Multiplex assay

The concentrations of selected helper T-cell type 1 (Th1), Th2, and Th17 cytokines and chemokines from BALF supernatant were measured with commercially available multiplex assays (EMD Millipore; Billerica, MA). For cytokine/chemokine sample measurements below the lower detection limit, results were assigned a value equal to the minimal detection limit for the specific assay to facilitate statistical analysis of the data.

Venous blood was collected onto sterile plastic tubes (BD Vacutainer^® SST™ Serum Separation Tubes, Becton-Dickenson; Franklin Lakes, NJ, USA) and allowed to coagulate for 120 min at room temperature before centrifugation (300× g for 10 min) and serum collection. Serum was immediately frozen and stored at −80 °C until analyzed. Repeated freezing and thawing were avoided. The samples were analyzed with Bio-Plex kits for IL-6, IL-11, IL-27(p28), sIL-6R (sCD126), LIF and IL-31 (Bio-Rad, Hercules, CA, USA), and Multiplex Assays (Millipore, Billerica, MA, USA) for CNTF and OSM. All samples were analyzed using Luminex^®200™ Bio-Rad platform with program version 6.1 and all analyses were performed in duplicates strictly according to the manufacturer’s instructions. CRP was analyzed using an immunoturbidimetric method provided by Roche (Basel, Switzerland), and during the entire period the lower limit of detection for CRP was 1 mg/L.

The plasma concentrations of glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP) and peptide YY (PYY) were determined using multiplex assays (Millipore Corporation).

Blood samples were obtained and centrifuged immediately to prevent the degradation of complement components, and the plasma was stored at −80°C within 1 h of blood collection. We measured the plasma levels of cytokines and the culture supernatants. We conducted multiple the assay system using Multiplex Assays (Millipore, Billerica, MA) according to the manufacturers' protocols.