Circadian rhythm-related genes: implication in autoimmunity and type 1 diabetes.

Recent gene association and functional studies have proven the implication of several circadian rhythm-related genes in diabetes. Diabetes has been related to variation in central circadian regulation and peripheral oscillation. Different transcriptional regulators have been identified. Circadian genes are clearly implicated in metabolic pathways, pancreatic function and in type 2 diabetes. Much less evidence has been shown for the link between circadian regulation and type 1 diabetes. The hypothesis that circadian genes are involved in type 1 diabetes is reinforced by findings that the immune system undergoes circadian variation and that several autoimmune diseases are associated with circadian genes. Recent findings in the non-obese diabetic mouse model pinpoint to specific mechanisms controlling type 1 diabetes by the clock-related gene Arntl2 in the immune system.

The targeting of ß-cells by T lymphocytes in human type 1 diabetes: clinical perspectives.

This review focuses on genes that control ß-cell targeting in autoimmune, type 1-dependent, diabetes (T1D) and on insulin as the major autoantigen recognized by T lymphocytes throughout the disease process. T1D associates with multiple gene variants. Beyond genes that predispose to general failure of immune tolerance to self, loci identified by the analysis of crosses between non-obese diabetic (NOD) and conventional mouse strains harbour genes that control ß-cell targeting or the deviation of autoimmunity towards other tissues. We report here the role of genes encoding co-activation molecules involved in the activation of T lymphocytes, ICOS and ICOS ligand (B7RP1). NOD mice which are deficient in either of these two molecules are protected from diabetes, but instead develop a neuromuscular autoimmune disease. We also report the characterization in humans of T lymphocytes that are specific for major ß-cell autoantigens, especially insulin. This opens the way towards new bioassays in the diagnosis of autoimmunity and towards autoantigen-specific immunotherapy in T1D. In order to develop a new preclinical model of T1D that would allow testing insulin epitopes to induce immune tolerance in vivo, we developed a mouse that is deficient in endogenous major histocompatibility complex class I and class II genes and deficient for the two murine insulin genes and that express human class I, class II and insulin genes.

Effect of dietary protein on post-prandial glucose in patients with type 1 diabetes.

BACKGROUND: In flexible insulin therapy, determination of the prandial insulin dose only takes into account the carbohydrate content of the evening meal, and not the protein content. Protein can, however, contribute to gluconeogenesis. We compared the glycaemic effect of a standard evening meal with that of a test evening meal enriched in protein. METHODS: The present study was conducted in 28 C-peptide negative patients with type 1 diabetes. Two evening meals that were similar in content, except that one was enriched by the addition of 300 g of 0%-fat fromage frais, were taken on two consecutive days. Insulin doses were maintained exactly the same before both evening meals. Patients were monitored with a continuous glucose-monitoring device. RESULTS: Patients ate similar quantities at both evening meals, except for protein (21.5 g more at the test evening meal). The preprandial insulin dose was 0.96 (0.4) U per 10 g carbohydrates. After correction for differences of interstitial glucose at the start of the evening meals, both interstitial and capillary glucose levels were similar after both evening meals, except for the late-post-prandial interstitial glucose level. CONCLUSIONS: We found no effect of dietary protein on post-prandial-, overnight- or late-night glucose levels in patients with type 1 diabetes. This confirms that dietary proteins need not be included in the calculation of prandial insulin dose.

Susceptibility to insulin dependent diabetes mellitus maps to a 4.1 kb segment of DNA spanning the insulin gene and associated VNTR.

Recent studies have demonstrated that a locus at 11p15.5 confers susceptibility to insulin dependent diabetes mellitus (IDDM). This locus has been shown to lie within a 19 kb region. We present a detailed sequence comparison of the predominant haplotypes found in this region in a population of French Caucasian IDDM patients and controls. Identification of polymorphisms both associated and unassociated with IDDM has allowed us to define further the region of association to 4.1 kb. Ten polymorphisms within this region are in strong linkage disequilibrium with each other and extend across the insulin gene locus and the variable number tandem repeat (VNTR) situated immediately 5' to the insulin gene. These represent a set of candidate disease polymorphisms one or more of which may account for the susceptibility to IDDM.

Zinc transporter (ZnT)8(186-194) is an immunodominant CD8+ T cell epitope in HLA-A2+ type 1 diabetic patients.

AIMS/HYPOTHESIS: Anti-zinc transporter (ZnT)8 autoantibodies are commonly detected in type 1 diabetic patients. We hypothesised that ZnT8 is also recognised by CD8(+) T cells and aimed to identify HLA-A2 (A*02:01)-restricted epitope targets. METHODS: Candidate epitopes were selected by ZnT8 plasmid DNA immunisation of HLA-A2/DQ8 transgenic mice and tested for T cell recognition in peripheral blood mononuclear cells of type 1 diabetic, type 2 diabetic and healthy participants by IFN-γ enzyme-linked immunospot. RESULTS: White HLA-A2(+) adults (83%) and children (60%) with type 1 diabetes displayed ZnT8-reactive CD8(+) T cells that recognised a single ZnT8(186-194) (VAANIVLTV) epitope. This ZnT8(186-194)-reactive fraction accounted for 50% to 53% of total ZnT8-specific CD8(+) T cells. Another sequence, ZnT8(153-161) (VVTGVLVYL), was recognised in 20% and 25% of type 1 diabetic adults and children, respectively. Both epitopes were type 1 diabetes-specific, being marginally recognised by type 2 diabetic and healthy participants (7-12% for ZnT8(186-194), 0% for ZnT8(153-161)). CONCLUSIONS/INTERPRETATION: ZnT8-reactive CD8(+) T cells are predominantly directed against the ZnT8(186-194) epitope and are detected in a majority of type 1 diabetic patients. The exceptional immunodominance of ZnT8(186-194) may point to common environmental triggers precipitating beta cell autoimmunity.

Pancreatic exocrine function in patients with diabetes.

BACKGROUND: Decreased function of the exocrine pancreas is frequent in patients with diabetes. Our aim was to investigate clinical correlates of pancreatic exocrine failure in patients with diabetes. PATIENTS AND METHODS: We investigated exocrine function by assaying both elastase-1 concentration and chymotrypsin activity in 667 patients. We conducted separate analysis on patients with Type 1 diabetes and patients with Type 2 diabetes. Patients were separated into three groups according to whether both elastase-1 concentration and chymotrypsin activity were normal, or one or both were altered. RESULTS: A total of 667 consecutive patients were analysed, including 195 with Type 1 and 472 with Type 2 diabetes. Elastase-1 concentration was <200 µg/g in 23% of the patients. Chymotrypsin activity was <6 U/g in 26% of the patients. In 66% of the patients elastase-1 concentration was >200 ug/g and chymotrypsin activity >6 U/g. One test was below threshold in 19%, both in 15%. In patients with Type 1 diabetes, the three groups defined by results of elastase-1 concentration and chymotrypsin activity differed with regard to duration of diabetes and prevalence of glutamic acid decarboxylase antibodies, but not BMI or HbA(1c) , or prevalence of retinopathy, neuropathy, nephropathy or vascular disease. In patients with Type 2 diabetes, the three groups differed with regard to BMI, use of insulin and vascular disease, but not known duration. CONCLUSION: Factors associated with pancreatic exocrine failure differ in patients with Type 1 diabetes compared with patients with type 2 diabetes. In patients with Type 2 diabetes, association of decreased pancreatic exocrine function with BMI and vascular disease suggests a role of pancreatic arteriopathy.

Immunology of Diabetes Society T-Cell Workshop: HLA class I tetramer-directed epitope validation initiative T-Cell Workshop Report-HLA Class I Tetramer Validation Initiative.

BACKGROUND: Identification of T-cell reactivity to ß-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D). METHODS: We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (<2 years) diagnosed HLA-A*02:01+ T1D patients using blind-coded HLA-A2 tetramers (TMrs) and pentamers (PMrs), encompassing two epitopes of preproinsulin (PPI; PPIA12-20 and PPIB10-18) and two epitopes of glutamic acid decarboxylase (GAD; GAD114-122 and GAD536-545). We also compared the readout of TMrs and PMrs with a CD8+ T-cell interferon-γ enzyme-linked immunospot assay. RESULTS: Despite the minute frequencies of autoreactive cells detected by TMrs/PMrs, most (73-77%) T1D patients had responses to one or more of the epitopes used. All four epitopes were recognized by T1D patients, with a prevalence ranging from 5 to 25%. TMrs and PMrs detected more positive responses to the ß-cell epitopes than CD8+ T-cell interferon-γ enzyme-linked immunospot. However, concordance between positive responses to TMrs and PMrs was limited. CONCLUSIONS: Using a multicentre blind-coded setup and three different T-cell assays, we have validated PPI and GAD epitopes as commonly recognized CD8+ T-cell targets in recently diagnosed T1D patients. Both TMrs and PMrs showed higher detection sensitivity than the CD8+ T-cell interferon-γ enzyme-linked immunospot assay. However, there are some important methodological issues that need to be addressed in using these sensitive techniques for detecting low frequency responses.

T cell-mediated inhibition of the transfer of autoimmune diabetes in NOD mice.

The nonobese diabetic (NOD) mouse has recently been introduced as a model for insulin-dependent diabetes mellitus. The role of regulatory T cells in the development of antipancreatic autoimmunity in this model remains unclear. To evaluate the presence of suppressive phenomena, we used disease transfer by spleen cells from diabetic NOD mice into preirradiated adult recipients as a model for accelerated disease. Suppressor phenomena were detected by testing the protection afforded by lymphoid cells from nondiabetic NOD mice against diabetes transfer in irradiated recipients. Transfer of diabetes was delayed by reconstituting recipients with spleen cells from nondiabetic NOD donors. The greatest protection against diabetes transfer was conferred by spleen cells from 8-wk-old nondiabetic female NOD mice. Depletion experiments showed that the protection was dependent on CD4+ cells. Protection was also detected within thymic cells from nondiabetic NOD mice and protection conferred by spleen cells was abrogated by thymectomy of nondiabetic female, but not male, NOD donors at 3 wk of age. These findings indicate that suppressive CD4+ T cells that are dependent on the presence of the thymus may delay the onset of diabetes in female diabetes-prone NOD mice.

Pancreatic islet beta cells drive T cell-immune responses in the nonobese diabetic mouse model.

The role of autoantigens and that of target organs in which tissue lesions develop remains elusive in most spontaneous models of autoimmune diseases. Whether the presence of target autoantigens is required for the recruitment of autoreactive lymphocytes is unknown in most cases. To evaluate the importance of islet cells in the development of autoimmunity in the nonobese diabetic (NOD) mouse, we generated beta cell-deprived mice by injecting a high dose of alloxan, a toxic agent specific for beta cells. In contrast with spleen cells from 6-mo-old naive NOD mice which transfer diabetes in irradiated 8-mo-old male recipients, spleen cells from age-matched NOD mice which received a single injection of alloxan at 3 wk of age did not transfer diabetes. With the exception of the ability to transfer diabetes, beta cell-deprived NOD mice showed maintained immune competence. Furthermore, sialitis developed with the expected intensity and prevalence in beta cell-deprived mice. Already committed "diabetogenic" spleen cells collected from spontaneously diabetic mice also showed a reduced capacity to transfer diabetes after their removal from the diabetic mice and transient "parking" in beta cell-deprived mice. Taken together, our data bring evidence that involvement of autoreactive T cells detected by the capacity to transfer diabetes requires the presence of target beta cells.

Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4+ and Lyt-2+ T cells.

We have developed a model of syngeneic adoptive transfer for type I diabetes mellitus of NOD mice. This model consists in injecting spleen cells from diabetic adult mice into newborn NOD recipients. 50% of recipients inoculated with 20 X 10(6) cells develop diabetes within the first 10 wk of life, at a time when none of the control littermates have yet become diabetic. The earliest successful transfers are observed at 3 wk of age, at a time when controls do not even exhibit histological changes in their pancreas. In addition we have shown that: (a) both males and females can be adoptively transferred, despite the fact that males rarely develop spontaneous diabetes in our colony; (b) diabetes transfer is a dose-dependent phenomenon that provides an in vivo assay for comparing the autoimmune potential of spleen cells from mice at various stages of their natural history; (c) the susceptibility of the recipients to the transfer is limited in time and declines after 3 wk; and (d) both L3T4+ and Lyt-2+ T cell subsets are necessary for the successful transfer. The neonatal syngeneic transfer provides an effective model for studies of the cellular events involved at regulatory and effector stages of autoimmune type I diabetes.

Decreased insulin response to glucose in islet cell antibody-negative siblings of type 1 diabetic children.

Measurement of beta-cell function is an important marker of progression to diabetes in individuals at risk for the disease. Although the peak incidence for the disease occurs before 17 years of age, normal values for insulin secretion were not available in this age group. We performed a simplified intravenous glucose tolerance test in 167 normal children, and in 98 islet cell antibody (ICA)-negative and 12 ICA-positive siblings of diabetic patients. Their age range was 1-16 yr. The first phase of insulin secretion, evaluated as the sum of plasma insulin concentrations at 1 and 3 min, increased with age and was significantly lower in ICA-negative siblings (86 +/- 6 microU/ml, P < 0.002) than in normal controls (115 +/- 6 microU/ml). This difference was not apparent before 8 yr of age. None of the ICA-negative siblings developed diabetes after an average of 4.5 yr. ICA-positive siblings at first study had a first phase insulin response similar to that of ICA negative siblings, but significantly lower than that of the normal controls (74 +/- 13 microU/ml, P < 0.02). The reason for the decreased insulin secretion in ICA-negative siblings is unknown, but could involve a defect in the growth of beta-cell mass or insulin secretion that could be part of the multifactorial pathogenesis of type 1 diabetes.

Effect of cyclosporin A treatment on the production of antibody in insulin-dependent (type I) diabetic patients.

Anti-islet cell and anti-insulin antibody production was studies over a 12-mo period in 82 recently diagnosed diabetics randomly receiving either cyclosporin or placebo. Cyclosporin had only minimal effects on the production of anti-islet cell antibodies whether directed to islet cytoplasmic (immunofluorescence) or membrane (cytotoxicity assay) antigens even in patients undergoing remission. These data suggest that these antibodies do not play a major role in the pathogenesis of the disease particularly since their (irregular) presence is not predictive of the clinical response to cyclosporin. Conversely, cyclosporin completely suppressed the synthesis of antibodies elicited by exogenous insulin irrespective of the insulin doses received, and decreased the autoantibody production against thyroid antigens, indicating that cyclosporin has variable effects on antibody production against various antigens.

Age-dependent HLA genetic heterogeneity of type 1 insulin-dependent diabetes mellitus.

The association of insulin-dependent diabetes mellitus (IDDM) with certain HLA alleles is well documented in pediatric patients. Whether a similar association is found in adult-on-set IDDM is not clear, although the disease occurs after the age of 20 in 50% of cases. HLA class II DRB1, DQA1, and DQB1 alleles were studied in 402 type I diabetics and 405 healthy controls (all Caucasian) using oligonucleotide typing after gene amplification. Alleles DRB1*03, DRB1*04, DQB1*0201, DQB1*0302, DQA1*0301, and DQA1*0501 were indeed enriched in diabetics and the highest relative risk was observed in patients carrying both the DRB1*03-DQB1*0201 and the DRB1*0402 or DRB1*0405-DQB1*0302 haplotypes. However none of these alleles, or specific residues, could alone account for the susceptibility to IDDM. Furthermore, there were major differences in HLA class II gene profiles according to the age of onset. Patients with onset after 15 yr (n = 290) showed a significantly higher percentage of non-DR3/non-DR4 genotypes than those with childhood onset (n = 112) and a lower percentage of DR3/4 genotypes. These non-DR3/non-DR4 patients, although presenting clinically as IDDM type 1 patients, showed a lower frequency of islet cell antibodies at diagnosis and a significantly milder initial insulin deficiency. These subjects probably represent a particular subset of IDDM patients in whom frequency increases with age. The data confirm the genetic heterogeneity of IDDM and call for caution in extrapolating to adult patients the genetic concepts derived from childhood IDDM.

B-cell responses in autoimmune diseases.

Autoantibodies are detected in most autoimmune diseases. Beyond their direct role in mediating tissue damage in some models, the characterization of several intracellular autoantigens and the study of the autoimmune B-cell repertoire favor an antigen-driven B-cell response in most autoimmune models studied.