Childhood screening for type 1 diabetes comparing automated multiplex Antibody Detection by Agglutination-PCR (ADAP) with single plex islet autoantibody radiobinding assays.

BACKGROUND: Two or more autoantibodies against either insulin (IAA), glutamic acid decarboxylase (GADA), islet antigen-2 (IA-2A) or zinc transporter 8 (ZnT8A) denote stage 1 (normoglycemia) or stage 2 (dysglycemia) type 1 diabetes prior to stage 3 type 1 diabetes. Automated multiplex Antibody Detection by Agglutination-PCR (ADAP) assays in two laboratories were compared to single plex radiobinding assays (RBA) to define threshold levels for diagnostic specificity and sensitivity. METHODS: IAA, GADA, IA-2A and ZnT8A were analysed in 1504 (54% females) population based controls (PBC), 456 (55% females) doctor's office controls (DOC) and 535 (41% females) blood donor controls (BDC) as well as in 2300 (48% females) patients newly diagnosed (1-10 years of age) with stage 3 type 1 diabetes. The thresholds for autoantibody positivity were computed in 100 10-fold cross-validations to separate patients from controls either by maximizing the χ2-statistics (chisq) or using the 98th percentile of specificity (Spec98). Mean and 95% CI for threshold, sensitivity and specificity are presented. FINDINGS: The ADAP ROC curves of the four autoantibodies showed comparable AUC in the two ADAP laboratories and were higher than RBA. Detection of two or more autoantibodies using chisq showed 0.97 (0.95, 0.99) sensitivity and 0.94 (0.91, 0.97) specificity in ADAP compared to 0.90 (0.88, 0.95) sensitivity and 0.97 (0.94, 0.98) specificity in RBA. Using Spec98, ADAP showed 0.92 (0.89, 0.95) sensitivity and 0.99 (0.98, 1.00) specificity compared to 0.89 (0.77, 0.86) sensitivity and 1.00 (0.99, 1.00) specificity in the RBA. The diagnostic sensitivity and specificity were higher in PBC compared to DOC and BDC. INTERPRETATION: ADAP was comparable in two laboratories, both comparable to or better than RBA, to define threshold levels for two or more autoantibodies to stage type 1 diabetes. FUNDING: Supported by The Leona M. and Harry B. Helmsley Charitable Trust (grant number 2009-04078), the Swedish Foundation for Strategic Research (Dnr IRC15-0067) and the Swedish Research Council, Strategic Research Area (Dnr 2009-1039). AL was supported by the DiaUnion collaborative study, co-financed by EU Interreg ÖKS, Capital Region of Denmark, Region Skåne and the Novo Nordisk Foundation.

Assisting the implementation of screening for type 1 diabetes by using artificial intelligence on publicly available data.

The type 1 diabetes community is coalescing around the benefits and advantages of early screening for disease risk. To be accepted by healthcare providers, regulatory authorities and payers, screening programmes need to show that the testing variables allow accurate risk prediction and that individualised risk-informed monitoring plans are established, as well as operational feasibility, cost-effectiveness and acceptance at population level. Artificial intelligence (AI) has the potential to contribute to solving these issues, starting with the identification and stratification of at-risk individuals. ASSET (AI for Sustainable Prevention of Autoimmunity in the Society; www.asset.healthcare ) is a public/private consortium that was established to contribute to research around screening for type 1 diabetes and particularly to how AI can drive the implementation of a precision medicine approach to disease prevention. ASSET will additionally focus on issues pertaining to operational implementation of screening. The authors of this article, researchers and clinicians active in the field of type 1 diabetes, met in an open forum to independently debate key issues around screening for type 1 diabetes and to advise ASSET. The potential use of AI in the analysis of longitudinal data from observational cohort studies to inform the design of improved, more individualised screening programmes was also discussed. A key issue was whether AI would allow the research community and industry to capitalise on large publicly available data repositories to design screening programmes that allow the early detection of individuals at high risk and enable clinical evaluation of preventive therapies. Overall, AI has the potential to revolutionise type 1 diabetes screening, in particular to help identify individuals who are at increased risk of disease and aid in the design of appropriate follow-up plans. We hope that this initiative will stimulate further research on this very timely topic.

Interaction Between Dietary Iron Intake and Genetically Determined Iron Overload: Risk of Islet Autoimmunity and Progression to Type 1 Diabetes in the TEDDY Study.

OBJECTIVE: To examine whether iron intake and genetically determined iron overload interact in predisposing to the development of childhood islet autoimmunity (IA) and type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: In The Environmental Determinants of Diabetes in the Young (TEDDY) study, 7,770 genetically high-risk children were followed from birth until the development of IA and progression to T1D. Exposures included energy-adjusted iron intake in the first 3 years of life and a genetic risk score (GRS) for increased circulating iron. RESULTS: We found a U-shaped association between iron intake and risk of GAD antibody as the first autoantibody. In children with GRS ≥2 iron risk alleles, high iron intake was associated with an increased risk of IA, with insulin as first autoantibody (adjusted hazard ratio 1.71 [95% CI 1.14; 2.58]) compared with moderate iron intake. CONCLUSIONS: Iron intake may alter the risk of IA in children with high-risk HLA haplogenotypes.

The Vbeta13 T Cell Receptor Monoclonal Antibody Reduces Hyaluronan and CD68+, CD3+, and CD8+ Cell Infiltrations to Delay Diabetes in Congenic BB DRLyp/Lyp Rats.

The depleting Vß13a T cell receptor monoclonal antibody (mAb) 17D5 prevents both induced and spontaneous autoimmune diabetes in BB rats. Here it was tested in congenic DRLyp/Lyp rats, all of which spontaneously developed diabetes. Starting at 40 days of age, rats were injected once weekly with either saline, His42 Vß16 mAb, or 17D5 mAb and monitored for hyperglycemia. Diabetes occurred in 100% (n = 5/5) of saline-treated rats (median age, 66 days; range 55-73), and in 100% (n = 6/6) of His42-treated rats (median age, 69 days; range 59-69). Diabetes occurred in fewer (n = 8/11, 73%) 17D5-treated rats at a later age (median 76 days, range 60-92). Three (27%) of the 17D5-treated rats were killed at 101-103 days of age without diabetes (17D5 no-diabetes rats). Survival analysis demonstrated that 17D5 mAb delayed diabetes onset. Saline- and His42-treated rats had severely distorted islets with substantial loss of insulin-positive cells. These rats exhibited prominent hyaluronan (HA) staining, with the intra-islet HA+ accumulations measuring 5,000 ± 2,400 µm2 and occupying 36 ± 12% of islet area, and severe (grade 4) insulitis with abundant infiltration by CD68+, CD3+, and CD8+ cells. The 17D5 mAb-treated rats with delayed diabetes onset exhibited less severe insulitis (predominantly grade 3). In contrast, the 17D5 no-diabetes rats had mostly normal islets, with insulin+ cells representing 76 ± 3% of islet cells. In these rats, the islet HA deposits were significantly smaller than in the diabetic rats; the intra-islet HA+ areas were 1,200 ± 300 µm2 and accounted for 8 ± 1% of islet area. Also, islet-associated CD68+ and CD3+ cells occurred less frequently (on average in 60 and 3% of the islets, respectively) than in the diabetes rats (present in >95% of the islets). No CD8+ cells were detected in islets in all 17D5 no-diabetes rats. We conclude that mAb 17D5 delayed diabetes in DRLyp/Lyp rats and markedly reduced expression of HA and concomitant infiltration of CD68+, CD3+, and CD8+ cells. Our findings underscore the importance of refining immune suppression in prevention or intervention clinical trials to use mAb reagents that are directed against specific T cell receptors.

Prospective virome analyses in young children at increased genetic risk for type 1 diabetes.

Viruses are implicated in autoimmune destruction of pancreatic islet ß cells, which results in insulin deficiency and type 1 diabetes (T1D)1-4. Certain enteroviruses can infect ß cells in vitro5, have been detected in the pancreatic islets of patients with T1D6 and have shown an association with T1D in meta-analyses4. However, establishing consistency in findings across studies has proven difficult. Obstacles to convincingly linking RNA viruses to islet autoimmunity may be attributed to rapid viral mutation rates, the cyclical periodicity of viruses7 and the selection of variants with altered pathogenicity and ability to spread in populations. ß cells strongly express cell-surface coxsackie and adenovirus receptor (CXADR) genes, which can facilitate enterovirus infection8. Studies of human pancreata and cultured islets have shown significant variation in enteroviral virulence to ß cells between serotypes and within the same serotype9,10. In this large-scale study of known eukaryotic DNA and RNA viruses in stools from children, we evaluated fecally shed viruses in relation to islet autoimmunity and T1D. This study showed that prolonged enterovirus B rather than independent, short-duration enterovirus B infections may be involved in the development of islet autoimmunity, but not T1D, in some young children. Furthermore, we found that fewer early-life human mastadenovirus C infections, as well as CXADR rs6517774, independently correlated with islet autoimmunity.

Cesarean Section on the Risk of Celiac Disease in the Offspring: The Teddy Study.

OBJECTIVE: Cesarean section (C-section) is associated with various immune-mediated diseases in the offspring. We investigated the relationship between mode of delivery and celiac disease (CD) and CD autoimmunity (CDA) in a multinational birth cohort. METHODS: From 2004 to 2010, infants from the general population who tested positive for HLA DR3-DQ2 or DR4-DQ8 were enrolled in The Environmental Determinants for Diabetes in the Young (TEDDY) study. Children were annually screened for transglutaminase autoantibodies, if positive, they are retested after 3 to 6 months and those persistently positive defined as CDA. Associations of C-section with maternal (age, education level, parity, pre-pregnancy weight, diabetes, smoking, weight gain during pregnancy) and child characteristics (gestational age, birth weight) were examined by Fisher exact test or Wilcoxon rank-sum test. Hazard ratios (HRs) for CDA or CD were calculated by Cox proportional hazard regression models. RESULTS: Of 6087 analyzed singletons, 1600 (26%) were born by C-section (Germany 38%, United States 37%, Finland 18%, Sweden 16%), and the remaining were born vaginally without instrumental support; 979 (16%) had developed CDA and 343 (6%) developed CD. C-section was associated with lower risk for CDA (hazard ratio [HR] = 0.85; 95% confidence interval [CI] 0.73, 0.99 P = 0.032) and CD (HR = 0.75; 95% CI 0.58, 0.98; P = 0.034). After adjusting for country, sex, HLA-genotype, CD in family, maternal education, and breast-feeding duration, significance was lost for CDA (HR = 0.91; 95% CI 0.78, 1.06; P = 0.20) and CD (HR = 0.85; 95% CI 0.65, 1.11; P = 0.24). Presurgical ruptured membranes had no influence on CDA or CD development. CONCLUSION: C-section is not associated with increased risk for CDA or CD in the offspring.

Early deficits in insulin secretion, beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats.

AIMS/HYPOTHESIS: Genetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available. METHODS: We used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells. RESULTS: DRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3 ± 121.3 vs 633.3 ± 148.7; p < 0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5-7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9 ± 1593.7 vs 4416.8 ± 1230.5 pg islet-1 h-1; p < 0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1 × 109 ± 9.5 × 107 vs 3.8 × 109 ± 5.8 × 107 µm3; p = 0.044) and small sized islets (1.6 × 109 ± 5.1 × 107 vs 1.4 × 109 ± 4.5 × 107 µm3; p = 0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 µl min-1 [g pancreas]-1; p = 0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats. CONCLUSIONS/INTERPRETATION: The present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.

Genetic and Environmental Interactions Modify the Risk of Diabetes-Related Autoimmunity by 6 Years of Age: The TEDDY Study.

OBJECTIVE: We tested the associations between genetic background and selected environmental exposures with respect to islet autoantibodies and type 1 diabetes. RESEARCH DESIGN AND METHODS: Infants with HLA-DR high-risk genotypes were prospectively followed for diabetes-related autoantibodies. Single nucleotide polymorphisms (SNPs) came from the Illumina ImmunoChip and environmental exposure data were by parental report. Children were followed to age 6 years. RESULTS: Insulin autoantibodies occurred earlier than GAD antibody (GADA) and then declined, while GADA incidence rose and remained constant (significant in HLA-DR4 but not in the DR3/3 children). The presence of SNPs rs2476601 (PTPN22) and rs2292239 (ERBB3) demonstrated increased risk of both autoantibodies to insulin (IAA) only and GADA only. SNP rs689 (INS) was protective of IAA only, but not of GADA only. The rs3757247 (BACH2) SNP demonstrated increased risk of GADA only. Male sex, father or sibling as the diabetic proband, introduction of probiotics under 28 days of age, and weight at age 12 months were associated with IAA only, but only father as the diabetic proband and weight at age 12 months were associated with GADA only. Mother as the diabetic proband was not a significant risk factor. CONCLUSIONS: These results show clear differences in the initiation of autoimmunity according to genetic factors and environmental exposures that give rise to IAA or GADA as the first appearing indication of autoimmunity.

Maternal use of dietary supplements during pregnancy is not associated with coeliac disease in the offspring: The Environmental Determinants of Diabetes in the Young (TEDDY) study.

Perinatal exposure to nutrients and dietary components may affect the risk for coeliac disease (CD). We investigated the association between maternal use of vitamin D, n-3 fatty acids (FA) and Fe supplements during pregnancy and risk for CD autoimmunity (CDA) and CD in the offspring. Children at increased genetic risk were prospectively followed from birth in The Environmental Determinants of Diabetes in the Young (TEDDY) study. CDA was defined as having persistently positive tissue transglutaminase autoantibodies (tTGA). Diagnosis of CD was either biopsy-confirmed or considered likely if having persistently elevated levels of tTGA>100 AU. Of 6627 enrolled children, 1136 developed CDA at a median 3·1 years of age (range 0·9-10) and 409 developed CD at a median 3·9 years of age (range 1·2-11). Use of supplements containing vitamin D, n-3 FA and Fe was recalled by 66, 17 and 94 % of mothers, respectively, at 3-4 months postpartum. The mean cumulative intake over the entire pregnancy was 2014 µg vitamin D (sd 2045 µg), 111 g n-3 FA (sd 303 g) and 8806 mg Fe (sd 7017 mg). After adjusting for country, child's human leucocyte antigen genotype, sex, family history of CD, any breast-feeding duration and household crowding, Cox's proportional hazard ratios did not suggest a statistically significant association between the intake of vitamin D, n-3 FA or Fe, and risk for CDA or CD. Dietary supplementation during pregnancy may help boost nutrient intake, but it is not likely to modify the risk for the disease in the offspring.

Type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM), also known as autoimmune diabetes, is a chronic disease characterized by insulin deficiency due to pancreatic ß-cell loss and leads to hyperglycaemia. Although the age of symptomatic onset is usually during childhood or adolescence, symptoms can sometimes develop much later. Although the aetiology of T1DM is not completely understood, the pathogenesis of the disease is thought to involve T cell-mediated destruction of ß-cells. Islet-targeting autoantibodies that target insulin, 65 kDa glutamic acid decarboxylase, insulinoma-associated protein 2 and zinc transporter 8 - all of which are proteins associated with secretory granules in ß-cells - are biomarkers of T1DM-associated autoimmunity that are found months to years before symptom onset, and can be used to identify and study individuals who are at risk of developing T1DM. The type of autoantibody that appears first depends on the environmental trigger and on genetic factors. The pathogenesis of T1DM can be divided into three stages depending on the absence or presence of hyperglycaemia and hyperglycaemia-associated symptoms (such as polyuria and thirst). A cure is not available, and patients depend on lifelong insulin injections; novel approaches to insulin treatment, such as insulin pumps, continuous glucose monitoring and hybrid closed-loop systems, are in development. Although intensive glycaemic control has reduced the incidence of microvascular and macrovascular complications, the majority of patients with T1DM are still developing these complications. Major research efforts are needed to achieve early diagnosis, prevent ß-cell loss and develop better treatment options to improve the quality of life and prognosis of those affected.

Identification of Non-HLA Genes Associated with Celiac Disease and Country-Specific Differences in a Large, International Pediatric Cohort.

OBJECTIVES: There are significant geographical differences in the prevalence and incidence of celiac disease that cannot be explained by HLA alone. More than 40 loci outside of the HLA region have been associated with celiac disease. We investigated the roles of these non-HLA genes in the development of tissue transglutaminase autoantibodies (tTGA) and celiac disease in a large international prospective cohort study. METHODS: A total of 424,788 newborns from the US and European general populations and first-degree relatives with type 1 diabetes were screened for specific HLA genotypes. Of these, 21,589 carried 1 of the 9 HLA genotypes associated with increased risk for type 1 diabetes and celiac disease; we followed 8676 of the children in a 15 y prospective follow-up study. Genotype analyses were performed on 6010 children using the Illumina ImmunoChip. Levels of tTGA were measured in serum samples using radio-ligand binding assays; diagnoses of celiac disease were made based on persistent detection of tTGA and biopsy analysis. Data were analyzed using Cox proportional hazards analyses. RESULTS: We found 54 single-nucleotide polymorphisms (SNPs) in 5 genes associated with celiac disease (TAGAP, IL18R1, RGS21, PLEK, and CCR9) in time to celiac disease analyses (10-4>P>5.8x10-6). The hazard ratios (HR) for the SNPs with the smallest P values in each region were 1.59, 1.45, 2.23, 2.64, and 1.40, respectively. Outside of regions previously associated with celiac disease, we identified 10 SNPs in 8 regions that could also be associated with the disease (P<10-4). A SNP near PKIA (rs117128341, P = 6.5x10-8, HR = 2.8) and a SNP near PFKFB3 (rs117139146, P<2.8x10-7, HR = 4.9) reached the genome-wide association threshold in subjects from Sweden. Analyses of time to detection of tTGA identified 29 SNPs in 2 regions previously associated with celiac disease (CTLA4, P = 1.3x10-6, HR = 0.76 and LPP, P = 2.8x10-5, HR = .80) and 6 SNPs in 5 regions not previously associated with celiac disease (P<10-4); non-HLA genes are therefore involved in development of tTGA. CONCLUSIONS: In conclusion, using a genetic analysis of a large international cohort of children, we associated celiac disease development with 5 non-HLA regions previously associated with the disease and 8 regions not previously associated with celiac disease. We identified 5 regions associated with development of tTGA. Two loci associated with celiac disease progression reached a genome-wide association threshold in subjects from Sweden.

Association of Early Exposure of Probiotics and Islet Autoimmunity in the TEDDY Study.

IMPORTANCE: Probiotics have been hypothesized to affect immunologic responses to environmental exposures by supporting healthy gut microbiota and could therefore theoretically be used to prevent the development of type 1 diabetes mellitus (T1DM)-associated islet autoimmunity. OBJECTIVE: To examine the association between supplemental probiotic use during the first year of life and islet autoimmunity among children at increased genetic risk of T1DM. DESIGN, SETTING, AND PARTICIPANTS: In this ongoing prospective cohort study that started September 1, 2004, children from 6 clinical centers, 3 in the United States (Colorado, Georgia/Florida, and Washington) and 3 in Europe (Finland, Germany, and Sweden), were followed up for T1DM-related autoantibodies. Blood samples were collected every 3 months between 3 and 48 months of age and every 6 months thereafter to determine persistent islet autoimmunity. Details of infant feeding, including probiotic supplementation and infant formula use, were monitored from birth using questionnaires and diaries. We applied time-to-event analysis to study the association between probiotic use and islet autoimmunity, stratifying by country and adjusting for family history of type 1 diabetes, HLA-DR-DQ genotypes, sex, birth order, mode of delivery, exclusive breastfeeding, birth year, child's antibiotic use, and diarrheal history, as well as maternal age, probiotic use, and smoking. Altogether 8676 infants with an eligible genotype were enrolled in the follow-up study before the age of 4 months. The final sample consisted of 7473 children with the age range of 4 to 10 years (as of October 31, 2014). EXPOSURES: Early intake of probiotics. MAIN OUTCOMES AND MEASURES: Islet autoimmunity revealed by specific islet autoantibodies. RESULTS: Early probiotic supplementation (at the age of 0-27 days) was associated with a decreased risk of islet autoimmunity when compared with probiotic supplementation after 27 days or no probiotic supplementation (hazard ratio [HR], 0.66; 95% CI, 0.46-0.94). The association was accounted for by children with the DR3/4 genotype (HR, 0.40; 95% CI, 0.21-0.74) and was absent among other genotypes (HR, 0.97; 95% CI, 0.62-1.54). CONCLUSIONS AND RELEVANCE: Early probiotic supplementation may reduce the risk of islet autoimmunity in children at the highest genetic risk of T1DM. The result needs to be confirmed in further studies before any recommendation of probiotics use is made.

Effects of Gluten Intake on Risk of Celiac Disease: A Case-Control Study on a Swedish Birth Cohort.

BACKGROUND & AIMS: Early nutrition may affect the risk of celiac disease. We investigated whether amount of gluten in diet until 2 years of age increases risk for celiac disease. METHODS: We performed a 1-to-3 nested case-control study of 146 cases, resulting in 436 case-control pairs matched for sex, birth year, and HLA genotype generated from Swedish children at genetic risk for celiac disease. Newborns were annually screened for tissue transglutaminase autoantibodies (tTGA). If tested tTGA positive, time point of seroconversion was determined from frozen serum samples taken every 3 months. Celiac disease was confirmed by intestinal biopsies. Gluten intake was calculated from 3-day food records collected at ages 9, 12, 18 and 24 months. Odds ratios (OR) were calculated through conditional logistic regression. RESULTS: Breastfeeding duration (median, 32 wk) and age at first introduction to gluten (median, 22 wk) did not differ between cases and tTGA-negative controls. At the visit before tTGA seroconversion, cases reported a larger intake of gluten than controls (OR, 1.28; 95% confidence interval [CI], 1.13-1.46; P = .0002). More cases than controls were found in the upper third tertile (ie, >5.0 g/d) before they tested positive for tTGA seroconversion than controls (OR, 2.65; 95% CI, 1.70-4.13; P < .0001). This finding was similar in children homozygous for DR3-DQ2 (OR, 3.19; 95% CI, 1.61-6.30; P = .001), heterozygous for DR3-DQ2 (OR, 2.24; 95% CI, 1.08-4.62; P = .030), and for children not carrying DR3-DQ2 (OR, 2.43; 95% CI, 0.90-6.54; P = .079). CONCLUSIONS: The amount of gluten consumed until 2 years of age increases the risk of celiac disease at least 2-fold in genetically susceptible children. These findings may be taken into account for future infant feeding recommendations.

Factors associated with longitudinal food record compliance in a paediatric cohort study.

OBJECTIVE: Non-compliance with food record submission can induce bias in nutritional epidemiological analysis and make it difficult to draw inference from study findings. We examined the impact of demographic, lifestyle and psychosocial factors on such non-compliance during the first 3 years of participation in a multidisciplinary prospective paediatric study. DESIGN: The Environmental Determinants of Diabetes in the Young (TEDDY) study collects a 3 d food record quarterly during the first year of life and semi-annually thereafter. High compliance with food record completion was defined as the participating families submitting one or more days of food record at every scheduled clinic visit. SETTING: Three centres in the USA (Colorado, Georgia/Florida and Washington) and three in Europe (Finland, Germany and Sweden). SUBJECTS: Families who finished the first 3 years of TEDDY participation (n 8096). RESULTS: High compliance was associated with having a single child, older maternal age, higher maternal education and father responding to study questionnaires. Families showing poor compliance were more likely to be living far from the study centres, from ethnic minority groups, living in a crowded household and not attending clinic visits regularly. Postpartum depression, maternal smoking behaviour and mother working outside the home were also independently associated with poor compliance. CONCLUSIONS: These findings identified specific groups for targeted strategies to encourage completion of food records, thereby reducing potential bias in multidisciplinary collaborative research.

Magnetic resonance imaging reveals altered distribution of hepatic fat in children with type 1 diabetes compared to controls.

INTRODUCTION: Children with type 1 diabetes have been identified as a risk group for non-alcoholic fatty liver disease (NAFLD). The aim was to compare total hepatic fat fraction and fat distribution across Couinaud segments in children with type 1 diabetes and controls and the relation of hepatic fat to plasma and anthropometric parameters. METHODS: Hepatic fat fraction and fat distribution across Couinaud segments were measured with magnetic resonance imaging (MRI) in 22 children with type 1 diabetes and 32 controls. Blood tests and anthropometric data were collected. RESULTS: No children had NAFLD. Children with type 1 diabetes had a slightly lower hepatic fat fraction (median 1.3%) than controls (median 1.8%), and their fat had a different segmental distribution. The fat fraction of segment V was the most representative of the liver as a whole. An incidental finding was that diabetes patients treated with multiple daily injections of insulin (MDI) had a fat distribution more similar to controls than patients with continuous subcutaneous insulin infusion (CSII). CONCLUSIONS: In children with type 1 diabetes, NAFLD may be less common than recent studies have suggested. Children with type 1 diabetes may have a lower fat fraction and a different fat distribution in the liver than controls. Diabetes treatment with MDI or CSII may affect liver fat, but this needs to be confirmed in a larger sample of patients. The heterogeneity of hepatic fat infiltration may affect results when liver biopsy is used for diagnosing fatty liver.

Lack of evidence for a role of islet autoimmunity in the aetiology of canine diabetes mellitus.

AIMS/HYPOTHESIS: Diabetes mellitus is one of the most common endocrine disorders in dogs and is commonly proposed to be of autoimmune origin. Although the clinical presentation of human type 1 diabetes (T1D) and canine diabetes are similar, the aetiologies may differ. The aim of this study was to investigate if autoimmune aetiology resembling human T1D is as prevalent in dogs as previously reported. METHODS: Sera from 121 diabetic dogs representing 40 different breeds were tested for islet cell antibodies (ICA) and GAD65 autoantibodies (GADA) and compared with sera from 133 healthy dogs. ICA was detected by indirect immunofluorescence using both canine and human frozen sections. GADA was detected by in vitro transcription and translation (ITT) of human and canine GAD65, followed by immune precipitation. Sections of pancreata from five diabetic dogs and two control dogs were examined histopathologically including immunostaining for insulin, glucagon, somatostatin and pancreas polypeptide. RESULTS: None of the canine sera analysed tested positive for ICA on sections of frozen canine or human ICA pancreas. However, serum from one diabetic dog was weakly positive in the canine GADA assay and serum from one healthy dog was weakly positive in the human GADA assay. Histopathology showed marked degenerative changes in endocrine islets, including vacuolisation and variable loss of immune-staining for insulin. No sign of inflammation was noted. CONCLUSIONS/INTERPRETATIONS: Contrary to previous observations, based on results from tests for humoral autoreactivity towards islet proteins using four different assays, and histopathological examinations, we do not find any support for an islet autoimmune aetiology in canine diabetes mellitus.

High plasma levels of islet amyloid polypeptide in young with new-onset of type 1 diabetes mellitus.

AIMS/HYPOTHESIS: Islet amyloid polypeptide (IAPP) is a beta cell hormone secreted together with insulin upon glucose stimulation. IAPP participates in normal glucose regulation, but IAPP is also known for its ability to misfold and form islet amyloid. Amyloid fibrils form through smaller cell toxic intermediates and deposited amyloid disrupts normal islet architecture. Even though IAPP and amyloid formation are much discussed in type 2 diabetes, our aim was to study the significance of IAPP in type 1 diabetes. RESULTS: Plasma IAPP levels in children and adolescents with newly diagnosed type 1 diabetes (n = 224) were analysed and concentrations exceeding 100 pmol/L (127.2-888.7 pmol/L) were found in 11% (25/224). The IAPP increase did not correlate with C-peptide levels. CONCLUSIONS/INTERPRETATION: Plasma levels of IAPP and insulin deviate in a subpopulation of young with newly-diagnosed type 1 diabetes. The determined elevated levels of IAPP might increase the risk for IAPP misfolding and formation of cell toxic amyloid in beta cells. This finding add IAPP-aggregation to the list over putative pathological factors causing type 1 diabetes.

A/H1N1 antibodies and TRIB2 autoantibodies in narcolepsy patients diagnosed in conjunction with the Pandemrix vaccination campaign in Sweden 2009-2010.

Narcolepsy is a lifelong sleep disorder related to hypocretin deficiency resulting from a specific loss of hypocretin-producing neurons in the lateral hypothalamic area. The disease is thought to be autoimmune due to a strong association with HLA-DQB1*06:02. In 2009 the World Health Organization (WHO) declared the H1N1 2009 flu pandemic (A/H1N1PDM09). In response to this, the Swedish vaccination campaign began in October of the same year, using the influenza vaccine Pandemrix(®). A few months later an excess of narcolepsy cases was observed. It is still unclear to what extent the vaccination campaign affected humoral autoimmunity associated with narcolepsy. We studied 47 patients with narcolepsy (6-69 years of age) and 80 healthy controls (3-61 years of age) selected after the Pandemrix vaccination campaign. The first aim was to determine antibodies against A/H1N1 and autoantibodies to Tribbles homolog 2 (TRIB2), a narcolepsy autoantigen candidate as well as to GAD65 and IA-2 as disease specificity controls. The second aim was to test if levels and frequencies of these antibodies and autoantibodies were associated with HLA-DQB1*06:02. In vitro transcribed and translated [(35)S]-methionine and -cysteine-labeled influenza A virus (A/California/04/2009/(H1N1)) segment 4 hemagglutinin was used to detect antibodies in a radiobinding assay. Autoantibodies to TRIB2, GAD65 and IA-2 were similarly detected in standard radiobinding assays. The narcolepsy patients had higher median levels of A/H1N1 antibodies than the controls (p = 0.006). A/H1N1 antibody levels were higher among the <13 years old (n = 12) compared to patients who were older than 30 years (n = 12, p = 0.014). Being HLA-DQB1*06:02 positive was associated with higher A/H1N1 antibody levels in both patients and controls (p = 0.026). Serum autoantibody levels to TRIB2 were low overall and high binders did not differ between patients and controls. We observed an association between levels of A/H1N1 antibodies and TRIB2 autoantibody levels particularly among the youngest narcolepsy patients (r = 0.819, p < 0.001). In conclusion, following the 2009 influenza pandemic vaccination, A/H1N1 antibody levels were associated with young age-at-onset narcolepsy patients positive for HLA-DQB1*06:02. The possibility that TRIB2 is an autoantigen in narcolepsy remains to be clarified. We could verify autoantibody responses against TRIB2 which needs to be determined in larger patient cohorts and control populations.

Biobreeding rat islets exhibit reduced antioxidative defense and N-acetyl cysteine treatment delays type 1 diabetes.

Islet-level oxidative stress has been proposed as a trigger for type 1 diabetes (T1D), and release of cytokines by infiltrating immune cells further elevates reactive oxygen species (ROS), exacerbating ß cell duress. To identify genes/mechanisms involved with diabetogenesis at the ß cell level, gene expression profiling and targeted follow-up studies were used to investigate islet activity in the biobreeding (BB) rat. Forty-day-old spontaneously diabetic lymphopenic BB DRlyp/lyp rats (before T cell insulitis) as well as nondiabetic BB DR+/+ rats, nondiabetic but lymphopenic F344lyp/lyp rats, and healthy Fischer (F344) rats were examined. Gene expression profiles of BB rat islets were highly distinct from F344 islets and under-expressed numerous genes involved in ROS metabolism, including glutathione S-transferase (GST) family members (Gstm2, Gstm4, Gstm7, Gstt1, Gstp1, and Gstk1), superoxide dismutases (Sod2 and Sod3), peroxidases, and peroxiredoxins. This pattern of under-expression was not observed in brain, liver, or muscle. Compared with F344 rats, BB rat pancreata exhibited lower GST protein levels, while plasma GST activity was found significantly lower in BB rats. Systemic administration of the antioxidant N-acetyl cysteine to DRlyp/lyp rats altered abundances of peripheral eosinophils, reduced severity of insulitis, and significantly delayed but did not prevent diabetes onset. We find evidence of ß cell dysfunction in BB rats independent of T1D progression, which includes lower expression of genes related to antioxidative defense mechanisms during the pre-onset period that may contribute to overall T1D susceptibility.

BB rat Gimap gene expression in sorted lymphoid T and B cells.

AIMS: The Gimap gene family has been shown to be integral to T cell survival and development. A frameshift mutation in Gimap5, one of seven members of the Gimap family, results in lymphopenia and is a prerequisite for spontaneous type 1 diabetes (T1D) in the BioBreeding (BB) rat. While not contributing to lymphopenia, the Gimap family members proximal to Gimap5, encompassed within the Iddm39 quantitative trait locus (QTL), have been implicated in T1D. We hypothesized that expression of the Gimap family members within the Iddm39 QTL, during thymocyte development as well as in peripheral T and B cells contribute to T1D. MAIN METHODS: Cell sorted subpopulations were analyzed by quantitative real time (qRT) PCR. KEY FINDINGS: Gimap4 expression was reduced in DR.(lyp/lyp) rat double negative, double positive and CD8 single positive (SP) thymocytes while expression of Gimap8, Gimap6, and Gimap7 was reduced only in CD8 SP thymocytes. Interestingly, expression of the entire Gimap gene family was reduced in DR.(lyp/lyp) rat peripheral T cells compared to non-lymphopenic, non-diabetic DR.(+/+) rats. With the exception of Gimap6, the Gimap family genes were not expressed in B cells from spleen and mesenteric lymph node (MLN). Expression of Gimap9 was only detected in hematopoietic cells of non B cell lineage such as macrophage, dendritic or NK cells. SIGNIFICANCE: These results suggest that lack of the Gimap5 protein in the DR.(lyp/lyp) congenic rat was associated with impaired expression of the entire family of Gimap genes and may regulate T cell homeostasis in the peripheral lymphoid organs.