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Diabetes constitutes a major public health problem, with dramatic consequences for patients. Both genetic and environmental factors were shown to contribute to the different forms of the disease. The monogenic forms, found both in humans and in animal models, specially help to decipher the role of key genes in the physiopathology of the disease. Here, we describe the phenotype of early diabetes in a colony of NOD mice, with spontaneous invalidation of Akt2, that we called HYP. The HYP mice were characterised by a strong and chronic hyperglycaemia, beginning around the age of one month, especially in male mice. The phenotype was not the consequence of the acceleration of the autoimmune response, inherent to the NOD background. Interestingly, in HYP mice, we observed hyperinsulinemia before hyperglycaemia occurred. We did not find any difference in the pancreas' architecture of the NOD and HYP mice (islets' size and staining for insulin and glucagon) but we detected a lower insulin content in the pancreas of HYP mice compared to NOD mice. These results give new insights about the role played by Akt2 in glucose homeostasis and argue for the ß cell failure being the primary event in the course of diabetes. [ABSTRACT FROM AUTHOR]

In humans, CXCR1 and CXCR2 are two homologous proteins that bind ELR+ chemokines. Both receptors play fundamental roles in neutrophil functions such as migration and reactive oxygen species production. Mouse Cxcr1 and Cxcr2 genes are located in an insulin-dependent diabetes genetic susceptibility locus. The non obese diabetic (NOD) mouse is a spontaneous well-described animal model for insulin-dependent type 1 diabetes. In this disease, insulin deficiency results from the destruction of insulin-producing beta cells by autoreactive T lymphocytes. This slow-progressing disease is dependent on both environmental and genetic factors. Here, we report descriptive data about the Cxcr1 gene in NOD mice. We demonstrate decreased expression of mRNA for Cxcr1 in neutrophils and CD4+ lymphocytes isolated from NOD mice compared to other strains, related to reduced NOD Cxcr1 gene promoter activity. Looking for Cxcr1 protein, we next analyze the membrane proteome of murine neutrophils by mass spectrometry. Although Cxcr2 protein is clearly found in murine neutrophils, we did not find evidence of Cxcr1 peptides using this method. Nevertheless, in view of recently-published experimental data obtained in NOD mice, we argue for possible Cxcr1 involvement in type 1 diabetes pathogenesis. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) treatment improves pathogenic outcome of autoimmune diseases by promoting tolerance. However, the mechanism behind this protective tolerance is not yet defined. Here, we show in a transgenic mouse model for autoimmune diabetes that ex vivo gaseous CO (gCO)-treated DCs loaded with pancreatic β-cell peptides protect mice from disease. This protection is peptide-restricted, independent of IL-10 secretion by DCs and of CD4+ T cells. Although no differences were observed in autoreactive CD8+ T-cell function from gCO-treated versus untreated DC-immunized groups, gCO-treated DCs strongly inhibited accumulation of autoreactive CD8+ T cells in the pancreas. Interestingly, induction of β1-integrin was curtailed when CD8+ T cells were primed with gCO-treated DCs, and the capacity of these CD8+ T cells to lyse isolated islet was dramatically impaired. Thus, immunotherapy using CO-treated DCs appears to be an original strategy to control autoimmune disease. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Islet-reactive CD8+ T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown. RESEARCH DESIGN AND METHODS--We took advantage of a recently validated islet-specific CD8+ T-cell γ-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2+ adult type 1 diabetic patients close to diagnosis and at a second time point 7-16 months later. RESULTS--CD8+ T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P = 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60-67 to 20% (P < 0.02). The previously subdominant IA-2206-214 and IGRP265273 peptides were newly targeted, thus becoming the immunodominant epitopes. CONCLUSIONS--Shifts both in frequency and in immunodominance of CD8+ T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements. Diabetes 57:1312-1320, 2008 [ABSTRACT FROM AUTHOR]

Despite the understanding that type 1 diabetes pathogenesis is mediated by T-cells, detection of these rare lymphocytes remains largely elusive. Suitable T-cell assays are highly needed, since they could offer preclinical diagnoses and immune surrogate end points for clinical trials. Although CD4+ T-cell assays have met with limited success, CD8+ T-cells are increasingly recognized as key actors in the diabetes of the NOD mouse. CD8+ T-cells are likely to play a role also in humans and may provide new markers of β-cell autoimmunity. Taking advantage of a panel of HLA-A2-restricted β-cell epitopes derived from preproinsulin, GAD, and islet glucose-6-phosphatase catalytic subunit-related protein (IGRP), we have implemented an islet-specific CD8+ T-cell interferon-γ enzyme-linked immunospot (ISL8Spot) assay. The ISL8Spot assay is capable of detecting and quantifying β-cell-reactive CD8+ T-cells directly ex vivo, without any preliminary expansion, using either fresh or frozen samples. Positive ISL8Spot responses separate new-onset diabetic and healthy samples with high accuracy (86% sensitivity, 91% specificity), using as few as five immunodominant epitopes. Moreover, sensitivity reaches 100% when the ISL8Spot assay is complemented by antibody determinations. Combination of CD8+ T-cell measurements with immune intervention strategies may open new avenues toward type 1 diabetes prediction and prevention. Diabetes 56:613-621, 2007 [ABSTRACT FROM AUTHOR]

Type 1 diabetes mellitus (T1DM) results from the destruction of β cells by autoantigen-specific T cells. In the non-obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I-restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B-chain and C-peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA-A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA-A2.1-transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA-transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA-2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot- and MHC tetramer-based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention. [ABSTRACT FROM AUTHOR]

Plasmid-DNA gene-gun immunization may be an efficient approach for investigating the role of skin dendritic cells (DCs) in type 1 diabetes (T1D) pathogenesis and the significance of the presentation of peptides that mimic autoantigenic epitopes in aggravating or modulating the autoimmune reaction. Gene-gun immunization has been described as producing longlasting immune responses elicited by skin DCs, especially Langerhans cells (LCs). Therefore, we tested the immune response and diabetes modulation in nonobese diabetic (NOD) mice and in control BALB/c mice, by gene-gun administration of plasmid-DNA encoding (1) human 65 kDa glutamic acid decarboxylase (hGAD65) mimicking the crucial mouse autoantigen GAD65 (similarity of 95.7%) or (2) β-galactosidase (βGAL) as a negative control. Expression of GAD and βGAL in skin of pc-GAD- and pc-LacZ-injected mice, respectively, was confirmed. It was surprising that both pc-LacZ-injected BALB/c and NOD mice exhibited a βGAL-specific Th1 immune response: spleen cells of pc-LacZ mice proliferated specifically to βGAL (P < 10-4) and secreted significant amounts of IFNγ (P < 10-4). pc-LacZ mice also developed a βGAL-specific Th1-related (IgG2a/2c) and Th2-related (IgG1) humoral response. Although pc-GAD BALB/c mice showed Th2-related GAD-specific IgG1 production and a significant secretion of IL4 (P < .03), pc-GAD NOD mice did not generate either an antibody response or a T cell response specific to GAD. Moreover, gene-gun immunization encoding hGAD65 did not clearly modulate diabetes onset in NOD mice. This absence of detectable GAD-specific response may implicate skin DC deficiencies in NOD mice. The gene-gun technique could thus provide an interesting model for studying skin DC abnormalities in NOD mice and their potential implication of presenting mimetic peptides that modulate the autoimmune response in T1D. [ABSTRACT FROM AUTHOR]

Abstract: Cyclophosphamide (CTX) was previously shown to induce the recruitment of immunosuppressive myeloid cells in mouse. In the non-obese diabetic (NOD) mouse, which develops spontaneously type I diabetes, CTX is widely known to accelerate the autoimmune process. Our data demonstrated that CTX actually did mobilize an immunosuppressive myeloid CD11b+ Ly-6G− population in the NOD mouse spleen in addition to a well-identified neutrophil CD11b+ Ly-6G+ population. CD11b+ Ly-6G− cells, in contrast with CD11b+ Ly-6G+ cells, were able to inhibit in vitro mitogen-induced syngeneic T cell proliferation. CD11b+ Ly-6G− cells represented a heterogeneous population mainly made of CD31hi cells and Ly-6C+ monocytes. Only these last ones supported the immunosuppressive in vitro activity and resembled circulating inflammatory monocytes according to flow cytometry, cytology and RT-PCR data. Although CD11b+ Ly-6G− Ly-6C+ cells exhibited immunosuppressive function in vitro, they were not able to control the autoimmune response following CTX injection. Our data show that these CTX-induced immunosuppressive myeloid cells actually behaved as very plastic cells in vitro. Likewise, in the model of prediabetic NOD/SCID mice, CD11b+ Ly-6G− Ly-6C+ were able to differentiate into CD11c+ cells after i.v. injection. Herein, we described a new mechanism by which CTX might induce diabetes acceleration in the NOD mouse. In summary, recruited immunosuppressive cells might participate in the immunopotentiating effect of CTX on the autoimmune response by their further differentiation into immunostimulatory cells. [Copyright &y& Elsevier]

Abstract: 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 (GAD90–98) 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 GAD90–98 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. GAD90–98 specific CD8+ lymphocytes lysed efficiently islet cells in vitro and transferred diabetes into NODSCID mice (100%). Finally, diabetes was accelerated greatly in 3-week-old female NOD mice injected i.p. with GAD90–98, strengthening the role of GAD-specific CTLs in diabetes pathogenesis. [Copyright &y& Elsevier]