FOT Technique Applied for Monitoring of COVID-19 Pneumonia Reveals Small Airways Involvement.

The fact that some SARS-CoV-2 pneumonia patients benefit from changing body position, and some from continuous positive airways pressure (CPAP), indicates the functional character of hypoxia. We hypothesize that such effects could be explained by the closure of small airways. To prove the hypothesis, we evaluated the patency of small airways in 30 oxygen-dependent, spontaneously breathing patients with SARS-CoV-2 pneumonia during their hospital stay using the FOT method and then compared the results with data obtained three months later. During the acute period, total resistance (R5) and peripheral resistance (R5-20) rose above the upper limit of normal (ULN) in 28% and 50% of all patients, respectively. Reactance indices X5, AX and Fres exceeded ULN in 55%, 68% and 66% of cases. Significant correlations were observed between PaO2/FiO2, the time spent in the hospital and R5, X5, AX and Fres. After 3 months, 18 patients were re-examined. During the hospital stay, 11 of them had risen above the upper limit of normal (ULN), for both resistance (R5-20) and reactance (X5, AX) values. Three months later, ULN for R5-20 was exceeded in only four individuals, but ULN for X5 and AX was exceeded in five individuals. Lung function examination revealed a combined restrictive/obstructive ventilatory failure and reduced CO transfer factor. We interpret these changes as lung tissue remodeling due to the process of fibrosis. We conclude that during acute period of SARS-CoV-2 pneumonia, dilated pulmonary blood vessels and parenchymal oedema induce functional closure of small airways, which in turn induce atelectasis with pulmonary right-to-left shunting, followed by the resulting hypoxemia.

Tumor necrosis factor­α ­308 G/A genetic polymorphism in patients with chronic obstructive pulmonary disease presenting with hyperactive airways.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. COPD is often diagnosed late in the disease leading to a delay in management. Notably, tumor necrosis factor-α (TNF-α) polymorphisms may serve an important role in the development of COPD. A single-center, case-control study was conducted to determine the presence of the TNF-α -308 G/A polymorphism among patients diagnosed with COPD presenting with hyperactive airways, patients without COPD presenting with hyperactive airways, and normal study participants without pulmonary comorbidities. Three genotypes: G/G (94%; 157/167), G/A (5%; 9/167) and A/A (1%; 1/167) were detected by quantitative PCR. The present study showed that the presence of the TNF-α -308 G/A polymorphism reduced the odds of having hyperactive airways with COPD by 29.3% and hyperactive airways without COPD by 26.3%. Multinomial logistic regression analysis showed that having the TNF-α -308 G/A polymorphism did not significantly reduce the odds of having hyperactive airways with COPD and without COPD compared to those with the G/G genotype. In conclusion, the presence of the TNF-α -308 G/A gene polymorphism showed no significant association with patients with COPD with or without hyperactive airways. The presence of the TNF-α -308 G/A polymorphism instead had a weak association with the reduction in the development of COPD regardless of the presence or absence of airway hyperactivity.

Airways epithelial exposure to Streptococcus pneumoniae in the presence of the alarmin IL-33 induces a novel subset of pro-inflammatory ILC2s promoting a mixed inflammatory response.

BACKGROUND: We have previously shown that asthma-like airways inflammation may be induced by topical exposure to respiratory tract pathogens such as S. pneumoniae (SP) in concert with epithelial alarmins such as IL-33. Details of the pathogenesis of this murine surrogate remain however unexplored. METHODS: Airways inflammation was induced by repeated, intranasal exposure of Il-4-/-, Rag1-/- and Rag2-/-Il2rg-/- mice (in which B lymphocyte IgE switching, adaptive and innate immunity are respectively ablated) as well as wild type mice to inactivated SP, IL-33 or both. Airways pathological changes were analysed, and the subsets and functions of locally accumulated ILC2s investigated by single cell RNA sequencing and flow cytometry. RESULTS: In the presence of IL-33, repeated exposure of the airways to inactivated SP caused marked eosinophil- and neutrophil-rich inflammation and local accumulation of ILC2s, which was retained in the Il-4-/- and Rag1-/- deficient mice but abolished in the Rag2-/-Il2rg-/- mice, an effect partly reversed by adoptive transfer of ILC2s. Single cell sequencing analysis of ILC2s recruited following SP and IL-33 exposure revealed a Klrg1+Ly6a+subset, expressing particularly elevated quantities of the pro-inflammatory cytokine IL-6, type 2 cytokines (IL-5 and IL-13) and MHC class II molecules, promoting type 2 inflammation as well as involved in neutrophil-mediated inflammatory responses. CONCLUSION: Local accumulation of KLRG1+Ly6a+ ILC2s in the lung tissue is a critical aspect of the pathogenesis of airways eosinophilic and neutrophil-rich inflammation induced by repeated exposure to SP in the presence of the epithelial alarmin IL-33.

Laryngeal microsurgery under Transnasal Humidified Rapid Insufflation Ventilatory Exchange.

Objective: Since 2015, Transnasal Humidified Rapid Insufflation Ventilatory Exchange (THRIVE) has been used in general anesthesia for preoxygenation or difficult exposure airway management. Its use offers new opportunities in laryngology. THRIVE increases apnea time and frees the access to the upper airway. However, its use may be less stable than orotracheal intubation. The main objective of this work was to evaluate the feasibility of laryngeal microsurgery under THRIVE including using Laser. Study Design: Retrospective. Setting: A total of N = 99 patients with laryngeal microsurgery (with or without CO2 laser) under THRIVE were included successively from January 1, 2020 to January 30, 2022. Method: Medical history, comorbidities, clinical and surgical data were extracted and analyzed. Two groups were constituted regarding the "success" (use of THRIVE along all the procedure) or the "failure" (need for an endotracheal tube) of the use of THRIVE during the procedure. Results: A failure occurred in N = 15/99 patients (15.2%) mainly due to refractory hypoxia. The odd ratios (OR) for THRIVE failure were: OR = 6.6 [2.9-35] for overweight (BMI >25 kg/m2); OR = 3.8 [1.7-18.7] for ASA score >2; OR = 4.7 [2.3-24.7] for the use of CO2 laser. Elderly patients and patients with pulmonary pathology were not statistically at greater risk of THRIVE failure. No adverse event was described. Conclusion: This work confirms the feasibility of laryngeal microsurgery under THRIVE, including with CO2 laser. Overweight, ASA >2 and lower fraction of inspired oxygen during CO2 laser use increased the risk for orotracheal intubation.

Causal associations between pediatric asthma and united airways disease: a two-sample Mendelian randomization analysis.

Background: Prior observational research has indicated a potential link between pediatric asthma and united airways disease (UAD). However, these findings could be subject to confounding factors and reverse causation. Therefore, our study utilizes Mendelian randomization (MR) method to further investigate the causal relationship between pediatric asthma and UAD. Methods: We conducted a comprehensive two-sample Mendelian randomization (MR) analysis to investigate the association between pediatric asthma and seven groups of UAD, including chronic sinusitis, chronic rhinitis, nasopharyngitis and pharyngitis, chronic diseases of tonsils and adenoids, chronic laryngitis and laryngotracheitis, chronic bronchitis, bronchiectasis, chronic obstructive pulmonary disease (COPD). The present study employed a range of methods for two-sample MR analysis, including inverse variance weighted (IVW), MR-Egger regression, Simple mode, weighted median, and weighted models. The conclusion of the MR analysis primarily relies on the IVW results, while other analytical methods are utilized as supplementary evidence to ensure result robustness in this MR analysis. And sensitivity analyses were conducted, including heterogeneity test, horizontal pleiotropy test, MR-PRESSO test, and leave-one-out analysis to validate the results. Results: The results of the MR analysis indicate significant causal effects of pediatric asthma on chronic rhinitis, nasopharyngitis and pharyngitis (IVW: OR = 1.15, 95%CI: 1.05-1.26, p-value = 0.003), chronic diseases of tonsils and adenoids (IVW: OR = 1.07, 95%CI: 1.00-1.15, p-value = 0.038), chronic bronchitis (IVW: OR = 1.51, 95%CI: 1.42-1.62, p-value <0.001), bronchiectasis (IVW: OR = 1.51, 95%CI: (1.30-1.75), p-value <0.001), and COPD (IVW: OR = 1.43, 95%CI: 1.34-1.51, p-value <0.001). However, no significant causal association was observed between pediatric asthma and chronic sinusitis (IVW: OR = 1.00, 95%CI: 1.00-1.00, p-value = 0.085), chronic laryngitis and laryngotracheitis (IVW: OR = 1.05, 95%CI: 0.90-1.21, p-value = 0.558). Conclusion: Our findings support a potential causal relationship between pediatric asthma and UAD, suggesting that pediatric asthma may be a potential risk factor for various UAD.

Targeted Stereology: Sampling heterogeneously distributed structures and lesions in the lung.

Stereology, the gold standard of lung morphometry, critically depends on sampling of tissue for analysis. Random sampling approaches guarantee each part of the organ an equal chance of being included in the analysis, hence they guarantee a representative sample of the whole. However, when biological or pathological structures of interest are rare and/or heterogeneously distributed over the whole lung, the random sampling approach can be inefficient or even result in meaningless data. In such cases, a targeted sampling approach can be useful which helps to relate the analytical items to an appropriate reference space. Targeted stereology greatly benefits from the increasing availability of multi-resolution imaging techniques at macroscopic and microscopic level as well as digital tools of segmentation. As such, the present article outlines two basic sampling scenarios: 1. In the first scenario, computed tomography and microscopy are subsequently used to segment the airway/arterial tree and perform stereological measurements on specific branches of the tree. 2. The second scenario deals with heterogeneous distribution of pathological lesions. This type of analysis can be divided into two stages: assessment of lesions of interest (LOI) within the lung and assessment of subcompartments within LOI. Taken together, targeted stereology has a thorough foundation in stereological theory and is not only able to significantly increase the efficiency of the analysis but also to yield new types of information that would be lost with the classical random sampling approach.

Impulse oscillometry in patients with persistent post-COVID-19 symptoms: A retrospective study.

Introduction: Impaired lung function has been observed in patients following COVID-19 infection, with studies reporting persistent lung volume and diffusing capacity impairments. Some studies have demonstrated significantly higher small airway resistance in COVID-19 positive cases. This retrospective study aims to examine impulse oscillometry (IOS) data of patients with persistent symptoms after COVID-19 infection, focusing on the relationship between time and symptoms. Material and Method: The study analyzed data from adult patients with persistent symptoms who underwent IOS testing within and after 84 days from the diagnosis date. Result: The results showed that patients within 84 days and those between 31 and 84 days had higher small airway resistance values, indicating peripheral airway disease. Patients with dyspnea exhibited higher IOS values compared to those with cough symptoms, suggesting more significant impairment in the peripheral airways. Conclusion: The study highlights the importance of using comprehensive diagnostic tools like IOS to assess respiratory impairments in post-COVID-19 patients, particularly in the small airways. Understanding the relationship between time and symptoms can provide valuable insights for the treatment of peripheral airway dysfunction in post-COVID-19 patients.

The relevance of eosinophils in chronic obstructive pulmonary disease: inflammation, microbiome and clinical outcomes.

Chronic obstructive pulmonary disease (COPD) is caused by the inhalation of noxious particles such as cigarette smoke. The pathophysiological features include airway inflammation, alveolar destruction and poorly reversible airflow obstruction. A sub-group of COPD patients have higher blood eosinophil counts (BECs), associated with an increased response to inhaled corticosteroids and increased biomarkers of pulmonary type 2 (T2) inflammation. Emerging evidence shows that COPD patients with increased pulmonary eosinophil counts have an altered airway microbiome. Higher BECs are also associated with increased lung function decline, implicating T2 inflammation in progressive pathophysiology in COPD. We provide a narrative review of the role of eosinophils and T2 inflammation in the pathophysiology of COPD, encompassing the lung microbiome, pharmacological targeting of T2 pathways in COPD, and the clinical use of BEC as a COPD biomarker.

[Home Respiratory Therapies: SEPAR Technical Document for Institutional Decision Support]. / Terapias respiratorias domiciliarias: documento técnico de la Sociedad Española de Neumología y Cirugía Torácica (SEPAR) de apoyo institucional a la toma de decisiones.

Home Respiratory Therapies (HRT) encompass a variety of treatments for patients with chronic respiratory diseases, administered at home over extended periods. The Service Providers of HRT (SPHRT) collaborate with hospital resources to address chronic respiratory diseases, acting as strategic partners. The contracting of HRT in the public health system involves contests where the SPHRT present technical and economic offers. The purpose of this document is to provide a technical basis that can assist professionals, SPHRT, and the administration in making decisions when calling for, evaluating, and deciding on such contests.

Meaning in Life: A Novel Factor for Promoting Wellbeing in COPD.

Research evidence indicates that meaning in life (MIL) is a meaningful outcome in individuals living with chronic illness, but evidence is lacking for chronic obstructive pulmonary disease (COPD). We hypothesized that MIL is independently associated with clinically meaningful outcomes for patients with COPD. We performed cross sectional analysis from a large cohort with moderate - severe COPD that participated in a home pulmonary rehabilitation study. MIL was measured using the Meaning in Life Questionnaire (MLQ), with domains of Presence and Search. The study included 340 participants: mean age 69 y/o (SD 9.4), 55.3% female, FEV1% 44.3% (SD 19.4) and mMRC dyspnea scale of 2.69 (SD 0.84). Patients with high MLQ Presence (vs low score) had meaningfully better scores (beyond the MCID) across all Chronic Respiratory Questionnaire domains Dyspnea, Fatigue, Emotions and Mastery (p ≤ 0.02), self-management (p ≤ 0.001), social support (p ≤ 0.001); anxiety (p ≤ 0.001) and depression (p ≤ 0.01) scores. When adjusting for age, sex, FEV1, mMRC, social support, and anxiety, MLQ Presence was independently associated with CRQ domains Fatigue, Emotions, and self-management (p ≤ 0.01). MLQ Search was independently associated with CRQ Dyspnea, Fatigue, and Mastery (p < 0.05). In patients with COPD, the perception that life has meaning or the willingness to search for MIL, is associated with the outcomes that patients consider most important. Our results are novel as MIL is a potentially modifiable outcome that could complement person-centered conversations during clinical visits, pulmonary rehabilitation, and health coaching.

Imaging of Small Airways Disease.

Bronchiolitis refers to a small airways disease and may be classified by etiology and histologic features. In cellular bronchiolitis inflammatory cells involve the small airway wall and peribronchiolar alveoli and manifest on CT as centrilobular nodules of solid or ground glass attenuation. Constrictive bronchiolitis refers to luminal narrowing by concentric fibrosis. Direct CT signs of small airway disease include centrilobular nodules and branching tree-in-bud opacities. An indirect sign is mosaic attenuation that may be exaggerated on expiratory CT and represent air trapping. Imaging findings can be combined with clinical and pathologic data to facilitate a more accurate diagnosis.

Cost-effectiveness of nasal high-flow in children with acute hypoxaemic respiratory failure.

AIM: A pilot randomised controlled trial assessed the early application of nasal high-flow (NHF) therapy compared with standard oxygen therapy (SOT), in children aged 0 to 16 years presenting to paediatric emergency departments with acute hypoxaemic respiratory failure (AHRF). The study estimated the need to escalate therapy and hospital length of stay in the NHF group compared with SOT. This sub-study then assessed the subsequent cost-effectiveness. METHODS: A decision tree-based model was developed, alongside the clinical study, to estimate cost-effectiveness, from the healthcare sector perspective. The primary health economics outcome is measured as incremental cost per length of hospital stay avoided. Incremental cost effectiveness ratios (ICER) measuring change in cost per change in length of stay, were obtained for four samples, depending on responder status and obstructive airways disease. These were (1) obstructive and responder, (2) non-obstructive and responder, (3) obstructive and non-responder and (4) non obstructive and non-responder. Bootstrapping of parameters accounted for uncertainty in estimates of cost and outcome. RESULTS: The ICER for patients randomised to NHF, indicated an additional A$367.20 for a lower hospital length of stay (in days) in the non-obstructive/non-responder sample. In the bootstrap sample, this was found to be cost effective above a willingness to pay threshold of A$10 000. The ICER was A$440.86 in the obstructive/responder sample and A$469.56 in the non-obstructive/responder sample - but both resulted in a longer length of stay. The ICER in the obstructive/non-responder sample was A$52 167.76, also with a longer length of stay, mainly impacted by a small sample of severe cases. CONCLUSION: As first-line treatment, NHF is unlikely to be cost-effective compared with SOT, but for non-obstructive patients who required escalation in care (non-obstructive non-responder), NHF is likely to be cost-effective if willingness-to-pay per reduced hospital length of stay is more than A$10 000 per patient.

Small Airways Obstruction and Mortality: Findings From the UK Biobank.

BACKGROUND: Small airways obstruction (SAO) is common in general populations. It has been associated with respiratory symptoms, cardiometabolic diseases, and progression to COPD over time. Whether SAO predicts mortality is largely unknown. RESEARCH QUESTION: Is spirometry-defined SAO associated with increased mortality? METHODS: Data were analyzed from 252,877 adult participants, aged 40 to 69 years at baseline, in the UK Biobank who had provided good-quality spirometry measurements. SAO was defined as the ratio of the forced expiratory volume in 3 s to the forced expiratory volume in 6 s (FEV6) less than the lower limit of normal. SAO was considered to be isolated if present when the FEV1/FEV6 ratio was normal (ie, greater than the lower limit of normal). A multivariable Cox regression model was used to assess the association of SAO, and isolated SAO, with all-cause and disease-specific mortality. Sex differences were investigated in these associations, and the primary analysis was repeated, excluding those who ever smoked. All models were adjusted for potential confounders such as sex, BMI, smoking status, smoking pack-years, assessment center, Townsend deprivation index, and ethnicity. RESULTS: A total of 59,744 participants with SAO were identified, of whom 24,004 had isolated SAO. A total of 5,009 deaths were reported over a median of 12.8 years of follow-up. Participants with SAO had increased all-cause (hazard ratio [HR], 1.31; 95% CI, 1.26-1.36), cardiovascular (HR, 1.39; 95% CI, 1.29-1.51), respiratory (HR, 2.20; 95% CI, 1.92-2.51), and neoplasm (HR, 1.23; 95% CI, 1.17-1.29) mortality risk. These associations were not modified by sex. However, in those who never smoked, only respiratory and cardiovascular mortality risk was associated with SAO. Isolated SAO was also associated with an increased mortality risk (HR, 1.14; 95% CI, 1.07-1.20). INTERPRETATION: Individuals with SAO have an increased risk of all-cause and disease-specific mortality. Further studies are needed to determine whether SAO causes mortality or is a marker of underlying disease.

Cross-sectional and retrospective study regarding the changes in the spatial position of the hyoid bone and upper airways before and after orthodontic therapy in developing patients with normocclusion.

OBJECTIVE: The objective was to study the effect of orthodontic therapy regarding the position of the hyoid bone and upper airways in nighty-one 6-21 year-old Caucasian patients with normoclussion. METHODS: Nineteen variables were analyzed by teleradiography. The differences were analyzed with the ANOVA test of repeated measures with an intra-group factor for two occasions. RESULTS: The statistical analysis revealed a greater influence on the variables Hy-MP (12.3 vs 14.2), Hps-C3 (25.2 vs 28.1), Hpi-C3 (28.8 vs 35.5), H°-C3 (14.4 vs 12.7), Hps-Rg (36.0 vs 42.7), Hpi-Rg (34.1 vs 39.7), USP (16.2 vs 20.2), MPP (12.9 vs 14.8), C3P (10.6 vs 12.8), PNS-Ba (43.5 vs 66.5), PtV-Ad (12.0 vs 17.1) and PtM-Ba (33.9 vs 35.9), and Ad2-SO (24.16 vs 20.87). DISCUSSION: Significant differences were observed between most of the airway and hyoid bone variables before and after orthodontic treatment in our Spanish pediatric population, especially in the nasopharynx.

Microscopic Small Airway Abnormalities Identified in Early Idiopathic Pulmonary Fibrosis In Vivo Using Endobronchial Optical Coherence Tomography.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) affects subpleural lung, but is considered to spare small airways. Micro-CT studies demonstrated small airway reduction in end-stage IPF explanted lungs, raising questions about small airway involvement in early-stage disease. Endobronchial optical coherence tomography (EB-OCT) is a volumetric imaging modality that detects microscopic features from subpleural to proximal airways. We use EB-OCT to evaluate small airways in early IPF and control subjects in vivo. METHODS: EB-OCT was performed in 12 IPF and 5 control subjects (matched by age, sex, smoking-history, height, BMI). IPF subjects had early disease with mild restriction (FVC: 83.5% predicted), diagnosed per current guidelines and confirmed by surgical biopsy. EB-OCT volumetric imaging was acquired bronchoscopically in multiple, distinct, bilateral lung locations (total: 97 sites). IPF imaging sites were classified by severity into affected (all criteria for UIP present) and less affected (some but not all criteria for UIP present) sites. Bronchiole count and small airway stereology metrics were measured for each EB-OCT imaging site. RESULTS: Compared to control subjects (mean: 11.2 bronchioles/cm3; SD: 6.2), there was significant bronchiole reduction in IPF subjects (42% loss; mean: 6.5/cm3; SD: 3.4; p=0.0039), including in IPF affected (48% loss; mean: 5.8/cm3; SD: 2.8; p<0.00001) and IPF less affected (33% loss; mean: 7.5/cm3; SD: 4.1; p=0.024) sites. Stereology metrics showed IPF affected small airways were significantly larger and more distorted/irregular than in IPF less affected sites and control subjects. IPF less affected and control airways were statistically indistinguishable for all stereology parameters (p=0.36-1.0). CONCLUSION: EB-OCT demonstrated marked bronchiolar loss in early IPF (between 30 and 50%), even in areas minimally affected by disease, compared to matched controls. These findings support small airway disease as a feature of early IPF, providing novel insight into pathogenesis and potential therapeutic targets.

Pulmonary rehabilitation in Iranian outpatients with mustard gas lung disease: a randomised controlled trial.

OBJECTIVE: People with mustard gas lung disease experience cough, sputum, breathlessness and exercise limitation. We hypothesised that pulmonary rehabilitation (PR) would be beneficial in this condition. DESIGN: An assessor-blind, two-armed, parallel-design randomised controlled clinical trial. SETTING: Secondary care clinics in Iran. PARTICIPANTS: 60 men with breathlessness due to respiratory disease caused by documented mustard gas exposure, mean (SD) age 52.7 (4.36) years, MRC dyspnoea score 3.5 (0.7), St. George's Respiratory Questionnaire (SGRQ) 72.3 (15.2). INTERVENTIONS: Participants were allocated either to a 6-week course of thrice-weekly PR (n=31) or to usual care (n=29), with 6-week data for 28 and 26, respectively. OUTCOME MEASURES: Primary endpoint was change in cycle endurance time at 70% baseline exercise capacity at 6 weeks. Secondary endpoints included 6 min walk distance, quadriceps strength and bulk, body composition and health status. For logistical reasons, blood tests that had been originally planned were not performed and 12-month follow-up was available for only a small proportion. RESULTS: At 6 weeks, cycle endurance time increased from 377 (140) s to 787 (343) s with PR vs 495 (171) s to 479 (159) s for usual care, effect size +383 (231) s (p<0.001). PR also improved 6 min walk distance+103.2 m (63.6-142.9) (p<0.001), MRC dyspnoea score -0.36 (-0.65 to -0.07) (p=0.016) and quality of life; SGRQ -8.43 (-13.38 to -3.48) p<0.001, as well as quadriceps strength+9.28 Nm (1.89 to 16.66) p=0.015. CONCLUSION: These data suggest that PR can improve exercise capacity and quality of life in people with breathlessness due to mustard gas lung disease and support the wider provision of this form of care. TRIAL REGISTRATION NUMBER: IRCT2016051127848N1.

Modeling airway persistent infection of Moraxella catarrhalis and nontypeable Haemophilus influenzae by using human in vitro models.

Non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) are two common respiratory tract pathogens often associated with acute exacerbations in Chronic Obstructive Pulmonary Disease (COPD) as well as with otitis media (OM) in children. Although there is evidence that these pathogens can adopt persistence mechanisms such as biofilm formation, the precise means through which they contribute to disease severity and chronicity remains incompletely understood, posing challenges for their effective eradication. The identification of potential vaccine candidates frequently entails the characterization of the host-pathogen interplay in vitro even though this approach is limited by the fact that conventional models do not permit long term bacterial infections. In the present work, by using air-liquid-interface (ALI) human airway in vitro models, we aimed to recreate COPD-related persistent bacterial infections. In particular, we explored an alternative use of the ALI system consisting in the assembly of an inverted epithelium grown on the basal part of a transwell membrane with the aim to enable the functionality of natural defense mechanisms such as mucociliary clearance and cellular extrusion that are usually hampered during conventional ALI infection experiments. The inversion of the epithelium did not affect tissue differentiation and considerably delayed NTHi or Mcat infection progression, allowing one to monitor host-pathogen interactions for up to three weeks. Notably, the use of these models, coupled with confocal and transmission electron microscopy, revealed unique features associated with NTHi and Mcat infection, highlighting persistence strategies including the formation of intracellular bacterial communities (IBCs) and surface-associated biofilm-like structures. Overall, this study demonstrates the possibility to perform long term host-pathogen investigations in vitro with the aim to define persistence mechanisms adopted by respiratory pathogens and individuate potential new vaccine targets.