The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays.

Vitamin D exerts multiple immunomodulatory functions and has been implicated in the etiology and treatment of several autoimmune diseases, including multiple sclerosis (MS). We have previously reported that in juvenile/adolescent rats, vitamin D supplementation protects from experimental autoimmune encephalomyelitis (EAE), a model of MS. Here we demonstrate that this protective effect associates with decreased proliferation of CD4+ T cells and lower frequency of pathogenic T helper (Th) 17 cells. Using transcriptome, methylome, and pathway analyses in CD4+ T cells, we show that vitamin D affects multiple signaling and metabolic pathways critical for T-cell activation and differentiation into Th1 and Th17 subsets in vivo. Namely, Jak/Stat, Erk/Mapk, and Pi3K/Akt/mTor signaling pathway genes were down-regulated upon vitamin D supplementation. The protective effect associated with epigenetic mechanisms, such as (i) changed levels of enzymes involved in establishment and maintenance of epigenetic marks, i.e., DNA methylation and histone modifications; (ii) genome-wide reduction of DNA methylation, and (iii) up-regulation of noncoding RNAs, including microRNAs, with concomitant down-regulation of their protein-coding target RNAs involved in T-cell activation and differentiation. We further demonstrate that treatment of myelin-specific T cells with vitamin D reduces frequency of Th1 and Th17 cells, down-regulates genes in key signaling pathways and epigenetic machinery, and impairs their ability to transfer EAE. Finally, orthologs of nearly 50% of candidate MS risk genes and 40% of signature genes of myelin-reactive T cells in MS changed their expression in vivo in EAE upon supplementation, supporting the hypothesis that vitamin D may modulate risk for developing MS. Vitamin D deficiency is one of the most consistently reported environmental factor in the etiology of multiple sclerosis (MS) (5), a chronic inflammatory disease of the CNS characterized by autoimmune destruction of myelin, axonal loss, and brain atrophy (6). Increased risk of developing MS has been described in carriers of rare and common variants of the CYP27B gene (7, 8), which encodes the enzyme that catalyzes the last step in converting vitamin D to its active form, from 25(OH)D 3 to 1,25 (OH) 2 D 3 . These studies imply a causal role of low vitamin D in MS, which has recently been further supported by Mendelian randomization studies in two large cohorts demonstrating that three genetic variants that associate with serum 25(OH)D 3 levels also associate with the risk of developing MS (9). However, high levels of vitamin D have been associated not only with the reduced risk of developing MS (10, 11) but also with the reduced risk for relapses, new brain lesions, and subsequent disability (12, 13). Moreover, it has been described that increased levels of vitamin D can reduce serum levels of . Most of what is known about the immunological mechanisms of vitamin D in MS comes from the studies in its animal model, experimental autoimmune encephalomyelitis (...

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BackgroundExperimental and natural human adenovirus-36 (Adv36) infection of multiple animal species results in obesity through increasing adipogenesis and lipid accumulation in adipocytes. Presence of Adv36 antibodies detected by serum neutralization assay has previously been associated with obesity in children and adults living in the USA, South Korea and Italy, whereas no association with adult obesity was detected in Belgium/the Netherlands nor among USA military personnel. Adv36 infection has also been shown to reduce blood lipid levels, increase glucose uptake by adipose tissue and skeletal muscle biopsies, and to associate with improved glycemic control in non-diabetic individuals.Principal FindingsUsing a novel ELISA, 1946 clinically well-characterized individuals including 424 children and 1522 non-diabetic adults, and 89 anonymous blood donors, residing in central Sweden representing the population in Stockholm area, were studied for the presence of antibodies against Adv36 in serum. The prevalence of Adv36 positivity in lean individuals increased from ∼7% in 1992–1998 to 15–20% in 2002–2009, which paralleled the increase in obesity prevalence. We found that Adv36-positive serology was associated with pediatric obesity and with severe obesity in females compared to lean and overweight/mildly obese individuals, with a 1.5 to 2-fold Adv36 positivity increase in cases. Moreover, Adv36 positivity was less common among females and males on antilipid pharmacological treatment or with high blood triglyceride level. Insulin sensitivity, measured as lower HOMA-IR, showed a higher point estimate in Adv36-positive obese females and males, although it was not statistically significant (p = 0.08).ConclusionUsing a novel ELISA we show that Adv36 infection is associated with pediatric obesity, severe obesity in adult females and lower risk of high blood lipid levels in non-diabetic Swedish individuals.