CTSH regulates ß-cell function and disease progression in newly diagnosed type 1 diabetes patients.

Over 40 susceptibility loci have been identified for type 1 diabetes (T1D). Little is known about how these variants modify disease risk and progression. Here, we combined in vitro and in vivo experiments with clinical studies to determine how genetic variation of the candidate gene cathepsin H (CTSH) affects disease mechanisms and progression in T1D. The T allele of rs3825932 was associated with lower CTSH expression in human lymphoblastoid cell lines and pancreatic tissue. Proinflammatory cytokines decreased the expression of CTSH in human islets and primary rat ß-cells, and overexpression of CTSH protected insulin-secreting cells against cytokine-induced apoptosis. Mechanistic studies indicated that CTSH exerts its antiapoptotic effects through decreased JNK and p38 signaling and reduced expression of the proapoptotic factors Bim, DP5, and c-Myc. CTSH overexpression also up-regulated Ins2 expression and increased insulin secretion. Additionally, islets from Ctsh(-/-) mice contained less insulin than islets from WT mice. Importantly, the TT genotype was associated with higher daily insulin dose and faster disease progression in newly diagnosed T1D patients, indicating agreement between the experimental and clinical data. In line with these observations, healthy human subjects carrying the T allele have lower ß-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of ß-cell function during progression of T1D and reinforce the concept that candidate genes for T1D may affect disease progression by modulating survival and function of pancreatic ß-cells, the target cells of the autoimmune assault.

HLA-typing, clinical, and immunological characterization of youth with type 2 diabetes mellitus phenotype from the German/Austrian DPV database.

AIM: To characterize the clinical and immunological features of HLA-typed youth with pediatric onset of type 2 diabetes mellitus (T2DM). METHOD: One hundred and seven patients with clinically diagnosed T2DM (aged ≤20 yr at diagnosis) were examined. DNA and serum, obtained after a median diabetes duration of 2.2 (Q1-Q3: 0.8-4.6) yr, were used for centralized HLA-typing and autoantibody (GADA, IA-2A, ZnT8A) measurements. RESULTS: 64.6% of patients were female and median age at diagnosis was 13.8 (Q1-Q3: 11.6-15.4) yr. Patients were obese [median body mass index-standard deviation score (BMI-SDS): 2.6 (2.0-3.1)], 88.0% had a family history of diabetes and 40.2% a migration background. Islet autoantibodies were detected in 16 (15.0%), among which 7 (6.5%) had multiple islet autoantibodies. Autoantibody positive patients had poorer metabolic control than autoantibody negative patients [glycosylated hemoglobin A1c (HbA1c): 8.1 (6.9-10.1) % vs. 6.6 (5.9-8.0) %; p = 0.033], while patients with HLA-DR genetic risk had higher BMI-SDS than those with HLA-DRXX [2.6 (2.4-3.7) vs. 2.4 (1.7-2.9); p = 0.007]. Metabolic syndrome (61.7%), microalbuminuria (13.4%), and retinopathy (3.9%) were diagnosed. Therapies used were lifestyle only (35.5%), oral anti-diabetics (OAD) only (43.3 %), insulin + OAD (15.9%) and insulin only (5.6%). Patients with ß-cell autoimmunity or HLA-DR genetic risk more frequently used insulin than confirmed T2DM patients (50.0 vs. 22.0%; p = 0.037) and less often had diabetic relatives (61.1 vs. 86.0%; p = 0.030). CONCLUSION: T2DM was confirmed in about 90% of patients while about 10% with ß-cell autoimmunity or HLA-DR genetic risk likely had either T1.5DM or 'double diabetes' or an unknown diabetes type.

Duration of type 2 diabetes strongly predicts all-cause and cardiovascular mortality in people referred for coronary angiography.

OBJECTIVE: Type 2 diabetes represents a major cardiovascular risk factor. However, few studies have addressed the impact of the disease duration on mortality. Thus, we aimed to investigate the predictive value of diabetes duration for all-cause and cardiovascular mortality in subjects undergoing coronary angiography. METHODS: We studied 2455 participants of the LUdwigshafen RIsk and Cardiovascular health study (1768 males/687 females). They had a mean ± standard deviation (SD) age of 63.1 ± 9.0 years (range: 40.0-79.9) and a mean ± SD body mass index of 27.7 ± 4.0 kg/m(2). 704 subjects were newly diagnosed with type 2 diabetes according to the 2010 criteria of the American Diabetes Association and 446 subjects had a known history of type 2 diabetes. The mean ± SD duration of the follow-up for all-cause and cardiovascular mortality was 7.4 ± 2.3 years. RESULTS: A total of 543 deaths occurred during the follow-up. Among these, 343 were accounted for by cardiovascular diseases. The duration of type 2 diabetes was strongly and positively correlated with all-cause and cardiovascular mortality (both P<0.001). The multivariate adjusted hazard ratios (95% confidence intervals) for cardiovascular mortality compared to subjects without diabetes were 1.76 (1.34-2.32), 2.86 (2.00-4.08), 2.96 (1.85-4.74), and 4.55 (3.24-6.39) for subjects with new onset type 2 diabetes and subjects with known type 2 diabetes (duration ≤ 5, >5 and ≤ 10, >10 years), respectively. CONCLUSIONS: The data emphasise the need to consider the diabetes duration for the prediction of mortality in subjects at intermediate to high cardiovascular risk.

Genetic analysis of adult-onset autoimmune diabetes.

OBJECTIVE: In contrast with childhood-onset type 1 diabetes, the genetics of autoimmune diabetes in adults are not well understood. We have therefore investigated the genetics of diabetes diagnosed in adults positive for autoantibodies. RESEARCH DESIGN AND METHODS: GAD autoantibodies (GADAs), insulinoma-associated antigen-2 antibodies (IA-2As), and islet cell autoantibodies were measured at time of diagnosis. Autoantibody-positive diabetic subjects (n = 1,384) and population-based control subjects (n = 2,235) were genotyped at 20 childhood-onset type 1 diabetes loci and FCRL3, GAD2, TCF7L2, and FTO. RESULTS: PTPN22 (1p13.2), STAT4 (2q32.2), CTLA4 (2q33.2), HLA (6p21), IL2RA (10p15.1), INS (11p15.5), ERBB3 (12q13.2), SH2B3 (12q24.12), and CLEC16A (16p13.13) were convincingly associated with autoimmune diabetes in adults (P ≤ 0.002), with consistent directions of effect as reported for pediatric type 1 diabetes. No evidence of an HLA-DRB1*03/HLA-DRB1*04 (DR3/4) genotype effect was obtained (P = 0.55), but it remained highly predisposing (odds ratio 26.22). DR3/4 was associated with a lower age at diagnosis of disease, as was DR4 (P = 4.67 × 10(-6)) but not DR3. DR3 was associated with GADA positivity (P = 6.03 × 10(-6)) but absence of IA-2A (P = 3.22 × 10(-7)). DR4 was associated with IA-2A positivity (P = 5.45 × 10(-6)). CONCLUSIONS: Our results are consistent with the hypothesis that the genetics of autoimmune diabetes in adults and children are differentiated by only relatively few age-dependent genetic effects. The slower progression toward autoimmune insulin deficiency in adults is probably due to a lower genetic load overall combined with subtle variation in the HLA class II gene associations and autoreactivity.

Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium.

BACKGROUND: and PURPOSE: To yield large amounts of DNA for many genotype analyses and to provide a renewable source of DNA, the Type 1 Diabetes Genetics Consortium (T1DGC) harvested DNA and peripheral blood mononuclear cells (PBMCs) from individuals with type 1 diabetes and their family members in several regions of the world. METHODS: DNA repositories were established in Asia-Pacific, Europe, North America, and the United Kingdom. To address region-specific needs, different methods and sample processing techniques were used among the laboratories to extract and to quantify DNA and to establish Epstein-Barr virus transformed cell lines. RESULTS: More than 98% of the samples of PBMCs were successfully transformed. Approximately 20-25 microg of DNA were extracted per mL of whole blood. Extraction of DNA from the cell pack ranged from 92 to 165 microg per cell pack. In addition, the extracted DNA from whole blood or transformed cells was successfully utilized in each regional human leukocyte antigen genotyping laboratory and by several additional laboratories performing consortium-wide genotyping projects. LIMITATIONS: Although the isolation of PBMCs was consistent among sites, the measurement of DNA was difficult to harmonize. CONCLUSIONS: DNA repositories can be established in different regions of the world and produce similar amounts of high-quality DNA for a variety of high-throughput genotyping techniques. Furthermore, even with the distances and time necessary for transportation, highly efficient transformation of PBMCs is possible. For future studies/trials involving several laboratories in different locations, the T1DGC experience includes examples of protocols that may be applicable. In summary, T1DGC has developed protocols that would be of interest to any scientific organization attempting to overcome the logistical problems associated with studies/trials spanning multiple research facilities, located in different regions of the world.

A proinsulin 74-90-derived protease-resistant, altered peptide ligand increases TGF-beta 1 secretion in PBMC from patients with type 1 diabetes mellitus.

Proinsulin is a major diabetes-associated autoantigen. APL have been shown to manipulate the immune response of T cells. Here, we generated a lysosomal protease-resistant proinsulin 74-90-derived APL using a CS-directed amino acid modification approach. These prAPL activated TGF-beta 1 secretion in proinsulin-reactive T cells from PBMC of patients with T1D. We provide evidence that proinsulin-derived prAPL modulate the cytokine signature of proinsulin-reactive T cells at a micromolar range by increasing anti-inflammatory cytokines, including TGF-beta 1. Thus, the use of prAPL is a promising tool to mitigate autoaggressive T cells.

Protease-resistant human GAD-derived altered peptide ligands decrease TNF-alpha and IL-17 production in peripheral blood cells from patients with type 1 diabetes mellitus.

Glutamic acid decarboxylase 65 (GAD) and proinsulin are major diabetes-associated autoantigens that drive autoreactive T cells. Altered peptide ligands (APL) have been proposed as reagents for the modification of autoimmune reactions. Here, we have prepared GAD-derived protease-resistant APL (prAPL) by cleavage site-directed modification. The resulting prAPL are resistant to lysosomal and serum proteases, bind with high-affinity to HLA-DRB1(*)0401 and have a prolonged half-life in the serum. GAD-derived prAPL significantly decreased the secretion of proinflammatory cytokines by a GAD-specific human T cell clone. Likewise, the production of IL-17, TNF-alpha, and secretion of IL-6 by peripheral blood lymphocytes from patients with type 1 diabetes mellitus (T1D) was reduced, when stimulated with both GAD and GAD-derived prAPL. Thus, prAPL with high affinity for HLA-DRB1(*)0401 mitigate the response of GAD-reactive human Th17 cells. The strategy of designing specific immunomodulatory protease-resistant altered peptide ligands provides the basis for novel avenues of therapeutic intervention.

Thyroid examination in highly radiation-exposed workers after the Chernobyl accident.

CONTEXT: Radioactive contamination from the Chernobyl nuclear accident that happened on the morning of 26th April 1986 had a major impact on thyroid health in the Belarus region. OBJECTIVE: Observational study of a cohort of 99 adults, most strongly exposed to ionizing radioactivity. DESIGN, SETTING AND PATIENTS: Observational study performed between 1998 and 2000. The cohort comprised 99 workers (92 male) of the Chernobyl nuclear power plant. Examination including physical examination, ultrasonography of the thyroid gland and measurement of serum free thyroxin (fT(4)), free triiodothyronine (fT(3)) and TSH. Anti-thyroperoxidase (anti-TPO), antithyroglobulin (anti-Tg) antibodies and thyroid stimulating immunoglobulin were also determined. MAIN OUTCOME MEASURES: The impact of exposure to high-dose radiation, including radioactive iodine, on the thyroid gland was examined. RESULTS: Levels of fT(4) in all probands were within the normal World Health Organization-defined range. Elevated levels of fT(3) were found in two workers (2%), high titres of anti-TPO and anti-Tg antibodies were present in four subjects (4%). Mild hypothyroidism was present in one patient. Enlargement of the thyroid gland was observed in 17 workers (17%). There was no evidence of clinically overt thyroid cancer. CONCLUSIONS: The Chernobyl accident showed surprisingly little impact on the thyroid in a cohort of workers strongly exposed to radiation. Our data suggest an age-dependent heterogeneity in response to the short-lived radioiodine isotopes and favours long-term follow-up analysis.

Lymphocytes of type 2 diabetic women carry a high load of stable chromosomal aberrations: a novel risk factor for disease-related early death.

OBJECTIVE: Diabetes is associated with an increased risk of death in women. Oxidative stress due to chronic hyperglycemia leads to the generation of reactive oxygen species and loss of chromosomal integrity. To clarify whether diabetes is a premature aging syndrome, we determined telomere erosion dynamics and occurrence of structural chromosomal aberrations in women of the Ludwigshafen Risk and Cardiovascular Health (LURIC) Study. RESEARCH DESIGN AND METHODS: Telomere lengths and karyotypes were examined in peripheral blood mononuclear cells. Regarding these parameters, surviving and deceased type 2 diabetic women of the LURIC study were compared with nondiabetic LURIC women with or without coronary heart disease and with healthy female control subjects. RESULTS: Significantly enhanced telomere attrition was seen in all LURIC subjects compared with healthy control subjects. Although the average telomere-length loss is equivalent to well >10 years of healthy aging, telomere erosion was not associated with outcome within the LURIC cohort. However, strikingly high numbers of stable chromosomal aberrations were found in type 2 diabetic women but not in LURIC disease control subjects or in healthy individuals. Furthermore, within the younger age- groups, deceased type 2 diabetes patients had significantly more marker chromosomes than the surviving type 2 diabetic patients. CONCLUSIONS: All women at high risk for cardiovascular death have accelerated telomere erosion, not caused by type 2 diabetes per se but likely linked to other risk factors, including dyslipidemia. By contrast, the occurrence of marker chromosomes is associated with type 2 diabetes and is a novel risk factor for type 2 diabetes-related early death.

DRB1*0401-restricted human T cell clone specific for the major proinsulin73-90 epitope expresses a down-regulatory T helper 2 phenotype.

Recently, we have identified proinsulin (P-Ins)(73-90) as an immunodominant T cell epitope of HLA-DRB1*0401 (DR4) subjects with beta-islet cell autoimmunity and of HLA-DR4/CD4 double-transgenic mice immunized with human P-Ins. We have compared the fine specificities of one human CD4 T cell clone and two mouse T cell hybridoma clones recognizing this epitope, and, although these three clones all recognized the same core region (LALEGSLQK), there were major differences in how they interacted with the peptide (p)/HLA complex, reflecting the fact that human P-Ins is a foreign antigen in the mouse and an autoantigen in the type 1 diabetes patient. The human T cell clone was forkhead transcription factor 3 (Foxp3)-positive, a marker for regulatory T cell lineages, and secreted predominantly IL-5, IL-10, and low levels of IFNgamma in response to P-Ins(73-90). This finding is compatible with the previously detected regulatory cytokine pattern in subjects with beta-cell autoimmunity. However, added N- or C-terminal amino acids drastically changed HLA and tetramer binding capacity as well as T cell reactivity and the cytokine phenotype of the P-Ins(73-90)-specific human CD4 T cell clone, suggesting a potential for this P-Ins epitope as a target for therapeutic intervention in HLA-DR4-positive humans with beta-islet cell autoimmunity or recent-onset type 1 diabetes.

Coexpression of CD25 and OX40 (CD134) receptors delineates autoreactive T-cells in type 1 diabetes.

T-cell-mediated loss of pancreatic beta-cells is the crucial event in the development of type 1 diabetes. The phenotypic characteristics of disease-associated T-cells in type 1 diabetes have not yet been defined. The negative results from two intervention trials (the Diabetes Prevention Trial-Type 1 Diabetes and the European Nicotinamide Diabetes Intervention Trial) illustrate the need for technologies to specifically monitor ongoing autoimmune reactions. We used fluorescence-activated cell sorter analysis to study surface marker expression on T-cell lines specific for two major type 1 diabetes autoantigens, GAD65 and proinsulin. We then applied this knowledge in a cross-sectional approach to delineate the phenotype of circulating memory T-cells. The autoreactive T-cells of patients could be distinguished from those of control subjects by their coexpression of CD25 and CD134. Autoantigen-specific T-cells that recognized multiple GAD65- and preproinsulin-derived peptides and coexpressed CD25(+)CD134(+) were confined to patients (n = 32) and pre-diabetic probands (n = 5). Autoantigen-reactive T-cells in control subjects (n = 21) were CD25(+)CD134(-) and recognized fewer autoantigen-derived peptides. Insulin therapy did not induce CD25(+)CD134(+) T-cells in type 2 diabetic patients. The coexpression of CD25 and the costimulatory molecule CD134 on memory T-cells provides a novel marker for type 1 diabetes-associated T-cell immunity. The CD134 costimulatory molecule may also provide a novel therapeutic target in type 1 diabetes.

Preproinsulin-specific CD8+ T cells secrete IFNgamma in human type 1 diabetes.

In animal models autoreactive CD8(+) T cells are crucial in the development of type 1 diabetes (T1D); however, their role in human T1D is still not known. To address the role of CD81 T cells we performed a pilot study by investigating CD8(+) T cell-mediated cytokine secretion after in vitro stimulation with 94 preproinsulin (PPI) peptides. We were able to show that CD8(+) T cells contribute to a strong IFNgamma reactivity against PPI in human T1D. Further investigations defining epitope specificity, cytokine secretion, and cytotoxic capacity are important to clarify their role in T1D development.

Th2 dominance of T helper cell response to preproinsulin in individuals with preclinical type 1 diabetes.

In human type 1 diabetes (T1D) autoantibodies to insulin precede clinical disease, while little is known about the contribution of insulin-specific T lymphocytes-in particular, T helper (Th) subsets. Here we have studied the in vivo primed cytokine response to preproinsulin in peripheral blood mononuclear cells (PBMCs) and two major Th cell subsets-CD45RO+ memory cells and CD45RA+ naive/resting cells-in 35 individuals with HLA-DRB1*04, DQB1*0302 diabetes risk marker: 12 patients with T1D, 12 autoantibody-positive (Ab+) individuals, and 11 healthy controls. Cytokine secretion (TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, and IL-10) was measured in the supernatants of the cultures stimulated with 21 overlapping preproinsulin peptides as well as proinsulin and insulin. In Ab+ individuals our results reveal higher IL-4 levels in CD45RO+ memory cells and higher IL-5 levels in CD45RA+ naive/resting cells, while higher IL-2 production was found in PBMCs. In contrast, in PBMCs of T1D patients higher IFN-gamma and IL-10 secretion was found. Our data delineate characteristic cytokine patterns in peripheral T lymphocytes from patients at different stages of the T1D development.