Silke Rosinger

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.

Pro- and anti-inflammatory cytokine production by autoimmune T cells against preproinsulin in HLA-DRB1*04, DQ8 Type 1 diabetes

Aims/hypothesisPreproinsulin is a target T cell autoantigen in human Type 1 diabetes. This study analyses the phenotype and epitope recognition of preproinsulin reactive T cells in subjects with a high genetic risk of diabetes [HLA-DRB1*04, DQ8 with Ab+ (autoantibody-positive) or without islet autoantibodies (control subjects)], and in HLA-matched diabetic patients.MethodsA preproinsulin peptide library approach was used to screen for cytokine profiles and epitope specificities in human peripheral blood lymphocytes, and CD4+CD45RA− and CD4+CD45RA+ T cell subfractions, representing memory and naive and recently primed T cells respectively.ResultsIn CD4+ T cell subsets we identified immunodominant epitopes and cytokine production patterns that differed profoundly between patients, Ab+ subjects and non-diabetic HLA-matched control subjects. In Ab+ subjects, a C-peptide epitope C13–29 and insulin B-chain epitope B11–27 were preferentially recognised, whereas insulin-treated Type 1 diabetic patients reacted to native insulin and B-chain epitope B1–16. In peripheral blood lymphocytes of Ab+ subjects, an increase in T helper (Th) 1 (IFNγ, IL-2) and Th2 (IL-4) cytokines was detectable, wheras in CD45RA+ and CD45RA− subsets, IL-4 and IL-10 phenotypes dominated, compatible with the contribution of non-CD4 cells to IFNγ content. In insulin-treated Type 1 diabetic patients, naive and recently primed CD4+ cells were characterised by increasd IFNγ, TNFα, and IL-5.Conclusions/interpretationOur data show that T cell reactivity to preproinsulin in CD45RA subsets is Th2-dominant in Ab+ subjects, challenging the Th1 paradigm in Type 1 diabetes. Characteristic immunodominant epitopes and cytokine patterns distinguish diabetic patients and Ab+ subjects from HLA-matched healthy individuals. This could prove useful in monitoring of T-cell immunity in clinical diabetes intervention trials.

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

Significance In type 1 diabetes (T1D), the insulin-producing pancreatic β-cells are destroyed by the immune system. Both genetic and environmental factors contribute to T1D risk. Candidate genes for T1D identified by genome-wide association studies have been proposed to act at both the immune system and the β-cell levels. This study shows that the risk variant rs3825932 in the candidate gene cathepsin H (CTSH) predicts β-cell function in both model systems and human T1D. Collectively, our data indicate that higher CTSH expression in β-cells may protect against immune-mediated damage and preserve β-cell function, thereby representing a possible therapeutic target. Our study reinforces the concept that candidate genes for T1D may affect disease progression by modulating survival and function of the β-cells. 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 β-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 β-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of β-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 β-cells, the target cells of the autoimmune assault.

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.

Protease-resistant human GAD-derived altered peptide ligands decrease TNF-α 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.

Preproinsulin-Specific CD8+ T Cells Secrete IFNγ in Human Type 1 Diabetes

Abstract: 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 IFNγ 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.

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

To characterize the clinical and immunological features of HLA‐typed youth with pediatric onset of type 2 diabetes mellitus (T2DM).

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 μg of DNA were extracted per mL of whole blood. Extraction of DNA from the cell pack ranged from 92 to 165 μg 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. Clinical Trials 2010; 7: S65—S74. http://ctj.sagepub.com

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.

Th2 Dominance of T Helper Cell Response to Preproinsulin in Individuals with Preclinical Type 1 Diabetes

Abstract: 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‐α, IFN‐γ, 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‐γ 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.