Proximal Airways Research Articles

Defining VOC signatures of Airway Epithelial Cells with PM2.5 Exposure.

Volatile organic compounds (VOCs) produced by the lung in response to exposure to environmental pollutants can be utilized to study their impact on lung health and function. Previously, we developed a method to measure VOCs emitted from well-differentiated tracheobronchial epithelial cells in vitro. Using this method, we exposed well-differentiated proximal (PECs) and distal airway epithelial cells (DECs) to varying doses of traffic-related air pollutants (TRAP) and wildfire particulates to determine specific VOC signatures after exposure. We utilized PM2.5 TRAP collected from the Caldecott tunnel in Oakland, CA and the 2018 Camp Fire to model "real-life" exposures. The VOCs were collected and extracted from Twisters and analyzed using gas chromatography-mass spectrometry (GC-MS). Exposure to both types of particulate matter (PM) resulted in specific VOC responses grouped by individual subjects with little overlap. Interestingly the VOCs produced by the PECs and DECs were also differentiated from each other. Our studies suggest that PM exposure induces a specific compartmentalized cellular response that can be exploited for future studies. This response is cell-type specific and potentially related to a phenotype we have yet to uncover.

Bronchoscopic Diagnosis of Severe Respiratory Infections

The diagnosis of severe respiratory infections in intensive care remains an area of uncertainty and involves a complex balancing of risks and benefits. Due to the frequent colonisation of the lower respiratory tract in mechanically ventilated patients, there is an ever-present possibility of microbiological samples being contaminated by bystander organisms. This, coupled with the frequency of alveolar infiltrates arising from sterile insults, risks over-treatment and antimicrobial-associated harm. The use of bronchoscopic sampling to obtain protected lower respiratory samples has long been advocated to overcome this problem. The use of bronchoscopy further enables accurate cytological assessment of the alveolar space and direct inspection of the proximal airways for signs of fungal infection or alternative pathologies. With a growing range of molecular techniques, including those based on nucleic acid amplification and even alveolar visualisation and direct bacterial detection, the potential for bronchoscopy is increasing concomitantly. Despite this, there remain concerns regarding the safety of the technique and its benefits versus less invasive sampling techniques. These discussions are reflected in the lack of consensus among international guidelines on the topic. This review will consider the benefits and challenges of diagnostic bronchoscopy in the context of severe respiratory infection.

Application research on asthma-COPD overlap using low-dose CT scan and quantitative analysis

The aim of this study was to assess the proximal airway remodeling, emphysema, and air trapping of asthma-COPD Overlap. 20 ACO patients, 55 mild to moderate COPD patients and 38 non-severe asthma patients were participated in low-dose dual phase CT scanning and pulmonary function test, comparative analysis was performed to identify differences in CT measurements among three groups. (Ⅰ) The average age and smoking index of ACO and mild to moderate COPD were both higher than non-severe asthma. ACO and mild to moderate COPD group had a higher proportion of males than non-severe asthma. (Ⅱ) In terms of pulmonary function test, FEV1 (%Pred), FEV1/FVC%, MEF25% (%Pred), MEF 50% (%Pred), MMEF (%Pred), and PEF (%Pred) in ACO were significantly reduced than those in non-severe asthma. (Ⅲ) On proximal airway parameters, ACO exhibited higher WA% and Pi10 compared to mild to moderate COPD. However, there was no statistically significant difference in WA% and Pi10 between ACO and non-severe asthma. (Ⅳ) On CT lung function, the emphysema index VI-910ex of ACO was significantly higher than non-severe asthma.Additionally, ACO demonstrated higher MLDex and VI-856ex compared to non-severe asthma. Compared with non-severe asthma, ACO is more common in male patients with older age and a longer history of smoking, which had more severe airflow obstruction and airway dysfunction than non-severe asthma.ACO showed more obvious proximal airway remodeling than mild to moderate COPD, and was more similar to non-severe asthma.The extent of emphysema and air trapping in ACO were more pronounced compared to non-severe asthma.

Flow interruption compared to forced oscillatory maneuvers and esophageal balloon/pneumotachography for measurement of respiratory resistance in the horse.

Pulmonary function testing is critical to the diagnosis of equine asthma (EA), an important cause of respiratory disease in the horse, but its clinical use has remained elusive, unfortunately, due to the complexity of reference methods, esophageal balloon/pneumotachography (EBP), and forced oscillatory mechanics (FOM), so we sought a noninvasive, portable method for use in horses through rapid interruption of airflow for equilibration of alveolar pressure with proximal airway pressure, termed flow interruption (FI). Resistance (RINT) was computed as the relationship between the change in pressure at the nose before and immediately after interruption and flow immediately before interruption. A pilot study in five healthy university-owned animals using EBP and FI showed good correspondence between the two methods: RINT (0.33 ± 0.05 cmH2O/L/s) and RL (0.31 ± 0.06 cmH2O/L/s). In two separate populations of client-owned horses, with random assignment of methods to FI versus EBP (n = 8), RINT showed good correlation with RL in horses (rs = 0.995, P = 0.0002) and accords with RL, with no significant difference between RINT and RL. Using FOM (n = 12), RINT (0.67 ± 0.31 cmH2O/L/s) has good correlation with RRS measured with FOM (r = 0.834, P = 0.0001), but is consistently smaller than RRS (0.74 ± 0.33 cmH2O/L/s). Histamine bronchoprovocation (HBP) was performed in a subset of these horses: FI classified one horse in six as less reactive than did EBP, and FI classified one horse in seven as less reactive than did FOM.NEW & NOTEWORTHY We developed and document for the first time the use of flow interruption for the rapid and noninvasive measurement of resistance in equine patients and demonstrated short- and long-term stability and accuracy in comparison with the reference methods.

Differentiation of CD166-positive hPSC-derived lung progenitors into airway epithelial cells.

The generation of lung epithelial cells through the directed differentiation of human pluripotent stem cells (hPSCs) in vitro provides a platform to model both embryonic lung development and adult airway disease. Here, we describe a robust differentiation protocol that closely recapitulates human embryonic lung development. Differentiating cells progress through obligate intermediate stages, beginning with definitive endoderm formation and then patterning into anterior foregut endoderm that yields lung progenitors (LPs) with extended culture. These LPs can be purified using the cell surface marker CD166 (also known as ALCAM), and further matured into proximal airway epithelial cells including basal cells, secretory cells and multiciliated cells using either an organoid platform or culture at the air-liquid interface (ALI). We additionally demonstrate that these hPSC-derived airway epithelial cells can be used to model Influenza A infection. Collectively, our results underscore the utility of CD166 expression for the efficient enrichment of LPs from heterogenous differentiation cultures and the ability of these isolated cells to mature into more specialized, physiologically relevant proximal lung cell types.

Deficiency of Secreted Phosphoprotein 1 Alleviates Hyperoxia-induced Bronchopulmonary Dysplasia in Neonatal Mice.

Bronchopulmonary dysplasia (BPD) is a common chronic lung disorder characterized by impaired proximal airway and bronchoalveolar development in premature births. Secreted phosphoprotein 1 (SPP1) is involved in lung development and lung injury events, while its role was not explored in BPD. For establishing the in vivo models of BPD, a mouse model of hyperoxia-induced lung injury was generated by exposing neonatal mice to hyperoxia for 7days after birth. Alveolar myofibroblasts (AMYFs) were treated with hyperoxia to establish the in vitro models of BPD. Based on the scRNA-seq analysis of lungs of mice housed under normoxia or hyperoxia conditions, mouse macrophages and fibroblasts were main different cell clusters between the two groups, and differentially expressed genes in fibroblasts were screened. Further GO and KEGG enrichment analysis revealed that these differentially expressed genes were mainly enriched in the pathways related to cell proliferation, apoptosis as well as the PI3K-AKT and ERK/MAPK pathways. SPP1 was found up-regulated in the lung tissues of hyperoxia mice. We also demonstrated the up-regulation of SPP1 in the BPD patients, the mouse model of hyperoxia-induced lung injury, and hyperoxia-induced cells. SPP1 deficiency was revealed to reduce the hyperoxia-induced apoptosis, oxidative stress and inflammation and increase the viability of AMYFs. In the mouse model of hyperoxia induced lung injury, SPP1 deficiency was demonstrated to reverse the hyperoxia-induced alveolar growth disruption, oxidative stress and inflammation. Overall, SPP1 exacerbates BPD progression in vitro and in vivo by regulating oxidative stress and inflammatory response via the PI3K-AKT and ERK/MAPK pathways, which might provide novel therapeutic target for BPD therapy.

Adverse effects of Mechanical Insufflation-Exsufflation in Mechanically Ventilated Patients in the Adult Intensive Care Unit - A Literature Review

Introduction Mechanical ventilation via an artificial airway can impede cough, predisposing the critically ill patient to retained secretions and an increased risk of respiratory complications. Physiotherapeutic techniques aimed at mobilising secretions and optimising airway clearance are often relied upon in this cohort. Mechanical Insufflation-Exsufflation (MI-E) is a cough augmentation device that utilises positive pressure, followed by a rapid switch to negative pressure, to simulate a cough and aid sputum clearance from proximal airways. To date, MI-E has been widely researched in the neuromuscular population, with emerging use in the critically ill. However, adverse effects associated with MI-E in intubated populations remains unknown. The aim of this review was to report on the incidence of adverse events associated with MI-E in acutely invasively ventilated critically ill patients. Methods An electronic search of online databases was conducted using AMED, CINAHL, Cochrane Library, EMBASE, MEDLINE, SPORTDiscuss and Web of Science. Additionally, the reference lists of relevant articles were hand searched for eligible studies. Studies including adult subjects (>18 years) who were invasively ventilated and receiving MI-E were deemed eligible for inclusion. The outcome of interest was adverse events. Studies were excluded if they were in a paediatric population, not written in English language and were editorials or conference papers. Quality was assessed using the Critical Skills Appraisal Programme tool. Results A total 77 citations were identified, five of which met the inclusion criteria: three randomised crossover studies, one randomised parallel-group open label trial and one case series (278 participants in total). All studies applied MI-E followed by endotracheal or tracheal suction. Insufflation and exsufflation pressures ranged from +30 to +50cmH2O and -30 to -50cmH2O across studies. Only two studies pre-defined an adverse event. Common reported measures included heart rate, systolic and diastolic blood pressure, and oxygen saturations. Two studies reported the occurrence of an adverse event (oxygen saturations, blood pressure and heart rate changes) but noted that changes were transient and not clinically significant. Overall, all studies concluded that MI-E was a safe intervention in this patient cohort. Conclusion Overall, the use of MI-E in the acutely intubated patient does not result in adverse events that are clinically significant. However, limitations to the evidence base should be acknowledged and include a lack of definition and variation in outcome measures used and small sample sizes across studies. Larger clinical trials are needed, to further evaluate the safety of MI-E on clinically important parameters that are more clearly defined.

Molecular distinctions of bronchoalveolar and alveolar organoids under differentiation conditions.

The bronchoalveolar organoid (BAO) model is increasingly acknowledged as an ex-vivo platform that accurately emulates the structural and functional attributes of proximal airway tissue. The transition from bronchoalveolar progenitor cells to alveolar organoids is a common event during the generation of BAOs. However, there is a pressing need for comprehensive analysis to elucidate the molecular distinctions characterizing the pre-differentiated and post-differentiated states within BAO models. This study established a murine BAO model and subsequently triggered its differentiation. Thereafter, a suite of multidimensional analytical procedures was employed, including the morphological recognition and examination of organoids utilizing an established artificial intelligence (AI) image tracking system, quantification of cellular composition, proteomic profiling and immunoblots of selected proteins. Our investigation yielded a detailed evaluation of the morphologic, cellular, and molecular variances demarcating the pre- and post-differentiation phases of the BAO model. We also identified of a potential molecular signature reflective of the observed morphological transformations. The integration of cutting-edge AI-driven image analysis with traditional cellular and molecular investigative methods has illuminated key features of this nascent model.

Structure-specific rigid dose accumulation dosimetric analysis of ablative stereotactic MRI-guided adaptive radiation therapy in ultracentral lung lesions.

Definitive local therapy with stereotactic ablative radiation therapy (SABR) for ultracentral lung lesions is associated with a high risk of toxicity, including treatment related death. Stereotactic MR-guided adaptive radiation therapy (SMART) can overcome many of the challenges associated with SABR treatment of ultracentral lesions. We retrospectively identified 14 consecutive patients who received SMART to ultracentral lung lesions from 10/2019 to 01/2021. Patients had a median distance from the proximal bronchial tree (PBT) of 0.38 cm. Tumors were most often lung primary (64.3%) and HILUS group A (85.7%). A structure-specific rigid registration approach was used for cumulative dose analysis. Kaplan-Meier log-rank analysis was used for clinical outcome data and the Wilcoxon Signed Rank test was used for dosimetric data. Here we show that SMART dosimetric improvements in favor of delivered plans over predicted non-adapted plans for PBT, with improvements in proximal bronchial tree DMax of 5.7 Gy (p = 0.002) and gross tumor 100% prescription coverage of 7.3% (p = 0.002). The mean estimated follow-up is 17.2 months and 2-year local control and local failure free survival rates are 92.9% and 85.7%, respectively. There are no grade ≥ 3 toxicities. SMART has dosimetric advantages and excellent clinical outcomes for ultracentral lung tumors. Daily plan adaptation reliably improves target coverage while simultaneously reducing doses to the proximal airways. These results further characterize the therapeutic window improvements for SMART. Structure-specific rigid dose accumulation dosimetric analysis provides insights that elucidate the dosimetric advantages of SMART more so than per fractional analysis alone.

ASSOCIATION OF ACID REFLUX AND LARYNGOMALACIA : A COMPREHENSIVE SYSTEMATIC REVIEW

Background: Laryngomalacia (LM) is a common cause of stridor in infants and young children. It is characterized by reflux episodes that are slightly acidic, which harm the supraglottic tissues and exacerbate airway collapse. Further investigation is required to ascertain the presence of the gastric proteolytic enzyme pepsin in the oral secretions of patients with less severe LM, as it has been proposed as a biomarker for both weakly acidic and nonacid reflux events. Methods: This systematic review focused on full-text English literature published between 2014 and 2024, adhering to PRISMA 2020 principles. Without a DOI, editorials and review papers that were published in the same journal as the submission were not accepted. ScienceDirect, PubMed, and SagePub were only a few of the web resources used to compile the literature. Result: Following a thorough screening process consisting of three stages, six papers were determined to have pertinent connections to our ongoing comprehensive investigation. Following that, the text as a whole was thoroughly studied and these sections received more attention. Conclusion: Recent research has revealed a link between laryngomalacia (LM) and pepsin, a diagnostic marker, but no significant differences have been found. Pepsin is a common comorbidity in LM patients and serves as a biomarker for airway reflux, but its harmful impact on proximal airways remains unclear.

Fisiologi Mukosiliar Bronkus

One of the defense systems possessed by the respiratory tract is mucociliary transport. In order for this system to work properly, coordination of 3 components is required, namely: the motile cila, the periciliar layer (PCL), and the mucus layer. The mucus layer will catch foreign particles that enter the respiratory tract. The motile cilia will push the mucus to keep moving towards the proximal airway until it finally reaches the oropharynx and is swallowed. Cilia movement is certainly influenced by PCL which is a medium for cilia to move. In some lung diseases, this component can be impaired so that the mucociliary transport process does not work effectively.

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

Rationale: Idiopathic pulmonary fibrosis (IPF) affects the subpleural lung but is considered to spare small airways. Micro-computed tomography (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. Objectives: In this study, EB-OCT was used to evaluate small airways in early IPF and control subjects invivo. Methods: EB-OCT was performed in 12 subjects with IPF and 5 control subjects (matched by age, sex, smoking history, height, and body mass index). Subjects with IPF had early disease with mild restriction (FVC: 83.5% predicted), which was 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 usual interstitial pneumonia present) and less affected (some but not all criteria for usual interstitial pneumonia present). Bronchiole count and small airway stereology metrics were measured for each EB-OCT imaging site. Measurements and Main Results: Compared with the number of bronchioles in control subjects (mean = 11.2/cm3; SD = 6.2), there was significant bronchiole reduction in subjects with IPF (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 that IPF-affected small airways were significantly larger, more distorted, and more 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). Conclusions: EB-OCT demonstrated marked bronchiolar loss in early IPF (between 30% and 50%), even in areas minimally affected by disease, compared with matched control subjects. These findings support small airway disease as a feature of early IPF, providing novel insight into pathogenesis and potential therapeutic targets.

Performance of the galactomannan test for the diagnosis of invasive pulmonary aspergillosis using non-invasive proximal airway samples

To diagnose invasive pulmonary aspergillosis (IPA), galactomannan (GM) detection in serum or bronchoalveolar lavage fluid (BALF) is widely used. However, the utility of proximal airway GM test (from induced sputum or tracheal aspirate) has not been well elucidated. In this retrospective cohort study, we evaluated the diagnostic performance of proximal airway GM in diagnosis of IPA including COVID-19 associated pulmonary aspergillosis (CAPA). Between January 2022 and January 2023, patients who had been tested for GM with clinical suspicion or for surveillance from any specimen (serum, induced sputum, tracheal aspirate, and BALF) were screened. IPA was diagnosed using EORTC/MSGERC criteria, and CAPA was diagnosed following the 2020 ECMM/ISHAM consensus criteria. Of 624 patients with GM results, 70 met the criteria for proven/probable IPA and 427 had no IPA. The others included possible IPA and chronic form of aspergillosis. The sensitivities and specificities of serum, proximal airway, and BALF GM for proven/probable IPA versus no IPA were 78.9% and 70.6%, 93.1% and 78.7%, and 78.6% and 91.0%, respectively. Areas under the receiver operating characteristic curve (AUCs) were 0.742 for serum GM, 0.935 for proximal airway GM, and 0.849 for BALF GM (serum GM vs proximal airway GM, p=0.014; proximal airway GM vs BALF GM, p=0.334; serum GM vs BALF GM, p=0.286). This study demonstrates that the performance of GM test from non-invasive proximal airway samples is comparable or even better than those from serum and distal airway sample (BALF).

Functional analysis of airway remodeling is related with fibrotic mediators in asthmatic children

Background Asthmatic children present variable degrees of airway inflammation, remodeling, and resistance, which correlate with disease control and severity. The chronic inflammatory process of the airway triggers airway remodeling, which reflects the degree of airway resistance. Pro-inflammatory and pro-fibrotic mediators are centrally involved in this process. Objective To investigate whether the levels of pulmonary and systemic pro-inflammatory and pro-fibrotic mediators present a correlation with the resistance of the respiratory system and of the proximal and distal airways. Methods 39 Asthmatic children (persistent mild and moderate) and 39 non-asthmatic children (both between 6 and 13 years old) were evaluated for anthropometric characteristics, lung function and mechanics, and pulmonary and systemic immune responses. Results Asthmatic children showed an increased number of blood eosinophils (p < 0.04), basophils (p < 0.04), monocytes (p < 0.002) and lymphocytes (p < 0.03). In addition, asthmatic children showed impaired lung function, as demonstrated by FEV1 (p < 0.0005) and FEV1/FVC (p < 0.004), decreased total resistance of the respiratory system (R5Hz; p < 0.009), increased resistance of the proximal airways (R20Hz; p < 0.02), increased elastance (Z5Hz; p < 0.02) and increased reactance (X5Hz; p < 0.002) compared to non-asthmatic children. Moreover, the following inflammatory factors were significantly higher in asthmatic than non-asthmatic children: GM-CSF in the breath condensate (BC) (p < 0.0001) and in the serum (p < 0.0001); TGF-beta in the BC (p < 0.0001) and in the serum (p < 0.004); IL-5 in the BC (p < 0.02) and in the serum (p < 0.01); IL-4 in the serum (p < 0.0002). Conclusions Impulse oscillometry is a sensitive method to detect airway resistance in persistent mild and moderate asthmatic children, an event followed by increased levels of pro-inflammatory and pro-fibrotic mediators.

Abstract 5657: The intricate relationship between genomic damage and epidemiological lung cancer risk in ever-smokers

Abstract Extensive epidemiological studies have illuminated the intricate relationship between tobacco smoking and lung cancer. Squamous cell carcinoma (LUSC), which is clinically tied to smoking, is formed in the proximal airway of the lung, whereas adenocarcinoma (LUAD), arising in the distal airway, has a weaker connection to tobacco and often occurs in never-smokers. The cessation of smoking promotes lung health and gradually lowers cancer risk. Dissecting the impact of smoking cessation on different lung cancer types in &amp;gt;650,000 individuals from UK Biobank and PLCO, we confirmed previous findings of LUSC risk diminishing more rapidly after quitting smoking, while LUAD risk endures over time. We previously found that normal bronchial epithelium in the proximal airway accumulates DNA damage proportionally to an individual’s smoking habits. However, our analysis identified a population of cells with near-normal mutation burden, evading tobacco-induced damage. We hypothesized that the reduction in LUSC risk with smoking cessation could be explained by the repopulation of the proximal airway by these cells. Here, we followed up on this finding by exploring the genomics of alveolar stem cells (ASCs), which are believed to be the origin of LUAD. Analyzing WGS data of &amp;gt;800 single-cell-derived organoids from 9 patients, we found that tobacco smoking is the major driver of increased mutation burden in ever- compared to never-smokers. More importantly, our results demonstrated no significant difference in the burden of tobacco-induced genomic damage between ex- and current-smokers, suggesting that DNA damage in the distal airway is both persistent and long-lasting. Using single-molecule sequencing in a cohort of 43 individuals with heterogeneous smoking histories confirmed this observation. Overall, contrasting this finding with the results from bronchial epithelium and the epidemiological lung cancer risk brings forth a genomic explanation for the differences in long-term cancer risk, ingrained in the abundance of near-normal mutation burden cells. By contextualizing our results within the carcinogenic process in the lung, we suggest that persistent genomic alterations are the major driver in the long-standing discussion about the reason for an epidemiological shift from LUSC to LUAD in the last 30 years. As the largest proportion of lung cancers occurs within ex-smokers, the persistent genomic damage in the distal airway will result in a relative increase in LUAD incidence compared to LUSC, likely explaining this clinical phenomenon. Citation Format: Moritz Jakob Przybilla, Amany Ammar, Hugh Selway-Clarke, Andrew R. Lawson, Hyunchul Jung, Adam Pennycuick, Kate Gowers, Ryan C. Khaw, Zoe Frazer, Kate Davies, Inigo Martincorena, Sam Janes, Peter J. Campbell. The intricate relationship between genomic damage and epidemiological lung cancer risk in ever-smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5657.

Similarity Network Analysis of the Adaptive Immune Response in the Proximal Airway.

Recent immunologic study of the adaptive immune repertoire in the subglottic airway demonstrated high-frequency T cell clones that do not overlap between individuals. However, the anatomic distribution and antigenic target of the T cell repertoire in the proximal airway mucosa remain unresolved. Single-cell RNA sequencing of matched scar and unaffected mucosa from idiopathic subglottic stenosis patients (iSGS, n = 32) was performed and compared with airway mucosa from healthy controls (n = 10). T cell receptor (TCR) sequences were interrogated via similarity network analysis to explore antigenic targets using the published algorithm: Grouping of Lymphocyte Interactions by Paratope Hotspots (GLIPH2). The mucosal T cell repertoire in healthy control airways consisted of highly expressed T cell clones conserved across anatomic subsites (trachea, bronchi, bronchioles, and lung). In iSGS, high-frequency clones were equally represented in both scar and adjacent non-scar tissue. Significant differences in repertoire structure between iSGS scar and unaffected mucosa was observed, driven by unique low-frequency clones. GLIPH2 results suggest low-frequency clones share targets between multiple iSGS patients. Healthy airway mucosa has a highly conserved T cell repertoire across multiple anatomic subsites. Similarly, iSGS patients have highly expressed T cell clones present in both scar and unaffected mucosa. iSGS airway scar possesses an abundance of less highly expanded clones with predicted antigen targets shared between patients. Interrogation of these shared motifs suggests abundant adaptive immunity to viral targets in iSGS airway scar. These results provide insight into disease pathogenesis and illuminate new treatment strategies in iSGS. NA Laryngoscope, 134:3245-3252, 2024.

Impact of biologics on lung hyperinflation in patients with severe asthma

BackgroundIn asthma, inflammation affects both the proximal and distal airways and can cause significant hyperinflation, which is thought to be a major cause of dyspnea. MethodsThis is a retrospective observational study evaluating the effect of three months of treatment with different biologic drugs (benralizumab, dupilumab and omalizumab) on pulmonary hyperinflation in a cohort of patients with severe asthma already receiving regular triple inhaled therapy. Changes in RV, RV/TLC ratio, FRC and FRC/TLC ratio were the primary efficacy measures. Secondary outcomes included FEV1, FVC, FEV1/FVC ratio, IC, IC/TLC ratio, asthma control test, the percentage of eosinophils in the blood and fractional FENO. ResultsBenralizumab led to significant changes (p < 0.001) in RV, RV/TLC, FRC, and FRC/TLC. Dupilumab demonstrated a notable reduction in RV (p = 0.017) and RV/TLC (p = 0.002), but the decreases in FRC and FRC/TLC were merely numerical and not as pronounced as those induced by benralizumab. Omalizumab's positive impact on RV (p = 0.057) and RV/TLC (p = 0.085), as well as FRC (p = 0.202) and FRC/TLC (p = 0.096), was also predominantly numerical, with a tendency towards efficacy, albeit excluding the effect on FRC. Treatment with biologics resulted in improvements in all other lung function parameters assessed and a decrease in FENO levels. ConclusionThis study, although limited by small sample size, lack of a placebo control, and unbalanced group sizes, suggests that biological agents are effective in reducing lung hyperinflation even after a relatively short treatment.

Characterisation of airway disease associated with Sjögren disease

ObjectiveAlthough airway disease associated with Sjögren’s disease (Sjo-AD) is common, it is poorly studied compared with interstitial lung disease (ILD). In this study, we aimed to assess factors associated with...

Culture and expansion of murine proximal airway basal stem cells

BackgroundThe stem cell characteristic makes basal cells desirable for ex vivo modeling of airway diseases. However, to date, approaches allowing them extensively in vitro serial expansion and maintaining bona fide stem cell property are still awaiting to be established. This study aims to develop a feeder-free culture system of mouse airway basal stem cells (ABSCs) that sustain their stem cell potential in vitro, providing an experimental basis for further in-depth research and mechanism exploration.MethodsWe used ROCK inhibitor Y-27632-containing 3T3-CM, MEF-CM, and RbEF-CM to determine the proper feeder-free culture system that could maintain in vitro stem cell morphology of mouse ABSCs. Immunocytofluorescence was used to identify the basal cell markers of obtained cells. Serial propagation was carried out to observe whether the stem cell morphology and basal cell markers could be preserved in this cultivation system. Next, we examined the in vitro expansion and self-renewal ability by evaluating population doubling time and colony-forming efficiency. Moreover, the differentiation potential was detected by an in vitro differentiation culture and a 3D tracheosphere assay.ResultsWhen the mouse ABSCs were cultured using 3T3-CM containing ROCK inhibitor Y-27632 in combination with Matrigel-coated culture dishes, they could stably expand and maintain stem cell-like clones. We confirmed that the obtained clones comprised p63/Krt5 double-positive ABSCs. In continuous passage and maintenance culture, we found that it could be subculture to at least 15 passages in vitro, stably maintaining its stem cell morphology, basal cell markers, and in vitro expansion and self-renewal capabilities. Meanwhile, through in vitro differentiation culture and 3D tracheosphere culture, we found that in addition to maintaining self-renewal, mouse ABSCs could differentiate into other airway epithelial cells such as acetylated tubulin (Act-Tub) + ciliated and MUC5AC + mucus-secreting cells. However, they failed to differentiate into alveoli epithelial cells, including alveolar type I and alveolar type II.ConclusionWe established an in vitro feeder-free culture system that allows mouse ABSCs to maintain their stem cell characteristics, including self-renewal and airway epithelium differentiation potential, while keeping up in vitro expansion stability.

Antenatal Endotoxin Induces Dysanapsis in Experimental Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, is characterized by impaired lung development with sustained functional abnormalities due to alterations of airways and the distal lung. Although clinical studies have shown striking associations between antenatal stress and BPD, little is known about the underlying pathogenetic mechanisms. Whether dysanapsis, the concept of discordant growth of the airways and parenchyma, contributes to late respiratory disease as a result of antenatal stress is unknown. We hypothesized that antenatal endotoxin (ETX) impairs juvenile lung function as a result of altered central airway and distal lung structure, suggesting the presence of dysanapsis in this preclinical BPD model. Fetal rats were exposed to intraamniotic ETX (10 μg) or saline solution (control) 2 days before term. We performed extensive structural and functional evaluation of the proximal airways and distal lung in 2-week-old rats. Distal lung structure was quantified by stereology. Conducting airway diameters were measured using micro-computed tomography. Lung function was assessed during invasive ventilation to quantify baseline mechanics, response to methacholine challenge, and spirometry. ETX-exposed pups exhibited distal lung simplification, decreased alveolar surface area, and decreased parenchyma-airway attachments. ETX-exposed pups exhibited decreased tracheal and second- and third-generation airway diameters. ETX increased respiratory system resistance and decreased lung compliance at baseline. Only Newtonian resistance, specific to large airways, exhibited increased methacholine reactivity in ETX-exposed pups compared with controls. ETX-exposed pups had a decreased ratio of FEV in 0.1 second to FVC and a normal FEV in 0.1 second, paralleling the clinical definition of dysanapsis. Antenatal ETX causes abnormalities of the central airways and distal lung growth, suggesting that dysanapsis contributes to abnormal lung function in juvenile rats.