female mice.RESULTS—The majority of GAD65-specific CD4+ T-cells isolated from 4- and 14-week-old NOD female mice were specific for peptides spanning amino acids 217–236 (p217) and 290–309 (p290). Surprisingly, 31% of the T-cell clones prepared from 14-week-old but not younger NOD mice were stimulated with both p217 and p290. These promiscuous T-cell clones recognized the two epitopes when naturally processed and presented, and this dual specificity was mediated by a single TCR. Furthermore, promiscuous T-cell clones demonstrated increased functional avidity and relative TCR binding affinity, which correlated with enhanced islet infiltration on adoptive transfer compared with that of monospecific T-cell clones.CONCLUSIONS—These results indicate that promiscuous recognition contributes to the development of GAD65-specific CD4+ T-cell clones in NOD mice. Furthermore, these findings suggest that T-cell promiscuity reflects a novel form of T-cell avidity maturation.

Equivalent Specificity of Peripheral Blood and Islet-Infiltrating CD8+ T Lymphocytes in Spontaneously Diabetic HLA-A2 Transgenic NOD Mice

CD8(+) T cells play an important role in the initiation of insulitis and in the destructive stage leading to insulin-dependent diabetes mellitus. A string of recent studies has led to the identification of numerous HLA-A2-restricted epitopes derived from pancreatic beta cell Ags. It is hoped that assays detecting responses of patient PBMC to such epitopes might be instrumental for early diagnosis of beta cell-directed autoimmunity and for monitoring trials of immunointervention. However, it remains unclear whether the results of assays studying PBMC reflect responses of islet-infiltrating lymphocytes, and to what extent they correlate with disease risk and/or activity. We have used female and male humanized NOD mice expressing HLA-A2 in addition to murine MHC class I molecules to study spontaneous responses of islet-infiltrating blood, spleen, and lymph node lymphocytes of various age groups to a panel of 16 epitopes. Twelve of these are restricted by HLA-A2, have previously been shown to be recognized by patient CTL, and have identical sequences in human and murine autoantigens. Using an IFN-gamma ELISPOT assay, we find highly similar hierarchies of epitope immunodominance in the different T cell compartments, including peripheral blood and pancreatic islets. Moreover, we demonstrate that most of the epitopes eliciting dominant responses in humans display similar status in the mouse model. These results emphasize the potential of humanized mice as tools for studying spontaneous autoimmune CTL responses, and they provide a strong rationale for the development and use of assays monitoring responses of CD8(+) PBMC in human type 1 diabetes.

T cell repertoire development of quasi‐public polyspecific clonal peaks in NOD mice

A variety of autoreactive T cells have been described in T1D with specificity for a diverse set of autoantigens. Here, we analyzed the T cell repertoires that arise spontaneously in pancreatic lymph nodes (PLN) from female NOD mice, using CDR3‐length spectroscopy. Quasi‐public expansions in Vβ5.2Jβ2.5 (168), Vβ10Jβ1.5 (90) and Vβ13Jβ2.1 (131) were found in 60% (6/10) and 50% (5/10) of 4w old NOD mice. At 10w of age, expansions in the rearrangements Vβ12Jβ2.5 (152) and Vβ5.1Jβ2.2 (198) were detected in 60% and 50% of mice, respectively. Also, expansions in the rearrangements Vβ9Jβ2.4 (97), Vβ12Jβ2.5 (152) and Vβ16Jβ2.2 (121) were detected in 50% of 16w old mice. Interestingly, at this age, we identified a dominant expansion in the clonotype Vβ12Jβ2.7 (170) in all 6 diabetic NOD mice analyzed. In contrast, no expansion of this clonotype was showed at 4w of age and only 2/10 mice showed expansions in this clonotype at 10w. Therefore, the Vβ12Jβ2.7 (170) could be considered a public T cell clone. To examine the peptide specificity, PLN T cells were expanded in vitro with anti‐CD3/anti‐CD28 beads and stimulated with peptides derived from GAD65, insulin or IGRP. Surprisingly, several of the resulting peaks were responsive to different peptides from more than one islet antigen. These dynamic repertoire shifts with age, featuring the emergence of some polyspecific T cells, warrant further detailed study. Supported by JDRF and DNRG

B Lymphocyte Depletion by CD20 Monoclonal Antibody Prevents Diabetes in Nonobese Diabetic Mice despite Isotype-Specific Differences in FcγR Effector Functions

NOD mice deficient for B lymphocytes from birth fail to develop autoimmune or type 1 diabetes. To assess whether B cell depletion influences type 1 diabetes in mice with an intact immune system, NOD female mice representing early and late preclinical stages of disease were treated with mouse anti-mouse CD20 mAbs. Short-term CD20 mAb treatment in 5-wk-old NOD female mice reduced B cell numbers by approximately 95%, decreased subsequent insulitis, and prevented diabetes in >60% of littermates. In addition, CD20 mAb treatment of 15-wk-old NOD female mice significantly delayed, but did not prevent, diabetes onset. Protection from diabetes did not result from altered T cell numbers or subset distributions, or regulatory/suppressor T cell generation. Rather, impaired CD4+ and CD8+ T cell activation in the lymph nodes of B cell-depleted NOD mice may delay diabetes onset. B cell depletion was achieved despite reduced sensitivity of NOD mice to CD20 mAbs compared with C57BL/6 mice. Decreased B cell depletion resulted from deficient FcgammaRI binding of IgG2a/c CD20 mAbs and 60% reduced spleen monocyte numbers, which in combination reduced Ab-dependent cellular cytotoxicity. With high-dose CD20 mAb treatment (250 microg) in NOD mice, FcgammaRIII and FcgammaRIV compensated for inadequate FcgammaRI function and mediated B cell depletion. Thereby, NOD mice provide a model for human FcgammaR polymorphisms that reduce therapeutic mAb efficacy in vivo. Moreover, this study defines a new, clinically relevant approach whereby B cell depletion early in the course of disease development may prevent diabetes or delay progression of disease.

Continuous stimulation of human glucagon-like peptide-1 (7–36) amide in a mouse model (NOD) delays onset of autoimmune type 1 diabetes

We examined the effect of glucagon-like peptide-1 (GLP-1) on the development of diabetes and islet morphology in NOD mice by administering GLP-1 to prediabetic mice. Eight-week-old female NOD mice were infused subcutaneously with human GLP-1 via a mini-osmotic pump for 4 or 8 weeks. In mice treated with GLP-1 for 4 weeks, blood glucose levels and body weight were measured. An intraperitoneal glucose tolerance test (IPGTT) and evaluation of insulitis score were also performed. Beta cell area, proliferation, apoptosis, neogenesis from ducts and subcellular localisation of forkhead box O1 (FOXO1) were examined by histomorphometrical, BrdU-labelling, TUNEL, insulin/cytokeratin and FOXO1/insulin double-immunostaining methods, respectively. Mice treated with human GLP-1 for 4 weeks had lower blood glucose levels until 2 weeks after completion of treatment, showing improved IPGTT data and insulitis score. This effect continued even after cessation of the treatment. In addition to the increase of beta cell neogenesis, BrdU labelling index was elevated (0.24 vs 0.13%, p < 0.001), while apoptosis was suppressed by 54.2% (p < 0.001) in beta cells. Beta cell area was increased in parallel with the translocation of FOXO1 from the nucleus to the cytoplasm. The onset of diabetes was delayed in mice treated with GLP-1 for 4 weeks, while mice treated with GLP-1 for 8 weeks did not develop diabetes by age 21 weeks compared with a 60% diabetes incidence in control mice at this age. Continuous infusion of human GLP-1 to prediabetic NOD mice not only induces beta cell proliferation and neogenesis, but also suppresses beta cell apoptosis and delays the onset of type 1 diabetes.

Bone Marrow Is a Preferential Homing Site for Autoreactive T-Cells in Type 1 Diabetes

The pancreatic microenvironment is considered to be the primary location of autoreactive T-cells in type 1 diabetes. Diabetogenic T-cells have also been detected in the spleens of NOD mice. However, it is not known whether bone marrow also contains T-cells specific for self-antigens in hosts with autoimmunity. In this study, we investigated whether autoreactive diabetogenic T-cells are present in the bone marrow of NOD mice. Bone marrow and splenic T-cells of female NOD mice were purified and tested for their cytokine secretion and proliferation in response to stimulation with immunodominant peptides of pancreatic beta-cells. The diabetogenic nature and homing properties of purified bone marrow T-cells were compared with those of splenic T-cells in NOD-Scid and wild-type mice. The bone marrow T-cells from both hyperglycemic and young euglycemic mice demonstrated profoundly higher proliferation and cytokine production in response to stimulation with beta-cell antigens than T-cells from spleen. Bone marrow T-cells showed rapid expansion and aggressive infiltration into pancreatic islets in NOD-Scid mice and induced hyperglycemia earlier than splenic T-cells. Adoptive transfer of bone marrow T-cells resulted in their trafficking predominantly to bone marrow and pancreatic lymph nodes. Our study demonstrates that a large number of diabetogenic T-cells are present in the bone marrow of female NOD mice and that these autoreactive T-cells can be detected long before clinical onset of the disease.

Sleep deprivation reduces the lymphocyte count in a non-obese mouse model of type 1 diabetes mellitus

The objective of the present study was to determine whether sleep deprivation (SD) would promote changes in lymphocyte numbers in a type 1 diabetes model (non-obese diabetic, NOD, mouse strain) and to determine whether SD would affect female and male NOD compared to Swiss mice. The number of lymphocytes in peripheral blood after 24 and 96 h of SD (by multiple platform method) or equivalent period of time in home-cage controls was examined prior to the onset of diabetes. SD for 96 h significantly reduced lymphocytes in male Swiss mice compared to control (8.6 ± 2.1 vs 4.1 ± 0.7 10³/µL; P < 0.02). In male NOD animals, 24- and 96-h SD caused a significant decrease of lymphocytes compared to control (4.4 ± 0.3 vs 1.6 ± 0.5; P < 0.001 and 4.4 ± 0.3 vs 0.9 ± 0.1 10³/µL; P < 0.00001, respectively). Both 24- and 96-h SD induced a reduction in the number of lymphocytes in female Swiss (7.5 ± 0.5 vs 4.5 ± 0.5, 4.4 ± 0.6 10³/µL; P < 0.001, respectively) and NOD mice (4 ± 0.6 vs 1.8 ± 0.2, 1.2 ± 0.4 10³/µL; P < 0.01, respectively) compared to the respective controls. Loss of sleep induced lymphopenia in peripheral blood in both genders and strains used. Since many cases of autoimmunity present reduced numbers of lymphocytes and, in this study, it was more evident in the NOD strain, our results suggest that SD should be considered a risk factor in the onset of autoimmune disorders.

Sleep deprivation reduces the lymphocyte count in a non-obese mouse model of type 1 diabetes mellitus

The objective of the present study was to determine whether sleep deprivation (SD) would promote changes in lymphocyte numbers in a type 1 diabetes model (non-obese diabetic, NOD, mouse strain) and to determine whether SD would affect female and male NOD compared to Swiss mice. The number of lymphocytes in peripheral blood after 24 and 96 h of SD (by multiple platform method) or equivalent period of time in home-cage controls was examined prior to the onset of diabetes. SD for 96 h significantly reduced lymphocytes in male Swiss mice compared to control (8.6 +/- 2.1 vs 4.1 +/- 0.7 10(3)/microL; P < 0.02). In male NOD animals, 24- and 96-h SD caused a significant decrease of lymphocytes compared to control (4.4 +/- 0.3 vs 1.6 +/- 0.5; P < 0.001 and 4.4 +/- 0.3 vs 0.9 +/- 0.1 10(3)/microL; P < 0.00001, respectively). Both 24- and 96-h SD induced a reduction in the number of lymphocytes in female Swiss (7.5 +/- 0.5 vs 4.5 +/- 0.5, 4.4 +/- 0.6 10(3)/microL; P < 0.001, respectively) and NOD mice (4 +/- 0.6 vs 1.8 +/- 0.2, 1.2 +/- 0.4 10(3)/microL; P < 0.01, respectively) compared to the respective controls. Loss of sleep induced lymphopenia in peripheral blood in both genders and strains used. Since many cases of autoimmunity present reduced numbers of lymphocytes and, in this study, it was more evident in the NOD strain, our results suggest that SD should be considered a risk factor in the onset of autoimmune disorders.

The Impact of B Lymphocyte Depletion on the Pathogenesis of Autoimmune Diabetes (131.28)

Abstract NOD B cell development is characterized by a defective homeostatic checkpoint in B cell maturation and selection. Having demonstrated that the progression of autoimmune diabetes requires antigen presentation by B-lymphocytes, we hypothesized that B cell depletion may prevent diabetes progression in NOD mice. Here, we employed two distinct approaches for in vivo B lymphocyte depletion: a cohort of adult hCD20 Tg NOD mice were treated with the B lymphocyte depleting mAb, Rituximab (anti-CD20) anda cohort of adult NOD mice were treated with a neutralizing Hamster anti-mouse mAb specific for the B cell survival factor, BLyS/BAFF. Treatment with both Rituximab (2mg/injection x3doses) and anti-BLyS mAb (100?g/injection x2 doses) led to B cell depletion within 10 days. BLyS/BAFF neutralization, using anti-BLyS, caused a profound depletion of mature/recirculating B cells in these mice and lasted for &amp;gt;30 days. Diabetes occurred within 2 weeks in all Rituximab treated female hCD20 Tg NOD mice (n=4); despite their early backcross generations (i.e., N4-6F1) onto the NOD genetic background. Conversely, adult female NOD/LtJ mice (n=20), B cell depleted using anti-BLyS/BAFF did not progress to diabetes for &amp;gt;30 days following treatment. Collectively, these results indicate that BLyS/BAFF neutralization may be required for the prevention of autoimmune diabetes following B lymphocyte depletion.

Expansion of GAD65–Specific Immunoregulatory Effector Cells by Biolistic-Mediated Gene Delivery Prevents Autoimmune Diabetes in NOD Mice (131.14)

Abstract Intramuscular (i.m.) vaccination with plasmid DNA (pDNA) encoding beta cell autoantigens such as glutamic acid decarboxylase 65 (GAD65) is an approach to suppress ongoing Type 1 diabetes (T1D) in NOD mice. However, protection is dependent on co-treatment with pDNAs encoding IL-4 and/or IL-10, and the subsequent induction of type 2 CD4+ T effectors. To enhance the therapeutic efficacy of pDNA vaccination, we explored epidermal delivery of pDNA via “gene gun”. Notably, reports have shown induction of a preferential type 2-like CD4+ T cell response when pDNA encoding antigen-only is delivered to the epidermis via gene gun. We hypothesized that biolistic delivery of pDNA would preferentially induce type 2 CD4+ T effectors and suppress established beta cell autoimmunity in NOD mice. Groups of 10 wk-old NOD female mice were treated four times with pDNA encoding GAD65-Ig (pGAD65) administered i.m. or delivered by gene gun on 1.6 uM gold particles. Whereas i.m. injection of pGAD65 resulted in preferential induction of GAD65-specific CD4+ Th1 cells and no protection against T1D, the majority of NOD mice treated with gene gun delivered pGAD65 remained diabetes-free and protection correlated with increased GAD65-specific IL-4 secreting CD4+ T cells. These results demonstrate that gene-gun delivered pDNA encoding beta cell autoantigen-only is an effective strategy to induce immunoregulatory CD4+ T cells and suppress T1D.

Suppression of autoimmune diabetes by peptide-MHC class II dimers (131.13)

Abstract Type 1 diabetes (T1D) is an autoimmune disease mediated by pathogenic beta cell-specific T cells. The use of antigen-specific based immunotherapies has been one strategy to selectively target beta cell-specific T cells leaving the remainder of the immune system intact. Administration with peptides derived from beta cell autoantigens such as glutamic acid decarboxylase 65 (GAD65) to NOD mice prevents T1D. However, as beta cell autoimmunity progresses the conditions for peptide treatment to suppress T1D become highly stringent. To enhance the efficacy of peptide treatment, recombinant IAg7-immunoglobulin (Ig) dimers covalently linked to GAD65 peptides (217–236, 286–309) or the mimetic BDC2.5 epitope (sBDC) were tested. Twelve week-old NOD female mice with established beta cell autoimmunity received a short course of IAg7-Ig dimers intravenously. Surprisingly, treatment with IAg7-Ig:sBDC accelerated the onset of diabetes in NOD mice due to expansion of sBDC-specific CD4+ Th1 cells. In marked contrast, the majority of NOD mice treated with IAg7-Ig:GADp217 or IAg7-Ig:GADp286 remained diabetes-free. Protection correlated with an increased frequency of IL-10 secreting immunoregulatory CD4+ T cells that blocked diabetes in a co-adoptive transfer model. These results demonstrate that treatment with a short-course of IAg7-Ig-GAD65 peptide dimers is an effective approach to suppress T1D.

Cyclophosphamide treatment of the NOD mouse reveals quasi-public dominant T cell clones (130.34)

Abstract To define the major components of the autoreactive T cell repertoire that arise spontaneously in the NOD mouse, we used CDR3 length spectroscopy to study the T cell repertoire in PLN from pre-diabetic NOD mice to identify public dominant clones; andto identify potentially pathogenic clones in islet-infiltrating cells that persist after Cyclophosphamide (Cyp) treatment. We found that any public T cell expansions were identified in PLN of female NOD mice at 4 and 10 weeks of age. Two expansions in the rearrangements Vb5.2/Jb2.5 and Vb10/Jb1.5 were found in 60% and 50% of 4 week old NOD mice. These expansions can be useful in the study of how the T cell repertoire can be modified after islet transplantation. After Cyp-treatment, the Vb4, Vb5.2, Vb10 and Vb12 families showed dominant expansions. Interestingly, we found a unique Vb4-"195 nucleotide" peak that was found in 10 out of 11 (91%) Cyp-treated mice. In contrast, this peak was not detected in untreated NOD mouse islets. In the Vb12 family, a "200" peak was prominent in 6 out of 10 (60%) Cyp-treated mice. These expansions may represent the more pathogenic clones. Currently, we are sequencing these expansions to establish whether the Cyp-induced expansions represent truly unique clones. We believe that these Cyp experiments will provide the possibility of distinguishing between regulatory and aggressive clones. This work was supported by JDRF 3-2005-336.

Treatment with myeloid suppressor cells in late stage autoimmune insulitis in order to prevent type 1 diabetes onset in the NOD mouse model

Objectives: Myeloid suppressor cells (MSC) are precursers of dendritic cells, which were well charactherized by our group in vitro and show strong immunosuppressive activity (1). We have shown earlier, that onset of type 1 diabetes can be prevented by i.v. injection of MSC in 8 week old female NOD mice. Here we investigate the effect of MSC in the prevention of diabetes onset in late stage autoimmune insulitis (12 week old female NOD mice).

CD8 + T lymphocytes specific for glutamic acid decarboxylase 90–98 epitope mediate diabetes in NOD SCID mouse

During the past decade, glutamic acid decarboxylase (GAD) has been considered a crucial β-cell autoantigen involved in type 1 diabetes in the NOD mouse and human. Recently, the etiological role of GAD has remained controversy. In the NOD mouse, some previous studies argued in favor of a regulatory role for GAD-specific CD4 + T cells, and no diabetogenic CD8 + T cells specific for GAD have been identified so far, discrediting the importance of GAD in β-cell injury. Here, we identified, in the NOD model, a relevant GAD CD8 + T cell epitope (GAD 90–98) using immunization with a plasmid encoding GAD, a protocol relying on in vivo processing of peptides from the autoantigenic protein. In pancreatic lymph nodes of naïve female NOD mice, CD8 + T lymphocytes recognizing GAD 90–98 peptide were detected during the initial phase of invasive insulitis (between 4 and 8 weeks of age), suggesting an important role for these cells in the first stage of the disease. GAD 90–98 specific CD8 + lymphocytes lysed efficiently islet cells in vitro and transferred diabetes into NOD SCID mice (100%). Finally, diabetes was accelerated greatly in 3-week-old female NOD mice injected i.p. with GAD 90–98, strengthening the role of GAD-specific CTLs in diabetes pathogenesis.

Systemic Expression of Heme Oxygenase-1 Ameliorates Type 1 Diabetes in NOD Mice

Heme oxygenase-1 (HO-1) is an enzyme with potent immunoregulatory capacity. To evaluate the effect of HO-1 on autoimmune diabetes, female NOD mice at 9 weeks of age received a single intravenous injection of a recombinant adeno-associated virus bearing HO-1 gene (AAV-HO-1; 0.5 x 10(10)-2.5 x 10(10) viruses/mouse). In a dose-dependent manner, HO-1 transduction reduced destructive insulitis and the incidence of overt diabetes examined over a 15-week period. HO-1-mediated protection was associated with a lower type 1 T-helper cell (Th1)-mediated response. Adaptive transfer experiments in NOD.scid mice demonstrated that splenocytes isolated from AAV-HO-1-treated mice were less diabetogenic. Flow cytometry analysis revealed no significant difference in the percentages of CD4(+)CD25(+) regulatory T-cells between saline-treated and AAV-HO-1-treated groups. However, the CD11c(+) major histocompatibility complex II(+) dendritic cell population was much lower in the AAV-HO-1-treated group. A similar protective effect against diabetes was observed in NOD mice subjected to carbon monoxide (CO) gas (250 ppm CO for 2 h, twice per week). These data suggest that HO-1 slows the progression to overt diabetes in pre-diabetic NOD mice by downregulating the phenotypic maturity of dendritic cells and Th1 effector function. CO appears to mediate at least partly the beneficial effect of HO-1 in this disease setting.

Long-lasting specific antibodies against P277 induced by mucosal administration of P277 repeat sequences carried by Hsp65 in the absence of adjuvants

To improve the weak immunogenicity of peptide P277, the recombinant expression plasmid pET28-Hsp65-6 × P277 was constructed by inserting 5 × P277 which was amplified by PCR into the vector pET28-Hsp65-P277. It was transformed into Escherichia coli BL21 (DE3) and the fusion protein (Hsp65-6 × P277) was expressed effectively as soluble protein after inducing by lactose. The fusion protein was purified by means of cell disruption, ammonium sulfate precipitation, double-distilled H 2O dialysis, DEAE52-cellulose column chromatography, and then used to immunize female NOD mice with three i.n. inoculations in the absence of adjuvants. Serum samples from the immunized mice were collected at 3 weeks interval. Antibodies against P277 and HSP65 were detected in immunized mice sera by enzyme-linked immunosorbent assay (ELISA) and Western blot. Specific antibodies were successfully induced and lasted for more than 20 weeks in animals immunized with the fusion protein via intranasal route even in the absence of adjuvants.

Young NOD Mice Show Increased Diabetes Sensitivity to Low Doses of Streptozotocin

In type 1 diabetes, environmentally induced early-limited beta cell damage may pre-empt the subsequent immune-mediated beta cell destruction. Low doses of streptozotocin (Stz), given early to diabetes-prone mice, may cause limited beta cell destruction during the early phase and precipitate diabetes. Here, we aimed to see if young NOD mice are more diabetes-sensitive to various multiple low doses of Stz than non-diabetes-prone mice. We also determined the molecular pathology of islets following administration of the diabetogen. Female NOD and CD-1 mice received 5 daily doses of Stz at day 21 (20, 30, and 40 mg/kg body weight; 18 mice per group) or diluent, and diabetes was monitored. Pancreas were studied histochemically and immunohistochemically at various time points after Stz administration. Following administration of Stz, NOD mice showed a much earlier onset and increased diabetes rate, at all three doses, than CD-1 mice. By day 80, the final diabetes rates following the 40, 30, and 20 mg dose in NOD mice were 95%, 85%, and 33%, respectively, compared with 33%, 28%, and 5.5%, respectively, in CD-1 mice. However, following the 20 mg dose, only 2 of the 12 remaining NOD mice developed the disease between 90 and 250 days compared with 19 of 24 NOD mice that did not receive Stz at day 21. Stz-administered NOD and CD-1 mice showed an initial loss of beta cells, with redistribution of islet endocrine cells, early macrophage infiltration, and increasing insulitis.

Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD mice

Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic beta-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice. HSCs were isolated from 6-8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3-4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer. Chimerism was established in all major lymphoid tissues, ranging from 5-15% in non-sorted bone marrow transplants to 20-45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific. We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner.