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Characterize and quantify epithelium in multiple etiologies of laryngotracheal stenosis (LTS) to better understand its role in pathogenesis.Controlled in vitro cohort study.Endoscopic brush biopsy samples of both normal (non-scar) and scar were obtained in four patients with idiopathic subglottic stenosis (iSGS) and four patients with iatrogenic LTS (iLTS). mRNA expression of basal, ciliary, and secretory cell markers were evaluated using quantitative PCR. Cricotracheal resection tissue samples (n = 5 per group) were also collected, analyzed using quantitative immunohistochemistry, and compared with rapid autopsy tracheal samples.Both iSGS and iLTS-scar epithelium had reduced epithelial thickness compared with non-scar control epithelium (P = .0009 and P = .0011, respectively). Basal cell gene and protein expression for cytokeratin 14 was increased in iSGS-scar epithelium compared with iLTS or controls. Immunohistochemical expression of ciliary tubulin alpha 1, but not gene expression, was reduced in both iSGS and iLTS-scar epithelium compared with controls (P = .0184 and P = .0125, respectively). Both iSGS and iLTS-scar had reductions in Mucin 5AC gene expression (P = .0007 and P = .0035, respectively), an epithelial goblet cell marker, with reductions in secretory cells histologically (P.0001).Compared with non-scar epithelium, the epithelium within iSGS and iLTS is morphologically abnormal. Although both iSGS and iLTS have reduced epithelial thickness, ciliary cells, and secretory cells, only iSGS had significant increases in pathological basal cell expression. These data suggest that the epithelium in iSGS and iLTS play a common role in the pathogenesis of fibrosis in these two etiologies of laryngotracheal stenosis.Tertiary referral center (2017-2020).NA Laryngoscope, 132:2194-2201, 2022.

Ghosh B, Majumder S, Acharyya S, Ghosh A, Saha S, Sarkar S, Chakraborty S, Bhattacharya M. 2022. Comparative phytochemical analysis of mature mango leaves from nineteen cultivars of Murshidabad district, India. Asian J Nat Prod Biochem 20: 48-55. Mango (Mangifera indica L.), "the king of fruits," is one of the most popular fruits in tropical regions. This research aimed to qualitatively and quantitatively screen major phytochemical groups present in the leaves of nineteen cultivars of mango tree extracted with three different solvents (petroleum benzene, acetone, methanol) belonging to different polarities and to determine their antibacterial activity against two Gram-positive (Bacillus subtilis, Staphylococcus aureus) and two Gram-negative (Escherichia coli, Klebsiella pneumoniae) bacteria. The antiradical scavenging activity was performed using a DPPH assay. In addition, total phenol and flavonoids in the leaf extracts were also quantitatively analyzed. Phytochemical investigations showed that mango leaf extracts contain phenol, tannin, protein, coumarin, terpenoid, alkaloid, steroid, and cardiac glycoside. The extracts also contain a variety of total phenols and flavonoids. Antiradical scavenging activity showed that polar solvents (methanol, acetone) are more potent than non-polar (petroleum benzene) extracts. Mango leaf extracts inhibit the growth of E. coli, K. pneumoniae, and S. aureus, but no inhibition zone against B. subtilis. Based on the phytochemical compounds and significant antioxidant and antibacterial properties of mango leaf extracts, mango leaves might be a potential source for developing pharmaceutical formulations and drugs.

Subclinical hypothyroidism (SCH) is a condition where serum thyroid-stimulating hormone (TSH) level is high, but the T3 and T4 are within normal level. SCH carries a risk of cardiovascular diseases or progression to overt hypothyroidism. Treatment becomes more necessary in case of older age groups and in females. Two cases of SCH treated with individualised homoeopathic medicine are presented and the improvement was significant showing reduction in the TSH levels with overall improvement in health.

Air-liquid interface (ALI) cultures are ex vivo models that are used extensively to study the epithelium of patients with chronic respiratory diseases. However, the in vitro conditions impose a milieu different from that encountered in the patient in vivo, and the degree to which this alters gene expression remains unclear. In this study we employed RNA sequencing to compare the transcriptome of fresh brushings of nasal epithelial cells with that of ALI-cultured epithelial cells from the same patients. We observed a strong correlation between cells cultured at the ALI and cells obtained from the brushed nasal epithelia: 96% of expressed genes showed similar expression profiles, although there was greater similarity between the brushed samples. We observed that while the ALI model provides an excellent representation of the in vivo airway epithelial transcriptome for mechanistic studies, several pathways are affected by the change in milieu.

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, characterized by a progressive decline in lung function, alveolar loss (emphysema), and airflow limitation due to excessive mucus secretion (chronic bronchitis), that can occur even after the injurious agent is removed. It is slated to rise to the 3rd leading cause of death due to chronic disease by 2030 globally, and the 4th leading cause of death due to chronic disease in the USA. While there is substantial evidence indicating loss of E-cadherin in the lung epithelium of patients with COPD, it is not known if this is causal to the disease. We investigated if loss of E-cadherin can result in lung disease using in both in vitro models of primary, differentiated human cells and in mouse models. Using a cell type-specific promoter using Cre/LoxP mice system to knock-out E- cadherin in ciliated and alveolar epithelial cell (Type 1 and Type 2) populations in adult mouse models, we determined that loss of E-cadherin caused airspace enlargement, as well as increased airway hyperresponsiveness indicating that it does have a causative role in causing COPD. Strategies to upregulate CDH1 (encodes for E-cadherin) in CHBEs and cigarette-smoke injured NHBEs can rescue the dysfunctional epithelium.

The airway epithelium is subjected to insults such as cigarette smoke (CS), a primary cause of chronic obstructive pulmonary disease (COPD) and serves as an excellent model to study cell plasticity. Here, we show that both CS-exposed and COPD-patient derived epithelia (CHBE) display quantitative evidence of cellular plasticity, with loss of specialized apical features and a transcriptional profile suggestive of partial epithelial-to-mesenchymal transition (pEMT), albeit with distinct cell motion indicative of cellular unjamming. These injured/diseased cells have an increased fraction of polymerized actin, due to loss of the actin-severing protein cofilin-1. We observed that decreasing polymerized actin restores the jammed state in both CHBE and CS-exposed epithelia, indicating that the fraction of polymerized actin is critical in unjamming the epithelia. Our kinetic energy spectral analysis suggests that loss of cofilin-1 results in unjamming, similar to that seen with both CS exposure and in CHBE cells. The findings suggest that in response to chronic injury, although epithelial cells display evidence of pEMT, their movement is more consistent with cellular unjamming. Inhibitors of actin polymerization rectify the unjamming features of the monolayer. This article has an associated First Person interview with the first author of the paper.

BackgroundTaking into consideration a recent surge of a lung injury condition associated with electronic cigarette use, we devised an in vitro model of sub-chronic exposure of human bronchial epithelial cells (HBECs) in air-liquid interface, to determine deterioration of epithelial cell barrier from sub-chronic exposure to cigarette smoke (CS), e-cigarette aerosol (EC), and tobacco waterpipe exposures (TW).MethodsProducts analyzed include commercially available e-liquid, with 0% or 1.2% concentration of nicotine, tobacco blend (shisha), and reference-grade cigarette (3R4F). In one set of experiments, HBECs were exposed to EC (0 and 1.2%), CS or control air for 10 days using 1 cigarette/day. In the second set of experiments, exposure of pseudostratified primary epithelial tissue to TW or control air exposure was performed 1-h/day, every other day, until 3 exposures were performed. After 16–18 h of last exposure, we investigated barrier function/structural integrity of the epithelial monolayer with fluorescein isothiocyanate–dextran flux assay (FITC-Dextran), measurements of trans-electrical epithelial resistance (TEER), assessment of the percentage of moving cilia, cilia beat frequency (CBF), cell motion, and quantification of E-cadherin gene expression by reverse-transcription quantitative polymerase chain reaction (RT-qPCR).ResultsWhen compared to air control, CS increased fluorescence (FITC-Dextran assay) by 5.6 times, whereby CS and EC (1.2%) reduced TEER to 49 and 60% respectively. CS and EC (1.2%) exposure reduced CBF to 62 and 59%, and cilia moving to 47 and 52%, respectively, when compared to control air. CS and EC (1.2%) increased cell velocity compared to air control by 2.5 and 2.6 times, respectively. The expression of E-cadherin reduced to 39% of control air levels by CS exposure shows an insight into a plausible molecular mechanism. Altogether, EC (0%) and TW exposures resulted in more moderate decreases in epithelial integrity, while EC (1.2%) substantially decreased airway epithelial barrier function comparable with CS exposure.ConclusionsThe results support a toxic effect of sub-chronic exposure to EC (1.2%) as evident by disruption of the bronchial epithelial cell barrier integrity, whereas further research is needed to address the molecular mechanism of this observation as well as TW and EC (0%) toxicity in chronic exposures.

Epithelial surfaces lining the lung serve as the primary environmental gaseous interface, and are subject to common life-limiting diseases, including COPD (Chronic Obstructive Pulmonary Disease). Despite the critical role of epithelial cells in pulmonary health and disease, quantitative models are lacking but are required given the large patient to patient variability to characterize the epithelial plasticity that follows injury. We have identified a series of assessments to quantitatively identify the changes that occur in the epithelium and to identify targets that reverse injury. The injured epithelium has decreased ciliary function and monolayer height, which in the case of cells derived from COPD patients results in an overall disorganization of structure. Injury causes the cells to shift to an unjammed state, with corresponding increases in the velocity correlation length implicating cell shape and stiffness as fundamental to the injury response. Specific inhibitors of actin polymerization (LatA), of MAPK/ERK kinase (U0126) and Nrf-2 pathway activation (CDDO-Me) push the epithelium back towards a jammed state with decreased cell movement and correlation length, as well as improve barrier function and CBF. These studies attest to cell intrinsic properties that allow for a transition to an unjammed state, and that quantitative phenotypic analysis can identify potential specific pharmacologic targets in a given patient and provide insight into basic mechanisms of cellular damage.One Sentence SummaryEnvironmental toxins undermine tissue integrity by manipulating transitions from jammed to unjammed states, thereby mimicking or inducing disease.

Additional file 1. Exposure of epithelial cell monolayer to EC or CS shows dysregulation of CBF. The heat map is an overlap of a single field of view of HBECs at ALI. Hz (Hertz). Panels: control air (a), EC aerosol with 0% nicotine (b), EC aerosol with 1.2% of nicotine (c), CS (d).

Additional file 2. Showed is the heat map analysis of a single image taken during recording time-lapse video of HBECs exposed to (left to right) control air, CS, EC (0%) nicotine and EC (1.2%) nicotine.

Electronic-cigarettes (ECs) are battery powered devices (containing ~7X1011 free radicals/puff), vaporizes flavors with/without nicotine, which may aid smokers in quitting or attenuating their tobacco habits. To date, health concerns of vaping EC use among healthy & COPD is limited and whether regular use has any effect on airway epithelium. We aim to investigate if vaping alters the structure & function of the airway epithelial cells. Primary human bronchial epithelia from 5 healthy donors (NHBE, Lonza & Epithelix) were differentiated on an air-liquid interface & exposed to popular EC flavored aerosols (Tobacco/Mango) with/without nicotine compared to clean air control for 10 days using a Vitrocell exposure system connected to a peristaltic pump (CRM81 puffing regimen). Epithelial function was assessed by cell permeability (FITC-dextran assay & TEER), number of cilia, & ciliary beat frequency (CBF). Effects on cell-cell adhesion were measured by quantifying junctional protein abundance & functional effects of altered adhesion were measured by computing cellular motion over time. We observed that vaping EC aerosols increased permeability, as well as decreased number of cilia & CBF of the airway epithelia (Fig.1). In addition, decreased cell-cell adhesion was quantified by increased cellular motion over time. Our data suggests that vaping EC disrupts structural and functional integrity of airway epithelia, resulting in airway inflammation & lung diseases.

Background:-Obstructive Airways Diseases (OADs) are one of the major causes of mortality and morbidity in India. Improper use of inhaler devices is by far most common reason for inadequate disease control. Treating physicians and nurses are main source of information on inhaler devices for patients in India. Aim: Through UPSIDE (U nderstanding of P ost graduate-PG Medicine, Undergraduate-UG Medicine and nursing S tudents about I nhaler DE vices) we aimed to understand level of knowledge and skills for using inhaler devices namely pMDI (pressurized metered dose inhalers) and pMDI with spacer among students from a tertiary care teaching hospital. Methods: An interviewer based questionnaire was administered which evaluated knowledge (7 points) and Skills (9 points for pMDI;10 for pMDI with spacer). Clean data was used for simple descriptive analysis using IBM SPSS Statistics 23. Results: Out of 377 study participants approached, 269(71.3%) consented. Less than 2% of the total undergraduate, post graduate medical and nursing students knew how to use pMDI or pMDI with spacer correctly. The results for their knowledge and beliefs are mentioned in Fig. 1. Conclusion: Less than 2% of doctors and nurses in a tertiary care teaching hospital know how to use an inhaler device correctly and 46% believe that inhalers are addictive. This study highlights the need to educate doctors and nurses in India about the proper use of inhaler devices.

Lower airway colonisation with species of potentially pathogenic bacteria (PPB) is associated with defective bacterial phagocytosis, in monocyte-derived macrophages (MDMs) and alveolar macrophages, from tobacco smoke-associated chronic obstructive pulmonary disease (S-COPD) subjects. In the developing world, COPD among nonsmokers is largely due to biomass smoke (BMS) exposure; however, little is known about PPB colonisation and its association with impaired innate immunity in these subjects.We investigated the PPB load (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa) in BMS-exposed COPD (BMS-COPD) subjects compared with S-COPD and spirometrically normal subjects. We also examined the association between PPB load and phagocytic activity of MDMs and lung function. Induced sputum and peripheral venous blood samples were collected from 18 healthy nonsmokers, 15 smokers without COPD, 16 BMS-exposed healthy subjects, 19 S-COPD subjects and 23 BMS-COPD subjects. PPB load in induced sputum and MDM phagocytic activity were determined using quantitative PCR and fluorimetry, respectively.Higher bacterial loads of S. pneumoniae, H. influenzae and P. aeruginosa were observed in BMS-COPD subjects. Increased PPB load in BMS-exposed subjects was significantly negatively associated with defective phagocytosis in MDMs and spirometric lung function indices (pIncreased PPB load in airways of BMS-COPD subjects is inversely associated with defective bacterial phagocytosis and lung function.

Culture independent techniques have allowed a better understanding of association between tobacco-smoke exposed COPD (TS-COPD) & lung microbiome, but there is limited knowledge about lung microbiome in non-smoker COPD (NS-COPD) subjects. We aimed to study lung microbiome in smoker & non-smoker COPD subjects and its association with lung function in rural Indian subjects. Induced sputum samples were obtained from 11 healthy-nonsmokers (H-Nonsmokers), 10 healthy-smokers (H-Smokers), 18 biomass-smoke exposed healthy (H-Biomass), 15 TS-COPD & 20 NS-COPD subjects. Samples were sequenced using V3 & V4 hypervariable region (Illumina MiSeq Platform) and taxanomic diversity information was obtained with QIIME pipeline, & R script package. Bacterial abundance was significantly higher in NS-COPD subjects compared to H-Nonsmokers, H-Biomass, & TS-COPD subjects (Fig.1A). Exposure to tobacco-smoke caused decrease in bacterial diversity (Fig.1B). Firmicutes, Bacteriodetes, Proteobacteria, Actinobcater & Fusobacter were the most prevalent phyla in our samples. Phylum Proteobacteria, which includes many pathogens showed higher abundance in COPD subjects (Fig.1C) & were negatively associated with lung function (Fig.1D). The impaired lung function observed in COPD is associated with in increased abundance of pathogenic microbiome & a decline in its diversity, which may have implications in COPD pathophysiology.

Dear editor We read the article by Porpodis et al1 with great interest. In this study, the authors have evaluated the effect of roflumilast on the phagocytic activity of systemic phagocytes in severe and very severe COPD patients by measuring the oxidative burst post-bacterial stimulation. The study group for this study involved 21 severe or very severe COPD patients who were administered roflumilast in addition to other COPD treatments such as long-acting beta-adrenoceptor agonists (LABA) + inhaled corticosteroids (ICS) + long-acting anti-muscarinic agent (LAMA) or ICS + LABA. Prior animal studies have reported that ICS impairs Klebsiella pneumoniae phagocytosis and decreases oxidative stress post-infection with bacteria.2 It would have been interesting to know the phagocytic efficacy of roflumilast in combination with other therapeutic drugs for instance, whether the phagocytic efficacy of the combination of roflumilast and ICS is more as compared to other combinations. This could have been possibly achieved by dividing the study group into subgroups such as ICS + roflumilast or LABA + roflumilast or LAMA + roflumilast or ICS + LABA + roflumilast. Porpodis et al1 also reported that roflumilast increases phagocytic activity, and this anti-inflammatory effect contributes toward increase in lung function parameters among COPD subjects.3 Previous studies have reported that ICS + LABA or ICS + LABA + LAMA improves the lung function. However, in this study there is no control group (group without roflumilast treatment) therefore, the improvement seen in lung function could be a combined effect of ICS + LABA, ICS + LABA + LAMA and roflumilast and not roflumilast alone. Additionally, it would have been interesting to know whether increase in phagocytosis by roflumilast can contribute toward improvement of exacerbations. It has been reported by Calverley et al4 that roflumilast decreases exacerbations among mild to moderate COPD patients, but there are no data available with reference to severe COPD patients. Lastly, the authors should have presented the lung function data in terms of actual values along with the percent predicted format, this would have made the results easy to comprehend.

Background: We have previously reported that monocytes-derived macrophages (MDM) from smoker COPD (S-COPD) subjects show defective bacterial phagocytosis. However, it is not known whether the same holds true for non-smoker COPD (NS-COPD) subjects. We aimed to study the phagocytic activity of MDM from NS-COPD subjects and compare it with S-COPD and healthy (H) subjects from India. Methods: Monocytes were derived from peripheral venous blood from 22 H subjects, 10 S-COPD subjects and 15 NS-COPD subjects (10 biomass fuel smoke exposed and 5 from other causes). The monocytes were cultured with complete media containing granulocyte-monocyte colony stimulating factor for 16-20 days to obtain MDM. 1X10 6 MDM/well were incubated with fluorescently-tagged, heat-killed S. pneumoniae (SP: 1.2X10 9 CFU/mL) and H. influenzae (HI: 1.5X10 8 CFU/mL) for 4 hours and fluorescence was measured to quantify the intracellular uptake of SP and HI. Results: Conclusion: Monocyte-derived macrophages of non-smoker COPD subjects show defective phagocytosis, which provides further evidence to suggest that there is a common susceptibility mechanism.

Background: India has a high burden of CRDs. Physicians in practice see a large number of patients daily for whom they can give little time. In this pilot study we aimed to identify the need and utility of additional skilled healthcare resource assistance for the proper management of CRD in India. Method: 1784 General Practitioners (GPs), Pulmonologists (Ps), Intensivists (Is) and Pediatricians (Peds) from our database were invited via email to participate in this observational study from across India. The study questionnaire consisted of doctor demographics, burden of CRD, time spent on each patient and questions assessing the need and utility of additional skilled assistance. 84 doctors consented and completed the study questionnaire. Simple descriptive analysis was performed. Results: Conclusion: A heavy workload of CRD patients and insufficient time and manpower necessitates a second tier of skilled healthcare providers to bridge this gap. There is a need to develop chronic respiratory disease managers in India who can contribute significantly to improving quality of patient care.

To the Editor: Ambade et al[1][1] conducted an observational study to evaluate the potential serum biomarkers in subjects with COPD. The authors assessed superoxide dismutase-3, glutathione peroxidase, catalase, ceruloplasmin ferroxidase activity, C-reactive protein, and surfactant protein D in