Umbilical cord blood DNA methylation in children who later develop type 1 diabetes.

AIMS/HYPOTHESIS: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. METHODS: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. RESULTS: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate <0.05. CONCLUSIONS/INTERPRETATION: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.

Coxsackievirus B1 infections are associated with the initiation of insulin-driven autoimmunity that progresses to type 1 diabetes.

AIMS/HYPOTHESIS: Islet autoimmunity usually starts with the appearance of autoantibodies against either insulin (IAA) or GAD65 (GADA). This categorises children with preclinical type 1 diabetes into two immune phenotypes, which differ in their genetic background and may have different aetiology. The aim was to study whether Coxsackievirus group B (CVB) infections, which have been linked to the initiation of islet autoimmunity, are associated with either of these two phenotypes in children with HLA-conferred susceptibility to type 1 diabetes. METHODS: All samples were from children in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study. Individuals are recruited to the DIPP study from the general population of new-born infants who carry defined HLA genotypes associated with susceptibility to type 1 diabetes. Our study cohort included 91 children who developed IAA and 78 children who developed GADA as their first appearing single autoantibody and remained persistently seropositive for islet autoantibodies, along with 181 and 151 individually matched autoantibody negative control children, respectively. Seroconversion to positivity for neutralising antibodies was detected as the surrogate marker of CVB infections in serial follow-up serum samples collected before and at the appearance of islet autoantibodies in each individual. RESULTS: CVB1 infections were associated with the appearance of IAA as the first autoantibody (OR 2.4 [95% CI 1.4, 4.2], corrected p = 0.018). CVB5 infection also tended to be associated with the appearance of IAA, however, this did not reach statistical significance (OR 2.3, [0.7, 7.5], p = 0.163); no other CVB types were associated with increased risk of IAA. Children who had signs of a CVB1 infection either alone or prior to infections by other CVBs were at the highest risk for developing IAA (OR 5.3 [95% CI 2.4, 11.7], p < 0.001). None of the CVBs were associated with the appearance of GADA. CONCLUSIONS/INTERPRETATION: CVB1 infections may contribute to the initiation of islet autoimmunity being particularly important in the insulin-driven autoimmune process.

Serum APOC1 levels are decreased in young autoantibody positive children who rapidly progress to type 1 diabetes.

Better understanding of the early events in the development of type 1 diabetes is needed to improve prediction and monitoring of the disease progression during the substantially heterogeneous presymptomatic period of the beta cell damaging process. To address this concern, we used mass spectrometry-based proteomics to analyse longitudinal pre-onset plasma sample series from children positive for multiple islet autoantibodies who had rapidly progressed to type 1 diabetes before 4 years of age (n = 10) and compared these with similar measurements from matched children who were either positive for a single autoantibody (n = 10) or autoantibody negative (n = 10). Following statistical analysis of the longitudinal data, targeted serum proteomics was used to verify 11 proteins putatively associated with the disease development in a similar yet independent and larger cohort of children who progressed to the disease within 5 years of age (n = 31) and matched autoantibody negative children (n = 31). These data reiterated extensive age-related trends for protein levels in young children. Further, these analyses demonstrated that the serum levels of two peptides unique for apolipoprotein C1 (APOC1) were decreased after the appearance of the first islet autoantibody and remained relatively less abundant in children who progressed to type 1 diabetes, in comparison to autoantibody negative children.

Permutation-based significance analysis reduces the type 1 error rate in bisulphite sequencing data analysis of human umbilical cord blood samples.

DNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2% of human CpG sites. To detect such associations outside these regions, we chose the bisulphite sequencing approach. We collected and curated clinical data on 200 newborn infants; whose umbilical cord blood samples were analysed with the reduced representation bisulphite sequencing (RRBS) method. A generalized linear mixed-effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables, such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis. We discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. The inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.

Serum 25-Hydroxyvitamin D Concentrations at Birth in Children Screened for HLA-DQB1 Conferred Risk for Type 1 Diabetes.

CONTEXT: Vitamin D has several effects on the immune system that might be of relevance for the pathogenesis of type 1 diabetes (T1D). OBJECTIVE: To evaluate whether umbilical cord serum concentrations of 25-hydroxy-vitamin D (25[OH]D) differ in children developing either islet autoimmunity (IA) or overt T1D during childhood and adolescence. DESIGN: Umbilical cord serum samples from 764 children born from 1994 to 2004 with HLA-DQB1 conferred risk for T1D participating in the Type 1 Diabetes Prediction and Prevention Study were analyzed for 25(OH)D using an enzyme immunoassay. SETTING: DIPP clinics in Turku, Oulu, and Tampere University Hospitals, Finland. PARTICIPANTS: Two hundred fifty children who developed T1D diabetes at a median age of 6.7 years (interquartile range [IQR] 4.0 to 10.1 years) and 132 additional case children who developed IA, i.e., positivity for multiple islet autoantibodies. Cases were matched for date of birth, gender, and area of birth with 382 control children who remained autoantibody negative. The median duration of follow up was 9.8 years (IQR 5.7 to 13.1 years). MAIN OUTCOME MEASURE: The median 25(OH)D concentrations. RESULTS: The median 25(OH)D concentration in cord serum was low [31.1 nmol/L (IQR 24.0 to 41.8); 88% <50 nmol/L], but not statistically different between children who developed T1D or IA and their control groups (P = 0.70). The levels were associated mainly with geographical location, year and month of birth, age of the mother, and maternal intake of vitamin D during pregnancy. CONCLUSIONS: The 25(OH)D concentrations at birth are not associated with the development of T1D during childhood.

Reduced ß-cell function in early preclinical type 1 diabetes.

OBJECTIVE: We aimed to characterize insulin responses to i.v. glucose during the preclinical period of type 1 diabetes starting from the emergence of islet autoimmunity. DESIGN AND METHODS: A large population-based cohort of children with HLA-conferred susceptibility to type 1 diabetes was observed from birth. During regular follow-up visits islet autoantibodies were analysed. We compared markers of glucose metabolism in sequential intravenous glucose tolerance tests between 210 children who were positive for multiple (≥2) islet autoantibodies and progressed to type 1 diabetes (progressors) and 192 children testing positive for classical islet-cell antibodies only and remained healthy (non-progressors). RESULTS: In the progressors, the first phase insulin response (FPIR) was decreased as early as 4-6 years before the diagnosis when compared to the non-progressors (P=0.001). The difference in FPIR between the progressors and non-progressors was significant (P<0.001) in all age groups, increasing with age (at 2 years: difference 50% (95% CI 28-75%) and at 10 years: difference 172% (95% CI 128-224%)). The area under the 10-min insulin curve showed a similar difference between the groups (P<0.001; at 2 years: difference 36% (95% CI 17-58%) and at 10 years: difference 186% (95% CI 143-237%)). Insulin sensitivity did not differ between the groups. CONCLUSIONS: FPIR is decreased several years before the diagnosis of type 1 diabetes, implying an intrinsic defect in ß-cell mass and/or function.

Imbalance of plasma amino acids, metabolites and lipids in patients with lysinuric protein intolerance (LPI).

BACKGROUND: Lysinuric protein intolerance (LPI [MIM 222700]) is an aminoaciduria with defective transport of cationic amino acids in epithelial cells in the small intestine and proximal kidney tubules due to mutations in the SLC7A7 gene. LPI is characterized by protein malnutrition, failure to thrive and hyperammonemia. Many patients also suffer from combined hyperlipidemia and chronic kidney disease (CKD) with an unknown etiology. METHODS: Here, we studied the plasma metabolomes of the Finnish LPI patients (n=26) and healthy control individuals (n=19) using a targeted platform for analysis of amino acids as well as two analytical platforms with comprehensive coverage of molecular lipids and polar metabolites. RESULTS: Our results demonstrated that LPI patients have a dichotomy of amino acid profiles, with both decreased essential and increased non-essential amino acids. Altered levels of metabolites participating in pathways such as sugar, energy, amino acid and lipid metabolism were observed. Furthermore, of these metabolites, myo-inositol, threonic acid, 2,5-furandicarboxylic acid, galactaric acid, 4-hydroxyphenylacetic acid, indole-3-acetic acid and beta-aminoisobutyric acid associated significantly (P<0.001) with the CKD status. Lipid analysis showed reduced levels of phosphatidylcholines and elevated levels of triacylglycerols, of which long-chain triacylglycerols associated (P<0.01) with CKD. CONCLUSIONS: This study revealed an amino acid imbalance affecting the basic cellular metabolism, disturbances in plasma lipid composition suggesting hepatic steatosis and fibrosis and novel metabolites correlating with CKD in LPI. In addition, the CKD-associated metabolite profile along with increased nitrite plasma levels suggests that LPI may be characterized by increased oxidative stress and apoptosis, altered microbial metabolism in the intestine and uremic toxicity.

Natural Development of Antibodies against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis Protein Antigens during the First 13 Years of Life.

Conserved protein antigens have been investigated as vaccine candidates against respiratory pathogens. We evaluated the natural development of antibodies against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis proteins during childhood. Serum samples were collected from 50 healthy children from their first months to age 13 years (median sampling interval, 6 months). We also analyzed serum samples from 24 adults. Serum IgG antibodies against eight pneumococcal proteins (Ply, CbpA, PspA 1 and 2, PcpA, PhtD, StkP-C, and PcsB-N), three H. influenzae proteins, and five M. catarrhalis proteins were measured using a multiplexed bead-based immunoassay. Antibody levels were analyzed using multilevel mixed-effects regression and Spearman's correlation. Antibody levels against pneumococcal proteins peaked at 3 to 5 years of age and then reached a plateau. Antibody levels against H. influenzae proteins peaked during the second year and then stabilized. Antibody levels against M. catarrhalis proteins peaked during the first year and then slowly decreased. Peak antibody levels during childhood were higher than those of adults. Correlations among pneumococcal antibody levels were highest among anti-CbpA, anti-PcpA, and anti-PhtD antibodies (r = 0.71 to 0.75; P < 0.001). The children presented 854 symptomatic respiratory infections on 586 occasions. Symptomatic respiratory infections did not improve prediction of antibody levels in the regression model. The maturation of immune responses against the investigated pneumococcal proteins shares similarities, especially among CbpA, PcpA, and PhtD. Antibody production against H. influenzae and M. catarrhalis proteins starts early in life and reaches peak levels earlier than antibody production against the pneumococcal proteins. Basal antibody levels are not related to the occurrence of symptomatic respiratory infections.

Dysfunction in macrophage toll-like receptor signaling caused by an inborn error of cationic amino acid transport.

Amino acids, especially arginine, are vital for the well-being and activity of immune cells, and disruption of amino acid balance may weaken immunity and predispose to infectious and autoimmune diseases. We present here a model of an inborn aminoaciduria, lysinuric protein intolerance (LPI), in which a single mutation in y(+)LAT1 cationic amino acid transporter gene SLC7A7 leads to a multisystem disease characterized by immunological complications, life-threatening pulmonary alveolar proteinosis and nephropathy. Macrophages are suggested to play a central role in LPI in the development of these severe secondary symptoms. We thus studied the effect of the Finnish y(+)LAT1 mutation on monocyte-derived macrophages where toll-like receptors (TLRs) act as the key molecules in innate immune response against external pathogens. The function of LPI patient and control macrophage TLR signaling was examined by stimulating the TLR2/1, TLR4 and TLR9 pathways with their associated pathogen-associated molecular patterns. Downregulation in expression of TLR9, IRF7, IRF3 and IFNB1 and in secretion of IFN-α was detected, suggesting an impaired response to TLR9 stimulation. In addition, secretion of TNF-α, IL-12 and IL-1RA by TLR2/1 stimulation and IL-12 and IL-1RA by TLR4 stimulation was increased in the LPI patients. LPI macrophages secreted significantly less nitric oxide than control macrophages, whereas plasma concentrations of inflammatory chemokines CXCL8, CXCL9 and CXCL10 were elevated in the LPI patients. In conclusion, our results strengthen the relevance of macrophages in the pathogenesis of LPI and, furthermore, suggest that cationic amino acid transport plays an important role in the regulation of innate immune responses.

Serum proteomes distinguish children developing type 1 diabetes in a cohort with HLA-conferred susceptibility.

We determined longitudinal serum proteomics profiles from children with HLA-conferred diabetes susceptibility to identify changes that could be detected before seroconversion and positivity for disease-associated autoantibodies. Comparisons were made between children who seroconverted and progressed to type 1 diabetes (progressors) and those who remained autoantibody negative, matched by age, sex, sample periodicity, and risk group. The samples represented the prediabetic period and ranged from the age of 3 months to 12 years. After immunoaffinity depletion of the most abundant serum proteins, isobaric tags for relative and absolute quantification were used for sample labeling. Quantitative proteomic profiles were then measured for 13 case-control pairs by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, a label-free LC-MS/MS approach was used to analyze depleted sera from six case-control pairs. Importantly, differences in abundance of a set of proteins were consistently detected before the appearance of autoantibodies in the progressors. Based on top-scoring pairs analysis, classification of such progressors was observed with a high success rate. Overall, the data provide a reference of temporal changes in the serum proteome in healthy children and children progressing to type 1 diabetes, including new protein candidates, the levels of which change before clinical diagnosis.

Antibodies to Lactobacilli and Bifidobacteria in young children with different propensity to develop islet autoimmunity.

The intestinal microbiota is essential to the maturation and homeostasis of the immune system. Immunoblot assays were used to establish the prevalence of serum IgG, IgM, and IgA antibodies specific for Bifidobacterium adolescentis, Bifidobacterium longum, and Lactobacillus rhamnosus GG proteins in young children presenting with or without type 1 diabetes (T1D). We demonstrated that children between the ages of 6 and 12 months had a substantial increase in the frequency of IgG antibodies specific for L. rhamnosus GG proteins. We measured IgG, IgM, and IgA class antibody reactivity against B. adolescentis DSM 20083, B. adolescentis DSM 20086, and B. longum DSM 20088 proteins demonstrating significantly higher IgA responses against B. adolescentis DSM 20083 strain proteins in children who developed islet autoimmunity and T1D later in life. B. adolescentis strains showed more IgM type antibodies in children who developed T1D later in life, but the difference was not statistically significant. B. longum proteins were recognized by IgG and IgA antibodies to a higher extent compared to other bacteria studied. These results confirm that differences in immune reactivity against some commensal strains in young children may represent a different risk factor for developing T1D.

Cofilin regulator 14-3-3zeta is an evolutionarily conserved protein required for phagocytosis and microbial resistance.

Phagocytosis is an ancient cellular process that plays an important role in host defense. In Drosophila melanogaster phagocytic, macrophage-like hemocytes recognize and ingest microbes. We performed an RNAi-based in vitro screen in the Drosophila hemocyte cell line S2 and identified Abi, cpa, cofilin regulator 14-3-3ζ, tlk, CG2765, and CG15609 as mediators of bacterial phagocytosis. Of these identified genes, 14-3-3ζ had an evolutionarily conserved role in phagocytosis: bacterial phagocytosis was compromised when 14-3-3ζ was targeted with RNAi in primary Drosophila hemocytes and when the orthologous genes Ywhab and Ywhaz were silenced in zebrafish and mouse RAW 264.7 cells, respectively. In Drosophila and zebrafish infection models, 14-3-3ζ was required for resistance against Staphylococcus aureus. We conclude that 14-3-3ζ is essential for phagocytosis and microbial resistance in insects and vertebrates.