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OBJECTIVE Previous findings suggest that there are age-related endotypes of type 1 diabetes with different underlying etiopathological mechanisms in those diagnosed at age RESEARCH DESIGN AND METHODS We used data from the Finnish Pediatric Diabetes Register to analyze characteristics of 6,015 children and adolescents diagnosed with type 1 diabetes between 2003 and 2019. We described and compared demographic data, clinical characteristics at diagnosis, autoantibody profiles, and HLA class II–associated disease risk between three groups formed based on age at diagnosis: RESULTS We found significant age-related differences in most of the characteristics analyzed. Children diagnosed at age CONCLUSIONS Our findings suggest that the heterogeneity of type 1 diabetes is associated with the underlying disease process and support the existence of distinct endotypes of type 1 diabetes related to age at diagnosis.

Genes in the HLA class II region include the most important inherited risk factors for type 1 diabetes (T1D) although also polymorphisms outside the HLA region modulate the predisposition to T1D. This study set out to confirm a recent observation in which a novel expression quantitative trait locus was formed by three single nucleotide polymorphisms (SNP) in the intron of HLA-DRA1 in DR3-DQ2 haplotypes. The SNPs significantly increased the risk for T1D in DR3-DQ2 homozygous individuals and we intended to further explore this association, in the Finnish population, by comparing two DR3-DQ2 positive genotypes. Cohorts with DR3-DQ2/DR3-DQ2 (N = 570) and DR3-DQ2/DR1-DQ5 (N = 1035) genotypes were studied using TaqMan analysis that typed for rs3135394, rs9268645 and rs3129877. The tri-SNP haplotype was significantly more common in cases than controls in the DR3-DQ2/DR3-DQ2 cohort (OR = 1.70 CI 95% = 1.15-2.51P = 0.007). However, no significant associations could be observed in the DR3-DQ2/DR1-DQ5 cohort. (c) 2021 The Authors. Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

Aim Infections in early childhood are common reasons to seek medical attention. This study compares the prevalence of infections, and the use of antibiotics and antipyretic-analgesics, in children from Finland, Estonia and Russian Karelia. Methods Children with a genetically increased risk for type 1 diabetes (N = 797) were observed from birth up to 3 years of age. Illnesses and medications were reported by parents continuously. All reported infections, antibiotics and antipyretic-analgesics were compared between Finland and Estonia, and to a lesser extent with Russian Karelia, due to poor study compliance. Results Compared with Estonians, Finns reported more infections during the first and second years of life. During the follow-up, Finnish children had 10 infections while Estonians only had 8 (p

Context Children with initial autoantibodies to either insulin (IAA) or glutamic acid decarboxylase (GADA) differ in peak age of seroconversion and have different type 1 diabetes (T1D) risk gene associations, suggesting heterogeneity in the disease process. Objective To compare the associations of age at seroconversion, HLA risk, and specificity of secondary autoantibodies with the progression of islet autoimmunity between children with either IAA or GADA as their first autoantibody. Design and methods A cohort of 15,253 children with HLA-associated increased risk of T1D participated in a follow-up program in which islet autoantibodies were regularly measured. The median follow-up time was 6.7 years. Spearman correlation, Kaplan-Meier survival plots, and Cox proportional-hazard models were used for statistical analyses. Results Persistent positivity for at least one of the tested autoantibodies was detected in 998 children; 388 of children progressed to clinical T1D. Young age at initial seroconversion was associated with a high probability of expansion of IAA-initiated autoimmunity and progression to clinical diabetes, whereas expansion of GADA-initiated autoimmunity and progression to diabetes were not dependent on initial seroconversion age. The strength of HLA risk affected the progression of both IAA- and GADA-initiated autoimmunity. The simultaneous appearance of two other autoantibodies increased the rate of progression to diabetes compared with that of a single secondary autoantibody among subjects with GADA-initiated autoimmunity but not among those with IAA as the first autoantibody. Conclusions Findings emphasize the differences in the course of islet autoimmunity initiated by either IAA or GADA supporting heterogeneity in the pathogenic process.

The T-cell receptor (TCR) repertoire is generated in a semistochastic process of gene recombination and pairing of TCRα to TCRβ chains with the estimated total TCR diversity of >108. Despite this high diversity, similar or identical TCR chains are found to recur in immune responses. Here, we analyzed the thymic generation of TCR sequences previously associated with recognition of self- and nonself-antigens, represented by sequences associated with autoimmune diabetes and HIV, respectively. Unexpectedly, in the CD4+ compartment TCRα chains associated with the recognition of self-antigens were generated in significantly higher numbers than TCRα chains associated with the recognition of nonself-antigens. The analysis of the circulating repertoire further showed that these chains are not lost in negative selection nor predominantly converted to the regulatory T-cell lineage. The high abundance of self-reactive TCRα chains in multiple individuals suggests that the human thymus has a predilection to generate self-reactive TCRα chains independently of the HLA-type and that the individual risk of autoimmunity may be modulated by the TCRβ repertoire associated with these chains. publishedVersion

Early childhood infections have been implicated in the development of immune-mediated diseases, such as allergies, asthma, and type 1 diabetes. We set out to investigate the immunomodulatory effects of early viral infections experienced before the age of one year on the peripheral regulatory T cell population (Treg) and circulating cytokines in a birth-cohort study of Estonian and Finnish infants. We show here a temporal association of virus infection with the expression of FOXP3 in regulatory T cells. Infants with rhinovirus infection during the preceding 30 days had a higher FOXP3 expression in Treg cells and decreased levels of several cytokines related to Th1 and Th2 responses in comparison to the children without infections. In contrast, FOXP3 expression was significantly decreased in highly activated (CD4+CD127-/loCD25+FOXP3high) regulatory T cells (TregFOXP3high) in the infants who had enterovirus infection during the preceding 30 or 60 days. After enterovirus infections, the cytokine profile showed an upregulation of Th1- and Th17-related cytokines and a decreased activation of CCL22, which is a chemokine derived from dendritic cells and associated with Th2 deviation. Our results reveal that immunoregulatory mechanisms are up-regulated after rhinovirus infections, while enterovirus infections are associated with activation of proinflammatory pathways and decreased immune regulation. publishedVersion

Objective We aimed to clarify the association of various HLA risk alleles with different types of autoantibodies initiating islet specific autoimmunity. Methods Follow-up cohorts from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study and children diagnosed with type 1 diabetes from the Finnish Pediatric Diabetes Register (FPDR) were analyzed for the presence of autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), IA-2 antigen (IA-2A) and zinc transporter 8 (ZnT8A) and genotyped for HLA DR/DQ alleles. In the DIPP study autoantibodies were regularly analyzed from birth up to 15 years of age. Results In the DIPP cohort 621 children developed one single persistent autoantibody, GADA in 284, IAA in 268 and IA-2A in 40 cases. Highly significant differences in the specificity of the first autoantibody were observed between HLA genotypes. Homozygotes for the DR3-DQ2 haplotype had almost exclusively GADA as the first autoantibody whereas a more even distribution between GADA and IAA was found in DR3-DQ2/DR4-DQ8 as well as DR3-DQ/x and DR4-DQ8/x genotypes (x referring to neutral haplotypes). In DR4-DQ8 positive genotypes with the DRB1*04:01 allele IAA was more often the first autoantibody than in DRB1*04:04 positive genotypes. Various neutral haplotypes also significantly affected the relative proportions of different initial autoantibodies. These findings were confirmed and expanded in a series of 1591 T1D children under the age of 10 years from FPDR. Conclusions These results emphasize the importance of HLA class II polymorphisms in the recognition of autoantigen epitopes in the initiation of various pathways of the autoimmune response. This article is protected by copyright. All rights reserved.

Although gut bacterial dysbiosis is recognized as a regulator of beta-cell autoimmunity, no data is available on fungal dysbiosis in the children at the risk of type 1 diabetes (T1D). We hypothesized that the co-occurrence of fungal and bacterial dysbiosis contributes to the intestinal inflammation and autoimmune destruction of insulin-producing beta-cells in T1D. Fecal and blood samples were collected from 26 children tested positive for at least one diabetes-associated autoantibody (IAA, GADA, IA-2A or ICA) and matched autoantibody-negative children with HLA-conferred susceptibility to T1D (matched for HLA-DQB1 haplotype, age, gender and early childhood nutrition). Bacterial 16S and fungal ITS2 sequencing, and analyses of the markers of intestinal inflammation, namely fecal human beta-defensin-2 (HBD2), calprotectin and secretory total IgA, were performed. Anti-Saccharomyces cerevisiae antibodies (ASCA) and circulating cytokines, IFNG, IL-17 and IL-22, were studied. After these analyses, the children were followed for development of clinical T1D (median 8 years and 8 months). Nine autoantibody positive children were diagnosed with T1D, whereas none of the autoantibody negative children developed T1D during the follow-up. Fungal dysbiosis, characterized by high abundance of fecal Saccharomyces and Candida, was found in the progressors, i.e., children with beta-cell autoimmunity who during the follow-up progressed to clinical T1D. These children showed also bacterial dysbiosis, i.e., increased Bacteroidales and Clostridiales ratio, which was, however, found also in the non-progressors, and is thus a common nominator in the children with beta-cell autoimmunity. Furthermore, the progressors showed markers of intestinal inflammation detected as increased levels of fecal HBD2 and ASCA IgG to fungal antigens. We conclude that the fungal and bacterial dysbiosis, and intestinal inflammation are associated with the development of T1D in children with beta-cell autoimmunity.

Exposure to environmental chemicals can modulate the developing immune system, but its role in the pathogenesis of type 1 diabetes is largely unexplored. Our objective was to study the levels of circulating concentrations of environmental pollutants during the first years of life and their associations with the later risk of diabetes-predictive autoantibodies. From two birth-cohort studies including newborn infants with HLA-conferred susceptibility to type 1 diabetes (FINDIA and DIABIMMUNE), we identified case children with at least one biochemical diabetes-associated autoantibody (n = 30–40) and from one to four autoantibody-negative controls per each case child matched for age, gender, diabetes-related HLA-risk, delivery hospital, and, in FINDIA, also dietary intervention group. Plasma levels of 13 persistent organic pollutants and 14 per- and polyfluorinated substances were analyzed in cord blood and plasma samples taken at the age of 12 and 48 months. Both breastfeeding and the geographical living environment showed association with circulating concentrations of some of the chemicals. Breastfeeding-adjusted conditional logistic regression model showed association between decreased plasma HBC concentration at 12-month-old children and the appearance of diabetes-associated autoantibodies (HR, 0.989; 95% Cl, 0.978–1.000; P = 0.048). No association was found between the plasma chemical levels and the development of clinical type 1 diabetes. Our results do not support the view that exposure to the studied environmental chemicals during fetal life or early childhood is a significant risk factor for later development of β-cell autoimmunity and type 1 diabetes. Electronic supplementary material The online version of this article (10.1007/s11356-018-3659-6) contains supplementary material, which is available to authorized users.

Background The etiology of type 1 diabetes (T1D) is largely unknown. Infections and microbial exposures are believed to play a role in the pathogenesis and in the development of islet autoimmunity in genetically susceptible individuals. Objective To assess the relationships between early childhood infections, islet autoimmunity, and progression to T1D in genetically predisposed children. Methods Children with human leukocyte antigen (HLA)-conferred disease susceptibility (N=790; 51.5% males) from Finland (n = 386), Estonia (n = 322), and Russian Karelia (n = 82) were observed from birth up to the age of 3 years. Children attended clinical visits at the age of 3, 6, 12, 18, 24, and 36 months. Serum samples for analyzing T1D-associated autoimmune markers were collected and health data recorded during the visits. Results Children developing islet autoimmunity (n = 46, 5.8%) had more infections during the first year of life (3.0 vs 3.0, mean rank 439.1 vs 336.2; P = .001) and their first infection occurred earlier (3.6 vs 5.0 months; P = .005) than children with no islet autoimmunity. By May 2016, 7 children (0.9%) had developed T1D (progressors). Compared with non-diabetic children, T1D progressors were younger at first infection (2.2 vs 4.9 months; P = .004) and had more infections during the first 2 years of life (during each year 6.0 vs 3.0; P = .001 and P = .027, respectively). By 3 years of age, the T1D progressors had twice as many infections as the other children (17.5 vs 9.0; P = .006). Conclusions Early childhood infections may play an important role in the pathogenesis of T1D. Current findings may reflect either differences in microbial exposures or early immunological aberrations making diabetes-prone children more susceptible to infections.

A declining first-phase insulin response (FPIR) is associated with positivity for multiple islet autoantibodies, irrespective of class II HLA DR-DQ genotype. We examined the associations of FPIR with genetic variants outside the HLA DR-DQ region in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study in children with and without multiple autoantibodies. Association between FPIR and class I alleles A*24 and B*39 and eight single nucleotide polymorphisms outside the HLA region were analyzed in 438 children who had one or more FPIR results available after seroconversion. Hierarchical linear mixed models were used to analyze repeated measurements of FPIR. In children with multiple autoantibodies, the change in FPIR over time was significantly different between those with various PTPN2 (rs45450798), FUT2 (rs601338), CTSH (rs3825932), and IKZF4 (rs1701704) genotypes in at least one of the models. In general, children carrying susceptibility alleles for type 1 diabetes experienced a more rapid decline in insulin secretion compared with children without susceptibility alleles. The presence of the class I HLA A*24 allele was also associated with a steeper decline of FPIR over time in children with multiple autoantibodies. Certain genetic variants outside the class II HLA region may have a significant impact on the longitudinal pattern of FPIR.

Children develop rapidly during the first years of life, and understanding the sources and associated levels of variation in the serum proteome is important when using serum proteins as markers for childhood diseases. The aim of this study was to establish a reference model for the evolution of a healthy serum proteome during early childhood. Label-free quantitative proteomics analyses were performed for 103 longitudinal serum samples collected from 15 children at birth and between the ages of 3–36 months. A flexible Gaussian process-based probabilistic modelling framework was developed to evaluate the effects of different variables, including age, living environment and individual variation, on the longitudinal expression profiles of 266 reliably identified and quantified serum proteins. Age was the most dominant factor influencing approximately half of the studied proteins, and the most prominent age-associated changes were observed already during the first year of life. High interindividual variability was also observed for multiple proteins. These data provide important details on the maturing serum proteome during early life, and evaluate how patterns detected in cord blood are conserved in the first years of life. Additionally, our novel modelling approach provides a statistical framework to detect associations between covariates and non-linear time series data.

OBJECTIVE This study assessed the relationship between autoantibodies against zinc transporter 8 (ZnT8A) and disease characteristics at diagnosis of type 1 diabetes and during the first 2 years. RESEARCH DESIGN AND METHODS Children, younger than 15 years of age (n = 723) who were newly diagnosed with diabetes, were analyzed for ZnT8A, other diabetes-associated autoantibodies, HLA DR-DQ alleles, and metabolic status, which was monitored by pH, plasma glucose, and occurrence of ketoacidosis at diagnosis and through follow-up of C-peptide concentrations, exogenous insulin dose, and glycosylated hemoglobin for 2 years after the diagnosis. RESULTS ZnT8A positivity was detected in 530 children (73%). Positivity for ZnT8A was associated with older age (median 8.9 vs. 8.2 years, P = 0.002) and more frequent ketoacidosis (24% vs. 15%, P = 0.013). Children carrying the HLA DR3 allele were less often ZnT8A positive (66% vs. 77%, P = 0.002) than others. ZnT8A-positive children had lower serum C-peptide concentrations (P = 0.008) and higher insulin doses (P = 0.012) over time than their ZnT8A-negative peers. CONCLUSIONS Positivity for ZnT8A at diagnosis seems to reflect a more aggressive disease process before and after diagnosis.

Background The study aimed to define the frequencies of type 1 diabetes-associated gene polymorphisms and their associations with various diabetes-associated autoantibodies in Egyptian children. Methods One hundred and one children with type 1 diabetes and 160 healthy controls from the same region were studied for HLA-DQB1, HLA-DQA1, and HLA-DRB1 (DR4 subtypes) alleles; for INS and protein tyrosine phosphatase, non-receptor type 22 gene polymorphisms (rs689 and rs2476601); and for diabetes-associated autoantibodies. Results Most children with diabetes (77.2%) were positive for the HLA-(DR3)-DQA1*05-DQB1*02 (DR3-DQ2) haplotype compared with 26.2% of the controls (OR = 9.5; p

Objectives We set out to determine the prevalence of tissue transglutaminase antibodies (anti-tTG) and celiac disease (CD) in children with newly diagnosed type 1 diabetes (T1D) and their first-degree relatives (FDR). The hypothesis was that the individuals with both diabetes and CD form a distinct subgroup in terms of human leukocyte antigen (HLA) class II genetics, islet autoantibodies, and clinical characteristics at diabetes diagnosis. Subjects and methods This population-based observational study included 745 index children with T1D and their 2692 FDR from the Finnish Pediatric Diabetes Register. CD was ascertained by registers, patient records, and screening anti-tTG positive individuals for further testing. Results Among the index children, 4.8% had anti-tTG at diabetes diagnosis, and at the end of the study 3.2% had CD. Among the relatives, 2.9% had anti-tTG (4.8% mothers, 2.4% fathers, and 2.1% siblings), and 2.5% had CD (4.6% mothers, 2.1% fathers, and 1.4% siblings). Anti-tTG and CD associated with the HLA DR3-DQ2 haplotype. The usual female predominance of CD patients was observed in relatives (70%) but not among index children (46%). The index children with both diseases had a lower number of detectable islet autoantibodies than those with diabetes alone. Conclusions The children with double diagnosis differed from those with diabetes alone in HLA genetics, humoral islet autoimmunity directed against fewer antigens, and in the lack of usual female preponderance among CD patients. Compared with 61% of the anti-tTG positive relatives, only 36% of anti-tTG positive index children developed CD implicating transient anti-tTG positivity at diagnosis of T1D.

Objective The relationship between patterns of islet autoantibodies at diagnosis and specificity of the first islet autoantibody at the initiation of autoimmunity was analyzed with the aim of identifying patterns informative of the primary autoantibodies. Methods Information about a single first autoantibody at seroconversion and autoantibody data at diagnosis were available for 128 children participating in the follow-up cohort of the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study. Autoantibody data at diagnosis and genotyping results were also obtained from children in the Finnish Pediatric Diabetes Register (FPDR). Results Insulin autoantibodies (IAA) were the most common primary antibodies (N = 68), followed by those for glutamic acid decarboxylase (GADA; N = 38), IA-2 antigen (IA-2A; N = 13), and zinc transporter 8 (ZnT8A; N = 9), whereas at diagnosis, IA-2A were most frequent (N = 103), followed by IAA (N = 78), ZnT8A (N = 73), and GADA (N = 71). Accordingly, the presence of many specific autoantibodies at diagnosis was due to the secondary antibodies appearing after primary antibodies, and in some cases, the primary autoantibody, most often IAA, had already disappeared at the time of diagnosis. Many of the autoantibody combinations present at diagnosis could be assembled into groups associated with either IAA or GADA as first autoantibodies. These combinations, in children diagnosed below the age of 10 years in the FPDR, were found to be strongly associated with risk genotypes in either INS (IAA first) or IKZF4-ERBB3 (GADA first) genes. Conclusions Autoantibody patterns at diagnosis may be informative on primary autoantibodies initiating autoimmunity in young children developing type 1 diabetes.

Background Advanced glycation end products (AGEs) and their interaction with the receptor for AGEs (RAGE) have been studied for their role in the pathogenesis and complications of type 1 diabetes. Decreased concentrations of soluble RAGE (sRAGE) have been reported in acute autoimmune inflammation. We set out to analyze the changes in sRAGE concentration during preclinical diabetes in children seroconverting to islet autoantibody positivity. Methods We measured serum concentrations of sRAGE in 168 children who progressed to clinical disease and 43 children who turned positive for at least 2 diabetes-associated autoantibodies but remained nondiabetic. We analyzed the sRAGE before seroconversion in the first autoantibody-positive sample and annually thereafter until the diagnosis of type 1 diabetes or end of follow-up. Results Both groups had similar sRAGE before seroconversion, but subsequently, sRAGE concentrations were lower (P

Genetic predisposition could be assumed to be causing clustering of autoimmunity in individuals and families. We tested whether HLA and non-HLA loci associate with such clustering of autoimmunity. We included 1,745 children with type 1 diabetes from the Finnish Pediatric Diabetes Register. Data on personal or family history of autoimmune diseases were collected with a structured questionnaire and, for a subset, with a detailed search for celiac disease and autoimmune thyroid disease. Children with multiple autoimmune diseases or with multiple affected first-or second-degree relatives were identified. We analysed type 1 diabetes related HLA class II haplotypes and genotyped 41 single nucleotide polymorphisms (SNPs) outside the HLA region. The HLA-DR4-DQ8 haplotype was associated with having type 1 diabetes only whereas the HLA-DR3-DQ2 haplotype was more common in children with multiple autoimmune diseases. Children with multiple autoimmune diseases showed nominal association with RGS1 (rs2816316), and children coming from an autoimmune family with rs11711054 (CCR3-CCR5). In multivariate analyses, the overall effect of non-HLA SNPs on both phenotypes was evident, associations with RGS1 and CCR3-CCR5 region were confirmed and additional associations were implicated: NRP1, FUT2, and CD69 for children with multiple autoimmune diseases. In conclusion, HLA-DR3-DQ2 haplotype and some non-HLA SNPs contribute to the clustering of autoimmune diseases in children with type 1 diabetes and in their families.

The prevalence of immune-mediated diseases, such as allergies and type 1 diabetes, is on the rise in the developed world. In order to explore differences in the gene expression patterns induced in utero in infants born in contrasting standards of living and hygiene, we collected umbilical cord blood RNA samples from infants born in Finland (modern society), Estonia (rapidly developing society) and the Republic of Karelia, Russia (poor economic conditions). The whole blood transcriptome of Finnish and Estonian neonates differed from their Karelian counterparts, suggesting exposure to toll-like receptor (TLR) ligands and a more matured immune response in infants born in Karelia. These results further support the concept of a conspicuous plasticity in the developing immune system: the environmental factors that play a role in the susceptibility/protection towards immune-mediated diseases begin to shape the neonatal immunity already in utero and direct the maturation in accordance with the surrounding microbial milieu. (C) 2014 The Authors. Published by Elsevier Inc.

OBJECTIVE We analyzed the relationship among soluble receptor for advanced glycation end products (sRAGEs), the clinical phenotype, HLA genotype, and risk-associated single nucleotide polymorphisms (SNPs) in the AGER gene in a large population of Finnish children with newly diagnosed type 1 diabetes. RESEARCH DESIGN AND METHODS Samples from 2,115 clinically phenotyped children RESULTS Children with type 1 diabetes and control subjects had similar sRAGE concentrations (1,171 vs. 1,153 pg/mL, P = 0.48). There was a correlation between age at diagnosis and serum sRAGE concentrations (r = 0.10, P < 0.001) among the patients but not among the control subjects. Children CONCLUSIONS Age and AGER polymorphisms are associated with the circulating sRAGE concentration among children with type 1 diabetes. The observations of reduced sRAGE concentrations in young children, in those with ketoacidosis, and in carriers of the high-risk HLA DR3/DR4 genotype suggest that decreased sRAGE concentration reflects a more aggressive disease phenotype.

OBJECTIVE To determine the frequency of newly diagnosed diabetic children with first- and second-degree relatives affected by type 1 diabetes and to characterize the effects of this positive family history on clinical markers, signs of β-cell autoimmunity, and HLA genotype in the index case. RESEARCH DESIGN AND METHODS Children (n = 1,488) with type 1 diabetes diagnosed under 15 years of age were included in a cross-sectional study from the Finnish Pediatric Diabetes Register. Data on family history of diabetes and metabolic decompensation at diagnosis were collected using a questionnaire. Antibodies to β-cell autoantigens (islet cell antibodies, insulin autoantibodies, GAD antibodies, and antibodies to the islet antigen 2 molecule) and HLA genotypes were analyzed. RESULTS A total of 12.2% of the subjects had a first-degree relative with type 1 diabetes (father 6.2%, mother 3.2%, and sibling 4.8%) and 11.9% had an affected second-degree relative. Children without affected relatives had lower pH (P < 0.001), higher plasma glucose (P < 0.001) and β-hydroxybutyrate concentrations (P < 0.001), a higher rate of impaired consciousness (P = 0.02), and greater weight loss (P < 0.001). There were no differences in signs of β-cell autoimmunity. The familial cases carried the HLA DR4-DQ8 haplotype more frequently than sporadic cases (74.0 vs. 67.0%, P = 0.02). CONCLUSIONS When the extended family history of type 1 diabetes is considered, the proportion of sporadic diabetes cases may be reduced to

Patients with type 1 diabetes have antibodies to ZnT8 protein is encoded by SLC30A8. The C-allele of the R325 W variant in SLC30A8 is associated with type 2 diabetes and reduced beta-cell function in non-diabetic subjects. Our aim was to assess the prevalence of ZnT8 autoantibodies (ZnT8A) in patients with adult-onset diabetes, and to characterize associations between ZnT8A and phenotype, as well as SLC30A8 and HLA-DQB1 genotypes.ZnT8A were analyzed in patients diagnosed with diabetes35 years (type 1 diabetes: n = 274; Latent autoimmune diabetes in adults (LADA): n = 294). SLC30A8 R325 W (rs13266634) and HLA-DQB1 alleles were genotyped in all patients and 537 non-diabetic control subjects.ZnT8A were significantly more prevalent in LADA (34.3%) compared to adult-onset type 1 diabetes (18.7%, p0.0001). Among the patients with adult-onset type 1 diabetes, ZnT8A were associated with shorter disease duration [4.4 (6.0) vs. 10.8 (11.2) years, p0.0001], whereas no such association was observed among patients with LADA. The SLC30A8 R325 W variant was associated with LADA with low GADA levels [SLC30A8 CC: OR (95% CI): 1.46 (1.00 - 2.13), p = 0.049], and reduced insulin secretion among the non-diabetic subjects and the patients with LADA.ZnT8A were more common and more persistent in patients with LADA compared to adult-onset type 1 diabetes, but their presence was not associated with specific phenotypic characteristics. The SLC30A8 CC genotype adds to the genetic heterogeneity of LADA linked to GADA reactivity.

Background: During the last several decades the prevalence of coeliac disease (CD) has increased worldwide. Aim: To compare the cumulative incidence of CD between Estonian and Finnish children and to identify the risk factors. Materials and methods: Children were recruited as part of the DIABIMMUNE Study. In the birth cohort (BC) 258 children from Estonia and 305 from Finland, and in the young children's cohort (YCC) 1363 and 1384 children were followed up, respectively. The diagnosis of CD was made in accordance with the ESPGHAN guidelines-the presence of IgA-tTG antibodies and small bowel villous atrophy. Results: During the study period 29 children developed CD. The cumulative incidence of CD was significantly higher in Finland (0.77% vs 0.27%; P = 0.01). No difference was seen between the children with CD and the controls in the duration of breastfeeding or the age at cereal introduction. The BC children with CD had had significantly more episodes of infections with fever by the age of 12 months compared to the controls (3.4 vs 1.4; P = 0.04). Conclusion: The 5-year cumulative incidence of childhood CD is significantly higher in Finland than in Estonia. Sequential infections early in life may increase the risk for developing CD. (C) 2016 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l.

Background We analysed the previously reported association of the HLA-A*24:02, B*18 and B*39 alleles with type 1 diabetes and diabetes associated autoimmunity in the Finnish population applying HLA-DR/DQ stratification. Materials & methods Haplotype transmission was analysed in 2424 nuclear families from the Finnish Paediatric Diabetes Register. Survival analysis was applied to study the development of islet autoantibodies and further progression to clinical diabetes in the prospective follow-up cohort from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. The subjects were genotyped for specific HLA class I alleles by sequence-specific hybridization using lanthanide labelled nucleotide probes. Results The HLA-B*39:06 allele was found almost exclusively on the (DR8)-DQB1*04 haplotype in which its presence changed the disease risk status of the whole haplotype from neutral to predisposing. The HLA-A*24:02 and the B*39:01 alleles increased the diabetes-associated risk of the DRB1*04:04-DQA1*03-DQB1*03:02 haplotype but the alleles were in linkage disequilibrium and no independent effect could be detected. Within the DIPP cohort, neither the A*24:02 nor the B*39:01 allele were associated with seroconversion but were in contrast associated with increased progression from seroconversion to clinical disease. Discussion & Conclusions The independent predisposing effect of the HLA-B*39:06 allele with type 1 diabetes was confirmed in the Finnish population but the association of the A*24:02 and B*39:01 alleles remained inconclusive whilst both A*24:02 and B*39:01 affected the progression rate from seroconversion to autoantibody positivity to overt type 1 diabetes.

Article, Mesencephalic astrocyte-derived neurotrophic factor (MANF) was recently shown to be essential for the survival and proliferation of pancreatic β-cells in mice, where deletion of MANF resulted in diabetes. The current study aimed at determining whether the concentration of circulating MANF is associated with the clinical manifestation of human type 1 diabetes (T1D). MANF expression in T1D or MANF levels in serum have not been previously studied. We developed an enzyme-linked immunosorbent assay (ELISA) for MANF and measured serum MANF concentrations from 186 newly diagnosed children and adolescents and 20 adults with longer-term T1D alongside with age-matched controls. In healthy controls the mean serum MANF concentration was 7.0 ng/ml. High MANF concentrations were found in children 1–9 years of age close to the diagnosis of T1D. The increased MANF concentrations were not associated with diabetes-predictive autoantibodies and autoantibodies against MANF were extremely rare. Patients with conspicuously high MANF serum concentrations had lower C-peptide levels compared to patients with moderate MANF concentrations. Our data indicate that increased MANF concentrations in serum are associated with the clinical manifestation of T1D in children, but the exact mechanism behind the increase remains elusive., published version, peerReviewed

The gut microbial community is dynamic during the first 3 years of life, before stabilizing to an adult-like state. However, little is known about the impact of environmental factors on the developing human gut microbiome. We report a longitudinal study of the gut microbiome based on DNA sequence analysis of monthly stool samples and clinical information from 39 children, about half of whom received multiple courses of antibiotics during the first 3 years of life. Whereas the gut microbiome of most children born by vaginal delivery was dominated by Bacteroides species, the four children born by cesarean section and about 20% of vaginally born children lacked Bacteroides in the first 6 to 18 months of life. Longitudinal sampling, coupled with whole-genome shotgun sequencing, allowed detection of strain-level variation as well as the abundance of antibiotic resistance genes. The microbiota of antibiotic-treated children was less diverse in terms of both bacterial species and strains, with some species often dominated by single strains. In addition, we observed short-term composition changes between consecutive samples from children treated with antibiotics. Antibiotic resistance genes carried on microbial chromosomes showed a peak in abundance after antibiotic treatment followed by a sharp decline, whereas some genes carried on mobile elements persisted longer after antibiotic therapy ended. Our results highlight the value of high-density longitudinal sampling studies with high-resolution strain profiling for studying the establishment and response to perturbation of the infant gut microbiome., National Human Genome Research Institute (U.S.) (grant 2U54HG003067-10), Juvenile Diabetes Research Foundation International, National Institutes of Health (U.S.) (grant U54 DK102557), National Institutes of Health (U.S.) (grant R01 DK092405), National Institutes of Health (U.S.) (grant P30 DK043351), Leona M. and Harry B. Helmsley Charitable Trust, Crohn's and Colitis Foundation of America

OBJECTIVE Dendritic cells (DCs) are largely responsible for the activation and fine-tuning of T-cell responses. Altered numbers of blood DCs have been reported in type 1 diabetes (T1D). We aimed at characterizing the less well-known phenotypic properties of DCs in T1D. RESEARCH DESIGN AND METHODS In a case-control setting, samples from a total of 90 children were studied by flow cytometry or by quantitative real-time PCR (qPCR). RESULTS We found decreased numbers of myeloid DCs (mDCs) (8.97 vs. 13.4 cells/μL, P = 0.009, n = 31) and plasmacytoid DCs (pDCs) (9.47 vs. 14.6 cells/μL, P = 0.018, n = 30) in recent-onset T1D. Using a panel of antibodies against functionally important DC markers, we detected a decreased expression of CC chemokine receptor 2 (CCR2) on mDCs (percentage above negative control, P = 0.002, n = 29) and pDCs (median intensity, P = 0.003, n = 30) from T1D patients. In an independent series of children, the reduced expression of CCR2 was confirmed by qPCR in isolated mDCs (P = 0.043, n = 20). Serum concentrations of CCR2 ligands monocyte chemotactic protein-1 and -3 did not differ between the groups. A trend for an enhanced responsiveness of the nuclear factor-κB pathway (P = 0.063, n = 39) was seen in mDCs from children with β-cell autoantibodies, which is possibly related to the reduced CCR2 expression, since CCR2 on mDCs was downregulated by nuclear factor-κB–activating agents. CONCLUSIONS Given the role of CCR2 in DC chemotaxis and in DC-elicited Th1 differentiation, our results may indicate a functionally important DC abnormality in T1D affecting the initiation and quality of immune responses.

ObjectiveTo characterize the humoral immune response to islet antigen 2 (IA-2) in patients with newly diagnosed type 1 diabetes (T1D), we compared the profile of epitope- and isotype-specific IA-2 antibodies (IA-2A) between children with a humoral immune response restricted to IA-2 and children with a broad response including insulin autoantibodies (IAA) and antibodies to glutamic acid decarboxylase (GADA) in addition to IA-2A.MethodsThe study subjects (n=100) were derived from a consecutive series of 1108 patients from the Finnish Pediatric Diabetes Register (investigators listed in the Appendix). Islet cell antibodies, IAA, GADA, total IA-2A levels, IA-2/IA-2β epitopes, and isotypes were measured, and human leukocyte antigen (HLA) genotypes were analyzed.ResultsThere were no significant differences between the two groups in the frequency or levels of epitope-specific IA-2A. Those with an IA-2-restrictive response tested positive more frequently for IgA-IA-2A (P=0.001), had higher titers of IgE-IA-2A (P=0.025), tested positive for more IA-2A isotypes than the broad responders (P=0.04), and carried the high-riskHLA-(DR4)-DQB1*0302haplotype more frequently than those with a broad antibody response (P=0.019).ConclusionsThese data show that children with newly diagnosed T1D, who test positive only for IA-2A out of the three molecular antibodies predictive of T1D, have a broader IA-2-specific isotype response and stronger association with the high-risk HLA haplotype than those testing positive for all three molecular antibodies. This may be indicative of a different pathogenetic mechanism in those with their humoral immune response restricted to IA-2 at the time of diagnosis.

Summary In this study we aim to describe the characteristics of non-diabetic organ donors with circulating diabetes-associated autoantibodies collected within the Nordic Network for Islet Transplantation. One thousand and thirty organ donors have been screened in Uppsala for antibodies against glutamic acid decarboxylase (GADA) and islet antigen-2 (IA-2A). The 32 non-diabetic donors that tested positive for GADA (3·3% of all non-diabetic donors) were studied in more detail, together with 32 matched controls. Mean age among the autoantibody-positive donors was 52·6 (range 21–74), family history of type 1 diabetes (T1D) was unknown, and no donor was genetically predisposed for T1D regarding the human leucocyte antigen (HLA) locus. Subjects were analysed for islet cell antibodies (ICA), insulin autoantibodies (IAA) and zinc transporter 8 antibodies (ZnT8A), and pancreas morphology and clinical data were examined. Eight non-diabetic donors tested positive for two antibodies and one donor tested positive for four antibodies. No insulitis or other signs of a diabetic process were found in any of the donors. While inflammatory cells were present in all donors, subjects with high GADA titres had significantly higher CD45 cell numbers in exocrine tissue than controls. The extent of fibrosis was more pronounced in autoantibody-positive donors, even in subjects with lower GADA titres. Notably, it is possible that events not related directly to T1D (e.g. subclinical pancreatitis) may induce autoantibodies in some cases.

OBJECTIVE Dietary advanced glycation end products (AGEs) and their interactions with the receptor for AGEs (RAGE) may play a role in the pathogenesis of type 1 diabetes. This study set out to assess whether there is any association of circulating concentrations of soluble RAGE (sRAGE), AGEs, and their ratio with the appearance of diabetes-associated autoantibodies in children progressing to clinical diabetes. RESEARCH DESIGN AND METHODS Serum concentrations of sRAGE, N-ε(carboxymethyl)lysine (CML) adducts, and the sRAGE/CML ratio were analyzed in children who progressed to type 1 diabetes. The samples were taken at four time points: before seroconversion, at the time of the first autoantibody-positive sample, at the time of the first sample positive for multiple (>2) autoantibodies, and close to the disease diagnosis. Samples of autoantibody-negative controls matched for age, sex, and HLA-conferred diabetes risk were analyzed at corresponding time points. RESULTS The prediabetic children had higher sRAGE concentrations before seroconversion (Pc = 0.03), at the appearance of multiple autoantibodies (Pc = 0.008), and close to diagnosis (Pc = 0.04). Close to diagnosis, the cases had lower CML concentrations than the controls (Pc = 0.004). Prediabetic children had a higher sRAGE/CML ratio than the controls before seroconversion (Pc = 0.008) and at diagnosis (Pc < 0.001). CONCLUSIONS Prediabetic children have higher concentrations of sRAGE and a higher sRAGE/CML ratio than healthy controls. Circulating sRAGE concentrations seem to decline with the appearance of diabetes-predictive autoantibodies in children progressing to type 1 diabetes. The higher sRAGE/CML ratio in prediabetic children may reflect a higher AGE scavenger capacity.

SummaryThe natural history of preclinical diabetes is partly characterized, but there is still limited information on the dynamics of the immune response to β-cell autoantigens during the course of preclinical disease. The aim of this work was to assess the maturation of the humoral immune response to the protein tyrosine phosphatase(PTP)-related proteins (IA-2 and IA-2β) in preclinical type I diabetes (TID). Forty-five children participating in the Finnish Type I Diabetes Prediction and Prevention (DIPP) Study who had seroconverted to IA-2 antibody positivity were analysed. Specific radiobinding assays were used to determine IA-2/IA-2β epitope-specific antibodies (the juxtamembrane (JM) region of IA-2, PTP-like domain and βPTP-like domain) and isotype-specific IA-2 antibodies. Individual areas under the curve (AUC) over the observation period were calculated for total IA-2 antibodies, each isotype and specific epitope responses. The children who progressed to TID tended to have an initial IA-2 JM epitope response more frequently (P = 0·06), and this response was more often dominant during the observation period (P

Upregulation of IL-17 immunity and detrimental effects of IL-17 on human islets have been implicated in human type 1 diabetes. In animal models, the plasticity of Th1/Th17 cells contributes to the development of autoimmune diabetes. In this study, we demonstrate that the upregulation of the IL-17 pathway and Th1/Th17 plasticity in peripheral blood are markers of advanced β cell autoimmunity and impaired β cell function in human type 1 diabetes. Activated Th17 immunity was observed in the late stage of preclinical diabetes in children with β cell autoimmunity and impaired glucose tolerance, but not in children with early β cell autoimmunity. We found an increased ratio of IFN-γ/IL-17 expression in Th17 cells in children with advanced β cell autoimmunity, which correlated with HbA1c and plasma glucose concentrations in an oral glucose tolerance test, and thus impaired β cell function. Low expression of Helios was seen in Th17 cells, suggesting that Th1/Th17 cells are not converted thymus-derived regulatory T cells. Our results suggest that the development of Th1/Th17 plasticity may serve as a biomarker of disease progression from β cell autoantibody positivity to type 1 diabetes. These data in human type 1 diabetes emphasize the role of Th1/Th17 plasticity as a potential contributor to tissue destruction in autoimmune conditions.

Objective: A humoral autoimmune response to glutamic acid decarboxylase (GAD65) is common both in patients with type 1 diabetes and in those with the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, while overt type 1 diabetes is relatively rarely diagnosed in APECED patients. The aim of this study was to assess whether this difference in the incidence of type 1 diabetes is associated with variability in the humoral immune response to GAD65, one of the major autoantigens in type 1 diabetes. Methods: Epitope- and isotype-specific GAD65 autoantibodies were analysed in 20 patients with APECED and 20 patients with newly diagnosed type 1 diabetes alone by radiobinding assays. Results: GAD65 autoantibodies targeted the middle and carboxy-terminal regions of GAD65 and occasionally the amino-terminal region in the APECED patients and comprised mainly the IgG1 subclass and less frequently the IgG2 and IgG4 subclasses. The profile of epitope- and isotype-specific GAD65 autoantibodies was similar in type 1 diabetes and APECED, except that IgG2 subclass antibodies were observed more often and at higher levels in the patients with type 1 diabetes alone (P < 0.05). None of the measured parameters separated APECED patients with type 1 diabetes from those without type 1 diabetes. Conclusion: APECED-associated humoral autoimmunity to GAD65 does not differ markedly from that observed in type 1 diabetes; only IgG2-GAD65 antibodies may be more closely associated with the latter entity.

To characterize the humoral immune response to the protein tyrosine phosphatase (PTP)-like autoantigen (IA-2) in preclinical type 1 diabetes (T1D), and to assess the utility of epitope and isotype-specific IA-2 antibody responses as surrogate markers for disease development, we analyzed these antibodies in 34 initially non-diabetic siblings of affected children derived from the "Childhood Diabetes in Finland" (DiMe) Study. Half of them presented with T1D during an average observation period of 8.7 years. Radiobinding assays were used to determine IA-2/IA-2 beta epitope-specific (the juxtamembrane region, JM; the PTP-like and the beta PTP-like domain) antibodies and isotype-specific (IgG1-4, IgA, IgE and IgM) IA-2 antibodies. Initially, 30 of the 34 siblings tested positive for epitope-specific antibodies. The siblings who progressed to clinical diabetes had IA-2 JM antibodies more often (P0.05) but IgE-IA-2 antibodies less frequently (P0.05) than the siblings who did not progress to T1D. During the identical follow-up time, the non-progressors had higher integrated titers of IgE-IA-2 antibodies (P=0.05). The occurrence of IgE-IA-2 antibodies was protective, since despite IA-2 JM antibodies, children with IgE-IA-2 antibodies did rarely progress to T1D. This study demonstrates that JM-reactive IA-2 antibodies are associated with an increased risk of progression to overt T1D, whereas an IgE response to IA-2 confers relative protection against clinical disease.

Enterovirus infections, implicated in the pathogenesis of type 1 diabetes in a number of studies, may precipitate the symptoms of clinical diabetes and play a role in the initiation of the beta-cell damaging process. The aim of this study was to evaluate whether cellular immune responses to enterovirus antigens are abnormal in children with type 1 diabetes. Lymphocyte proliferation responses to enterovirus antigens were analyzed in 41 children with new-onset type 1 diabetes, 23 children with type 1 diabetes for 4-72 months, and healthy control children in subgroups matched for HLA-DQB1 risk alleles, sex, and age. Children with diabetes for 4-72 months more often had T-cell responses to the Coxsackievirus B4-infected cell lysate antigen than children with new-onset diabetes (P < 0.01) or control children (P < 0.01). Responses to recombinant nonstructural protein 2C of Coxsackievirus B4 were also more frequent in children with type 1 diabetes for 4-72 months when compared with control subjects (P = 0.03), whereas the responses to purified Coxsackievirus B4 and recombinant VP0 protein, which did not contain nonstructural proteins, did not differ. These data suggest that T-cell responses to Coxsackievirus B4 proteins and particularly to the antigens containing the nonstructural proteins of the virus are increased in children with type 1 diabetes after the onset of the disease. However, in children with new-onset diabetes, responses were normal or even decreased. This phenomenon was specific for enteroviruses and could be caused by trapping of enterovirus-specific T-cells in the pancreas.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a recessively inherited, progressive neurologic disease caused by mutations in the CLN3 gene, coding for a transmembrane protein with unknown function.1 The symptoms include visual failure, noticed around the age of 4 to 7 years, mental decline, epilepsy, parkinsonism, psychiatric symptoms, and sleeping difficulties. Thus far, no curative treatment is available and the disease leads to an early death at the age of 15 to 30 years. Autoantibodies to glutamic acid decarboxylase 65 (GAD65) were detected in the sera of patients with JNCL and in CLN3 knock-out mice.2 As a biologic sign of these antibodies, decreased GAD65 activity and increased levels of glutamate were observed in the mice brains. Together these findings suggest that an immune-mediated reaction to GAD65 may be involved in the pathogenesis of JNCL. Our aim was to test the effect of immunosuppressive therapy on the GAD65-antibodies, symptoms, and disease progression in patients with JNCL. ### Methods. Four girls and four boys with JNCL (table) were included in this 1-year study, approved by the Ethics Committee of the Hospital for Children and Adolescents, University of Helsinki. View this table: Table Patients, their GAD65-autoantibodies, verbal IQ, and weight for height ratio in percentage at the beginning (0 mo) and end …

ObjectiveTo examine the use of vitamin D supplements during infancy among the participants in an international infant feeding trial.DesignLongitudinal study.SettingInformation about vitamin D supplementation was collected through a validated FFQ at the age of 2 weeks and monthly between the ages of 1 month and 6 months.SubjectsInfants (n 2159) with a biological family member affected by type 1 diabetes and with increased human leucocyte antigen-conferred susceptibility to type 1 diabetes from twelve European countries, the USA, Canada and Australia.ResultsDaily use of vitamin D supplements was common during the first 6 months of life in Northern and Central Europe (>80 % of the infants), with somewhat lower rates observed in Southern Europe (>60 %). In Canada, vitamin D supplementation was more common among exclusively breast-fed than other infants (e.g. 71 % v. 44 % at 6 months of age). Less than 2 % of infants in the USA and Australia received any vitamin D supplementation. Higher gestational age, older maternal age and longer maternal education were study-wide associated with greater use of vitamin D supplements.ConclusionsMost of the infants received vitamin D supplements during the first 6 months of life in the European countries, whereas in Canada only half and in the USA and Australia very few were given supplementation.

The role of the intestinal microbiota as a regulator of autoimmune diabetes in animal models is well-established, but data on human type 1 diabetes are tentative and based on studies including only a few study subjects. To exclude secondary effects of diabetes and FILA risk genotype on gut microbiota, we compared the intestinal microbiota composition in children with at least two diabetes-associated autoantibodies (n = 18) with autoantibody-negative children matched for age, sex, early feeding history, and HLA risk genotype using pyrosequencing. Principal component analysis indicated that a low abundance of lactate-producing and butyrate-producing species was associated with beta-cell autoimmunity. In addition, a dearth of the two most dominant Bifidobacterium species, Bifidobacterium adolescentis and Bifidobacterium pseudocatenulatum, and an increased abundance of the Bacteroides genus were observed in the children with beta-cell autoimmunity. We did not find increased fecal calprotectin or IgA as marker of inflammation in children with beta-cell autoimmunity. Functional studies related to the observed alterations in the gut microbiome are warranted because the low abundance of bifidobacteria and butyrate-producing species could adversely affect the intestinal epithelial barrier function and inflammation, whereas the apparent importance of the Bacteroides genus in development of type 1 diabetes is insufficiently understood. Diabetes 62:1238-1244, 2013

OBJECTIVE Despite promising results from studies on mouse models, intranasal insulin failed to prevent or delay the development of type 1 diabetes in autoantibody-positive children with HLA-conferred disease susceptibility. To analyze whether the insulin dose was inadequate to elicit an immunomodulatory response, we compared the changes observed in insulin antibody (IA) affinity and isotypes after treatment with nasal insulin or placebo. RESEARCH DESIGN AND METHODS Ninety-five children (47 in the placebo group and 48 in the insulin group of the total of 224 children randomized for the trial) with HLA-conferred susceptibility to type 1 diabetes derived from the intervention arm of the Finnish Type 1 Diabetes Prediction and Prevention study were included in these analyses. Blood samples drawn before or at the beginning of the treatment and after treatment for 3 and 6 months were analyzed for IA affinity and isotype-specific IAs (IgG1–4, IgA, IgM, and IgE). RESULTS IgG3- and IgA-IA levels (P = 0.031 and 0.015, respectively) and the number of IgG3-IA–positive subjects (P = 0.022) were significantly higher at 6 months after the initiation of the treatment in the insulin group. No significant differences were observed between the two groups in IA affinity or other IA isotypes. CONCLUSIONS The insulin dose administered induced a modest change in the IA isotype profile. The lack of impact of nasal insulin on IA affinity implies that the immune response of study subjects was already mature at the beginning of the intervention.

Molecular mimicry is one of the mechanisms by which enterovirus infections have been postulated to have a role in the pathogenesis of type 1 diabetes. Immunogenic epitopes in enterovirus capsid protein VP1 and procapsid protein VP0 have sequence similarities with diabetes-associated epitopes in tyrosine phosphatase IA-2/IAR and heat shock protein 60. In the present study, documented enterovirus infection was shown to induce humoral responses, that in 7% and 1% of patients cross-reacted with the known diabetes-associated epitopes in tyrosine phosphatase IAR and heat shock protein 60, respectively. In contrast, none of the children vaccinated against poliomyelitis had antibodies to the diabetes-associated epitope of tyrosine phosphatases IA-2/IAR. The antibody response studied in serum samples from six patients with coxsackievirus A9 infection was mainly targeted to capsid protein VP1. Coxsackievirus A9 infection induced antibodies cross-reacted with one epitope in heat shock protein 60, but not with epitopes derived from other autoantigens. Most diabetic children had high levels of antibodies to both coxsackievirus and poliovirus derived VP1 peptides but the pattern of reactivity did not differ from that seen in healthy children. The reactivity of linear epitopes derived from autoantigens was low in general and associated with the presence of multiple autoantibodies in the patients. Some linear auto-epitopes derived from tyrosine phosphatase IA-2, glutamic acid decarboxylase 65, preproinsulin, and heat shock protein 60 were recognized by sera from diabetic patients, but not by sera from healthy children. In conclusion, enteroviruses may induce immune responses that react with islet cell autoantigens, which is a concern when a putative inactivated enterovirus vaccine is considered.

Recent evidence from serum metabolomics indicates that specific metabolic disturbances precede β-cell autoimmunity in humans and can be used to identify those children who subsequently progress to type 1 diabetes. The mechanisms behind these disturbances are unknown. Here we show the specificity of the pre-autoimmune metabolic changes, as indicated by their conservation in a murine model of type 1 diabetes. We performed a study in non-obese prediabetic (NOD) mice which recapitulated the design of the human study and derived the metabolic states from longitudinal lipidomics data. We show that female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, normoglycemia, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum group. Together, the findings indicate that autoimmune diabetes is preceded by a state of increased metabolic demands on the islets resulting in elevated insulin secretion and suggest alternative metabolic related pathways as therapeutic targets to prevent diabetes., Author Summary We have recently found that distinct metabolic disturbances precede β-cell autoimmunity in children who later progress to type 1 diabetes (T1D). Here we performed a murine study using non-obese diabetic (NOD) mice that recapitulated the protocol used in human, followed up by independent studies where NOD mice were studied in relation to risk of diabetes progression. We found that young female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum subgroup. The metabolic phenotypes observed in our study could be relevant as end points for studies investigating T1D pathogenesis and/or responses to interventions. By proceeding from a clinical study via metabolomics and modeling to an experimental model using a similar study design, then evolving further to tissue-specific studies, we hereby also present a conceptually novel approach to reversed translation that may be useful in future therapeutic studies in the context of prevention and treatment of T1D as well as of other diseases characterized by long prodromal periods.