Coronary artery lesion (CAL) was defined by internal diameter of artery >3.0 mm (<5 years); >4.0 mm (≥5 years) or coronary artery aneurysms. Patients with KD were divided into the KD-CAL+ group (n = 16) and the KD-CAL− group (n = 30) according to the echocardiographic examination results (Tables 1 and 2). Thirty age-matched healthy children (NC) (16 males and 14 females; mean age: 24.0 ± 16.4 months; age range: 1.1–4.3 years) were enrolled GSK1120212 concentration into this study. Informed

consent was obtained from their parents, and the study was approved by the medical ethics hospital committee. Venous blood (5 ml) was taken from patients with KD and normal controls using ethylene diaminetetraacetic acid (EDTA) Na2 as anti-coagulant. Blood samples were analysed immediately without stimulation of mitogens or culture in vitro unless particularly indicated. The whole blood (2 ml) was prepared for flow cytometric analysis. According to the manufacturer’s instructions, CD14+T cells were immediately isolated from peripheral blood by microbead (Dynal 111.49D, US). Plasma was obtained after centrifugation and stored at −80 °C see more for measurement of the enzyme-linked immunosorbent assay (ELISA). Purified

cells were identified as >97% with FCM, while results of cell activity were >95% by 0.05% trypan blue staining. The antibodies CD3-FITC, CD8-PC5, CD56-PC5, CD14-PC5, NKG2A-PE and mouse IgG1-PE were obtained from Beckman Coulter, Inc. (Miami, FL, USA). NKG2D-PE, MICA-PE and ULBP-1-PE were purchased from eBioscience. (San Diego, CA, USA).Whole blood (100 μl) was incubated with relevant

antibodies for 30 min at 4 °C. After incubation, red blood cells were lysed using Red Blood Cell Lysis Buffer,, and the remaining white blood cells were washed twice with phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) and 0.1% NaN3 (hereafter, PBS–0.2% BSA–0.1% NaN3). Immediately afterwards, expression of cell surface markers was analysed by flow cytometric analysis using an Epics-XL4 cytometer equipped with expo32 adc software (Beckman Coulter, San Diego, CA, USA). Data are presented as proportions of cells expressing antigen (%) and/or the relative levels of antigen Protein kinase N1 levels assessed by the median fluorescence intensity (MFI). Total RNA from CD14+ mononuclear cells (MC) was prepared using Versagene RNA Kit (Gentra 0050C, US) according to the manufacture’s instruction. DNase I (0050D; Gentra) was used to eliminate the trace DNA during extraction. Isolated total RNA integrity was verified by an average optical density (OD) OD260/OD286 absorption to cDNA with oligodeoxythymidylic acid (oligo-dT) primer, using RevertAid™ H Minus Moloney murine leukaemia virus (MMLV) reverse transcriptase (K1632#; Fermentas, Vilnius, Lithuania). Negative control samples (no first-strand synthesis) were prepared by performing reverse transcription reaction in the absence of reverse transcriptase.