The RNA concentration and purity was measured by a spectrophotometer (ND-1000; NanoDrop Technologies Inc.). Reverse transcription was performed with TaqMan reverse transcription reagents (Applied Biosystems). Quantitative PCR was performed using StepOnePlus instrumentation (Applied Talazoparib price Biosystems) with TaqMan Fast Universal PCR Master Mix and predesigned FAM-labelled gene expression assay reagents (Applied Biosystems). Selected cytokines and transcription factors were IL-17A (cat. no. Hs00174383_m1), FoxP3 (Hs00203958_m1), RORc (cat. no. Hs01076112_m1) and IFN-γ (Hs00174143_m1). Ribosomal 18 s RNA served as the endogenous control (Hs99999901_s1). The quantities of target gene

expression were analysed by a comparative threshold cycle (Ct) method (as recommended by Applied Biosystems). An exogenous cDNA pool calibrator was collected from phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) and considered as an interassay standard to which normalized samples were compared. ΔCt stands for the difference between the Ct of the marker gene and Ct of the 18S gene, whereas ΔΔCt is the difference between the ΔCt of the sample and ΔCt of the calibrator. Calculation of 2−ΔΔCt then gives the relative amount of target gene in the sample compared with the calibrator, both normalized Hormones antagonist to

an endogenous control (18S). For presentations the relative amounts (2−ΔΔCt) of target genes were multiplied by a factor 1000 and expressed as relative units. If the samples Ct value for target gene did not reach quantitative MTMR9 level, then an artificial value that was half the lowest quantitative value in relative units was given to the sample. We cultured small intestinal biopsy samples from 23 patients with untreated CD (of which six also had T1D) and 10 reference children (five positive for TGA) for 72 h in RPMI-5% human AB serum and measured the concentration

of IL-17, Il-1β and IL-6 secreted in the culture supernatants by using flow-cytometric bead array (Bender Medsystems, Vienna, Austria). Samples below the detection limit (or the cut-off level) of the method were considered as undetectable, but were given half the cut-off value to enable statistical analyses. The human colon adenocarcinoma cell line (CaCo-2) was obtained from American Type Culture Collection (ATCC) (Teddington, UK). Cells were grown in Eagle’s minimal essential medium (Sigma) containing 10% heat-activated and sterile filtered fetal bovine serum (FBS) supplemented with penicillin (0·1 g/l) and streptomycin (0·15 g/l) at + 37°C and 5% CO2. CaCo-2 cells were grown in a 75 cm2 flask for 6 days and were thereafter plated into sterile 48-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and grown for 4 days at a density of 1·5 × 105 cells per well and a final volume of 0·5 ml/well. The cells were incubated for 5 h with recombinant human IL (rhIL)-17 (1 or 50 pg/ml; cat. no.