Insulitis In Non-obese Diabetic Mice Research Articles

Excess Salt Intake Activates IL-21-Dominant Autoimmune Diabetogenesis via a Salt-Regulated Ste20-Related Proline/Alanine-Rich Kinase in CD4 T Cells.

The fundamental mechanisms whereby a diet affects susceptibility to or modifies autoimmune diseases are poorly understood. Despite excess dietary salt intake acts as a risk factor for autoimmune diseases, little information exists on the impact of salt intake on type 1 diabetes. To elucidate the potential effect of high-salt intake on autoimmune diabetes, non-obese diabetic (NOD) mice were fed with a high-salt diet (HSD) or a normal-salt diet from 6 to 12 weeks of age and monitored for diabetes development. Our results revealed that HSD accelerated diabetes progression with more severe insulitis in NOD mice in a CD4+ T cell-autonomous manner when compared to NSD group. Moreover, expression of IL-21 and SPAK in splenic CD4+ T cells from HSD-fed mice was significantly upregulated. Accordingly, we generated T cell-specific SPAK knockout (CKO) NOD mice and demonstrated that SPAK deficiency in T cells significantly attenuated diabetes development in NOD mice by downregulating IL-21 expression in CD4+ T cells. Furthermore, HSD-triggered diabetes acceleration was abolished in HSD-fed SPAK CKO mice when compared to HSD-fed NOD mice, suggesting an essential role of SPAK in salt-exacerbated T cell pathogenicity. Finally, pharmacological inhibition of SPAK activity using a specific SPAK inhibitor (closantel) in NOD mice ameliorated diabetogenesis, further illuminating the potential of a SPAK-targeting immunotherapeutic approach for autoimmune diabetes. Here, we illustrate that a substantial association between salt sensitivity and the functional impact of SPAK on T cell pathogenicity serves as a central player linking high-salt intake influences to immune-pathophysiology of diabetogenesis in NOD mice.

Interferon-α promotes MHC I antigen presentation of islet βcells through STAT1-IRF7 pathway in type 1 diabetes.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease. Increased incidence of T1D was reported in patients receiving IFN-α treatment. However, the exact mechanisms of IFN-α that facilitate the pathogenesis of T1D are not fully understood. To explore the mechanism of IFN-α on the immune system and islets, non-obese diabetic (NOD) mice were injected with IFN-α and the progression of autoimmune insulitis was assessed by haematoxylin and eosin (HE) staining, immunohistochemical and flow cytometry analysis. Transcriptional profiling of islets treated with IFN-α was explored by RNA-seq. IFN-α induced antigen presentation was evaluated by qRT-PCR, western blot and immunofluorescence, and key transcription factors were inhibited by small interfering RNAs (siRNAs). Our data show that IFN-α contributed to the progression of autoimmune insulitis in NOD mice by promoting the proliferation of CD8+ T cells. IFN-α upregulated antigen presentation related genes MHC I, TAP1, B2M, PSMB8, NLRC5 and transcriptional regulator STAT1, STAT2, IRF7 at a time and dose-dependent manner. The silence of STAT1 or STAT2 both weakened IFN-α-induced increase of antigen presenting related molecules. IRF7 was also merely influenced by STAT1 silence. The knockdown of IRF7 decreased the IFN-α induced expressions of TAP1, PSMB8 and MHC I and prevented the expression of STAT2 but not STAT1. Our study demonstrated that STAT1-IRF7-MHC I complex axis were crucial for IFN-α signalling in islets, and created positive feedback through IRF7-STAT2 cascade amplifying signals which accelerated the process of T1D.

A Novel Liposome Formulation Carrying Both an Insulin Peptide and a Ligand for Invariant Natural Killer T Cells Induces Accumulation of Regulatory T Cells to Islets in Nonobese Diabetic Mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of pancreatic β cells by autoantigen-reactive diabetogenic cells. Antigen-specific therapies using islet autoantigens for restoring immune tolerance have emerged as promising approaches for the treatment of T1D but have been unsuccessful in humans. Herein, we report that RGI-3100-iB, a novel liposomal formulation carrying both α-galactosylceramide (α-GalCer), which is a representative ligand for invariant natural killer T (iNKT) cells, and insulin B chain 9–23 peptide, which is an epitope for CD4+ T cells, could induce the accumulation of regulatory T cells (Tregs) in islets in a peptide-dependent manner, followed by the remarkable prevention of diabetes onset in nonobese diabetic (NOD) mice. While multiple administrations of a monotherapy using either α-GalCer or insulin B peptide in a liposomal formulation was confirmed to delay/prevent T1D in NOD mice, RGI-3100-iB synergistically enhanced the prevention effect of each monotherapy and alleviated insulitis in NOD mice. Immunopathological analysis showed that Foxp3+ Tregs accumulated in the islets in RGI-3100-iB-treated mice. Cotransfer of diabetogenic T cells and splenocytes of NOD mice treated with RGI-3100-iB, but not liposomal α-GalCer encapsulating an unrelated peptide, to NOD-SCID mice resulted in the prevention of diabetes and elevation of Foxp3 mRNA expression in the islets. These data indicate that the migration of insulin B-peptide-specific Tregs to islet of NOD mice that are involved in the suppression of pathogenic T cells related to diabetes onset and progression could be enhanced by the administration of liposomes containing α-GalCer and insulin B peptide.

Pancreatic ductal cell antigens are important in the development of invasive insulitis in Non-Obese Diabetic mice

Type 1 Diabetes (T1D) is an autoimmune disease in which insulin producing beta cells of the pancreas are selectively destroyed. Glial Fibrillary Acidic Protein (GFAP) expressed in peri-islet Schwann cells (pSCs) and in the ductal cells of the pancreas is one of the candidate autoantigens for T1D. Immune responses to GFAP expressing cell types precede the islet autoimmunity in Non-Obese Diabetic (NOD) mice. By removing MHC class I from GFAP expressing cell types, we tested the role of autoantigens presented by these cell types in the development of invasive insulitis. Our findings indicate that antigens expressed by pancreatic ductal cells are important in the development of invasive insulitis in NOD mice.

MHC-mismatched mixed chimerism mediates thymic deletion of cross-reactive autoreactive T cells and prevents insulitis in nonobese diabetic mice.

Type 1 diabetic NOD mice have defects in both thymic negative selection and peripheral regulation of autoreactive T cells, and induction of mixed chimerism can effectively reverse these defects. Our recent studies suggest that MHC-mismatched mixed chimerism mediates negative selection of autoreactive thymocytes in wild-type NOD and TCR-transgenic NOD.Rag1(+/+).BDC2.5 mice. However, it remains unknown how mismatched I-A(b) MHC class II can mediate deletion of autoreactive T cells positively selected by I-A(g7). In the present study, we directly tested the hypothesis that mismatched MHC class II in mixed chimeras mediates deletion of cross-reactive autoreactive thymocytes. We first identify that transgenic BDC2.5 T cells from NOD.Rag1(+/+).BDC2.5 but not NOD.Rag1(-/-).BDC2.5 mice possess cross-reactive TCRs with endogenous TCRα-chains; MHC-mismatched H-2(b) but not matched H-2(g7) mixed chimerism mediates thymic deletion of the cross-reactive transgenic T cells in NOD.Rag1(+/+).BDC2.5 mice. Second, by transplanting T cell-depleted (TCD) bone marrow (BM) cells from NOD.Rag1(+/+).BDC2.5 or NOD.Rag1(-/-).BDC2.5 mice into lethally irradiated MHC-mismatched H-2(b) C57BL/6 or MHC-matched congenic B6.H-2(g7) recipients, we demonstrate that NOD.Rag1(+/+).BDC2.5 BM-derived cross-reactive transgenic T cells, but not NOD.Rag1(-/-).BDC2.5 BM-derived non-cross-reactive transgenic T cells, can be positively selected in MHC-mismatched H-2(b) thymus. Third, by cotransplanting NOD.Rag1(+/+).BDC2.5 TCD BM cells with BM cells from MHC-mismatched T cell-deficient C57BL/6 mice into lethally irradiated MHC-matched B6.H-2(g7) recipients, we demonstrate that thymic deletion of the cross-reactive transgenic T cells is dependent on MHC-mismatched donor BM-derived APCs but not on donor BM-derived T cells. Taken together, our studies indicate that MHC-mismatched mixed chimerism can mediate thymic deletion of cross-reactive autoreactive T cells that express more than one TCR.

Killer Treg cells ameliorate inflammatory insulitis in non-obese diabetic mice through local and systemic immunomodulation

Treg cells endowed with enhanced killing activity through decoration with Fas-ligand (FasL) protein (killer Treg) have been effective in delay of hyperglycemia in prediabetic non-obese diabetic (NOD) mice. In this study, we assessed the therapeutic efficacy of these cells, harvested from age-matched euglycemic NOD donors, on the course of disease in new-onset diabetics. One dose of 4 × 10(6) killer Treg cells stabilized blood glucose associated with increased insulin levels in 5 of 9 mice and partially reversed the severity of islet inflammation, whereas naive Treg cells did not modulate the course of disease significantly. Killer Treg cells were shown to operate through induction of cell apoptosis within the pancreatic lymph nodes, resulting in reduced efficiency of adoptive disease transfer to NOD/SCID recipients. A second mechanism of action consisted of increased fractions of CD4(+)CD25(-)FoxP3(+) T cells in the pancreas and all lymphoid organs. Immunomodulation with FasL rather than Treg cells enhanced the expression of CD25 and FoxP3 in the thymus, suggesting a possible contribution of thymic output to prolonged stabilization of the glucose levels. Autologous Treg cells evolve as excellent vehicles for targeted delivery of FasL as an immunomodulatory protein, which delete pathogenic cells at the site of inflammation and induce systemic dominance of suppressor subsets.

Daintain/AIF-1 (Allograft Inflammatory Factor-1) accelerates type 1 diabetes in NOD mice

A large body of experimental evidence suggests that cytokines trigger pancreatic β-cell death in type 1 diabetes mellitus. Daintain/AIF-1 (Allograft Inflammatory Factor-1), a specific marker for activated macrophages, is accumulated in the pancreatic islets of pre-diabetic BB rats. In the present study, we demonstrate that daintain/AIF-1 is released into blood and the levels of daintain/AIF-1 in the blood of type 1 diabetes-prone non-obese diabetic (NOD) mice suffering from insulitis are significantly higher than that in healthy NOD mice. When injected intravenously into NOD mice, daintain/AIF-1 stimulates white blood cell proliferation, increases the concentrations of blood glucose, impairs insulin expression, up-regulates nitric oxide (NO) production in pancreases and accelerates diabetes in NOD mice, while the antibody against daintain/AIF-1 delays or prevents insulitis in NOD mice. These results imply daintain/AIF-1 triggers type 1 diabetes probably via arousing immune cells activation and induction of NO production in pancreas of NOD mice.

IFN-γ induced by IL-12 administration prevents diabetes by inhibiting pathogenic IL-17 production in NOD mice

Interleukin 12 (IL-12) is a pivotal Th1-associated cytokine and a potent immunoregulatory molecule. However, the role of IL-12 in inducing immune tolerance that prevents insulitis and inhibits type 1 diabetes (T1D) remains unknown. The aim of this study was to investigate whether intermittent administration of IL-12 could prevent the development of T1D in nonobese diabetic (NOD) mice. We examined whether IL-12 treatment prevented diabetes by injecting different doses of IL-12 into NOD mice and compared the incidence of diabetes and insulitis in NOD mice with the incidence in control mice. Furthermore, we investigated the potential mechanisms of IL-12-mediated prevention of diabetes and insulitis. The expression of pro-inflammatory and immunoregulatory cytokines was measured before and following therapeutic administration of IL-12 in NOD mice. Our data demonstrated that both the absolute number and the function of DCs were impaired in NOD mice and that the levels of the Th17-associated pro-inflammatory cytokines, IL-1β, IL-6 and IL-23, were elevated in NOD mice compared with age-matched BALB/c and C57BL/6 mice. However, treatment of NOD mice with IL-12 suppressed insulitis and increased the number of healthy islets, and the levels of IL-17, IL-1β, IL-6 and IL-23 were significantly decreased. Moreover, IL-12 treatment of NOD mice induced the secretion of IFN-γ, a potent inhibitor of Th17 cells. These data indicated that intermittent administration of IL-12 prevented diabetes by inducing IFN-γ, suppressing the pathogenic IL-17-producing cells and reducing the expression of Th17-associated pro-inflammatory cytokines. Our results suggest a promising strategy for the treatment of human T1D and other Th17 cell-mediated autoimmune diseases.

Imaging of Insulitis in NOD Mice with IL-2-Gd-DTPA and 1.5 T MRI

To explore the possibility to use 1.5 T MRI in imaging of accumulating gadolinium (Gd) into inflamed en- docrine pancreas (insulitis), we compared intravenously delivered IL-2-Gd-DTPA and Gd-DTPA (Magne- vist®) in phenotypically healthy 12 weeks old female non-obese diabetic (NOD) mice. At 1 to 48 h after the injection mice were sacrificed and the pancreas-to-background ratio was determined using MRI. In addition Gd concentration of the pancreas and plasma were measured. The pancreas-to-plasma ratio of Gd was 25-fold 24 h after the i.v. injection of IL-2-Gd-DTPA. At the same time the pancreas-to-background ratio measured with 1.5T MRI was 1.5-times higher in the NOD mice receiving IL-2-Gd-DTPA than Magnevist® (271 ± 37 vs. 183 ± 0,1, P = 0.04) indicating that sufficient differences may exist between IL-2-Gd-DTPA accumulating in the pancreas and in the surrounding tissues to support the use of MRI for imaging of pan- creatic insulitis.

Pancreatitis aguda secundaria a azatioprina versus pancreatitis autoinmune en un paciente con colitis ulcerosa

Several studies have shown that transplantation of embryonic stem cells into diabetic animals either improved or normalized blood glucose levels. In this study, we examined the dose-dependent effect of early (prediabetic stage) intravenous administration of human umbilical cord blood (HUCB) mononuclear cells on blood glucose levels, survival, and insulitis in nonobese diabetic (NOD) mice with autoimmune type 1 diabetes. The results show that mice treated with HUCB cells significantly lowered their blood glucose levels and increased their lifespan, as compared with untreated mice. Also, a significant reduction in insulitis was observed in treated than in untreated mice. The mice that received the highest dosage (200 × 106) of cells had greater reduction in blood glucose levels and the degree of insulitis than the mice that received lower dosage (100–150 × 106) of cells. Prolonged lifespan in the former group of mice seems to be related to better control of blood glucose levels. Thus, administration of HUCB cells in the prediabetic stage without any immunosuppression improves type 1 diabetes by protecting the islets from insulitis in NOD mice.

Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts-oral administration protects against development of insulitis in non-obese diabetic mice

Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts-oral administration protects against development of insulitis in non-obese diabetic mice

Suppression of Type 1 Diabetes in NOD Mice by Bifunctional Peptide Inhibitor: Modulation of the Immunological Synapse Formation

The aim of this work was to design and utilize a bifunctional peptide inhibitor called glutamic acid decarboxylase-bifunctional peptide inhibitor to suppress the progression of type 1 diabetes in non-obese diabetic mice. The hypothesis is that glutamic acid decarboxylase-bifunctional peptide inhibitor binds simultaneously to major histocompatibility complex-II and intercellular adhesion molecule type 1 on antigen-presenting cell and inhibits the immunological synapse formation during T-cell-antigen-presenting cell interactions. Glutamic acid decarboxylase-bifunctional peptide inhibitor was composed of a major epitope of the type 1 diabetes-associated antigen, glutamic acid decarboxylase 65 kDa, covalently linked to a peptide derived from CD11a of lymphocyte function-associated antigen-1. The suppression of insulitis and type 1 diabetes was evaluated using non-obese diabetic and non-obese diabetic severe combined immunodeficiency mice. Glutamic acid decarboxylase-bifunctional peptide inhibitor had the capacity to suppress invasive insulitis in non-obese diabetic mice. CD4+ T-cells isolated from glutamic acid decarboxylase-bifunctional peptide inhibitor treated mice also suppressed insulitis and hyperglycemia when transferred with diabetogenic non-obese diabetic spleen cells into non-obese diabetic severe combined immunodeficiency recipients. As predicted, the glutamic acid decarboxylase-bifunctional peptide inhibitor cross-linked a significant fraction of major histocompatibility complex class-II molecules to intercellular adhesion molecule type 1 molecules on the surface of live antigen-presenting cell. Intravenous injection of the glutamic acid decarboxylase-bifunctional peptide inhibitor elicited interleukin-4-producing T-cells in non-obese diabetic mice primed against the glutamic acid decarboxylase-epitope peptide. Together, the results indicate that glutamic acid decarboxylase-bifunctional peptide inhibitor induces interleukin-4-producing regulatory cells but does not expand the glutamic acid decarboxylase-specific Th2 population. Given that Th2 effector cells can cause pathology, the glutamic acid decarboxylase-bifunctional peptide inhibitor may represent a novel mechanism to induce interleukin-4 without Th2-associated pathology.

Reovirus delays diabetes onset but does not prevent insulitis in nonobese diabetic mice.

Mice infected with reovirus develop abnormalities in glucose homeostasis. Reovirus strain type 3 Abney (T3A) was capable of systemic infection of nonobese diabetic (NOD) mice, an experimental model of autoimmune diabetes. Reovirus antigen was detected in pancreatic islets of T3A-infected mice, and primary cultures of pancreatic islets from NOD mice supported T3A growth. Significantly fewer T3A-infected animals compared to uninfected controls developed diabetes. However, despite the alteration in diabetes penetrance, insulitis was evident in T3A-infected mice. These results suggest that viral infection of NOD mice alters autoimmune responses to beta-cell antigens and thereby delays development of diabetes.

Synthetic glycolipid OCH prevents insulitis and diabetes in NOD mice

Non-obese diabetic (NOD) mice develop diabetes mediated by pathogenic T-helper type 1 (Th1) cells. Vα14 Natural killer (NKT) cells are a unique lymphocyte subtype implicated in the regulation of autoimmunity and a good source of protective Th2 cytokines. We recently developed a Th2-skewing NKT cell ligand, OCH. OCH, a sphingosine truncated derivative of α-galactosylceramide (α-GC), stimulates NKT cells to selectively produce Th2 cytokines. Here we show that OCH prevented the development of diabetes and insulitis in NOD mice. The suppression of insulitis by OCH was more profound compared to α-GC. Infiltration of T cells, B cells and macrophages into islets is inhibited in OCH-treated NOD mice. OCH-mediated suppression of diabetes is associated with Th2 bias of anti-islet antigen response and increased IL-10 producing cells among islet-infiltrating leukocytes. Considering the non-polymorphic and well conserved features of the CD1d molecule in mice and humans, these findings not only support the proposed role of NKT cells in the regulation of self-tolerance but also highlight the potential use of OCH for therapeutic intervention in type I diabetes.

Effect of human umbilical cord blood cells on glycemia and insulitis in type 1 diabetic mice

Several studies have shown that transplantation of embryonic stem cells into diabetic animals either improved or normalized blood glucose levels. In this study, we examined the dose-dependent effect of early (prediabetic stage) intravenous administration of human umbilical cord blood (HUCB) mononuclear cells on blood glucose levels, survival, and insulitis in nonobese diabetic (NOD) mice with autoimmune type 1 diabetes. The results show that mice treated with HUCB cells significantly lowered their blood glucose levels and increased their lifespan, as compared with untreated mice. Also, a significant reduction in insulitis was observed in treated than in untreated mice. The mice that received the highest dosage (200 × 10 6) of cells had greater reduction in blood glucose levels and the degree of insulitis than the mice that received lower dosage (100–150 × 10 6) of cells. Prolonged lifespan in the former group of mice seems to be related to better control of blood glucose levels. Thus, administration of HUCB cells in the prediabetic stage without any immunosuppression improves type 1 diabetes by protecting the islets from insulitis in NOD mice.

Histochemical analysis of lymphatic endothelial cells in the pancreas of non-obese diabetic mice.

We studied the relationship between insulitic development and function-structural changes of pancreatic lymphatics in non-obese diabetic (NOD) mice using combined 5'-nucleotidase (5'-Nase) enzyme histochemical and secondary lymphoid tissue chemokine (SLC/CCL21) immunohistochemical methods. Interlobular lymphatic vessels were positive for 5'-Nase throughout the pancreas, and dependent on both blood vessels and pancreatic ducts. Intralobular initial lymphatics were rare and occasionally ran in the neighbourhood of islets. During the non-insulitic stage, the 5'-Nase-reactive product was evenly distributed on the surface of lymphatic endothelial cells (LECs) with weak expression of CCL21. The activity of 5'-Nase on lymphatic vessels became slightly reduced as insulitis developed. The increasing blood glucose values appeared to be consistent with an increasing CCL21 expression by the endothelial lining, especially on the surface of LECs adjacent to the infiltrated islets and tissues. Lymphocytes and dendritic cells (DCs) were frequently located in the connective tissue, surrounding the lymphatic wall with deposition of 5'-Nase precipitates. As the infiltration became severe, lymphocytes and DCs accumulated within lymphatic vessels and expressed high levels of CCL21. The most significant finding was that many DCs adhered to lymphatic vessels, transmigrating via the thin and indented endothelial walls. The activity of 5'-Nase was increased on the adhesion surface between DCs (or lymphocytes) and LECs. The latter were characterized by open intercellular junctions and obvious cytoplasmic protrusions. These results suggest that LECs closely interact with DCs and lymphocytes, and play a key role in the migration of DCs and lymphocytes via lymphatic vessels during the pathological processes of insulitis in NOD mice.

Overexpression of bcl-2 in T cells affects insulitis in the nonobese diabetic mouse.

The nonobese diabetic (NOD) mouse is a useful model for human autoimmune diabetes. The gene for the anti-apoptotic protein Bcl-2 has previously been suggested as a probable susceptibility candidate for the NOD mouse disease. In this study, we investigated how overexpression of Bcl-2 in lymphocytes might affect insulitis in NOD mice. A bcl-2 transgene expressed constitutively under the SV40-promoter and the 5'Igh enhancer, Emu, was bred onto NOD background. Two bcl-2 transgenic NOD strains were produced and analysed, one with overexpression of Bcl-2 on only B cells and the other with overexpression of Bcl-2 on both B and T cells. Subsequent to verification of expression pattern and functionality of the transgene, insulitis intensity was investigated in different backcross generations of the two transgenic strains. Overexpression of Bcl-2 on both B and T cells leads to a statistically significant protection of the mice from insulitis compared with normal littermates. Overexpression of Bcl-2 on only B cells, on the other hand, does not have any statistically significant effect on insulitis. Possible mechanisms for the effect of Bcl-2 on insulitis in NOD mice are discussed.

Reversal of spontaneous autoimmune insulitis in nonobese diabetic mice by soluble lymphotoxin receptor.

One striking feature of spontaneous autoimmune diabetes is the prototypic formation of lymphoid follicular structures within the pancreas. Lymphotoxin (LT) has been shown to play an important role in the formation of lymphoid follicles in the spleen. To explore the potential role of LT-mediated microenvironment in the pathogenesis of insulin-dependent diabetes mellitus (IDDM), an LTbeta receptor-immunoglobulin fusion protein (LTbetaR-Ig) was administered to nonobese diabetic mice. Early treatment with LTbetaR-Ig prevented insulitis and IDDM, suggesting that LT plays a critical role in the insulitis development. LTbetaR-Ig treatment at a late stage of the disease also dramatically reversed insulitis and prevented diabetes. Moreover, LTbetaR-Ig treatment prevented the development of IDDM by diabetogenic T cells in an adoptive transfer model. Thus, LTbetaR-Ig can disassemble the well established lymphoid microenvironment in the islets, which is required for the development and progression of IDDM.

Degradable polymeric carrier for the delivery of IL-10 plasmid DNA to prevent autoimmune insulitis of NOD mice.

Recently, we have reported that biodegradable poly [alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) can condense and protect plasmid DNA from DNase I. In this study, we investigated whether the systemic administration of pCAGGS mouse IL-10 (mIL-10) expression plasmid complexed with PAGA can reduce the development of insulitis in non-obese diabetic (NOD) mice. PAGA/mIL-10 plasmid complexes were stable for more than 60 min, but the naked DNA was destroyed within 10 min by DNase I. The PAGA/DNA complexes were injected into the tail vein of 3-week-old NOD mice. Serum mIL-10 level peaked at 5 days after injection, and could be detected for more than 9 weeks. The prevalence of severe insulitis on 12-week-old NOD mice was markedly reduced by the intravenous injection of PAGA/DNA complex (15.7%) compared with that of naked DNA injection (34.5%) and non-treated controls (90.9%). In conclusion, systemic administration of pCAGGS mIL-10 plasmid/PAGA complexes can reduce the severity of insulitis in NOD mice. This study shows that the PAGA/DNA complex has the potential for the prevention of autoimmune diabetes mellitus. Gene Therapy (2000) 7, 2099-2104.

Importance of GAD65 peptides and I-Ag7 in the development of insulitis in nonobese diabetic mice.

Insulin-dependent diabetes mellitus (IDDM) develops in nonobese diabetic (NOD) mice through the destruction of the B cells in pancreatic Langerhans islets by islet autoantigen-specific T cells. The islet autoantigen glutamic acid decarboxylase 65 (GAD65) is thought to be a major target autoantigen in IDDM. In the present report, we established GAD65-specific T-cell clones using overlapping peptides that cover the amino acid sequences of mouse GAD65. T-cell epitopes of GAD65 were characterized by proliferation and binding assays using various analogue peptides and wild-type or mutant I-Ag7 transfectants. The efficacy of the peptide vaccine in IDDM was determined by administering T-cell epitope peptides to NOD mice and evaluating the histopathology of their insulitis. We obtained two types of T-cell clone, one specific for peptide p316-335 and another specific for p531-545 of GAD65. The p531-545 site has already been identified, but we report the p316-335 site for the first time. T-cell clones recognized those peptides in the wild-type I-Ag7 but not in the mutant I-Ag7 in which the serine at position 57 of the beta-chain was replaced by an aspartic acid. Both the p316-335 and p531-545 peptides bound weakly to I-Ag7. Some peptides with amino acid substitutions had antagonistic activity, and administration of a large amount of wild-type peptide reduced the severity of insulitis in NOD mice. Our results suggest that peptide vaccine therapy may be useful in autoimmune diseases, including IDDM.