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To the Editor: Exposure to radiation may result in late adverse effects. Here we describe the consequences of irradiation for the endocrine system, particularly the parathyroid glands, in a cohort of 61 “liquidators,” or cleanup workers, who participated in the effort to contain the contamination at the Chernobyl nuclear power plant in Ukraine subsequent to the 1986 explosion. For these persons, who were among the most heavily irradiated workers on the site, we conducted annual clinical follow-up for 24 years, until 2009. For purposes of comparison, we recruited 687 healthy controls in Swabia, Germany, with the use of a population-based . . .

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.

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. 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. 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)). 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.

Coexpression of CD25 and OX40 (CD134) Receptors Delineates Autoreactive T-cells in Type 1 Diabetes Josef Endl 1 , Silke Rosinger 2 , Barbara Schwarz 1 , Sven-Olaf Friedrich 3 , Gregor Rothe 3 , Wolfram Karges 2 , Michael Schlosser 4 , Thomas Eiermann 5 , Dolores J. Schendel 6 and Bernhard O. Boehm 2 1 Roche Diagnostics, Penzberg, Germany 2 Division of Endocrinology and Diabetes, Department of Internal Medicine, Ulm University, Ulm, Germany 3 Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg, Germany 4 Institute of Pathophysiology, Ernst Moritz Arndt University of Greifswald, Greifswald, Germany 5 Department of Transfusion Medicine and Bone Marrow Transplantation Center, University Hospital Eppendorf, Hamburg, Germany 6 Institute of Molecular Immunology, GSF-National Research Center for Environment and Health, Munich, Germany Address correspondence and reprint requests to Prof. Bernhard O. Boehm, MD, Division of Endocrinology and Diabetes, Ulm, University, Robert-Koch-Str. 8, D-89081 Ulm, Germany. E-mail: bernhard.boehm{at}medizin.uni-ulm.de Abstract T-cell–mediated loss of pancreatic β-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. APC, antigen-presenting cell FACS, fluorescence-activated cell sorter IA-2, insulinoma-associated protein 2 ICA, islet cell autoantibody IL-2, interleukin-2 mAb, monoclonal antibody PBMC, peripheral blood mononuclear cell PPI, preproinsulin TT, tetanus toxoid Footnotes J.E., S.R., and D.J.S. contributed equally to this study. Accepted October 7, 2005. Received March 24, 2005. DIABETES

Recently, we have identified proinsulin $(P-Ins)_{73-90}$ as an immunodominant T cell epitope of HLA-DRB1*0401 (DR4) subjects with β-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 IFNy in response to $P-Ins_{73-90}$. This finding is compatible with the previously detected regulatory cytokine pattern in subjects with β-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-DR4positive humans with β-islet cell autoimmunity or recent-onset type 1 diabetes.

Lymphocytes of Type 2 Diabetic Women Carry a High Load of Stable Chromosomal Aberrations A Novel Risk Factor for Disease-Related Early Death Bernhard O. Boehm 1 , Peter Möller 2 , Josef Högel 3 , Bernhard R. Winkelmann 4 , Wilfried Renner 5 , Silke Rosinger 1 , Ursula Seelhorst 6 , Britta Wellnitz 6 , Winfried März 5 , Julia Melzner 2 and Silke Brüderlein 2 1 Division of Endocrinology and Diabetes, Graduate School Molecular Endocrinology and Diabetes, Ulm University, Ulm, Germany 2 Institute of Pathology, Ulm University, Ulm, Germany 3 Institute of Human Genetics, Ulm University, Ulm, Germany 4 Cardiology Group, Frankfurt-Sachsenhausen, Germany 5 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria 6 Ludwigshafen Risk and Cardiovascular Health Study, Freiburg, Germany Corresponding author: Silke Brüderlein, silke.bruederlein{at}uniklinik-ulm.de Abstract 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. Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 23 July 2008. B.O.B. and P.M. contributed equally to this work. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted July 17, 2008. Received February 25, 2008. DIABETES

Glatiramer acetate is used therapeutically in multiple sclerosis but also known for adverse effects including elevated coronary artery disease (CAD) risk. The mechanisms underlying the cardiovascular side effects of the medication are unclear. Here, we made use of the chromosomal variation in the genes that are known to be affected by glatiramer treatment. Focusing on genes and gene products reported by drug-gene interaction database to interact with glatiramer acetate we explored a large meta-analysis on CAD genome-wide association studies aiming firstly, to investigate whether variants in these genes also affect cardiovascular risk and secondly, to identify new CAD risk genes. We traced association signals in a 200-kb region around genomic positions of genes interacting with glatiramer in up to 60 801 CAD cases and 123 504 controls. We validated the identified association in additional 21 934 CAD cases and 76 087 controls. We identified three new CAD risk alleles within the TGFB1 region on chromosome 19 that independently affect CAD risk. The lead SNP rs12459996 was genome-wide significantly associated with CAD in the extended meta-analysis (odds ratio 1.09, p = 1.58×10-12). The other two SNPs at the locus were not in linkage disequilibrium with the lead SNP and by a conditional analysis showed p-values of 4.05 × 10-10 and 2.21 × 10-6. Thus, studying genes reported to interact with glatiramer acetate we identified genetic variants that concordantly with the drug increase the risk of CAD. Of these, TGFB1 displayed signal for association. Indeed, the gene has been associated with CAD previously in both in vivo and in vitro studies. Here we establish genome-wide significant association with CAD in large human samples.

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.

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. 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. 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. 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.

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