*2.3. Bronchofiberoscopy and Airway Sample Collection*

Bronchofiberoscopy was also carried out in asthma patients only, according to the guidelines of the American Thoracic Society [24], using the bronchofiberoscope BF 1T180 (Olympus, Shinjuku, Japan) with local anesthesia (2% lidocaine) and with mild sedation (2.5–5 mg of midazolam, 0.05–0.1 mg of fentanyl i.v.). Bronchoalveolar lavage (BAL) was performed with 200 mL of 0.9% saline administered to the right middle lobe bronchus, and 2 or 3 endobronchial biopsies were taken from the right lower lobe (the carina between B9 and B10) during the procedure. The differential of BAL fluid cells was analyzed by using May–Grunwald–Giemsa-stained cytospin preparations (Thermo Scientific, Walthman, MA, USA; 1000 cells counted). The results were shown as a percentage of all inflammatory cells (except for epithelial cells). The BAL fluid supernatant was aliquoted and stored at −70 ◦C until analyzed.

Endobronchial biopsy specimens were formalin-fixed (Sigma-Aldrich, Saint Luis, MO, USA) and prepared for histological examination (e.g., hematoxylin-and-eosin staining) as previously described [23]. The microscope slides were photographed with a Nikon D5300 camera attached to the Zeiss Axioscope microscope with a 100× oil immersion lens. Images were analyzed by AnalySIS 3.2 software (Soft Imaging System GmbH, Muenster, Germany). The RBM thickness was measured along the airway epithelium layer, according to the orthogonal intercept method suggested by Ferrando et al. [25], using arbitrary distance units. For each patient, at least 30 individual RBM measurements were evaluated at intervals of 9.5 μm. The results were expressed as a harmonic mean, as defined in our previous publication [23].
