The INNODIA Type 1 Diabetes Natural History Study: a European cohort of newly diagnosed children, adolescents and adults.

AIMS/HYPOTHESIS: Type 1 diabetes is an heterogenous condition. Characterising factors explaining differences in an individual's clinical course and treatment response will have important clinical and research implications. Our aim was to explore type 1 diabetes heterogeneity, as assessed by clinical characteristics, autoantibodies, beta cell function and glycaemic outcomes, during the first 12 months from diagnosis, and how it relates to age at diagnosis. METHODS: Data were collected from the large INNODIA cohort of individuals (aged 1.0-45.0 years) newly diagnosed with type 1 diabetes, followed 3 monthly, to assess clinical characteristics, C-peptide, HbA1c and diabetes-associated antibodies, and their changes, during the first 12 months from diagnosis, across three age groups: <10 years; 10-17 years; and ≥18 years. RESULTS: The study population included 649 individuals (57.3% male; age 12.1±8.3 years), 96.9% of whom were positive for one or more diabetes-related antibodies. Baseline (IQR) fasting C-peptide was 242.0 (139.0-382.0) pmol/l (AUC 749.3 [466.2-1106.1] pmol/l × min), with levels increasing with age (p<0.001). Over time, C-peptide remained lower in participants aged <10 years but it declined in all age groups. In parallel, glucose levels progressively increased. Lower baseline fasting C-peptide, BMI SD score and presence of diabetic ketoacidosis at diagnosis were associated with lower stimulated C-peptide over time. HbA1c decreased during the first 3 months (p<0.001), whereas insulin requirement increased from 3 months post diagnosis (p<0.001). CONCLUSIONS/INTERPRETATION: In this large cohort with newly diagnosed type 1 diabetes, we identified age-related differences in clinical and biochemical variables. Of note, C-peptide was lower in younger children but there were no main age differences in its rate of decline.

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.

Influence of sphingolipid enzymes on blood glucose levels, development of diabetes, and involvement of pericytes.

AIMS: Sulfatide is a chaperone for insulin manufacturing in beta cells. Here we explore whether the blood glucose values normally could be associated with this sphingolipid and especially two of its building enzymes CERS2 and CERS6. Both T1D and T2D have low blood sulfatide levels, and insulin resistance on beta cells at clinical diagnosis. Furthermore, we examined islet pericytes for sulfatide, and beta-cell receptors for GLP-1, both of which are related to the insulin production. MATERIALS AND METHODS: We examined mRNA levels in islets from the DiViD and nPOD studies, performed genetic association analyses, and histologically investigated pericytes in the islets for sulfatide. RESULTS: Polymorphisms of the gene encoding the CERS6 enzyme responsible for synthesising dihydroceramide, a precursor to sulfatide, are associated with random blood glucose values in non-diabetic persons. This fits well with our finding of sulfatide in pericytes in the islets, which regulates the capillary blood flow in the islets of Langerhans, which is important for oxygen supply to insulin production. In the islets of newly diagnosed T1D patients, we observed low levels of GLP-1 receptors; this may explain the insulin resistance in their beta cells and their low insulin production. In T2D patients, we identified associated polymorphisms in both CERS2 and CERS6. CONCLUSIONS: Here, we describe several polymorphisms in sulfatide enzymes related to blood glucose levels and HbA1c in non-diabetic individuals. Islet pericytes from such persons contain sulfatide. Furthermore, low insulin secretion in newly diagnosed T1D may be explained by beta-cell insulin resistance due to low levels of GLP-1 receptors.

Clinical care advice for monitoring of islet autoantibody positive individuals with presymptomatic type 1 diabetes.

BACKGROUND/AIM: Type 1 diabetes is an autoimmune disease that involves the development of autoantibodies against pancreatic islet beta-cell antigens, preceding clinical diagnosis by a period of preclinical disease activity. As screening activity to identify autoantibody-positive individuals increases, a rise in presymptomatic type 1 diabetes individuals seeking medical attention is expected. Current guidance on how to monitor these individuals in a safe but minimally invasive way is limited. This article aims to provide clinical guidance for monitoring individuals with presymptomatic type 1 diabetes to reduce the risk of diabetic ketoacidosis (DKA) at diagnosis. METHODS: Expert consensus was obtained from members of the Fr1da, GPPAD, and INNODIA consortia, three European diabetes research groups. The guidance covers both specialist and primary care follow-up strategies. RESULTS: The guidance outlines recommended monitoring approaches based on age, disease stage and clinical setting. Individuals with presymptomatic type 1 diabetes are best followed up in specialist care. For stage 1, biannual assessments of random plasma glucose and HbA1c are suggested for children, while annual assessments are recommended for adolescents and adults. For stage 2, 3-monthly clinic visits with additional home monitoring are advised. The value of repeat OGTT in stage 1 and the use of continuous glucose monitoring in stage 2 are discussed. Primary care is encouraged to monitor individuals who decline specialist care, following the guidance presented. CONCLUSIONS: As type 1 diabetes screening programs become more prevalent, effective monitoring strategies are essential to mitigate the risk of complications such as DKA. This guidance serves as a valuable resource for clinicians, providing practical recommendations tailored to an individual's age and disease stage, both within specialist and primary care settings.

Multi-omics analysis reveals drivers of loss of ß-cell function after newly diagnosed autoimmune type 1 diabetes: An INNODIA multicenter study.

AIMS: Heterogeneity in the rate of ß-cell loss in newly diagnosed type 1 diabetes patients is poorly understood and creates a barrier to designing and interpreting disease-modifying clinical trials. Integrative analyses of baseline multi-omics data obtained after the diagnosis of type 1 diabetes may provide mechanistic insight into the diverse rates of disease progression after type 1 diabetes diagnosis. METHODS: We collected samples in a pan-European consortium that enabled the concerted analysis of five different omics modalities in data from 97 newly diagnosed patients. In this study, we used Multi-Omics Factor Analysis to identify molecular signatures correlating with post-diagnosis decline in ß-cell mass measured as fasting C-peptide. RESULTS: Two molecular signatures were significantly correlated with fasting C-peptide levels. One signature showed a correlation to neutrophil degranulation, cytokine signalling, lymphoid and non-lymphoid cell interactions and G-protein coupled receptor signalling events that were inversely associated with a rapid decline in ß-cell function. The second signature was related to translation and viral infection was inversely associated with change in ß-cell function. In addition, the immunomics data revealed a Natural Killer cell signature associated with rapid ß-cell decline. CONCLUSIONS: Features that differ between individuals with slow and rapid decline in ß-cell mass could be valuable in staging and prediction of the rate of disease progression and thus enable smarter (shorter and smaller) trial designs for disease modifying therapies as well as offering biomarkers of therapeutic effect.

High-Throughput Human Complement C3 N-Glycoprofiling Identifies Markers of Early Onset Type 1 Diabetes Mellitus in Children.

Recently, it was shown that children at the onset of type 1 diabetes (T1D) have a higher proportion of oligomannose glycans in their total plasma protein N-glycome compared to their healthy siblings. The most abundant complement component, glycoprotein C3, contains two N-glycosylation sites occupied exclusively by this type of glycans. Furthermore, complement system, as well as C3, was previously associated with T1D. It is also known that changes in glycosylation can modulate inflammatory responses, so our aim was to characterize the glycosylation profile of C3 in T1D. For this purpose, we developed a novel high-throughput workflow for human C3 concanavalin A lectin affinity enrichment and subsequent LC-MS glycopeptide analysis which enables protein-specific N-glycosylation profiling. From the Danish Childhood Diabetes Register, plasma samples of 61 children/adolescents newly diagnosed with T1D and 84 of their unaffected siblings were C3 N-glycoprofiled. Significant changes of C3 N-glycan profiles were found. T1D was associated with an increase in the proportion of unprocessed glycan structures with more mannose units. A regression model including C3 N-glycans showed notable discriminative power between children with early onset T1D and their healthy siblings with area under curve of 0.879. This study confirmed our previous findings of plasma high-mannose glycan changes in a cohort of recent onset T1D cases, suggesting the involvement of C3 N-glycome in T1D development. Our C3 glycan-based discriminative model could be valuable in assessment of T1D risk in children.

Does rapid sequence divergence preclude RNA structure conservation in vertebrates?

Accelerated evolution of any portion of the genome is of significant interest, potentially signaling positive selection of phenotypic traits and adaptation. Accelerated evolution remains understudied for structured RNAs, despite the fact that an RNA's structure is often key to its function. RNA structures are typically characterized by compensatory (structure-preserving) basepair changes that are unexpected given the underlying sequence variation, i.e., they have evolved through negative selection on structure. We address the question of how fast the primary sequence of an RNA can change through evolution while conserving its structure. Specifically, we consider predicted and known structures in vertebrate genomes. After careful control of false discovery rates, we obtain 13 de novo structures (and three known Rfam structures) that we predict to have rapidly evolving sequences-defined as structures where the primary sequences of human and mouse have diverged at least twice as fast (1.5 times for Rfam) as nearby neutrally evolving sequences. Two of the three known structures function in translation inhibition related to infection and immune response. We conclude that rapid sequence divergence does not preclude RNA structure conservation in vertebrates, although these events are relatively rare.

Integrated glycomics and genetics analyses reveal a potential role for N-glycosylation of plasma proteins and IgGs, as well as the complement system, in the development of type 1 diabetes.

AIMS/HYPOTHESIS: We previously demonstrated that N-glycosylation of plasma proteins and IgGs is different in children with recent-onset type 1 diabetes compared with their healthy siblings. To search for genetic variants contributing to these changes, we undertook a genetic association study of the plasma protein and IgG N-glycome in type 1 diabetes. METHODS: A total of 1105 recent-onset type 1 diabetes patients from the Danish Registry of Childhood and Adolescent Diabetes were genotyped at 183,546 genetic markers, testing these for genetic association with variable levels of 24 IgG and 39 plasma protein N-glycan traits. In the follow-up study, significant associations were validated in 455 samples. RESULTS: This study confirmed previously known plasma protein and/or IgG N-glycosylation loci (candidate genes MGAT3, MGAT5 and ST6GAL1, encoding beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase, alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 gene, respectively) and identified novel associations that were not previously reported for the general European population. First, novel genetic associations of IgG-bound glycans were found with SNPs on chromosome 22 residing in two genomic intervals close to candidate gene MGAT3; these include core fucosylated digalactosylated disialylated IgG N-glycan with bisecting N-acetylglucosamine (GlcNAc) (pdiscovery=7.65 × 10-12, preplication=8.33 × 10-6 for the top associated SNP rs5757680) and core fucosylated digalactosylated glycan with bisecting GlcNAc (pdiscovery=2.88 × 10-10, preplication=3.03 × 10-3 for the top associated SNP rs137702). The most significant genetic associations of IgG-bound glycans were those with MGAT3. Second, two SNPs in high linkage disequilibrium (missense rs1047286 and synonymous rs2230203) located on chromosome 19 within the protein coding region of the complement C3 gene (C3) showed association with the oligomannose plasma protein N-glycan (pdiscovery=2.43 × 10-11, preplication=8.66 × 10-4 for the top associated SNP rs1047286). CONCLUSIONS/INTERPRETATION: This study identified novel genetic associations driving the distinct N-glycosylation of plasma proteins and IgGs identified previously at type 1 diabetes onset. Our results highlight the importance of further exploring the potential role of N-glycosylation and its influence on complement activation and type 1 diabetes susceptibility.

Human pathways in animal models: possibilities and limitations.

Animal models are crucial for advancing our knowledge about the molecular pathways involved in human diseases. However, it remains unclear to what extent tissue expression of pathways in healthy individuals is conserved between species. In addition, organism-specific information on pathways in animal models is often lacking. Within these limitations, we explore the possibilities that arise from publicly available data for the animal models mouse, rat, and pig. We approximate the animal pathways activity by integrating the human counterparts of curated pathways with tissue expression data from the models. Specifically, we compare whether the animal orthologs of the human genes are expressed in the same tissue. This is complicated by the lower coverage and worse quality of data in rat and pig as compared to mouse. Despite that, from 203 human KEGG pathways and the seven tissues with best experimental coverage, we identify 95 distinct pathways, for which the tissue expression in one animal model agrees better with human than the others. Our systematic pathway-tissue comparison between human and three animal modes points to specific similarities with human and to distinct differences among the animal models, thereby suggesting the most suitable organism for modeling a human pathway or tissue.

Children at onset of type 1 diabetes show altered N-glycosylation of plasma proteins and IgG.

AIMS/HYPOTHESIS: Individual variation in plasma N-glycosylation has mainly been studied in the context of diabetes complications, and its role in type 1 diabetes onset is largely unknown. Our aims were to undertake a detailed characterisation of the plasma and IgG N-glycomes in patients with recent onset type 1 diabetes, and to evaluate their discriminative potential in risk assessment. METHODS: In the first part of the study, plasma and IgG N-glycans were chromatographically analysed in a study population from the DanDiabKids registry, comprising 1917 children and adolescents (0.6-19.1 years) who were newly diagnosed with type 1 diabetes. A follow-up study compared the results for 188 of these participants with those for their 244 unaffected siblings. Correlation of N-glycan abundance with the levels and number of various autoantibodies (against IA-2, GAD, ZnT8R, ZnT8W), as well as with sex and age at diagnosis, were estimated by using general linear modelling. A disease predictive model was built using logistic mixed-model elastic net regression, and evaluated using a 10-fold cross-validation. RESULTS: Our study showed that onset of type 1 diabetes was associated with an increase in the proportion of plasma and IgG high-mannose and bisecting GlcNAc structures, a decrease in monogalactosylation, and an increase in IgG disialylation. ZnT8R autoantibody levels were associated with higher IgG digalactosylated glycan with bisecting GlcNAc. Finally, an increase in the number of autoantibodies (which is a better predictor of progression to overt diabetes than the level of any individual antibody) was accompanied by a decrease in the proportions of some of the highly branched plasma N-glycans. Models including age, sex and N-glycans yielded notable discriminative power between children with type 1 diabetes and their healthy siblings, with AUCs of 0.915 and 0.869 for addition of plasma and IgG N-glycans, respectively. CONCLUSIONS/INTERPRETATION: We defined N-glycan changes accompanying onset of type 1 diabetes, and developed a predictive model based on N-glycan profiles that could have valuable potential in risk assessment. Increasing the power of tests to identify individuals at risk of disease development would be a considerable asset for type 1 diabetes prevention trials.

Diabetes complications and extracellular vesicle therapy.

Diabetes is a chronic disorder characterized by dysregulated glycemic conditions. Diabetic complications include microvascular and macrovascular abnormalities and account for high morbidity and mortality rates in patients. Current clinical approaches for diabetic complications are limited to symptomatic treatments and tight control of blood sugar levels. Extracellular vesicles (EVs) released by somatic and stem cells have recently emerged as a new class of potent cell-free therapeutic delivery packets with a great potential to treat diabetic complications. EVs contain a mixture of bioactive molecules and can affect underlying pathological processes in favor of tissue healing. In addition, EVs have low immunogenicity and high storage capacity while maintaining nearly the same regenerative and immunomodulatory effects compared to current cell-based therapies. Therefore, EVs have received increasing attention for diabetes-related complications in recent years. In this review, we provide an outlook on diabetic complications and summarizes new knowledge and advances in EV applications. Moreover, we highlight recommendations for future EV-related research.

Genetic association study in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) identifies several potential risk loci.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disease of unknown etiology and pathogenesis, which manifests in a variety of symptoms like post-exertional malaise, brain fog, fatigue and pain. Hereditability is suggested by an increased disease risk in relatives, however, genome-wide association studies in ME/CFS have been limited by small sample sizes and broad diagnostic criteria, therefore no established risk loci exist to date. In this study, we have analyzed three ME/CFS cohorts: a Norwegian discovery cohort (N = 427), a Danish replication cohort (N = 460) and a replication dataset from the UK biobank (N = 2105). To the best of our knowledge, this is the first ME/CFS genome-wide association study of this magnitude incorporating 2532 patients for the genome-wide analyses and 460 patients for a targeted analysis. Even so, we did not find any ME/CFS risk loci displaying genome-wide significance. In the Norwegian discovery cohort, the TPPP gene region showed the most significant association (rs115523291, P = 8.5 × 10-7), but we could not replicate the top SNP. However, several other SNPs in the TPPP gene identified in the Norwegian discovery cohort showed modest association signals in the self-reported UK biobank CFS cohort, which was also present in the combined analysis of the Norwegian and UK biobank cohorts, TPPP (rs139264145; P = 0.00004). Interestingly, TPPP is expressed in brain tissues, hence it will be interesting to see whether this association, with time, will be verified in even larger cohorts. Taken together our study, despite being the largest to date, could not establish any ME/CFS risk loci, but comprises data for future studies to accumulate the power needed to reach genome-wide significance.

Differences in insulin sensitivity in the partial remission phase of childhood type 1 diabetes; a longitudinal cohort study.

AIMS: Studies suggest that type 1 diabetes (T1D) contributes to impaired insulin sensitivity (IS). Most children with T1D experience partial remission but the knowledge regarding the magnitude and implications of impaired IS in this phase is limited. Therefore, we investigate the impact of IS on the partial remission phase. METHODS: In a longitudinal study of children and adolescents, participants were seen at three clinical visits during the first 14.5 months after diagnosis of T1D. Partial remission was defined as IDAA1c (HbA1c (%) + 4*daily insulin dose) ≤ 9. Beta-cell function was considered significant by a stimulated c-peptide > 300 pmol/L. Participants were characterized by (i) remission or non-remission and (ii) stimulated c-peptide levels above or below 300 pmol/L. IS, body mass index (BMI), total body fat, sex, age, pubertal status and ketoacidosis at onset were compared. RESULTS: Seventy-eight children and adolescents aged 3.3-17.7 years were included. At 14.5 months post-diagnosis, 54.5% of the participants with stimulated c-peptide > 300 pmol/L were not in partial remission. Participants not in remission had significant lower IS 2.5 (p = 0.032), and 14.5 (p = 0.022) months after diagnosis compared to participants in partial remission with similar c-peptide levels. IS did not fluctuate during the remission phase. CONCLUSIONS: A number of children and adolescents have impaired IS in the remission phase of paediatric T1D and are not in remission 14.5 months after diagnosis despite stimulated c-peptide > 300 pmol/L.

The metabolic syndrome is frequent in children and adolescents with type 1 diabetes compared to healthy controls.

OBJECTIVE: There is a rise in overweight and obesity among children and adolescents with type 1 diabetes (T1D) in parallel with the rise in the metabolic syndrome (MetS) among children and adolescents. The aim of the study was to describe the prevalence and characteristics of MetS in children and adolescents with T1D compared to their healthy counterparts. RESEARCH DESIGN AND METHODS: The study includes two Danish cohorts; (i) the Copenhagen cross sectional cohort 2016 of 277 children and adolescents with T1D that attend the pediatric outpatient clinic at a large hospital in greater Copenhagen and (ii) the CHAMPS-study DK which is a population-based cohort study of Danish children and adolescents (control cohort). Participants were categorized to have MetS if at least two of the following criteria were met: (i) systolic and/or diastolic blood pressure ≥ 90th percentile, (ii) waist circumference ≥90th percentile, and (iii) triglyceride ≥90th percentile and/or HDL ≤10th percentile. RESULTS: The prevalence of children with Mets in the T1D cohort was higher than in the control cohort (p = 0.002). Moreover, participants with T1D had MetS at a lower level of BMI (p < 0.001) and waist circumference (p < 0.001) than participants with MetS from the control cohort (z-scores = 0.90 and 1.51). Participants with MetS were younger than the other T1D participants (median 12.8 [9.9,14.8] vs. median 14.6 [11.2,16.9] years, p = 0.006). CONCLUSIONS: Children and adolescents with T1D have an increased risk of MetS compared to healthy controls and clinicians and caretakers should consider early prevention and health promotion strategies.

Reference serum percentile values of adiponectin, leptin, and adiponectin/leptin ratio in healthy Danish children and adolescents.

The adipokines adiponectin and leptin play key roles in human metabolic regulation and have gained great attention as biomarkers for various metabolic pathologies. Though, pediatric reference values are few and needed. This study aims to establish age- and sex-specific adipokine reference percentiles based on healthy Danish school children. Further, it elucidates sex-specific differences in associations between z-scores of examined adipokines and metabolic variables. Serum adiponectin and serum leptin from 853 observations of healthy Danish schoolchildren aged 8-17 years (median 10.0) were quantified by immunoassays. Age- and sex-specific adipokine reference percentiles were calculated cross-sectionally using the LMS method, and adipokine z-scores were calculated from the fitted model. Multiple linear regression models were used to examine sex-specific differences in associations between adipokine z-scores and various metabolic variables. Girls had a higher median value of adiponectin (11.31 vs. 10.65 µg/mL, p < .001) and leptin (2.30 vs. 1.00 ng/mL, p < .001) and a lower median value of adiponectin/leptin ratio (4.64 vs. 10.76, p < .001) compared to boys. Sex-specific differences were found in associations between adiponectin z-score and HDL (p = .010), between leptin z-score and waist circumference z-score (p = .027) and LDL (p = .048), and between adiponectin/leptin ratio z-scores and waist circumference z-score (p = .044) and LDL (p = .040). Reference percentiles of adiponectin, leptin, and adiponectin/leptin ratio are presented in this paper. To our knowledge, this study is the first to demonstrate sex-specific differences in associations between adipokine z-scores and waist circumference z-score and lipids, respectively in healthy children and adolescents.

The effect of saffron supplementation on glycemic parameters: An overview of systematic reviews.

Due to the widespread use of herbal medicine and evidence pointing to the health benefits of saffron supplementation, this review was performed to evaluate the effects of saffron supplementation on glycemic parameters and lipid profiles based on previous reviews. Relevant articles were retrieved from various databases, which included PubMed, Scopus, ProQuest, Web of Science, Embase, and Cochrane until 2020, with no date restrictions. The quality of the included reviews was assessed using the Assessment of Multiple Systematic Reviews (AMSTAR) checklist. Finally, of 877 obtained articles, eight reviews meeting the inclusion criteria were included for analysis. Among the eight included reviews, seven articles were meta-analyses. In addition, one review had an average quality while seven had a good quality. A narrative description of the included reviews was performed, while a network meta-analysis was not conducted. A brief review of the results was reported according to the weighted mean difference and mean difference. Seven included reviews assessed the effects of saffron or crocin supplementation on glycemic parameters, and six examined these effects on lipid profile parameters. Almost half of the articles reported significant effects of these supplements on glycemic parameters and lipid profiles. Taken together, results suggest that saffron supplementation may improve glycemic and lipid profile parameters; however, further high-quality studies are needed to confirm the clinical efficacy of saffron on glycemic parameters and lipid profiles.

Peptide Antibody Reactivity to Homologous Regions in Glutamate Decarboxylase Isoforms and Coxsackievirus B4 P2C.

Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.

Genetic predisposition in the 2'-5'A pathway in the development of type 1 diabetes: potential contribution to dysregulation of innate antiviral immunity.

AIMS/HYPOTHESIS: The incidence of type 1 diabetes is increasing more rapidly than can be explained by genetic drift. Viruses may play an important role in the disease, as they seem to activate the 2'-5'-linked oligoadenylate (2'-5'A) pathway of the innate antiviral immune system. Our aim was to investigate this possibility. METHODS: Innate antiviral immune pathways were searched for type 1 diabetes-associated polymorphisms using genome-wide association study data. SNPs within ±250kb flanking regions of the transcription start site of 64 genes were examined. These pathways were also investigated for type 1 diabetes-associated RNA expression profiles using laser-dissected islets from two to five tissue sections per donor from the Diabetes Virus Detection (DiViD) study and the network of Pancreatic Organ Donors (nPOD). RESULTS: We found 27 novel SNPs in genes nominally associated with type 1 diabetes. Three of those SNPs were located upstream of the 2'-5'A pathway, namely SNP rs4767000 (p = 1.03 × 10-9, OR 1.123), rs1034687 (p = 2.16 × 10-7, OR 0.869) and rs739744 (p = 1.03 × 10-9, OR 1.123). We also identified a large group of dysregulated islet genes in relation to type 1 diabetes, of which two were novel. The most aberrant genes were a group of IFN-stimulated genes. Of those, the following distinct pathways were targeted by the dysregulation (compared with the non-diabetic control group): OAS1 increased by 111% (p < 1.00 × 10-4, 95% CI -0.43, -0.15); MX1 increased by 142% (p < 1.00 × 10-4, 95% CI -0.52, -0.22); and ISG15 increased by 197% (p = 2.00 × 10-4, 95% CI -0.68, -0.18). CONCLUSIONS/INTERPRETATION: We identified a genetic predisposition in the 2'-5'A pathway that potentially contributes to dysregulation of the innate antiviral immune system in type 1 diabetes. This study describes a potential role for the 2'-5'A pathway and other components of the innate antiviral immune system in beta cell autoimmunity.

The identification and functional annotation of RNA structures conserved in vertebrates.

Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization, and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for conserved RNA structures (CRSs), leveraging structure-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ∼516,000 human genomic regions containing CRSs. We find that a substantial fraction of human-mouse CRS regions (1) colocalize consistently with binding sites of the same RNA binding proteins (RBPs) or (2) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared expression and shared structure despite low abundance and low sequence identity. About 30,000 CRS regions are located near coding or long noncoding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3' ends have significantly increased expression levels over their nonstructured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality.

Increased levels of inflammatory factors are associated with severity of polyneuropathy in type 1 diabetes.

OBJECTIVE: Distal symmetrical polyneuropathy (DSPN) is a severe common long-term complication of type 1 diabetes caused by impaired sensory-motor nerve function. As chronic low-grade inflammation may be involved in the pathogenesis of DSPN, we investigated the circulating levels of inflammatory markers in individuals with type 1 diabetes with and without DSPN. Furthermore, we determined to what extent these factors correlated with different peripheral sensory nerve functions. DESIGN: Cross-sectional study. PATIENTS: The study included 103 individuals with type 1 diabetes with (n = 50) and without DSPN (n = 53) as well as a cohort of healthy controls (n = 21). MEASUREMENTS: Circulating levels of various inflammatory markers (cytokines, chemokines and soluble adhesion molecules) were determined in serum samples by Luminex multiplexing technology. Peripheral sensory nerve testing, for example vibration, tactile and thermal perception, was assessed by standardized procedures. RESULTS: The cytokines IL-1α, IL-4, IL-12p70, IL-13, IL-17A and TNF-α; the chemokine MCP-1; and the adhesion molecule E-selectin were significantly increased in individuals with type 1 diabetes with DSPN compared to those without DSPN (P < .001). These observations were independent of age, sex, BMI, disease duration and blood pressure. Additionally, higher serum concentrations of cytokines and chemokines were associated with higher vibration and tactile perception thresholds, but not with heat tolerance threshold. CONCLUSIONS: Individuals with type 1 diabetes and concomitant DSPN display higher serum levels of several inflammatory markers. These findings support that systemic low-grade inflammation may play a role in the pathogenesis of DSPN.