Immunostained cells were analysed using a fluorescence activated cell sorter [(FACS)Calibur, Becton Dickinson, San Jose, CA, USA]. Analysis of the Th17 cell population was GPCR Compound Library purchase performed

by gating on CD3+CD8– T cells. Total RNA was extracted from PBMCs or TMCs using TRIzol reagent (Invitrogen). Total RNA was isolated and reverse transcription was performed according to the manufacturer’s instructions (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed by triplicate using Bio-Rad SYBR green super mix (Bio-Rad, Hercules, CA, USA). Primer sequences were as follows: retinoic acid-related orphan receptor γt (RORγt), sense, 5′-CCTGGGCTCCTCGCCTGACC-3′, anti-sense, 5′-TCTCTCTGCCCTCAGCCTTGCC-3′; and β-actin, sense, 5′-CACGAAACTACCTTCAACTCC-3′, anti-sense, 5′-CATACTCCTGCTTGCTGATC-3′. Samples were run in triplicate, and their relative expression was determined by normalizing to the expression level of β-actin. Data were analysed using Bio-Rad CFX Manager software. In the case of TMCs, leptin, IL-17 and RORγt cDNA products were amplified by PCR with

the following primer sequences: leptin, sense, 5′-TCCTGGGCTCCACCCCATCC-3′, anti-sense, 5′-TGCAGAGACCCCTGCAGCCT-3′; and IL-17, sense, 5′-CAAGACTGAACACCGACTAAG-3′, anti-sense, 5′-TCTCCAAAGGAAGCCTGA-3′. Amplified products were electrophoresed on 2% agarose gel (Invitrogen), stained with ethidium bromide and visualized with ultraviolet transilluminator. One-way analysis of variance (anova) was performed to determine whether there was an overall statistically significant change among the groups, and the post-test comparison was carried out using Bonferroni’s test. Student’s Ulixertinib mouse 2-hydroxyphytanoyl-CoA lyase unpaired t-test was performed as appropriate. Correlations between variables were determined by Spearman’s correlation coefficient. Data were analysed with GrapPad Prism version 5 software. We first compared the basal plasma leptin levels of 27 female HT patients with 22 age-, sex- and BMI-matched female healthy controls. It was found that HT patients showed an increase of leptin which was at the border of statistical significance (P = 0·06, Fig. 1a). Subsequently, we analysed the correlation

between the level of plasma leptin and BMI in HT patients and healthy controls. The results showed that plasma leptin correlated positively with BMI in healthy controls, but no correlation was observed in HT patients (Fig. 1b,c). Furthermore, the level of leptin in culture of CD4+ T cells from HT patients was higher than that from healthy controls (Fig. 1d). Flow cytometric analysis revealed that an increased proportion of Th17 cells from peripheral blood mononuclear cells (PBMCs) was observed in HT patients compared with healthy controls (Fig. 2a,b). There were no statistically significant correlations between plasma leptin concentrations and the percentage of Th17 cells or the level of RORγt in HT patients (Fig. 2c,d).