Bronchial Brushing Research Articles

Radiomultiomics: quantitative CT clusters of severe asthma associated with multi-omics.

Lung quantitative computed tomographic (qCT) severe asthma clusters have been reported, but their replication and underlying disease mechanisms are unknown. We identified and replicated qCT clusters of severe asthma in two independent asthma cohorts and determined their association with molecular pathways. We used consensus clustering on qCT measurements of airway and lung CT scans, performed in 105 severe asthmatic adults from the U-BIOPRED cohort. The same qCT measurements were used to replicate qCT clusters in a subsample of the ATLANTIS asthma cohort (n=97). We performed integrated enrichment analysis using blood, sputum, bronchial biopsies, bronchial brushings and nasal brushings transcriptomics and blood and sputum proteomics to characterize radiomultiomic-associated clusters (RACs). qCT clusters and clinical features in U-BIOPRED were replicated in the matched ATLANTIS cohort. In the U-BIOPRED cohort, RAC1 (n=30) was predominantly female with elevated BMI, mild airflow limitation, normal qCT parameters and upregulation of the complement pathway. RAC2 (n=34) subjects had a lower degree of airflow limitation, airway wall thickness and dilatation, with upregulation of proliferative pathways, including neurotrophic receptor tyrosine kinase 2/tyrosine kinase receptor B (NTRK2/TRKB), and down-regulation of semaphorin pathways. RAC3 (n=41) showed increased lung attenuation area and air trapping, severe airflow limitation, hyperinflation, and upregulation of cytokine signaling and signaling by interleukin pathways, and matrix metallopeptidase 1, 2 and 9. U-BIOPRED severe asthma qCT clusters were replicated in a matched independent asthmatic cohort and associated with specific molecular pathways. Radiomultiomics might represent anovel strategy to identify new molecular pathways in asthma pathobiology.

Single cell sequencing reveals cellular landscape alterations in the airway mucosa of patients with pulmonary long COVID.

To elucidate the important cellular and molecular drivers of pulmonary long COVID, we generated a single-cell transcriptomic map of the airway mucosa using bronchial brushings from patients with long COVID who reported persistent pulmonary symptoms.Adults with and without long COVID were recruited from the general community in Greater Vancouver, Canada. The cohort was divided into those with pulmonary long COVID (PLC), which was defined as persons with new or worsening respiratory symptoms following at least one year from their initial acute SARS-CoV-2 infection (N=9); and control subjects defined as SARS-CoV-2 infected persons whose acute respiratory symptoms had fully resolved or individuals who had no history of acute COVID-19 (N=9). These participants underwent bronchoscopy from which a single cell suspension was created from bronchial brush samples and then sequenced.A total of 56 906 cells were recovered for the downstream analysis, with 34 840 cells belonging to the PLC group, which strikingly showed a unique cluster of neutrophils in the PLC group (p<0.05). Ingenuity Pathway Analysis revealed that the neutrophil degranulation pathway was enriched across epithelial cell clusters. Differential gene expression analysis between the PLC and control groups demonstrated upregulation of inflammatory chemokines and epithelial barrier dysfunction across epithelial cell clusters, as well as over-expression of mucin genes across secretory cell clusters.In conclusion, a single-cell transcriptomic landscape of the small airways suggest that neutrophils may play a significant role in mediating the chronic small airway inflammation driving pulmonary symptoms of long COVID.

Nasal epithelial gene expression identifies relevant asthma endotypes in the ATLANTIS study

IntroductionAsthma is an inflammatory airways disease encompassing multiple phenotypes and endotypes. Several studies suggested gene expression in nasal epithelium to serve as a proxy for bronchial epithelium, being a non-invasive...

Airway epithelial cell response to RSV is mostly impaired in goblet and multiciliated cells in asthma

BackgroundIn patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbation by infecting the epithelial layer of the airways, inducing subsequent immune response. The type I interferon antiviral...

Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations.

Hyper IgE syndrome (STAT3-HIES), also known as Job's syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense. To identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection. STAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1β) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Mucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1β expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1β exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a γ-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. STAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.

Deep multiomic profiling reveals molecular signatures that underpin preschool wheeze and asthma

Wheezing in childhood is prevalent, with over one-half of all children experiencing at least 1 episode by age 6. The pathophysiology of wheeze, especially why some children develop asthma while others do not, remains unclear. This study addresses the knowledge gap by investigating the transition from preschool wheeze to asthma using multiomic profiling. Unsupervised, group-agnostic integrative multiomic factor analysis was performed using host/bacterial (meta)transcriptomic and bacterial shotgun metagenomic datasets from bronchial brush samples paired with metabolomic/lipidomic data from bronchoalveolar lavage samples acquired from children 1-17 years old. Two multiomic factors were identified: one characterizing preschool-aged recurrent wheeze and another capturing an inferred trajectory from health to wheeze and school-aged asthma. Recurrent wheeze was driven by type 1-immune signatures, coupled with upregulation of immune-related and neutrophil-associated lipids and metabolites. Comparatively, progression toward asthma from ages 1 to 18 was dominated by changes related to airway epithelial cell gene expression, type 2-immune responses, and constituents of the airway microbiome, such as increased Haemophilus influenzae. These factors highlighted distinctions between an inflammation-related phenotype in preschool wheeze, and the predominance of airway epithelial-related changes linked with the inferred trajectory toward asthma. These findings provide insights into the differential mechanisms driving the progression from wheeze to asthma and may inform targeted therapeutic strategies.

Digital Image Comparison of Cellular Yield in Bronchial Brushing: Pre- and Post-Biopsy Lavage Cytology

Introduction: Bronchoscopy is a useful diagnostic tool capable of performing core biopsy, forceps biopsy, bronchoalveolar lavage, and bronchial brushing. This study compares the cellularity of bronchial cytology including pre- and post-biopsy lavage by digital image analysis, aiming to increase diagnostic and tumor yield by optimizing the sequence and combination of bronchial biopsy and cytology. Methods: Alveolar macrophage, bronchial epithelium, and tumor cell cellularity from liquid-based cytology preparations of bronchial brushing and pre-biopsy and post-biopsy bronchoalveolar lavage were annotated on digitized whole-slide images and compared. Secondary analysis on the relationship of tumor cell and non-lesional cell yield was performed. Results: Overall, 118 cytology specimens from 43 patients were retrieved in total. Bronchial epithelium count was higher in pre-biopsy than post-biopsy lavage (p < 0.01) but not for alveolar macrophages nor tumor cell (p > 0.05). Tumor cell count was higher for bronchial brushing cytology samples than lavage (p = 0.018). The alveolar macrophage count was higher in post-biopsy lavage than bronchial brushing (p = 0.033); otherwise, brushing showed consistently higher bronchial epithelium and tumor cell counts. There were 33 false negative (tumor cell absent) specimens, and the combination of bronchial brushing and pre-biopsy lavage yielded the lowest false negative cases. Correlation between bronchial epithelium and alveolar macrophage counts with tumor cell count was weak (correlation coefficient = −0.168–0.203) except for post-biopsy lavage (correlation coefficient = 0.412–0.479, p < 0.05). Conclusion: Bronchial brushing yields a greater amount of tumor cell than lavage, and timing lavage before or after core biopsy does not affect tumor cell yield. Combining bronchial brushing and pre-biopsy lavage results in the lowest false negative rate.

Comparison and review of abrasive bronchial brushing versus non-abrasive aspiration, lavage and washing – Higher sensitivity but with risk of over-diagnosis for bronchial brushing

Bronchial exfoliative cytology is classified as non-abrasive (washing, aspiration and bronchoalveolar lavage) and abrasive (brushing). Brush abrasion dislodges epithelial cells but can induce bleeding and cytomorphologic artifacts. In this study, the largest cohort to date of bronchial cytology specimens were referenced against bronchial biopsy as the reference standard. Findings in the study will be useful for selecting biopsy modality and reducing necessary procedural risks. All consecutive bronchial cytology and bronchial biopsy from 1995 to 2022 were retrieved. The diagnoses were reviewed and categorized into five-tiered diagnostic categories to compare diagnostic agreement and concordance. Review of 14,148 specimens yielded 3963 non-abrasive, 2378 abrasive cytology specimens matched to biopsy, with 4355 matches between non-abrasive and abrasive cytology specimens. Agreement between non-abrasive and abrasive cytology was moderate (κ = 0.580), and similar when referenced against biopsy (κ = 0.456 (non-abrasive), κ = 0.498 (abrasive)). Abrasive bronchial cytology showed a higher percentage of malignant diagnosis (20.95 % vs. 12.63 %, p < 0.001) and over-diagnosis rate (36.40 % vs. 29.79 %, p < 0.001), but higher sensitivity (0.747 vs. 0.572, p = 0.002). For subgroup analysis of transbronchial biopsies, matched abrasive cytology showed higher discordant rates (p < 0.05) and lower accuracy (0.907 vs. 0.873, p = 0.020). With the added bleeding risk associated with brushing, abrasive techniques may only be preferable in cases with clinical or bronchoscopic suspicion of malignancy, in particular endobronchial mucosal lesions. For routine bronchoscopy, non-abrasive bronchial cytology appears to be adequate.

Exhaled breath condensate contains extracellular vesicles (EVs) that carry miRNA cargos of lung tissue origin that can be selectively purified and analyzed.

Lung diseases, including lung cancer, are rising causes of global mortality. Despite novel imaging technologies and the development of biomarker assays, the detection of lung cancer remains a significant challenge. However, the lung communicates directly with the external environment and releases aerosolized droplets during normal tidal respiration, which can be collected, stored and analzsed as exhaled breath condensate (EBC). A few studies have suggested that EBC contains extracellular vesicles (EVs) whose microRNA (miRNA) cargos may be useful for evaluating different lung conditions, but the cellular origin of these EVs remains unknown. In this study, we used nanoparticle tracking, transmission electron microscopy, Western blot analyses and super resolution nanoimaging (ONi) to detect and validate the identity of exhaled EVs (exh-EVs). Using our customizable antibody-purification assay, EV-CATCHER, we initially determined that exh-EVs can be selectively enriched from EBC using antibodies against three tetraspanins (CD9, CD63 and CD81). Using ONi we also revealed that some exh-EVs harbour lung-specific proteins expressed in bronchiolar Clara cells (Clara Cell Secretory Protein [CCSP]) and Alveolar Type II cells (Surfactant protein C [SFTPC]). When conducting miRNA next generation sequencing (NGS) of airway samples collected at five different anatomic levels (i.e., mouth rinse, mouth wash, bronchial brush, bronchoalveolar lavage [BAL] and EBC) from 18 subjects, we determined that miRNA profiles of exh-EVs clustered closely to those of BAL EVs but not to those of other airway samples. When comparing the miRNA profiles of EVs purified from matched BAL and EBC samples with our three tetraspanins EV-CATCHER assay, we captured significant miRNA expression differences associated with smoking, asthma and lung tumor status of our subjects, which were also reproducibly detected in EVs selectively purified with our anti-CCSP/SFTPC EV-CATCHER assay from the same samples, but that confirmed their lung tissue origin. Our findings underscore that enriching exh-EV subpopulations from EBC allows non-invasive sampling of EVs produced by lung tissues.

Abstract 748: Impact of oral supplementation of sulforaphane for 12 months on lung cancer-related gene expression in former smokers at high risk for lung cancer: Results from a randomized placebo-controlled phase II clinical trial

Abstract Introduction: Higher consumption of dietary sulforaphane (SFN), a phytochemical found in many cruciferous vegetables, has been shown to be associated with lower risk of lung cancer (LC). SF activates the NRF2-pathway, which initiates cellular detoxification, antioxidant, and anti-inflammatory responses. To date, no chemoprevention trial for LC has examined the impact of SFN on LC-related gene expression (GE) as an endpoint in nasal and bronchial tissue. The objective of our study was to examine whether oral intake of SF for 12 months impacts the GE of previously identified LC- and premalignant lesions (PML)-related genes in bronchial and nasal brushing samples. Methods: A randomized, placebo-controlled clinical trial (NCT03232138) was conducted in former smokers with ≥ 30 pack-years and quitting smoking for 1-10 years in Western Pennsylvania, USA. Forty-three subjects were randomly assigned to the SFN treatment (n = 21) or placebo group (n = 22) in 2018-2022. Each participant was instructed to take 4 Avmacol® or identical placebo capsules BID for 12 months. Each Avmacol® capsule contained 15 mg of glucoraphanin, which yielded a daily internal dose of 120 μmol SFN. Nasal swabs and bronchial brushings were obtained at baseline and after 12 months. Among the 37 participants who completed the study, RNA was extracted from their nasal swabs and bronchial brushings. We constructed single sample gene signature scores (GSS) using the GSVA algorithm for the following: 1) the NRF2-pathway, 2) genes associated with LC risk in bronchial biopsies, and 3) genes associated with PML. The genes in these sets were stratified by their up- or down-regulation in LC or PML. We evaluated the association between the interaction of SFN supplementation over time (pre-/post-treatment) on GSS using linear mixed models, adjusted for the fixed effects of age at baseline, RNA integrity number (RIN), packyears of smoking, and random effect of participant. Results: GE data was available for 31 participant pairs with bronchial and/or nasal samples. Within nasal tissue, SFN treatment increased both the GSSs of the NRF2-pathway (p=0.0089) and the downregulated LC-risk genes (p=0.0047), and decreased the upregulated LC-risk genes (p=0.012) over time, whereas SFN had no significant impact on GSSs of the NRF2-pathway and LC-risk genes in the bronchial tissue. No association between SFN treatment over time and the PML-related GSS was observed in nasal or bronchial tissue. Conclusion: Our initial analysis indicates that SFN treatment over 12 months impacted LC-associated GE in nasal tissue but not in bronchial tissue from former smokers. The discrepancy results may be due to small sample size and additional studies are warranted to confirm if SFN has direct impact on LC-risk related genes in bronchial tissue (NIH grant No. R01CA213123). Citation Format: Kathryn Demanelis, Avrum Spira, Yohana Kefella, Renwei Wang, Jennifer Adams-Haduch, Thomas W. Kensler, Jennifer E. Beane, David O. Wilson, Jian-Min Yuan. Impact of oral supplementation of sulforaphane for 12 months on lung cancer-related gene expression in former smokers at high risk for lung cancer: Results from a randomized placebo-controlled phase II clinical trial [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 748.

Abstract 4781: Evaluation of a TP53 biomarker for lung cancer risk in nasal brushings and peripheral blood

Abstract Background and Purpose: Early detection of lung cancer through annual screening with low-dose computed tomography (LDCT) scans enables potentially curative surgical or stereotactic body radiation therapy (SBRT) treatment and thereby significantly reduces mortality. However, LDCT screening among those currently eligible based primarily on age and smoking criteria yields a large number of false-positive findings and many lung cancers occur among individuals who currently are ineligible. A biomarker that identifies lung cancer risk independent from that caused by age and cigarette smoking might increase specificity and increase the number of people eligible for LDCT screening. Recently we demonstrated that a biomarker measuring TP53 somatic mutations in bronchial brush biopsy specimens from grossly normal airway epithelium was significantly associated with cancer status independent of age and smoking status. Additionally, the TP53 biomarker was synergistic with traditional risk score models (e.g. PLCOM2012 risk score). The goal of this pilot study was to test the TP53 biomarker in nasal brush biopsy specimens, a potential surrogate tissue obtained using less invasive collection methods. Methods: Nasal brushings from 10 lung cancer and 11 control subjects were collected at the University of Toledo and Vanderbilt University Medical Center under approved institutional protocols. Genomic DNA (gDNA) was extracted with the Qiagen AllPrep DNA/RNA kit using a modified protocol. Amplifiable copies of gDNA were measured using quantitative PCR. For each test a sample comprising 25,000 amplifiable copies of gDNA was combined with 50,000 copies of a TP53 internal standard and a complexity control, then PCR amplified, purified and barcoded. Samples were pooled to create two NGS libraries, each with approximately equal numbers of cases and controls, and sequenced on an Illumina MiSeq (Two V3 flow cells, 2 x 250 paired-end). Bioinformatic analysis was conducted using Qiagen CLC Workbench. Variants were called using the Basic Variant Detection Tool. Poisson Exact Test was utilized to determine the significance of endogenous variants relative to internal standard variants at the same base position and a Bonferroni correction was applied to minimize false discovery. Comparison of variant numbers between cases and controls was conducted using an f-test followed by a paired t-test. Results and Conclusions: Consistent with previous results in airway epithelium, in this pilot study the TP53 mutation biomarker measured in nasal brushing was associated with lung cancer (p=0.0085). Measurement of the TP53 biomarker in nasal brush specimens in a larger case-control study currently is underway along with a pilot study in gDNA from peripheral blood. Citation Format: Erin L. Crawford, Daniel J. Craig, Patrick T. Gorman, Mohamed Omballi, Rami Ahmad, Eric L. Grogan, Stephen A. Deppen, James C. Willey. Evaluation of a TP53 biomarker for lung cancer risk in nasal brushings and peripheral blood [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 4781.

Quercetin improves epithelial regeneration from airway basal cells of COPD patients.

Airway basal cells (BC) from patients with chronic obstructive pulmonary disease (COPD) regenerate abnormal airway epithelium and this was associated with reduced expression of several genes involved in epithelial repair. Quercetin reduces airway epithelial remodeling and inflammation in COPD models, therefore we examined whether quercetin promotes normal epithelial regeneration from COPD BC by altering gene expression. COPD BC treated with DMSO or 1 µM quercetin for three days were cultured at air/liquid interface (ALI) for up to 4 weeks. BC from healthy donors cultured at ALI were used as controls. Polarization of cells was determined at 8 days of ALI. The cell types and IL-8 expression in differentiated cell cultures were quantified by flow cytometry and ELISA respectively. Microarray analysis was conducted on DMSO or 1 µM quercetin-treated COPD BC for 3 days to identify differentially regulated genes (DEG). Bronchial brushings obtained from COPD patients with similar age and disease status treated with either placebo (4 subjects) or 2000mg/day quercetin (7 subjects) for 6 months were used to confirm the effects of quercetin on gene expression. Compared to placebo-, quercetin-treated COPD BC showed significantly increased transepithelial resistance, more ciliated cells, fewer goblet cells, and lower IL-8. Quercetin upregulated genes associated with tissue and epithelial development and differentiation in COPD BC. COPD patients treated with quercetin, but not placebo showed increased expression of two developmental genes HOXB2 and ELF3, which were also increased in quercetin-treated COPD BC with FDR < 0.001. Active smokers showed increased mRNA expression of TGF-β (0.067) and IL-8 (22.0), which was reduced by 3.6 and 4.14 fold respectively after quercetin treatment. These results indicate that quercetin may improve airway epithelial regeneration by increasing the expression of genes involved in epithelial development/differentiation in COPD. This study was registered at ClinicalTrials.gov on 6-18-2019. The study number is NCT03989271.

Comparison between upper and lower airway microbiome profiles in chronic rhinosinusitis patients.

Dysregulation of the airway microbiota is thought to contribute to airway inflammation in both chronic rhinosinusitis (CRS) and asthma. However, the relationship between the upper and lower airway microbiome remains unclear. Sinus and lung brushes were collected from 29 CRS participants undergoing sinus surgery. DNA was extracted and submitted for 16s rRNA microbiome sequencing. Alpha and beta diversity metrics, taxonomic composition, and differences between individual taxa were compared for paired sinus and bronchial samples. Twenty-three out of 29 participants had sufficient samples for analysis. The mean (standard deviation) age was 51.59 (14.57) years, and 10 (44%) patients were female. Twelve (52%) patients had comorbid asthma. Sinus brushes had significantly higher alpha diversity indexes (Shannon and Faith) compared to bronchial brushes (p<0.001). Beta diversity metrics were significantly different between the sinus and bronchial samples. Principal coordinate analysis showed no clustering of paired nasal and bronchial samples. Sinus brushes had significantly more Lawsonella, Corynebacterium, and Staphylococcus compared to bronchia brushes, while the latter were enriched in Tropheryma and Sphingomonas, among others (false discovery rate [FDR]-adjusted p<0.01). Finally, CRS patients with comorbid asthma had significantly higher Pseudomonas and Peptoniphilus in sinus brushes and lower Prevotella in bronchial brushes when compared to non-asthmatics (FDR-adjusted p<0.01). The sinus and bronchial bacterial microbiomes differ in important ways. Our study suggests that migration of bacteria from the sinus into the lower airways is unlikely in patients with CRS.

Alternative Splicing Is a Major Factor Shaping Transcriptome Diversity in Mild and Severe Chronic Obstructive Pulmonary Disease.

The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.

Symbiotic microbial communities in various locations of the lung cancer respiratory tract along with potential host immunological processes affected.

Lung cancer has the highest mortality rate among all cancers worldwide. The 5-year overall survival rate for non-small cell lung cancer (NSCLC) is estimated at around 26%, whereas for small cell lung cancer (SCLC), the survival rate is only approximately 7%. This disease places a significant financial and psychological burden on individuals worldwide. The symbiotic microbiota in the human body has been significantly associated with the occurrence, progression, and prognosis of various diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Studies have demonstrated that respiratory symbiotic microorganisms and their metabolites play a crucial role in modulating immune function and contributing to the pathophysiology of lung cancer through their interactions with the host. In this review, we provide a comprehensive overview of the microbial characteristics associated with lung cancer, with a focus on the respiratory tract microbiota from different locations, including saliva, sputum, bronchoalveolar lavage fluid (BALF), bronchial brush samples, and tissue. We describe the respiratory tract microbiota's biodiversity characteristics by anatomical region, elucidating distinct pathological features, staging, metastasis, host chromosomal mutations, immune therapies, and the differentiated symbiotic microbiota under the influence of environmental factors. Our exploration investigates the intrinsic mechanisms linking the microbiota and its host. Furthermore, we have also provided a comprehensive review of the immune mechanisms by which microbiota are implicated in the development of lung cancer. Dysbiosis of the respiratory microbiota can promote or inhibit tumor progression through various mechanisms, including DNA damage and genomic instability, activation and regulation of the innate and adaptive immune systems, and stimulation of epithelial cells leading to the upregulation of carcinogenesis-related pathways.

Bronchoscopic bronchial brush cytology: an underutilized modality for diagnosing lung cancer in resource limited facilities: a case series of nine patients

Lung cancer incidence is on the rise with increasing industrialization across the globe. Early diagnosis is the key to a better prognosis. In the current scenario, in developing countries, the majority are diagnosed late, when surgical or curable treatment is not possible, and only palliative treatment options are left. Bronchoscopy is the most widely used modality for the diagnosis of lung cancer. Perhaps, all its accessories are not widely used. Bronchial brush is an easy, cost effective, easily available, without significant complication rates, safe, feasible, with high specificity which offers early preliminary report where biopsy is not possible. It should be used along with biopsy to increase the yield and accuracy in all suspected lung cancer patients who require bronchoscopy.

Utility of rapid on-site evaluation (ROSE) using toluidine blue stain in respiratory cytology

Introduction and Aim: Cytology has gained importance as a diagnostic test in pulmonary lesions, especially in patients presenting with advanced malignancy which are surgically non-resectable. Although cytopathological diagnosis may be limited, its role cannot be underestimated considering the minimal invasive nature of the technique. Single slide (SS) Rapid on-site evaluation (ROSE) will aid in adequacy assessment and diagnosis of pulmonary lesions on cytopathology. Materials and Methods: We conducted a prospective study. SS ROSE was done on transbronchial fine needle aspiration and bronchial brushing specimens using toluidine blue staining. The slides were microscopically evaluated for adequacy and for diagnosis. In case of inadequate smears, a repeat was suggested. The diagnosis was compared with the diagnosis rendered on remaining slides which were processed using conventional cytology. Results: The study includes 82 cases for ROSE. A correlation of 100% was found between ROSE diagnosis and final cytology diagnosis. Out of 82 cases, 24 cases were malignancies and 3 were granulomatous lesions on ROSE. The remaining were negative for atypical/malignant cells. 2nd pass was advised in 21 cases due to the inadequacy of the diagnostic material in 1st pass. Out of 21 cases advised for 2nd pass, 11 were found to be malignant. A statistically significant p value of 0.05 was obtained. Conclusion: In our study, we found that ROSE using toluidine blue stain is simple, yet a very useful procedure to reduce the number of passes, the need for repeat bronchoscopy procedure and helpful in suggesting more samples for ancillary tests and rapid cytopathological diagnosis.

Membrane lipid composition of bronchial epithelial cells influences antiviral responses during rhinovirus infection

ABSTRACT Lipids and their mediators have important regulatory functions in many cellular processes, including the innate antiviral response. The aim of this study was to compare the lipid membrane composition of in vitro differentiated primary bronchial epithelial cells (PBECs) with ex vivo bronchial brushings and to establish whether any changes in the lipid membrane composition affect antiviral defense of cells from donors without and with severe asthma. Using mass spectrometry, we showed that the lipid membrane of in vitro differentiated PBECs was deprived of polyunsaturated fatty acids (PUFAs) compared to ex vivo bronchial brushings. Supplementation of the culture medium with arachidonic acid (AA) increased the PUFA-content to more closely match the ex vivo membrane profile. Rhinovirus (RV16) infection of AA-supplemented cultures from healthy donors resulted in significantly reduced viral replication while release of inflammatory mediators and prostaglandin E2 (PGE2) was significantly increased. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthases, suppressed RV16-induced PGE2 release and significantly reduced CXCL-8/IL-8 release from AA-supplemented cultures indicating a link between PGE2 and CXCL8/IL-8 release. In contrast, in AA-supplemented cultures from severe asthmatic donors, viral replication was enhanced whereas PTGS2 expression and PGE2 release were unchanged and CXCL8/IL-8 was significantly reduced in response to RV16 infection. While the PTGS2/COX-2 pathway is initially pro-inflammatory, its downstream products can promote symptom resolution. Thus, reduced PGE2 release during an RV-induced severe asthma exacerbation may lead to prolonged symptoms and slower recovery. Our data highlight the importance of reflecting the in vivo lipid profile in in vitro cell cultures for mechanistic studies.

Blastomycosis: An Underrecognized Climate Sensitive Fungal Pathogen Causing Pneumonia in 52-year-old Immunocompetent Women from Southwestern Ohio—A Case Report and Literature Review

Blastomycosis, formerly known as Gilchrist disease, Chicago disease, North American blastomycosis, or Namekagos fever, is an uncommon but underappreciated fungal infection seen mostly in immunocompetent people, caused by the fungi Blastomyces. Blastomyces dermatitidis and Blastomyces gilchristii are the most common species responsible for most infections in the United States of America. Blastomycosis can cause a wide variety of diseases, the most common being pneumonia as the lungs are usually the main primary entrance source of infection after inhalation of Blastomyces spores. However, dissemination from the lungs to other organs such as skin, bones, genitourinary, and central nervous system can occur. We describe a 52-year-old female who was admitted to the hospital for unresolved pneumonia after failure to respond to multiple outpatient antibiotic treatments for a presumed diagnosis of community-acquired pneumonia, presenting with right-sided chest pain, fever, cough and weight loss for three weeks duration. Her initial Chest-X ray and CT chest revealed right middle lobe infiltrate, while her laboratory studies were significant for elevated white blood cells (WBC). The patient underwent diagnostic bronchoscopy which showed erythema of the right middle lobe bronchus with purulent secretion. Furthermore, cytology examination of specimens from bronchoalveolar lavage and bronchial brush from right middle lobe with (PAP) satin on thin prep and H&amp;E stain on the cell block revealed an abundance of broad-base budding yeast consistent with Blastomyces organism, which was confirmed by molecular testing with DNA probe assay. Her serum and urine Blastomyces antigens were positive, above the limit of quantification. The patient was started on antifungal therapy with Amphotericin-B intravenously. In the process, her fever resolved and her respiratory symptoms slowly improved. She was discharged home with oral itraconazole which was to be continued for 6-12 months as an outpatient.

Evaluation of the diagnostic yield of lung cancer on various Bronchoscopic modalities

Background: The application of cytological techniques in identifying malignant respiratory tract lesions has been widely praised as a significant achievement. Flexible fiberoptic bronchoscopy has greatly transformed respiratory cytology, making procedures such as bronchial brushings, broncho-alveolar lavage, and bronchial forceps biopsy more convenient, accessible, and popular. Methodology: All the suspected cases of lung cancer from 1st Jan, 2020 - 31st Dec, 2022 fulfilling inclusion criteria who have undergone flexible bronchoscopy in our center have been analyzed and enrolled in the study. Results: Out of 90 patients, 85 were found to be malignant. Squamous cell carcinoma was the most common carcinoma (31.1%) followed by adenocarcinoma (26.7%), non-small Cell Lung Carcinoma (17.8%) and small cell carcinoma (12.2%), majority of the cases were of 6-7th decade of life and were smoker. Sensitivities of Bronchoalveolar Lavage (BAL), Bronchial brush, Endobronchial forceps Biopsy (EBB), Transbronchial Lung Biopsy (TBLB) and Transbronchial Needle Aspiration (TBNA) were found to be 32%, 87%, 83%, 55% and 69% respectively. Diagnostic accuracy of BAL, bronchial brush, EBB, TBLB and TBNA were found to be 36%, 87%, 83%, 55% and 61% respectively. Conclusion: With a greater sensitivity (87%) and diagnostic accuracy (87%), bronchial brushing shows great promise as a convenient cytological technique suitable for screening ambiguous cases and early detection of lung cancer, as it saves the time required for processing biopsy samples.