Differential expression of mast cells in the small airways and alveolar septa of current smokers and patients with small airway disease and COPD.

Background: COPD patients suffer from dysregulated and suppressed immune functionality, determined by their loss of degranulating capacity. Here we provide crucial information on the presence of degranulated mast cells (MCs) in COPD airways and demonstrate their relationship to lung physiology and airway remodelling. Methods: Small airway lung resections from non-smoking controls (NC), normal lung function smokers (NLFS), small airway disease (SAD), and mild-to-moderate COPD current smokers (COPD-CS) and ex-smokers (COPD-ES) were dual immuno-stained with MC tryptase and degranulation marker lysosome-associated membrane protein (LAMP)-1. Total MCs, degranulating MCs and non-MCs were enumerated in small airway epithelium and subepithelium, and in alveolar septa. Results: In the small airway wall subepithelial areas, COPD-CS and COPD-ES patients had significantly lower MCs than the NC group (p<0.05), although the numbers were considerably higher in the small airway epithelium (p<0.01). Degranulating non-MCs were higher in SAD (p<0.05) than in COPD in the small airway subepithelium. In contrast, there were significant increases in total MCs (degranulated and non-degranulated) and degranulated non-MCs in the alveolar septum of COPD patients compared with the NC group (p<001). The lower numbers of MCs in the subepithelium correlated with lower forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) and forced expiratory flow at 25-75% of FVC (FEF25-75%), higher smoking rates in COPD patients, and increased small airway wall thickness and extracellular matrix. The increase in MCs in the alveolar septum negatively correlated with FEF25-75%. Conclusions: This study is the first to assess the differential pattern of MC, degranulating MC and non-MC populations in the small airways and alveoli of COPD patients. The spatial positioning of the MCs within the airways showed variable correlations with lung function.

Endothelial to mesenchymal transition is an active process in smokers and patients with early COPD contributing to pulmonary arterial pathology.

Background: We have previously reported pulmonary arterial remodelling in smokers and patients with early COPD, which can be attributed to endothelial to mesenchymal transition (EndMT). In this study, we aimed to evaluate if EndMT is an active mechanism in smokers and COPD. Methods: Immunohistochemical staining for the EndMT biomarkers CD31, N-cadherin, vimentin and S100A4 was done on lung resection tissue from 49 subjects. These comprised 15 nonsmoker controls (NC), six normal lung function smokers (NLFS), nine patients with small airway disease (SAD), nine current smokers with mild-moderate COPD (COPD-CS) and 10 ex-smokers with COPD (COPD-ES). Pulmonary arteries were analysed using Image ProPlus software v7.0. Results: We noted reduced junctional CD31+ endothelial cells (p<0.05) in the intimal layer of all smoking groups compared to NC. We also observed increased abundance of the mesenchymal markers N-cadherin (p<0.05) and vimentin (p<0.001) in all smoking groups and across all arterial sizes versus NC, except for N-cadherin in large arteries in COPD-CS. The abundance of S100A4 correlated with arterial thickness (small: r=0.29, p=0.05; medium: r=0.33, p=0.03; large: r=0.35, p=0.02). Vimentin in the small arterial wall negatively correlated with forced expiratory volume in 1 s/forced vital capacity (r= -0.35, p=0.02) and forced expiratory flow rate at 25-75% of forced vital capacity (r= -0.34, p=0.03), while increased cytoplasmic CD31 abundance in the intimal layer of medium and large arteries negatively correlated with predicted diffusing capacity of the lung for carbon monoxide (medium: r= -0.35, p=0.04; large: r= -0.39, p=0.03). Conclusion: This is the first study showing the acquisition of mesenchymal traits by pulmonary endothelial cells from NLFS, SAD and mild-moderate COPD patients through EndMT. This informs on the potential early origins of pulmonary hypertension in smokers and patients with early COPD.

TGF-ß1, pSmad-2/3, Smad-7, and ß-Catenin Are Augmented in the Pulmonary Arteries from Patients with Idiopathic Pulmonary Fibrosis (IPF): Role in Driving Endothelial-to-Mesenchymal Transition (EndMT).

Background: We have previously reported that endothelial-to-mesenchymal transition (EndMT) is an active process in patients with idiopathic pulmonary fibrosis (IPF) contributing to arterial remodelling. Here, we aim to quantify drivers of EndMT in IPF patients compared to normal controls (NCs). Methods: Lung resections from thirteen IPF patients and eleven NCs were immunohistochemically stained for EndMT drivers, including TGF-ß1, pSmad-2/3, Smad-7, and ß-catenin. Intima, media, and adventitia were analysed for expression of each EndMT driver in pulmonary arteries. Computer- and microscope-assisted Image ProPlus7.0 image analysis software was used for quantifications. Results: Significant TGF-ß1, pSmad-2/3, Smad-7, and ß-catenin expression was apparent across all arterial sizes in IPF (p < 0.05). Intimal TGF-ß1, pSmad-2/3, Smad-7, and ß-catenin were augmented in the arterial range of 100-1000 µm (p < 0.001) compared to NC. Intimal TGF-ß1 and ß-catenin percentage expression showed a strong correlation with the percentage expression of intimal vimentin (r' = 0.54, p = 0.05 and r' = 0.61, p = 0.02, respectively) and intimal N-cadherin (r' = 0.62, p = 0.03 and r' = 0.70, p = 0.001, respectively). Intimal TGF-ß1 and ß-catenin expression were significantly correlated with increased intimal thickness as well (r' = 0.52, p = 0.04; r' = 0.052, p = 0.04, respectively). Moreover, intimal TGF-ß1 expression was also significantly associated with increased intimal elastin deposition (r' = 0.79, p = 0.002). Furthermore, total TGF-ß1 expression significantly impacted the percentage of DLCO (r' = -0.61, p = 0.03). Conclusions: This is the first study to illustrate the involvement of active TGF-ß/Smad-2/3-dependent and ß-catenin-dependent Wnt signalling pathways in driving EndMT and resultant pulmonary arterial remodelling in patients with IPF. EndMT is a potential therapeutic target for vascular remodelling and fibrosis in general in patients with IPF.

Epithelial-mesenchymal transition changes in nonsmall cell lung cancer patients with early COPD.

Background: Epithelial-mesenchymal transition (EMT) might be central to lung cancer development in smokers and COPD. We illustrate EMT changes in a broader demographic of patient groups who were diagnosed with nonsmall cell lung cancer (adenocarcinoma and squamous cell carcinoma). These included COPD current and ex-smokers, patients with small airway disease and normal lung function smokers compared to normal controls. Methods: We had access to surgically resected small airway tissue from 46 subjects and assessed for airway wall thickness and immunohistochemically for the EMT biomarkers E-cadherin, N-cadherin, S100A4, vimentin and epidermal growth factor receptor (EGFR). All tissue analysis was done with a computer and microscope-assisted Image-Pro Plus 7.0 software. Results: Airway wall thickness significantly increased across all pathological groups (p<0.05) compared to normal controls. Small airway epithelial E-cadherin expression markedly decreased (p<0.01), and increases in N-cadherin, vimentin, S100A4 and EGFR expression were observed in all pathological groups compared to normal controls (p<0.01). Vimentin-positive cells in the reticular basement membrane, lamina propria and adventitia showed a similar trend to epithelium across all pathological groups (p<0.05); however, such changes were only observed in reticular basement membrane for S100A4 (p<0.05). Vimentin was higher in adenocarcinoma versus squamous cell carcinoma; in contrast, S100A4 was higher in the squamous cell carcinoma group. EGFR and N-cadherin expression in both phenotypes was markedly higher than E-cadherin, vimentin and S100A4 (p<0.0001). Conclusion: EMT is an active process in the small airway of smokers and COPD diagnosed with nonsmall cell lung cancer, contributing to small airway remodelling and cancer development as seen in these patients.

Airway inflammatory changes in the lungs of patients with asthma-COPD overlap (ACO): a bronchoscopy endobronchial biopsy study.

BACKGROUND: Although asthma and chronic obstructive pulmonary disease (COPD) are two distinct chronic airway inflammatory diseases, they often co-exist in a patient and the condition is referred to as asthma-COPD overlap (ACO). Lack of evidence regarding the inflammatory cells in ACO airways has led to their poor prognosis and treatment. The objective of this endobronchial biopsy (EBB) study was to enumerate inflammatory cellular changes in the airway wall of ACO compared with asthma, COPD current smokers (CS) and ex-smokers (ES), normal lung function smokers (NLFS), and non-smoker controls (HC). METHODS: EBB tissues from 74 patients were immunohistochemically stained for macrophages, mast cells, eosinophils, neutrophils, CD8+ T-cells and CD4+ T-cells. The microscopic images of stained tissues were evaluated in the epithelium, reticular basement membrane (RBM) cells/mm RBM length, and lamina propria (LP) cells/mm2 up to a depth of 120 µM using the image analysis software Image-Pro Plus 7.0. The observer was blinded to the images and disease diagnosis. Statistical analysis was performed using GraphPad Prism v9. RESULTS: The tissue macrophages in ACO were substantially higher in the epithelium and RBM than in HC (P < 0.001 for both), COPD-ES (P < 0.001 for both), and -CS (P < 0.05 and < 0.0001, respectively). The ACO LP macrophages were significantly higher in number than COPD-CS (P < 0.05). The mast cell numbers in ACO were lower than in NLFS (P < 0.05) in the epithelium, lower than COPD (P < 0.05) and NLFS (P < 0.001) in RBM; and lower than  HC (P < 0.05) in LP. We noted lower eosinophils in ACO LP than HC (P < 0.05) and the lowest neutrophils in both ACO and asthma. Furthermore, CD8+ T-cell numbers increased in the ACO RBM than HC (P < 0.05), COPD-ES (P < 0.05), and NLFS (P < 0.01); however, they were similar in number in epithelium and LP across groups. CD4+ T-cells remained lower in number across all regions and groups. CONCLUSION: These results suggest that the ACO airway tissue inflammatory cellular profile differed from the contributing diseases of asthma and COPD with a predominance of macrophages.

Transforming growth factor-ß1 and SMAD signalling pathway in the small airways of smokers and patients with COPD: potential role in driving fibrotic type-2 epithelial mesenchymal transition.

Background: COPD is a common disease characterized by respiratory airflow obstruction. TGF-ß1 and SMAD pathway is believed to play a role in COPD pathogenesis by driving epithelial mesenchymal transition (EMT). Methods: We investigated TGF-ß1 signalling and pSmad2/3 and Smad7 activity in resected small airway tissue from patients with; normal lung function and a smoking history (NLFS), current smokers and ex-smokers with COPD GOLD stage 1 and 2 (COPD-CS and COPD-ES) and compared these with normal non-smoking controls (NC). Using immunohistochemistry, we measured activity for these markers in the epithelium, basal epithelium, and reticular basement membrane (RBM). Tissue was also stained for EMT markers E-cadherin, S100A4 and vimentin. Results: The Staining of pSMAD2/3 was significantly increased in the epithelium, and RBM of all COPD groups compared to NC (p <0.0005). There was a less significant increase in COPD-ES basal cell numbers compared to NC (p= 0.02). SMAD7 staining showed a similar pattern (p <0.0001). All COPD group levels of TGF-ß1 in the epithelium, basal cells, and RBM cells were significantly lower than NC (p <0.0001). Ratio analysis showed a disproportionate increase in SMAD7 levels compared to pSMAD2/3 in NLFS, COPD-CS and COPD-ES. pSMAD negatively correlated with small airway calibre (FEF25-75%; p= 0.03 r= -0.36). EMT markers were active in the small airway epithelium of all the pathological groups compared to patients with COPD. Conclusion: Activation of the SMAD pathway via pSMAD2/3 is triggered by smoking and active in patients with mild to moderate COPD. These changes correlated to decline in lung function. Activation of the SMADs in the small airways is independent of TGF-ß1, suggesting factors other than TGF-ß1 are driving these pathways. These factors may have implications for small airway pathology in smokers and COPD through the process of EMT, however more mechanistic work is needed to prove these correlations.

Endothelial-to-mesenchymal transition: a precursor to pulmonary arterial remodelling in patients with idiopathic pulmonary fibrosis.

Background: We have previously reported arterial remodelling in patients with idiopathic pulmonary fibrosis (IPF) and suggested that endothelial-to-mesenchymal transition (EndMT) might be central to these changes. This study aims to provide evidence for active EndMT in IPF patients. Methods: Lung resections from 13 patients with IPF and 15 normal controls (NCs) were immunostained for EndMT biomarkers: vascular endothelial cadherin (VE-cadherin), neural cadherin (N-cadherin), S100A4 and vimentin. Pulmonary arteries were analysed for EndMT markers by using computer- and microscope-assisted image analysis software Image ProPlus7.0. All the analysis was done with observer blinded to subject and diagnosis. Results: Increased expression of mesenchymal markers N-cadherin (p<0.0001), vimentin (p<0.0001) and S100A4 (p<0.05) was noted with downregulation of junctional endothelial VE-cadherin (p<0.01) in the intimal layer of the arteries from patients with IPF compared to NCs. Cadherin switch was observed in IPF patients, showing increase in endothelial N-cadherin and decrease in VE-cadherin (p<0.01). There was also VE-cadherin shift from junctions to cytoplasm (p<0.01), effecting endothelial cell integrity in patients with IPF. In IPF, individual mesenchymal markers vimentin and N-cadherin negatively correlated with diffusing capacity of the lungs for carbon monoxide (r'= -0.63, p=0.03 and r'= -0.66, p=0.01). Further, N-cadherin positively correlated with arterial thickness (r'=0.58, p=0.03). Conclusion: This is the first study to demonstrate active EndMT in size-based classified pulmonary arteries from IPF patients and potential role in driving remodelling changes. The mesenchymal markers had a negative impact on the diffusing capacity of the lungs for carbon monoxide. This work also informs early origins of pulmonary hypertension in patients with IPF.

Arterial remodelling in smokers and in patients with small airway disease and COPD: implications for lung physiology and early origins of pulmonary hypertension.

Introduction: Pulmonary vascular remodelling in chronic obstructive pulmonary disease (COPD) has detrimental consequences for lung physiology. The aim of our study was to provide a comprehensive size-based morphometric quantification of pulmonary arterial remodelling in smokers and in patients with small airway disease (SAD) or COPD. Method: Movat's pentachrome staining was performed on lung resections for 46 subjects: 12 never-smoker normal controls (NC), six normal lung function smokers (NLFS), nine patients with SAD, nine patients with mild-to-moderate COPD who were current smokers (COPD-CS) and 10 patients with mild-to-moderate COPD who were ex-smokers (COPD-ES). Following a size-based classification of pulmonary arteries, image analysis software was used to measure their number, total wall thickness, individual layer thickness and elastin percentage. Results: All pathological groups showed decreased numbers of pulmonary arteries compared with the NC group in all artery sizes. Arterial wall thickness was greater in NLFS and COPD-CS than in NC. Thickness in COPD-ES was decreased compared with COPD-CS. Intimal thickness was greater in all pathological groups in all arterial sizes than in the NC group. Medial thickness was also greater in small and medium arteries. Intimal thickness of larger arteries in COPD-CS correlated negatively to forced expiratory volume in 1 s/forced vital capacity (FVC) % and forced expiratory flow at 25-75% of FVC. Elastin deposition in small arteries was greatest in COPD-CS. Intimal elastin deposition had a more negative correlation with intimal thickness in NLFS and SAD than in COPD-CS. Conclusion: Smoking, SAD and mild-to-moderate COPD are associated with pruning and a decrease in the number of pulmonary arteries, increased wall thickness and variable elastin deposition. These changes were associated with worse airway obstruction.

Differential airway remodeling changes were observed in patients with asthma COPD overlap compared to patients with asthma and COPD alone.

Management of patients with asthma COPD overlap (ACO) is clinically challenging due to insufficient evidence of pathological changes in these patients. In this cross-sectional study, we evaluated airway remodeling in endobronchial biopsies from a total of 90 subjects, which included 12 ACO, 14 patients with asthma, 12 COPD exsmokers (ES), 11 current smokers (CS), 28 healthy controls (HC), and 13 normal lung function smokers (NLFS). Tissue was stained with Masson's trichrome. Epithelium, goblet cells, reticular basement membrane (RBM), cellularity, lamina propria (LP), and smooth muscle (SM) changes were measured using Image-Pro Plus v7 software. Differential airway remodeling pattern was seen in patients with ACO. A limited change was noted in the ACO epithelium compared with other pathological groups. RBM was substantially thicker in patients with ACO than in HC (P < 0.0002) and tended to be thicker than in patients with asthma and NLFS. The total RBM cells were higher in ACO than in the HC (P < 0.0001), COPD-CS (P = 0.0559), -ES (P = 0.0345), and NLFS (P < 0.0002), but did not differ from patients with asthma. Goblet cells were higher in the ACO than in the HC (P = 0.0028) and COPD-ES (P = 0.0081). The total LP cells in ACO appeared to be higher than in HC, COPD-CS, and NLFS but appeared to be lower than in patients with asthma. Finally, SM area was significantly lower in the ACO than in patients with asthma (P = 0.001), COPD-CS (=0.0290), and NLFS (P = 0.0011). This first comprehensive study suggests that patients with ACO had distinguishable tissue remodeling that appeared to be more severe than patients with asthma and COPD. This study will help in informed decision-making for better patient management in clinical practice.

Development of an Australia and New Zealand Lung Cancer Clinical Quality Registry: a protocol paper.

INTRODUCTION: Lung cancer is the leading cause of cancer mortality, comprising the largest national cancer disease burden in Australia and New Zealand. Regional reports identify substantial evidence-practice gaps, unwarranted variation from best practice, and variation in processes and outcomes of care between treating centres. The Australia and New Zealand Lung Cancer Registry (ANZLCR) will be developed as a Clinical Quality Registry to monitor the safety, quality and effectiveness of lung cancer care in Australia and New Zealand. METHODS AND ANALYSIS: Patient participants will include all adults >18 years of age with a new diagnosis of non-small-cell lung cancer (NSCLC), SCLC, thymoma or mesothelioma. The ANZLCR will register confirmed diagnoses using opt-out consent. Data will address key patient, disease, management processes and outcomes reported as clinical quality indicators. Electronic data collection facilitated by local data collectors and local, state and federal data linkage will enhance completeness and accuracy. Data will be stored and maintained in a secure web-based data platform overseen by registry management. Central governance with binational representation from consumers, patients and carers, governance, administration, health department, health policy bodies, university research and healthcare workers will provide project oversight. ETHICS AND DISSEMINATION: The ANZLCR has received national ethics approval under the National Mutual Acceptance scheme. Data will be routinely reported to participating sites describing performance against measures of agreed best practice and nationally to stakeholders including federal, state and territory departments of health. Local, regional and (bi)national benchmarks, augmented with online dashboard indicator reporting will enable local targeting of quality improvement efforts.

Vascular remodelling in idiopathic pulmonary fibrosis patients and its detrimental effect on lung physiology: potential role of endothelial-to-mesenchymal transition.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible fibrotic interstitial lung disease. We performed size-based quantitation of pulmonary arterial remodelling in IPF and examined the role of endothelial-to-mesenchymal transition (EndMT) and effects on lung physiology. Methods: Resected lung tissues from 11 normal controls (NCs), and 13 IPF patients were differentially stained using the Movat Pentachrome technique. Size-based classification for pulmonary arteries was conducted in NC and IPF tissues. For each pulmonary artery, arterial size, luminal diameter, thickness of the intima, media and adventitia, and elastin deposition were quantified using Image ProPlus7.0 software. In addition, immunohistochemical staining was performed for EndMT markers and collagen. Results: Large and medium-size arterial numbers were significantly reduced in IPF compared to NCs (p<0.0001). Intima thickness was highest in the arterial range of 200-399 µm and 600-1000 µm (p<0.0001), while medial and adventitial thickness was significant across 200-1000 µm (p<0.05) compared to NC. Medial thickness was found to significantly affect the diffusing capacity of the lungs for carbon monoxide (D LCO) (r=-0.8, p=0.01). Total arterial elastin in IPF was higher across all arterial ranges except 100-199 µm in IPF than in NC, with the greatest differences in 200-399 µm (p<0.001) and 600-1000 µm (p<0.001). Total elastin also negatively correlated with D LCO (r'=-0.63, p=0.04) in IPF. An increase in EndMT markers and collagen type I/ IV was observed. Conclusions: This is the first study demonstrating size-based differences in pulmonary arteries in IPF and its detrimental effect on lung physiology. The process of EndMT might be central to these vascular remodelling changes and could be a potential novel therapeutic target.

Angiotensin-Converting Enzyme 2 (ACE2), Transmembrane Peptidase Serine 2 (TMPRSS2), and Furin Expression Increases in the Lungs of Patients with Idiopathic Pulmonary Fibrosis (IPF) and Lymphangioleiomyomatosis (LAM): Implications for SARS-CoV-2 (COVID-19) Infections.

We previously reported higher ACE2 levels in smokers and patients with COPD. The current study investigates if patients with interstitial lung diseases (ILDs) such as IPF and LAM have elevated ACE2, TMPRSS2, and Furin levels, increasing their risk for SARS-CoV-2 infection and development of COVID-19. Surgically resected lung tissue from IPF, LAM patients, and healthy controls (HC) was immunostained for ACE2, TMPRSS2, and Furin. Percentage ACE2, TMPRSS2, and Furin expression was measured in small airway epithelium (SAE) and alveolar areas using computer-assisted Image-Pro Plus 7.0 software. IPF and LAM tissue was also immunostained for myofibroblast marker α-smooth muscle actin (α-SMA) and growth factor transforming growth factor beta1 (TGF-ß1). Compared to HC, ACE2, TMPRSS2 and Furin expression were significantly upregulated in the SAE of IPF (p < 0.01) and LAM (p < 0.001) patients, and in the alveolar areas of IPF (p < 0.001) and LAM (p < 0.01). There was a significant positive correlation between smoking history and ACE2 expression in the IPF cohort for SAE (r = 0.812, p < 0.05) and alveolar areas (r = 0.941, p < 0.01). This, to our knowledge, is the first study to compare ACE2, TMPRSS2, and Furin expression in patients with IPF and LAM compared to HC. Descriptive images show that α-SMA and TGF-ß1 increase in the IPF and LAM tissue. Our data suggests that patients with ILDs are at a higher risk of developing severe COVID-19 infection and post-COVID-19 interstitial pulmonary fibrosis. Growth factors secreted by the myofibroblasts, and surrounding tissue could further affect COVID-19 adhesion proteins/cofactors and post-COVID-19 interstitial pulmonary fibrosis. Smoking seems to be the major driving factor in patients with IPF.

SARS-CoV-2 (COVID-19) Adhesion Site Protein Upregulation in Small Airways, Type 2 Pneumocytes, and Alveolar Macrophages of Smokers and COPD - Possible Implications for Interstitial Fibrosis.

Background: Smokers and patients with COPD are highly susceptible to SARS-CoV-2 infection, leading to severe COVID-19. Methods: This cross-sectional study involved resected lung tissues from 16 patients with GOLD stage I or II COPD; of which 8 were current smokers COPD (COPD-CS), and 8 ex-smokers COPD (COPD-ES), 7 normal lung function smokers (NLFS), 9 patients with small airways disease (SAD), and 10 were never-smoking normal controls (NC). Immunostaining for ACE2, Furin, and TMPRSS2 was performed and analysed for percent expression in small airway epithelium (SAE) and counts for positively and negatively stained type 2 pneumocytes and alveolar macrophages (AMs) were done using Image ProPlus V7.0. Furthermore, primary small airway epithelial cells (pSAEC) were analysed by immunofluorescence after exposure to cigarette smoke extract (CSE). Results: ACE2, Furin, and TMPRSS2 expression significantly increased in SAE and type 2 pneumocytes in all the subjects (except Furin for NLFS) compared to NC (p < 0.001). Similar significance was observed for ACE2 positive AM (p < 0.002), except COPD-ES, which decreased in ACE2 positive AMs (p < 0.003). Total type 2 pneumocytes and AMs significantly increased in the pathological groups compared to NC (p < 0.01), except SAD (p = 0.08). However, AMs are significantly reduced in COPD-ES (p < 0.003). Significant changes were observed for tissue co-expression of Furin and TMPRSS2 with ACE2 in SAE, type 2 pneumocytes and AMs. These markers also negatively correlated with lung function parameters, such as FEV1/FVC % predicted, FEF25-75%, DLCO% predicted. A strong co-localisation and expression for ACE2 (p < 0.0001), Furin (p < 0.01), and TMPRSS2 (p < 0.0001) was observed in pSAEC treated with 1% CSE than controls. Discussion: The increased expression of ACE2, TMPRSS2 and Furin, in the SAE, type 2 pneumocytes and AMs of smokers and COPD are detrimental to lung function and proves that these patient groups could be more susceptible to severe COVID-19 infection. Increased type 2 pneumocytes suggest that these patients are vulnerable to developing post-COVID-19 interstitial pulmonary fibrosis or fibrosis in general. There could be a silently developing interstitial pathology in smokers and patients with COPD. This is the first comprehensive study to report such changes.

Pathogenesis, clinical features of asthma COPD overlap, and therapeutic modalities.

Both asthma and COPD are heterogeneous diseases identified by characteristic symptoms and functional abnormalities, with airway obstruction common in both diseases. Asthma COPD overlap (ACO) does not define a single disease but is a descriptive term for clinical use that includes several overlapping clinical phenotypes of chronic airways disease with different underlying mechanisms. This literature review was initiated to describe published studies, identify gaps in knowledge, and propose future research goals regarding the disease pathology of ACO, especially the airway remodeling changes and inflammation aspects. Airway remodeling occurs in asthma and COPD, but there are differences in the structures affected and the prime anatomic site at which they occur. Reticular basement membrane thickening and cellular infiltration with eosinophils and T-helper (CD4+) lymphocytes are prominent features of asthma. Epithelial squamous metaplasia, airway wall fibrosis, emphysema, bronchoalveolar lavage (BAL) neutrophilia, and (CD8+) T-cytotoxic lymphocyte infiltrations in the airway wall are features of COPD. There is no universally accepted definition of ACO, nor are there clearly defined pathological characteristics to differentiate from asthma and COPD. Understanding etiological concepts within the purview of inflammation and airway remodeling changes in ACO would allow better management of these patients.

Electronic Cigarette Aerosol Is Cytotoxic and Increases ACE2 Expression on Human Airway Epithelial Cells: Implications for SARS-CoV-2 (COVID-19).

Tobacco smoking has emerged as a risk factor for increasing the susceptibility to infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via increased expression of angiotensin-converting enzyme-2 (ACE2) in the lung, linked to coronavirus disease 2019 (COVID-19) development. Given the modifiable nature of electronic cigarettes and the delivery of high concentrations of nicotine, we investigate whether electronic cigarette vaping has the potential to increase susceptibility to SARS-CoV-2 infection. We exposed BEAS-2B cells (bronchial epithelium transformed with Ad12-SV40 2B) and primary small airway epithelial cells (SAECs) to electronic cigarette aerosol condensates produced from propylene glycol/vegetable glycerin or commercially bought e-liquid (±added nicotine) and cigarette smoke extract to investigate if electronic cigarette exposure, like cigarette smoke, increases the expression of ACE2 in lung epithelial cells. In BEAS-2B cells, cytotoxicity (CCK-8), membrane integrity (LDH), and ACE2 protein expression (immunofluorescence) were measured for both 4- and 24 h treatments in BEAS-2B cells and 4 h in SAECs; ACE2 gene expression was measured using quantitative polymerase chain reaction (qPCR) for 4 h treatment in BEAS-2B cells. Nicotine-free condensates and higher concentrations of nicotine-containing condensates were cytotoxic to BEAS-2B cells. Higher LDH release and reduced membrane integrity were seen in BEAS-2B cells treated for 24 h with higher concentrations of nicotine-containing condensates. ACE2 protein expression was observably increased in all treatments compared to cell controls, particularly for 24 h exposures. ACE2 gene expression was significantly increased in cells exposed to the locally bought e-liquid condensate with high nicotine concentration and cigarette smoke extract compared with cell controls. Our study suggests that vaping alone and smoking alone can result in an increase in lung ACE2 expression. Vaping and smoking are avoidable risk factors for COVID-19, which, if avoided, could help reduce the number of COVID-19 cases and the severity of the disease. This is the first study to utilize electronic cigarette aerosol condensates, novel and developed in our laboratory, for investigating ACE2 expression in human airway epithelial cells.

Diagnostic approaches in COVID-19: clinical updates.

INTRODUCTION: COVID-19 is a recent emerging pandemic whose prognosis is still unclear. Diagnostic tools are the main players that not only indicate a possible infection but can further restrict the transmission and can determine the extent to which disease progression would occur. AREAS COVERED: In this paper, we have performed a narrative and critical review on different technology-based diagnostic strategies such as molecular approaches including real-time reverse transcriptase PCR, serological testing through enzyme-linked immunosorbent assay, laboratory and point of care devices, radiology-based detection through computed tomography and chest X-ray, and viral cell cultures on Vero E6 cell lines are discussed in detail to address COVID-19. This review further provides an overview of emergency use authorized immunodiagnostic and molecular diagnostic kits and POC devices by FDA for timely and efficient conduction of diagnostic tests. The majority of the literature cited in this paper is collected from guidelines on protocols and other considerations on diagnostic strategies of COVID-19 issued by WHO, CDC, and FDA under emergency authorization. EXPERT OPINION: Such information holds importance to the health professionals in conducting error-free diagnostic tests and researches in producing better clinical strategies by addressing the limitations associated with the available methods.

Dysregulation of endocytic machinery and ACE2 in small airways of smokers and COPD patients can augment their susceptibility to SARS-CoV-2 (COVID-19) infections.

Lungs of smokers and chronic obstructive pulmonary disease (COPD) are severely compromised and are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attack. The dangerous combination of enhanced SARS-CoV-2 attachment receptor protein ACE2 along with an increase in endocytic vacuoles will enable viral attachment, entry, and replication. The objective of the study was to identify the presence of SARS-CoV-2 host attachment receptor angiotensin-converting enzyme-2 (ACE2) along with endocytic vacuoles, early endosome antigen-1 (EEA1), late endosome marker RAB7, cathepsin-L, and lysosomal associated membrane protein-1 (LAMP-1) as lysosome markers in the airways of smokers and COPD patients. The study design was cross-sectional and involved lung resections from 39 patients in total, which included 19 patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I or GOLD stage II COPD, of which 9 were current smokers with COPD (COPD-CS) and 10 were ex-smokers with COPD (COPD-ES), 10 were normal lung function smokers, and 10 were never-smoking normal controls. Immunostaining for ACE2, EEA1, RAB7, and cathepsin-L was done. A comparative description for ACE2, EEA1, RAB7, and cathepsin-L expression pattern is provided for the patient groups. Furthermore, staining intensity for LAMP-1 lysosomes was measured as the ratio of the LAMP-1-stained areas per total area of epithelium or subepithelium, using Image ProPlus v7.0 software. LAMP-1 expression showed a positive correlation to patient smoking history while in COPD LAMP-1 negatively correlated to lung function. The active presence of ACE2 protein along with endocytic vacuoles such as early/late endosomes and lysosomes in the small airways of smokers and COPD patients provides evidence that these patient groups could be more susceptible to COVID-19.

Clinical Application of Forced Oscillation Technique (FOT) in Early Detection of Airway Changes in Smokers.

The forced oscillation technique (FOT) is a non-invasive method to assess airway function by emitting oscillatory signals into the respiratory tract during tidal ventilation. This opinion piece discusses the current use, trialled modification and future directions in utilizing FOT as a novel diagnostic tool for early detection of small airway changes in smokers. The published evidence to date has shown that FOT parameters could be a sensitive diagnostic tool to detect early respiratory changes in smokers. Multiple frequencies and the frequency dependence of resistance and reactance can provide the most valuable and early information regarding smoking induced changes in airways. Considering its non-invasiveness, lower level of discomfort to patients than spirometry, feasibility, and cost effectiveness, it could be the first-choice diagnostic technique for detection of early respiratory changes in smokers. The finding of FOT could further be supported and correlated with inflammatory markers.