Study Population

Following informed and written consent broncho-alveolar lavage (BAL) was performed during a fibreoptic bronchoscopy with sterile 0.9% (w/v) saline solution. Bronchoscopy was performed for medical reasons only, but the subjects included gave their consent to include BAL fluid (BALF) for study purpose. The BALF retrieved (without blood stain) were prepared as previously described.11,12 Healthy lungs were confirmed among all 13 control subjects (4 ex-smokers and 9 never-smokers) by a negative history of respiratory discomfort and a negative lung examination and were further confirmed by negative findings on bronchoscopy (including cultures), chest X-ray, and lung function tests. The 15 patients included with CAL (7 ex-smokers and 8 never-smokers) were 5 subjects with sarcoidosis, 4 subjects with COPD, 3 subjects with asthma and 3 subjects with fibrosis due to either cryptogenic organizing pneumonia (COP), rheumatoid arthritis (RA) or idiopathic pulmonary fibrosis (IPF). All these subjects fulfilled the criteria for CAL on dynamic spirometry post-dilatation, that is an FEV₁/FVC ratio <0.7, and demonstrated stable disease without clinical signs of ongoing exacerbation or infection. In addition, cultures on BALF were negative. A cell count of each BALF was also performed. Lung volumes post-dilatation (>15 min after inhalation of 0.6 mg salbutamol) was measured with a spirometer using Hedenström as a reference. Smoking habits were recorded as exposure to tobacco smoke oxidatively stress cells and lysosomes. Use of medications and supplements, including those with lysosomotropic properties,17 was also recorded. Particularly, attention was paid to ongoing use of proton-pump inhibitors (PPIs), since these drugs may theoretically interfere on LMP in this experimental model.