Sputum collection.
cough
STERILE, CAVITY
The healthy volunteers were asked to fast for 4 to 6 h and clean their oral cavity, including brushing teeth and gargling with 10 mL 0.9% saline for 60 s at our laboratory immediately before the sputum induction. The oral wash samples were collected. The sterile saline was used as an oral wash control. Then, they were subjected to inhalation of 4.5% sterile hypertonic saline nebulized from an inhalation delivery (PARI, Germany) for 10 min and endeavored to cough throughout the process. Most subjects could expectorate an adequate sample (sputum of ≥1 mL with as little saliva as possible) after the first inhalation. For those who failed to meet the requirements for sputum, we extended the duration of aerosol inhalation cumulatively up to 60 min with a pause every 10 min and allowed them to cough for the second time later. Sputum was collected in sterile containers and weighed, and 2 mL of 0.1% DTT (Sigma-Aldrich, Poole, UK) in phosphate-buffered saline (PBS) was added for liquefaction. The 0.1% DTT-PBS in sputum collection cups and sterile saline in the atomization device served as negative-control samples. After incubation at room temperature for 15 min on a rolling mixer, all the samples were stored in sterile tubes at −80°C immediately until processing. The sputum samples were collected at the day before drug administration (D0), the day after the treatment course was completed (D4), and 14, 30, and 60 days postdosing. We asked the participants to maintain a consistent time for each sample collection day.