Primers for IL-17, IL-1β, IL-6, IL-23, TGF-β1 and β-actin were designed according to the sequences published in GenBank, and the primers’ sequences are shown below: IL-17 forward 5′-AATTCTGAGGACAAGAACTTCCC-3′ and IL-17 reverse 5′-ATAGTCTAACTGCTTTGGGGAGTG-3′; IL-1β forward 5′-GCTGATGGCC CTAAACAGATGAA-3′ and IL-1β reverse 5′-TGAAGCCCTTGCTGTAGTGGTG-3′; IL-6 forward 5′ -AATTCGGTACATCCTCGA-3′ and IL-6 reverse 5′ -AACAAC AATCTGAGGTGCCC-3′; TGF-β1 forward 5′-AGCGACTCGCCAGAGTGGT TA-3′ and TGF-β1 reverse 5′-GCAGTGTGTTATCCCTGCTGTCA-3′; IL-23 forward 5′-GCAGCCTGAGGGTCACCACT-3′

and IL-23 reverse 5′-GGCGGCTACAGCC ACAAA-3′; and β-actin SCH727965 clinical trial forward 5′-CTGTCCACCTTCCAGCAGATGT-3′ and β-actin reverse 5′-CGCAACTAAGTCATAGTCCGCC-3′. IL-17, IL-1β, IL-6, IL-23 and TGF-β1 levels were normalized by the levels of β-actin in an individual sample and were analysed by using the 2-standard curve method. Cytokine assays.  By using commercially available ELISA kits, serum levels of IL-1β, IL-6, IL-23, IL-17A and TGF-β1 were measured according buy Obeticholic Acid to the protocols provided by the manufacturer (eBioscience, San Diego,

CA, USA), and all samples were assessed in triplicate. Flow cytometry.  The PBMCs were isolated from peripheral blood of the study subjects. Cells were stimulated for 5 h with 50 ng/ml PMA, 1 μg/ml ionomycin (Sigma, StLouis, MO, USA) and 2 μm monensin (Enzo, Plymouth, PA, USA). Upon harvest, cells were first surface-stained with fluorescein isothiocyanate–conjugated anti-human CD4 antibodies for 15 min, then fixed and permeabilized with Perm/Fix solution Methane monooxygenase and finally stained intracellularly with phycoerythrin (PE)-conjugated anti-human IL-17A antibodies or PE-conjugated anti-human FoxP3, respectively. Isotope controls were used to ensure antibody specificity. All antibodies were from eBioscience (San Diego). Data were acquired and analysed with FACSCalibur flow cytometer and cellquest software (BD Biosciences, San Jose, CA, USA). AChR antibodies assay.  The concentration of anti-AChR antibodies

was detected by enzyme-linked immunosorbent assay by using a human-AChR-Abs ELISA Kit (R&D, Minneapolis, MN, USA) according to the manufacturer’s protocol. Optical density (OD) values were obtained at 450 nm. The assay range is 20–500 pmol/l, and the concentration value above 20 was considered positive. Statistical analysis.  Statistical analysis was performed by using spss version 19.0 for Windows software (SPSS Inc., Chicago, IL, USA). The data were first analysed by one-way anova. The post hoc analyses were carried out by using a Bonferroni/Dunn multiple-comparison tests. The relationships between any two indices were analysed with Pearson’s correlation coefficient test. Any P values <0.05 were considered to be statistically significant.