Cubic regression-based degree of correction predicts the performance of whole bisulfitome amplified DNA methylation analysis.

Epigenetic mechanisms, including DNA methylation, are important determinants in development and disease. There is a need for technologies capable of detecting small variations in methylation levels in an accurate and reproducible manner, even if only limited amounts of DNA are available (which is the case in many studies in humans). Quantitative methylation analysis of minute DNA amounts after whole bisulfitome amplification (qMAMBA) has been proposed as an alternative, but this technique has not been adequately standardized and no comparative study against conventional methods has been performed, that includes a wide range of methylation percentages and different target assays. We designed an experiment to compare the performance of qMAMBA and bisulfite-treated genomic (non-amplified) DNA pyrosequencing. Reactions were performed in duplicate for each technique in eight different target genes, using nine artificially constructed DNA samples with methylation levels ranging between 0% and 100% with intervals of 12.5%. Cubic polynomial curves were plotted from the experimental results and the real methylation values and the resulting equation was used to estimate new corrected data points. The use of the cubic regression-based correction benefits the accuracy and the power of discrimination in methylation studies. Additionally, dispersion of the new estimated data around a y = x line (R ( 2) ) served to fix a cutoff that can discriminate, with a single 9-point curve experiment, whether whole bisulfitome amplification and subsequent qMAMBA can produce accurate methylation results. Finally, even with an optimized reagent kit, DNA samples subjected to whole bisulfitome amplification enhance the preferential amplification of unmethylated alleles, and subtle changes in methylation levels cannot be detected confidently.

TH17 (and TH1) signatures of intestinal biopsies of CD patients in response to gliadin.

Celiac disease (CD) is an immunological disorder caused by intolerance to ingested gliadin and other cereal prolamins that has been included in the T(H)1-dominated group of diseases, where IL-12 induced IFNgamma is the major proinflamatory signal. Recently, another linage of T cells has been described, namely T(H)17, characterized by production of IL-17, that differentiate in response to TGFbeta and IL-6 and participate in the pathogenesis of several autoimmune diseases. Using RT-PCR analysis of gene expression, we analyzed the presence of T(H)1 (IL-12 and IFNgamma) and T(H)17 (TGFbeta, IL-6, IL-17A, IL-17F and IL-23) related cytokines in intestinal biopsies from CD patients with active disease compared to remission and from treated patients after acute, in vitro re-exposure to gliadin. Potent T(H)1 and T(H)17 responses were present in the active stage of the disease, whereas short incubation of normalized biopsies with gliadin did not increase the expression of the effector cytokines, although a tendency of upregulation for both T(H)1 and T(H)17 promoting factors was observed, suggestive of a reactivation of proinflammatory pathways. These results place CD into the group of autoimmune disorders in which T(H)17 cells also participate, although the relative importance of each T cell response and their role in the initial events of the disease need further investigation.