Tests, Pulmonary Function

Under a protocol approved by the Weill Cornell Medical College Institutional Review Board, healthy nonsmokers were recruited for this study. The subjects were confirmed to be nonsmokers by urine levels of nicotine (<2 ng/ml) and cotinine (<5 ng/ml) with normal pulmonary function tests and chest X-ray. Following written informed consent, flexible bronchoscopy was used to collect large airway epithelial cells by brushing the epithelium [47 (link)-49 (link)]. Basal cells (BC) were subsequently purified from the total airway epithelium brushings by trypsinization of the cells and selective culturing of BC on T25 cm² plastic tissue culture flasks as previously described [4 (link),50 (link)]. The airway epithelial cells collected by brushing were pelleted by centrifugation (250 × g, 5 min) and disaggregated by resuspension in 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) for 5 min, at 37°C. Trypsinization was stopped by addition of HEPES buffered saline, (Lonza, Basel, Switzerland) supplemented with 15% fetal bovine serum (FBS; GIBCO-Invitrogen, Carlsbad, CA), and the cells were again pelleted at 250 × g, 5 min. The pellet was resuspended with 5 ml of phosphate buffered saline, pH 7.4 (PBS), at 23°C, then centrifuged at 250 × g, 5 min. Following centrifugation, the PBS was removed and the cells resuspended in 5 ml of Bronchial Epithelial Growth Media (BEGM, Lonza, CA) and 5 × 10⁵ cells plated in T25 flasks in 5 ml of BEGM and maintained in a humidified atmosphere of 5% CO₂ at 37°C. The next day, unattached cells were removed by changing the medium and thereafter, every 2 days. Following 7–8 days of culture, when the cells were 70% confluent, they were characterized by immunohistochemical staining of tryspinized cytopreps using cell type specific markers as being >99% BC (KRT5⁺, TP63⁺, CD151⁺, β-tubulin IV^-, MUC5AC^-, TFF3^-, CC10^-, chromogranin A^- and N-cadherin^-) and when put on air-liquid interface (ALI) culture, were capable of differentiating into a mucociliary epithelium [4 (link)]. To passage the cells, the primary BC were seeded at a cell density of 3000 cells/cm² in BEGM. The following day, the media was replaced with fresh BEGM and thereafter, every 2 days.

The anthropometric characteristics were recorded as described by Stavrou et al. (16 (link)). Tanita MC-980 (Arlington Heights, IL, USA) was used for body composition assessment. We performed the 6MWT, as described in the ATS guidelines, to assess the functional status of the patients (17 (link)). The parameters O₂ saturation (SpO₂), heart rate (HR) (Nonin 9590 Onyx Vantage, USA), blood pressure (Mac, Tokyo, Japan), and self-assessed lower limb fatigue and dyspnea (via Borg Scale CR-10) (18 (link)) were recorded at predetermined time-points of the 6MWT (16 (link)). The handgrip strength test was performed using an electronic dynamometer (Camry EH 101, South El Monte, CA, USA) (19 (link)). Patients were asked to perform as many repetitions as possible at a self-regulated pace (safe and comfortable) from a sitting-to-standing position while the arms were crossed at the shoulders so as not to use them as support to assess lower limb strength (30-s Sit-to-Stand test) (20 (link)). Blood sampling of 10 mL peripheral venous blood for the determination of reactive oxygen metabolites (d-ROMs test, free radical analytical system, FRAS5, Parma, Italy) was performed 20 min before physical fitness tests (21 (link)). Pulmonary function tests were performed according to the ATS/ERS guidelines (22 (link)) in the sitting position using a MasterScreen-CPX pneumotachograph (VIASYS HealthCare, Germany). Prior to physical fitness tests, all patients answered questionnaires to measure the quality and patterns of sleep using the Pittsburgh Sleep Quality Index (PSQI) (23 (link)), cognitive impairment was assessed using the Montreal Cognitive Assessment (MoCA) (24 (link)), STOP-Bang for stratification for obstructive sleep apnea risk (25 (link)), and (iv) work ability index (WAI) to investigate the ability to return to work without restrictions (26 ).
A stationary seated bike (Toorx, Chrono Line, BRX R 300) with bluetooth capabilities was used for the measurements. It was connected to the VR application, the Meta Quest 2 (Facebook Technologies, LCC, Hacker Way, Menlo Park, CA, USA) device headset and controllers, and a computer (27 (link)). This VR training system is called VRADA (VR exercise App for Dementia and Alzheimer's patients) version 4.1 and has been developed by ORAMA-VR and Biomechanical Solutions Engineering based on interviews with older people with mild cognitive impairment. The application of the VR training system includes cognitive exercises with simple math calculations and requests users to observe and count animals that appear in their VR to enhance cognitive health and motivational techniques to address the issue of low motivation for exercise. The system gives an opportunity for each participant to choose their exercise duration, landscape in which they will cycle (forest, beach, or snowy landscape), motivating words that they want to hear during their performance (“Calmly,” “I can,” “I will do it well,” “Very nice,” or no words) and the music to enjoy while cycling. VR controllers with raycast were used as a selection mechanism that allows the user to select an answer by pointing the ray at the button and pressing the trigger button at the controller. Moreover, participants received feedback during their performance, such as indications about cycling time, distance, and speed, and could self-monitor their performance using screen-provided data. They were requested to cycle at a constant speed of between 15 and 20 km/h¹. Simultaneously, speed and distance were recorded every 45 s. At the end of the cycling procedure, participants were informed their scores in math questions, the distance they covered, and they were asked to answer four more questions assessing if they were tired, if they liked the way they exercised, how many animals they saw, and if they repeated the motivational word. In this study, all participants performed the exercise in the forest.
The HR, SpO₂, and self-assessment of lower limb fatigue and dyspnea were performed before and at the end of each exercise condition (VR, no-VR, SSE-VR, and 6MWT) for each patient.

Patients who were 18 years of age or older and had a clinical diagnosis of CVA [Global Initiative for Asthma (GINA) 2018 criteria (14 (link), 15 (link))] for at least 8 weeks were included. The diagnosis of CVA was made by a respiratory physician based on history, symptoms, signs, and pulmonary function test. The other inclusion criteria for patients were as follows: (1) <75 years old. (2) Any gender or ethnicity. (3) All patients signed an informed consent form. The exclusion criteria were as follows: (1) Cough caused by pneumonia, upper respiratory tract infections, chronic obstructive pulmonary disease, interstitial fibrosis, or other extra-pulmonary diseases. (2) Patients with known hypersensitivity to lidocaine, vitamin B₁₂, or betamethasone. (3) Comorbidity, includes chronic pulmonary, cardiovascular, renal, neurologic, or other systemic disease. (4) Long-term use of oral glucocorticoids within the last 3 weeks. (5) Smoking within the past 6 months. (6) Pregnancy.