Isolated

immune cells were incubated with primary VX-770 cell line antibodies (fluorescence labelled, 1 µg/ml; isotype IgG was used as control) on ice for 30 min (for the intracellular staining, cells were fixed with 1% paraformaldehyde on ice for 30 min and incubated with permealization reagents for 30 min on ice). The stained cells were analysed using a fluorescence activated cell sorter (FACSarray; BD Bioscience, San Jose, CA, USA). Data were analysed with FlowJo software. Nasal mucosal cryosections were fixed with acetone for 20 min. After blocking with 2% bovine serum albumin for 30 min, the sections were incubated with primary antibodies (1 µg/ml, or isotype IgG as control) at 4°C overnight. Sections were incubated learn more with horseradish peroxidase-labelled secondary antibodies (1:300) for 1 h at room temperature. Washing with phosphate-buffered saline (PBS)

was performed after incubation. Sections were observed under a microscope. Surgically removed nasal tissue was cut into small pieces (2 × 2 × 2 mm) and treated with predigestion solution [1 × Hanks's balanced salt solution (HBSS) containing 5 mm ethylenediamine tetraacetic acid (EDTA) and 1 mm dithiothreitol (DTT)] at 37°C for 30 min under slow rotation. The tissue was collected by centrifugation (300 g for 10 min) and incubated in digestion solution (0·05 g of collagenase D, 0·05 g of DNase I and 0·3 g of dispase II in 100 ml of 1 × PBS) at 37°C for 60 min under slow rotation. Cells were filtered with a cell strainer. Isolation of CD4+ T cells was performed with commercial magnetic cell sorting kits. The purity of the isolated CD4+ T cells was more than selleck chemical 95%, as checked by flow cytometry.

Data are presented as the means ± standard deviation. Differences between two groups were evaluated with Student’s t-test; data among three or more groups were evaluated with analysis of variance (anova). Bonferroni adjustment was applied to post-hoc group comparisons when required. Two-variable correlation analysis was performed when necessary. A P < 0·05 was accepted as a significant criterion. Emerging evidence indicates that Treg functional deficiency or a decrease in Treg numbers plays a critical role in the pathogenesis of allergic disorders [15,16]. However, an increase in Treg numbers in allergic patients has also been reported [17]. Considering that the difference might result from allergic patients complicating with other disorders, 40 AR patients with or without NP (20 AR/NP, 20 AR; male 20, female 20; age: 22–58 years) were recruited into this study. Ten patients with chronic non-allergic rhinitis (CR) were recruited as a control group. All the AR patients showed a positive response to the challenge with mite antigen Der p1 (Der, in brief) and high serum Der-specific IgE levels (Fig. S1). These 50 patients also had inferior turbinate hyperplasia that did not respond well to conventional medical treatment; turbinatectomy was performed for these 50 patients.