Balance of GAD65-specific IL-10 production and polyclonal Th1-type response in type 1 diabetes.

It has been proposed that cytokine responses of memory CD4+ cells change from a T-helper 2 (Th2)-to a T-helper 1 (Th1)-dominant response as the disease progresses in non-obese diabetic (NOD) mice. However, the regulation of Th1/Th2 balance in spontaneous diabetes development in this model is not well understood. In this study, higher glutamic acid decarboxylase 65 (GAD65)-specific IL-10 production was observed at 10-12 weeks in NOD mice, and a marked increase of Th1-type response (IFN-gamma production) upon polyclonal (anti-CD3 antibody) stimulation was observed just before diabetes development along with a decline of GAD65-specific IL-10 production. Moreover, there was a clear negative correlation between IL-10 level upon GAD65 stimulation and log(IFN-gamma) level upon anti-CD3 antibody stimulation (r=-0.999, p<0.001). These results suggest that the balance between GAD65-specific IL-10 production and polyclonal Th1-type response may regulate the onset of hyperglycemia in type 1 diabetes in NOD mice.     

Inhalation of glutamic acid decarboxylase 65-derived peptides can protect against recurrent autoimmune but not alloimmune responses in the non-obese diabetic mouse

Systemic administration of islet-derived antigens has been shown to protect against diabetes in the non-obese diabetic (NOD) mouse by the induction of antigen-specific regulatory T cells. Bystander regulation to related and unrelated islet-derived antigens (intramolecular and intermolecular recognition) in this context is recognized. We tested if intranasal administration of glutamic acid decarboxylase 65 (GAD 65)-derived peptides could protect against both autoimmune and, through bystander regulation, alloimmune responses in a NOD mouse model. Spontaneously diabetic female NOD mice underwent islet transplantation from either C57Bl/6 or NOD islet donors. Islet recipients were treated with intranasal GAD 65-derived peptides or control (ovalbumin) peptide pre- and post-transplantation. In-vitro analysis of the effect of inhalation was defined using lymph node proliferation assays and supernatant analysis for cytokines. GAD 65-derived peptide inhalation resulted in significant protection against recurrent autoimmune disease, with the generation of an interleukin (IL)-10-producing immune phenotype in a syngeneic islet transplant model. This phenotype, however, was not robust enough to protect against alloimmune responses. Inhalation of GAD-derived peptides induces an immunoregulatory response that protects against recurrent autoimmune, but not alloimmune responses in the NOD mouse.     

Absence of significant Th2 response in diabetes-prone non-obese diabetic (NOD) mice.

Accumulating evidence suggests that Th1 T cells play a pivotal role in the development of autoimmune diabetes. Conversely, promoting a Th2 response inhibits disease progression. However, it has not been determined whether Th2 cells are regulatory T cells that fail at the time of diabetes development in naive non-diabetic NOD mice. Therefore, in order to evaluate cytokine secretion by spleen and islet infiltrating T cells in NOD mice at different stages of the autoimmune process, we developed an ELISPOT assay that detects IL-2, IL-4, and interferon-gamma (IFN-gamma) secretion in vitro at the single-cell level. We showed that, whatever the age considered, IFN-gamma is predominantly secreted, and that no IL-4-secreting cells are detected in the islets of male and female NOD mice. Spleen cells from 8-week-old female NOD mice, which include regulatory suppressor T cells, do not secrete IL-4, either upon presentation of islet cell antigens in vitro, or after transfer in vivo, but do secrete IFN-gamma. IFN-gamma secretion by T cells from diabetic mice results from CD4 but not CD8 T cells in transfer experiments into NOD/severe combined immunodeficient (SCID) recipients. These results suggest that (i) detection of regulatory CD4 T cells in NOD mice is not paralleled by a Th2 response; (ii) beta cell destruction does not depend on a switch from a Th2 to a Th1-type response; and (iii) CD8 T cells do not participate in induction of diabetes by secreting IFN-gamma.     

Regulatory Th2-type T cell Lines Against Insulin and GAD Peptides Derived from Orally- and Nasally-Treated NOD Mice Suppress Diabetes

Non-obese diabetic (NOD) mice spontaneously develop diabetes. Ourselves and others have previously shown that oral and nasal administration of insulin or glutamic acid decarboxylase (GAD) suppresses development of diabetes in the NOD mouse and that this suppression appears secondary to the generation of regulatory T cells that act by secreting anti-inflammatory cytokines such as IL-4 and TGF-beta. In the present study, we analysed cytokine patterns associated with mucosal administration of insulin B-chain, B-chain peptide 10-24 and GAD peptide 524-543 and derived lines and clones from mucosally-treated animals. Mice were fed five times (400-600 microg/feed) or nasally-treated three times (60 microg/application), and 2 days after the last treatment were immunized in the footpad with the mucosally administered antigen in CFA. Primary immune responses in the popliteal lymph node were measured 10 days after immunization and lines and clones were then established from the primary cultures. There was significantly less IFN-gamma production in mucosally-treated mice associated with increased production of IL-10 and TGF-beta. The nature of the antigen appeared to determine cytokine production as the B-chain given either orally or nasally primed for TGF-beta responses, whereas mucosally administered B-chain peptide 10-24 primed for IL-10. T cell clones, established from draining lymph nodes of fed or nasally-treated animals, secreted IL-4, IL-10 and TGF-beta whereas those from non-fed mice secreted IL-2 and IFN-gamma. Transfer of Th1 lines with splenocytes from diabetic NOD mice into NOD or NOD/SCID animals accelerated diabetes, whereas transfer of Th2 lines suppressed the development of diabetes. Our results further support a role for Th2-type cells in the regulation of diabetes in NOD mice.     

HLA-DQ8 transgenic and NOD mice recognize different epitopes within the cytoplasmic region of the tyrosine phosphatase-like molecule, IA-2

Type 1 diabetes mellitus is strongly associated with HLA-DQ8 in humans and I-A(g7) in the NOD mouse. The disease is characterized by loss of tolerance to auto-antigens such as GAD, insulin, and the protein tyrosine phosphatase-like molecule, IA-2. We identified T cell epitopes on the intracytoplasmic region of IA-2 by immunizing DQ8/NOD, DQ8/B10, and NOD mice with overlapping 18 mer peptides in CFA. We identified four peptides presented both by DQ8 and NOD, five DQ8 specific peptides, and six NOD specific peptides. Both mouse lines failed to respond to ten peptides. We demonstrated MHC class II and CD4 restriction of proliferative responses using appropriate blocking antibodies. To understand the role of non-MHC genes in the generation of immune response to the islet auto-antigen, we evaluated cytokine secretion following immunization of DQ8 transgenic mice with strongly immunogenic peptides. The NOD background resulted in increased secretion of cytokines. In conclusion, we have identified IA-2 peptides that induce lymphoproliferative responses in DQ8 transgenic and NOD mice and shown that these peptides stimulate production of Th1 and Th2 cytokines.     

Analysis of mercury-induced immune activation in nonobese diabetic (NOD) mice

In susceptible mice, the heavy metal ion mercury is able to induce a strong immune activation, which resembles a T helper 2 (Th2) type of immune response and is characterized by a polyclonal B cell activation, formation of high levels of IgG1 and IgE antibodies, production of autoantibodies of different specificities and development of renal IgG deposits. In the present study, we analysed the in vivo effects of mercury in nonobese diabetic (NOD) mice, which is believed to develop a spontaneous Th1 cell-mediated autoimmune diabetes similar to type 1 diabetes in humans. Three weeks of treatment with mercury induced a strong Th2 like immune/autoimmune response in NOD mice. This response was characterized by an intensive increase in splenic IgG1 antibody secreting cells, a marked elevation in serum IgE levels, a substantial increase in splenic IL-4 mRNA, but a significant decrease in splenic IFN-gamma mRNA. Mercury-induced IgG1 antibodies were mainly against ssDNA, TNP and thyroglobulin, but not against nucleolar antigen. Moreover, mercury-injected NOD mice developed high titres of IgG1 deposits in the kidney glomeruli. We further tested if the generated Th2 response could interfere with the development of insulitis and diabetes in NOD mice. We found that three weeks of treatment with mercury was also able to significantly suppress the development of insulitis and postpone the onset of diabetes in these mice. Thus, mercury-induced immune activation can counter-regulate the Th1 cell-mediated autoimmune responses and confer a partial protection against autoimmune diabetes in NOD mice.     

In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-gamma/TNF-alpha

Immunization with mycobacterial preparation such as Bacille Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) prevents the onset and recurrence of type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we explored the mechanism underlying the down-regulation of diabetogenic T cells by BCG treatment. We found that the potential of splenocytes from BCG-immunized diabetic NOD mice to adoptively transfer diabetes was significantly impaired. BCG immunization sequentially induced the production of TNF-alpha, IFN-gamma and IL-4 by splenocytes, increased the expression of Fas(high) (Apo-1/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis. The primary role of IFN-gamma and TNF-alpha in BCG-immunotherapy was demonstrated by (i) reversing the immune regulatory effect of BCG by in vivo treatment with neutralizing anti-cytokine antibodies, (ii) inducing effect similar to BCG by treatment with these cytokines. We show that Fas and TNF are two pathways in BCG-induced apoptosis of diabetogenic T cells, since in vitro blocking FasL or TNFR1 with antibody reduced T cell apoptosis and increased T cell proliferative response. In addition, TNF-alpha and agonistic anti-Fas antibody had a synergistic effect on the in vitro apoptosis of diabetogenic T cells. Our results suggest that BCG down-regulates destructive autoimmunity by TNF-alpha/IFN-gamma-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways. These studies provide a novel mechanism for blocking disease recurrence and immune modulating effect of BCG immunization in type 1 diabetes.     

Mechanisms of Mycobacterium avium-induced resistance against insulin-dependent diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice: role of Fas and Th1 cells

NOD mice spontaneously develop autoimmune diabetes. One of the manipulations that prevent diabetes in NOD mice is infection with mycobacteria or immunization of mice with mycobacteria-containing adjuvant. Infection of NOD mice with Mycobacterium avium, done before the mice show overt diabetes, results in permanent protection of the animals from diabetes and this protective effect is associated with increased numbers of CD4⁺ T cells and B220⁺ B cells. Here, we investigate whether the M. avium-induced protection of NOD mice from diabetes was associated with changes in the expression of Fas (CD95) and FasL by immune cells, as well as alterations in cytotoxic activity, interferon-gamma (IFN-γ) and IL-4 production and activation of T cells of infected animals. Our data indicate that protection of NOD mice from diabetes is a Th1-type response that is mediated by up-regulation of the Fas–FasL pathway and involves an increase in the cytotoxicity of T cells. These changes are consistent with induction by the infection of regulatory T cells with the ability of triggering deletion or anergy of peripheral self-reactive lymphocytes that cause the autoimmune disease of NOD mice.     

Th17 cells promote pancreatic inflammation but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells

IDDM is characterized by leukocyte invasion to the pancreatic tissues followed by immune destruction of the islets. Despite the important function of Th17 cells in other autoimmune disease models, their function in IDDM is relatively unclear. In this study, we found association of elevated Th17 cytokine expression with diabetes in NOD mice. To understand the function of Th17 cells in IDDM, we differentiated islet‐reactive BDC2.5 TcR transgenic CD4⁺ cells in vitro into Th17 cells and transferred them into NOD.scid and neonate NOD mice. NOD.scid recipient mice developed rapid onset of diabetes with extensive insulitic lesions, whereas in newborn NOD mice, despite extensive insulitis, most recipient mice did not develop diabetes. Surprisingly, BDC2.5⁺ cells recovered from diabetic NOD.scid mice, in comparison with those from neonate NOD mice, showed predominant IFN‐γ over IL‐17 expression, indicating conversion of donor cells into Th1 cells. Moreover, diabetes progression in NOD.scid recipients was dependent on IFN‐γ while anti‐IL‐17 treatment reduced insulitic inflammation. These results indicate that islet‐reactive Th17 cells promote pancreatic inflammation, but only induce IDDM upon conversion into IFN‐γ producers.     

A multivalent vaccine for type 1 diabetes skews T cell subsets to Th2 phenotype in NOD mice

Previous studies by our group, using an experimental autoimmune thyroiditis (EAT) model in Strain 13 inbred guinea pigs, resulted in T cell-mediated delayed hypersensitivity; however, autoantibodies proved not to be cytotoxic to thyroid epithelial cells in the presence or absence of complement proteins. Albeit, T cell-mediated lymphocyte cytotoxicity began to diminish sharply concomitantly with increasing titers of circulating autoantibodies, indicating a skewing of the self-reactive response and amelioration of the EAT. Furthermore, immunization of guinea pigs with thyroglobulin in incomplete Freund's adjuvant (IFA) generated a high titer of antithyroglobulin antibodies and proved to inhibit thyroiditis. These observations indicated that the shift in the immune response from Th1 to Th2 and the production of antibodies were likely responsible for ameliorating EAT. Based upon these results, we extrapolated our studies to design a multivalent vaccine, which shows promise in preventing/reversing T1D in NOD mice. A small pilot study was conducted in which a total of 34 mice, 20 non-immunized controls and 14 immunized with syngeneic islet lysate, were monitored for mean day to diabetes for a total of 28 weeks. Immunization of NOD animals with syngeneic islet lysates resulted in a significant delay in diabetes onset (P < 0.001) as compared to non-immunized controls. To further assess the vaccine's efficacy, robustness, and delay of disease, a large-scale experiment was conducted and monitored for 32 weeks using 106 mice, 64 non-immunized controls and 42 immunized with syngeneic islet lysate. At the end of the study, 90% of the non-immunized group developed diabetes, while less than 25% of the immunized group became diabetic (P < 0.0001). The protective effect, as a result of vaccination, correlated with an increase in the levels of IL-10 and IL-4 cytokines as well as a skewing to Th2-dependent isotype antibodies in serum. Strikingly, adoptive transfer of spleen cells from immunized animals into NOD.scid recipients provided protection against transfer of diabetes by diabetogenic spleen cells. The results of this study provide evidence that vaccination with islet lysate leads to a Th2-dependent skewing of the immune response to islet beta cells as a possible mechanism of protection. This strategy may be implemented as a possible vaccination protocol for arresting and/or preventing T1D in patients.     

DNA vaccination encoding glutamic acid decarboxylase can enhance insulitis and diabetes in correlation with a specific Th2/3 CD4 T cell response in non-obese diabetic mice

DNA vaccination encoding beta cell autoantigens has been shown very recently to prevent type I diabetes in non-obese diabetic (NOD) mice. However, DNA vaccination encoding microbial or reporter antigens is known to induce specific long-lasting CD4 Th1 and strong cytolytic CD8 T cell responses. As this immune phenotype is associated strongly with beta cell destruction leading to diabetes, we have chosen to study the effects of plasmids encoding glutamic acid decarboxylase (GAD), a crucial beta cell autoantigen, in female NOD mice that developed a 'moderate' diabetes incidence. In the present study, 3-week-old female NOD mice were vaccinated twice in tibialis muscles with plasmid-DNA encoding 65-kDa GAD or betagalactosidase. In GAD-DNA immunized mice, diabetes cumulative incidence (P < 3.10(-3)) and insulitis (P < 7.10(-3)) increased significantly. Simultaneously, DNA immunization induced GAD-specific CD4 T cells secreting interleukin (IL)-4 (P < 0.05) and transforming growth factor (TGF)-beta (P = 0.03). These cells were detected in spleen and in pancreatic lymph nodes. Furthermore, vaccination produced high amounts of Th2 cytokine-related IgG1 (P < 3.10(-3)) and TGF-beta-related IgG2b to GAD (P = 0.015). Surprisingly, diabetes onset was correlated positively with Th2-related GAD-specific IgG1 (P < 10(-4)) and TGF-beta-related IgG2b (P < 3.10(-3)). Moreover, pancreatic lesions resembled Th2-related allergic inflammation. These results indicate, for the first time, that GAD-DNA vaccination could increase insulitis and diabetes in NOD mice. In addition, our study suggests that Th2/3 cells may have potentiated beta cell injury.     

Attenuation of Th1 response through galectin‐9 and T‐cell Ig mucin 3 interaction inhibits autoimmune diabetes in NOD mice

Galectin‐9 (gal‐9), widely expressed in many tissues, regulates Th1 cells and induces their apoptosis through its receptor, T‐cell Ig mucin 3, which is mainly expressed on terminally differentiated Th1 cells. Type 1 diabetes is a Th1‐dominant autoimmune disease that specifically destroys insulin‐producing β cells. To suppress the Th1 immune response in the development of autoimmune diabetes, we overexpressed gal‐9 in NOD mice by injection of a plasmid encoding gal‐9. Mice treated with gal‐9 plasmid were significantly protected from diabetes and showed less severe insulitis compared with controls. Flow cytometric analyses in NOD‐T1/2 double transgenic mice showed that Th1‐cell population in spleen, pancreatic lymph node and pancreas was markedly decreased in gal‐9 plasmid‐treated mice, indicating a negative regulatory role of gal‐9 in the development of pathogenic Th1 cells. Splenocytes from gal‐9 plasmid‐treated mice were less responsive to mitogenic stimulation than splenocytes from the control group. However, adoptive transfer of splenocytes from gal‐9‐treated or control mice caused diabetes in NOD/SCID recipients with similar kinetics, suggesting that gal‐9 treatment does not induce active tolerance in NOD mice. We conclude that gal‐9 may downregulate Th1 immune response in NOD mice and could be used as a therapeutic target in autoimmune diabetes.     

Modulation of diabetes in NOD mice by GAD65-specific monoclonal antibodies is epitope specific and accompanied by anti-idiotypic antibodies

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells. Here we show that administration of a human monoclonal antibody (b96.11) specific to the 65-kDa isoform of glutamate decarboxylase (GAD65) to prediabetic non-obese diabetic (NOD) mice significantly delays the onset of autoimmune diabetes. We found this effect to be epitope-specific, as only b96.11 showed this therapeutic property, while a GAD65-specific human monoclonal control antibody (b78) derived from the same patient, but specific to a different determinant of GAD65, had no significant effect on the progression of disease. Administration of b96.11 or b78 to NOD mice was accompanied by the generation of anti-idiotypic antibodies. Importantly, the induced anti-idiotypic antibodies were specific for the immunizing antibody and blocked the binding of GAD65 by the respective antibody. These findings suggest a potential role for the internal image of the GAD65 determinant recognized by b96.11 in the anti-idiotypic antibody, supporting an immunomodulatory role for GAD65-specific autoantibodies, as originally postulated by Jerne.     

Regulatory cytokine production stimulated by DNA vaccination against an altered form of glutamic acid decarboxylase 65 in nonobese diabetic mice

Nonobese diabetic (NOD) mice develop a T-cell dependent autoimmune form of diabetes, in which glutamic acid decarboxylase 65 (GAD65) is an important islet target antigen. Intramuscular DNA vaccination with a plasmid encoding native GAD65 (a cytosolic antigen) did not significantly alter the incidence of diabetes, but vaccination against an altered form of GAD65 with a signal peptide (spGAD), which is secreted in vitro, was protective. The preventive effect was further enhanced by repeated injections of the spGAD plasmid. Following DNA injection into muscle GAD65 was expressed for several months, and this was not accompanied by an inflammatory response. Immunization against GAD65 was not associated with substantial alterations in cytokine production by splenic lymphocytes stimulated with immunogenic GAD65 peptides. In contrast, spGAD induced increased secretion of both interleukin 10 and interferon gamma and a striking decrease in the interferon gamma/interleukin 10 ratio in culture supernatants. Similarly, spGAD-immunized mice had higher serum interleukin 10 levels and lower serum interferon gamma levels than other groups, suggesting a systemic effect. In nondiabetic mice there was increased basal production of transforming growth factor beta(1), which was enhanced by antigenic stimulation. These alterations in regulatory cytokine production were apparent both early and late after the treatment was initiated. These findings suggest that DNA vaccination against spGAD protects NOD mice by increasing regulatory cytokine production.     

Immunization of non-obese diabetic (NOD) mice with glutamic acid decarboxylase-derived peptide 524–543 reduces cyclophosphamide-accelerated diabetes

NOD mice constitute a model for studying the prevention of human autoimmune type 1 diabetes. Glutamic acid decarboxylase (GAD) could be a key antigen involved in this disease, and GAD65 peptide 524–543 has been implicated in early T cell response in young NOD mice. We performed two i.p. injections of GAD peptide 524–543 (100 μg at each injection), together with Freund*s incomplete adjuvant (FIA), into female NOD mice at 30 and 45 days old. Diabetes was accelerated 2 weeks later by a single injection of cyclophosphamide (CY), which acts against suppressive mechanisms. Treatment with GAD 524–543 peptide delayed the onset of diabetes and reduced its incidence (28% versus 60%; P<0·001) compared with control mice injected with FIA alone, or GAD peptide 534–553, or an irrelevant peptide. In the same group, the severity of lymphocytic inflammation of pancreatic islets was reduced (P<0·03). Up to 3 months after peptide injections, a strong splenocytic proliferative response occurred in immunized NOD mice against the immunizing peptide alone (but not against a panel of seven other GAD65-derived peptides). After peptide challenge of splenocytes in vitro, protection against CY-accelerated diabetes was associated with higher peptide-specific production of T helper type 2 (Th2)-associated interleukins 4 and 10, whereas Th1-associated interferon-gamma and IL-2 were proportionally less represented. During cotransfer, T splenocytes from GAD 524–543-immunized mice were able to reduce the capacity of T cells from diabetic donors to transfer the disease adoptively (P<0·01), demonstrating the generation of cellular mechanisms that actively suppress the disease. It is concluded that immunization of NOD mice with GAD65 peptide 524–543 can counteract CY-accelerated diabetes, possibly through active cellular suppression linked to a shift of Th1/Th2 balance toward the production of Th2 cytokines such as IL-4 and IL-10. This study provides additional support for the notion that GAD, and more precisely its epitope 524–543, could be one of the key targets for the pathogenesis of type 1 diabetes in NOD mice, as well as for the efficacy of disease-specific peptide therapy in type 1 diabetes.     

Bacille Calmette–Guérin/DNAhsp65 prime‐boost is protective against diabetes in non‐obese diabetic mice but not in the streptozotocin model of type 1 diabetes

Type I diabetes is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this study was to evaluate the prophylactic potential of a prime‐boost strategy involving bacille Calmette–Guérin (BCG) and the pVAXhsp65 vaccine (BCG/DNAhsp65) in diabetes induced by streptozotocin (STZ) in C57BL/6 mice and also in spontaneous type 1 diabetes in non‐obese diabetic (NOD) mice. BCG/DNAhsp65 vaccination in NOD mice determined weight gain, protection against hyperglycaemia, decreased islet inflammation, higher levels of cytokine production by the spleen and a reduced number of regulatory T cells in the spleen compared with non‐immunized NOD mice. In the STZ model, however, there was no significant difference in the clinical parameters. Although this vaccination strategy did not protect mice in the STZ model, it was very effective in NOD mice. This is the first report demonstrating that a prime‐boost strategy could be explored as an immunomodulatory procedure in autoimmune diseases.     

Antibody combination therapy targeting CD25, CD70 and CD8 reduces islet inflammation and improves glycaemia in diabetic mice

Destruction of pancreatic islets in type 1 diabetes is caused by infiltrating, primed and activated T cells. In a clinical setting this autoimmune process is already in an advanced stage before intervention therapy can be administered. Therefore, an effective intervention needs to reduce islet inflammation and preserve any remaining islet function. In this study we have investigated the role of targeting activated T cells in reversing autoimmune diabetes. A combination therapy consisting of CD25-, CD70- and CD8-specific monoclonal antibodies was administered to non-obese diabetic (NOD) mice with either new-onset diabetes or with advanced diabetes. In NOD mice with new-onset diabetes antibody combination treatment reversed hyperglycaemia and achieved long-term protection from diabetes (blood glucose <13·9 mmol/l) in >50% of mice. In contrast, in the control, untreated group blood glucose levels continued to increase and none of the mice were protected from diabetes (P < 0·0001). Starting therapy early when hyperglycaemia was relatively mild proved critical, as the mice with advanced diabetes showed less efficient control of blood glucose and shorter life span. Histological analysis (insulitis score) showed islet preservation and reduced immune infiltration in all treated groups, compared to their controls. In conclusion, antibody combination therapy that targets CD25, CD70 and CD8 results in decreased islet infiltration and improved blood glucose levels in NOD mice with established diabetes.     

Inhibition of diabetes in NOD mice by human pregnancy factor

Clinical symptoms of Th1 mediated autoimmune diseases regress in many patients during pregnancy. A prominent feature of pregnancy is the presence of human chorionic gonadotrophin hormone (hCG) in blood and urine. In this report we tested the effect of clinical grade hCG (c-hCG) on the development of diabetes, a Th1 mediated autoimmune disease, in nonobese diabetic (NOD) mice. We show that treatment of NOD mice with c-hCG before the onset of clinical symptoms lowered the increased blood glucose levels, reversed the established inflammatory infiltrate of pancreatic tissue, and profoundly inhibited the development of diabetes for prolonged time. c-hCG also induced profound inhibition of the functional activity (i.e. production of IFN-γ) of Th1 cells. Transfer of spleen cells from c-hCG-treated NOD mice into immunocompromised NOD.SCID mice inhibited the development of diabetes in these otherwise nontreated mice. This shows that the treatment of the donor NOD mice induced persistent changes in the immune system. The antidiabetic activity of c-hCG was not caused by heterodimeric hCG or its subunits. Instead, this antidiabetic activity resided in a fraction of c-hCG preparation that contains a 400–2000 Dalton natural (immuno) modulatory pregnancy factor (NMPF).