Chronic airway diseases, like asthma or COPD, are characterized by excessive acetylcholine release and airway remodeling. The aim of this study was to investigate the long-term effect of muscarinic agonists on the phenotype and proliferation of rabbit tracheal airway smooth muscle cells (ASMCs).

Mechanically ventilated (MV) patients may present airway inflammation and elevated secretion production. However, it is unknown whether cell and/or protein counts in bronchial samples may be useful to evaluate their clinical condition. Our aim was to standardize sampling and propose a new mechanical mucus dissolution in Tracheal-Bronchial secretions. In all patients, bronchial lining fluid aspiration (BLF), Bronchoalveolar lavage (BAL) and Bronchial Washings (BW40, BW5) were performed, while visible bronchial secretions were obtained via bronchoscopy (VBS) and blinded, via a common catheter for tracheobronchial aspiration (AC). Mucus was mechanically or DTT dissolved and cell number was count. Protein, albumin and TNF-α levels were measured, in mucus dissolved samples from control and MV patients. Cell number and protein levels were elevated in mucus dissolved compared to non-dissolved, or DTT dissolved. Cell number and TNF-α levels were elevated in MV patients compared to controls, while protein levels were lower in MV patients. Differences in cell and protein levels were observed in samples acquired using different sampling technics. Therefore, mechanical mucus dissolution provides a proper sample for evaluation, and the sampling technic used can influence the sample’s characteristics.

Small extracellular vesicles (sEVs) released by cells during lung injury, act as cell-to-cell messengers involved in inflammation. Blood circulating sEVs are candidates as diagnostic biomarkers and therapeutic target in mechanically ventilated(MV) patients. However, their cell source heterogeneity is a significant challenge. This study aimed to develop a new protocol to isolate sEVs from bronchial secretions obtained by bronchoscopy or tracheal tube blind aspiration in MV patients. We have investigated the relevance of variables as post-intubation sampling time(day-0,day-7) and sampled lung region(upper, lower). Samples from diagnostic bronchoscopies of non-intubated patients were used as controls. 2ml of bronchial liquid, collected from patients(approval 55366/23-12-22) were exported to the Imperial College Tissue Bank (HTA-12275 R23038)for sEV analyses. sEVs were isolated by ultrafiltration and size exclusion chromatography, using Exospin-EXO350 columns. sEV concentration and size distribution was evaluated by nanoparticle tracking analysis (NTA) using Nanosight-NS3000. sEV size distribution was similar in all samples (p=ns). Regardless of sample region sEVs increased at day-0 (3285*10⁶±299particles/ml, p<0.05)in MV patients, compared to controls (1344*10⁶±245particles/ml). More specifically, sEVs increased in the aspiration samples (4392*10⁶±337particles/ml, p<0.01), or samples from upper lung (3942*10⁶±539particles /ml, p<0.05) of MV patients compared to controls (1344*10⁶±245particles/ml). In summary sEVs increase in the MV bronchial secretions. Further studies should investigate the sEV diagnostic and prognostic potential at admission in Intensive Care Units.