Endotypes of Paediatric Cough-Do They Exist and Finding New Techniques to Improve Clinical Outcomes.

Chronic cough is a common symptom of many childhood lung conditions. Given the phenotypic heterogeneity of chronic cough, better characterization through endotyping is required to provide diagnostic certainty, precision therapies and to identify pathobiological mechanisms. This review summarizes recent endotype discoveries in airway diseases, particularly in relation to children, and describes the multi-omic approaches that are required to define endotypes. Potential biospecimens that may contribute to endotype and biomarker discoveries are also discussed. Identifying endotypes of chronic cough can likely provide personalized medicine and contribute to improved clinical outcomes for children.

Cough Hypersensitivity Syndrome: Why Its Use Is Inappropriate in Children.

In children and adults, chronic cough is a common symptom presenting to health professionals worldwide. It is internationally accepted that children with chronic cough should be managed with pediatric specific management guidelines. The newly proposed clinical entity of 'cough hypersensitivity syndrome' has gained significant attention in adult literature. Given the significant differences between childhood and adult chronic cough, including in respiratory physiology and anatomy, and cough sensitivity, we address the suitability of the use of cough hypersensitivity syndrome in children. We explore these differences between childhood and adult chronic cough, explain what cough hypersensitivity is and highlight why the term cough hypersensitivity syndrome should not be used in children.

Optimal isolation of extracellular vesicles from pleural fluid and profiling of their microRNA cargo.

Pleural effusion occurs in both benign and malignant pleural disease. In malignant pleural effusions, the diagnostic accuracy and sensitivity of pleural fluid cytology is less than perfect, particularly for the diagnosis of malignant pleural mesothelioma, but also in some cases for the diagnosis of metastatic pleural malignancy with primary cancer in the lung, breast or other sites. Extracellular vesicles (EVs) carry an enriched cargo of microRNAs (miRNAs) which are selectively packaged and differentially expressed in pleural disease states. To investigate the diagnostic potential of miRNA cargo in pleural fluid extracellular vesicles (PFEVs), we evaluated methods for isolating the extracellular vesicle (EV) fraction including combinations of ultracentrifugation, size-exclusion chromatography (SEC) and ultrafiltration (10 kDa filter unit). PFEVs were characterized by total and EV-associated protein, nanoparticle tracking analysis and visualisation by transmission electron microscopy. miRNA expression was analyzed by Nanostring nCounter® in separate EV fractions isolated from pleural fluid with or without additional RNA purification by ultrafiltration (3 kDa filter unit). Optimal PFEV yield, purity and miRNA expression were observed when PFEV were isolated from a larger volume of pleural fluid processed through combined ultracentrifugation and SEC techniques. Purification of total RNA by ultrafiltration further enhanced the detectability of PFEV miRNAs. This study demonstrates the feasibility of isolating PFEVs, and the potential to examine PFEV miRNA cargo using Nanostring technology to discover disease biomarkers.

Plasma Extracellular Vesicle miRNA Profiles Distinguish Chronic Obstructive Pulmonary Disease Exacerbations and Disease Severity.

Purpose: Molecular biomarkers for chronic obstructive pulmonary disease (COPD) severity have been difficult to identify. We aimed to assess extracellular vesicle miRNAs' potential as a blood biomarker in discriminating disease severity in participants with COPD. Patients and Methods: Plasma extracellular vesicles (EVs) were obtained from two COPD cohorts (n = 20 during an exacerbation event, n = 20 during stable state), with varying disease severity (GOLD stages). The miRCURY LNA miRNA Serum/Plasma assay, specific to 179 targets, was used to evaluate EV miRNA expression. The miRNAs that were significantly dysregulated were further assessed for discriminatory power using ROC curve analysis, as well as their role in relevant biological pathways. Results: One miRNA was significantly dysregulated between moderate GOLD participants compared to severe/very severe GOLD participants, with an AUC of 0.798, p = 0.01 for miR-374b-5p. Five miRNAs were significantly dysregulated between exacerbating and stable COPD participants, with miR-223-3p resulting in the highest AUC (0.755, p = 0.006) for a single miRNA, with a combination of three miRNAs (miR-92b-3p, miR-374a-5p and miR-106b-3p) providing the highest discriminatory power (AUC 0.820, p = 0.001). The "cytokine-cytokine receptor interaction" (hsa04060 pathway) was the most significant KEGG pathway enriched for three out of the five miRNAs associated with COPD exacerbations. Conclusion: This initial small-scale study suggests that the bioactive cargo (miRNAs) in plasma EVs holds specific biological information for the severity of airflow obstruction and COPD exacerbations, warranting further investigation.

Burden of Care for Children with Bronchiectasis from Parents/Carers Perspective.

Bronchiectasis is a neglected chronic respiratory condition. In children optimal appropriate management can halt the disease process, and in some cases reverse the radiological abnormality. This requires many facets, including parental/carer bronchiectasis-specific knowledge, for which there is currently no such published data. Further, the importance of patient voices in guiding clinical research is becoming increasingly appreciated. To address these issues, we aimed to describe the voices of parents of children with bronchiectasis relating to (a) burden of illness and quality of life (QoL), (b) their major worries/concerns and (c) understanding/management of exacerbations. The parents of 152 children with bronchiectasis (median age = 5.8 years, range 3.5-8.4) recruited from the Queensland Children's Hospital (Australia) completed questionnaires, including a parent-proxy cough-specific QoL. We found that parents of children with bronchiectasis had impaired QoL (median 4.38, range 3.13-5.63) and a high disease burden with median 7.0 (range 4.0-10.0) doctor visits in 12-months. Parental knowledge varied with only 41% understanding appropriate management of an exacerbation. The highest worry/concern expressed were long-term effects (n = 42, 29.8%) and perceived declining health (n = 36, 25.5%). Our study has highlighted the need for improved education, high parental burden and areas of concern/worry which may inform development of a bronchiectasis-specific paediatric QoL tool.

E-cigarettes induce toxicity comparable to tobacco cigarettes in airway epithelium from patients with COPD.

BACKGROUND: The health effects of e-cigarettes in patients with pre-existing lung disease are unknown. The aim of this study was to investigate whether aerosols from a fourth-generation e-cigarette produces similar in-vitro cytotoxic, DNA damage and inflammatory effects on bronchial epithelial cells (BECs) from patients with COPD, as cigarette smoke. METHODS: BECs from patients with COPD who underwent surgery for lung cancer and comparator (immortalised 16HBE) cells were grown at air liquid interface (ALI). BECs were exposed to aerosols from a JUUL® e-cigarette (Virginia Tobacco and Menthol pods at 5% nicotine strength) or reference 3R4F cigarette for 30 min at ALI. Cell cytotoxicity, DNA damage and inflammation were measured. RESULTS: In response to the Virginia Tobacco and Menthol flavoured e-cigarette aerosols, COPD BECs showed comparable LDH release (cell cytotoxicity, p = 0.59, p = 0.67 respectively), DNA damage (p = 0.41, p = 0.51) and inflammation (IL-8, p = 0.20, p = 0.89 and IL-6, p = 0.24, p = 0.93), to cigarette smoke. 16HBE cells also showed comparable cellular responses to cigarette smoke. CONCLUSION: In airway cells from patients with COPD, aerosols from a fourth-generation e-cigarette were associated with similar toxicity to cigarette smoke. These results have potential implications for the safety of e-cigarette use in patients with lung disease.

Plasma Extracellular Vesicle miRNAs Can Identify Lung Cancer, Current Smoking Status, and Stable COPD.

Lung cancer remains the leading cause of cancer related mortality worldwide. We aimed to test whether a simple blood biomarker (extracellular vesicle miRNAs) can discriminate between cases with and without lung cancer. METHODS: plasma extracellular vesicles (EVs) were isolated from four cohorts (n = 20 in each): healthy non-smokers, healthy smokers, lung cancer, and stable COPD participants. EV miRNA expression was evaluated using the miRCURY LNA miRNA Serum/Plasma assay for 179 specific targets. Significantly dysregulated miRNAs were assessed for discriminatory power using ROC curve analysis. RESULTS: 15 miRNAs were differentially expressed between lung cancer and healthy non-smoking participants, with the greatest single miRNA being miR-205-5p (AUC 0.850), improving to AUC 0.993 in combination with miR-199a-5p. Moreover, 26 miRNAs were significantly dysregulated between lung cancer and healthy smoking participants, with the greatest single miRNA being miR-497-5p (AUC 0.873), improving to AUC 0.953 in combination with miR-22-5p; 14 miRNAs were significantly dysregulated between lung cancer and stable COPD participants, with the greatest single miRNA being miR-27a-3p (AUC 0.803), with two other miRNAs (miR-106b-3p and miR-361-5p) further improving discriminatory power (AUC 0.870). CONCLUSION: this case control study suggests miRNAs in EVs from plasma holds key biological information specific for lung cancer and warrants further prospective assessment.

Extracellular vesicles in chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterised by chronic inflammation and significant airflow obstruction that is not fully reversible, and is one of the leading causes of morbidity and mortality worldwide. Extracellular vesicles (EVs) (including apoptotic bodies, microvesicles and exosomes) are small membrane-bound vesicles released by nearly all cell types and can be found in various bodily fluids including blood, sputum and urine. EVs are key mediators in cell-cell communication due to their ability to exchange information to recipient cells, influencing physiological and pathological conditions using their bioactive cargo (DNA, RNA, miRNA, proteins and other metabolites). Therefore the main aim of this review is to highlight recent evidence of the potential use of EVs as diagnostic and therapeutic biomarkers for COPD managements, as well as EVs potential role in COPD pathogenesis. As EVs have been under intense investigation as diagnostic and therapeutic biomarkers for lung disease, in relation to COPD, key studies have identified EVs as potential biomarkers to distinguish exacerbations from stable state, and to characterise COPD phenotypes. EVs are also linked to key inflammatory mediators in COPD progression. In addition, bacteria and their EV cargo influence the lung microenvironment. Further recent therapeutic approaches and advances have seen EVs bioengineered as novel drug delivery vehicles, which could potentially have clinical utility for lung diseases such as COPD.

Chronic obstructive pulmonary disease (COPD) and lung cancer: common pathways for pathogenesis.

Chronic obstructive pulmonary disease (COPD) and lung cancer comprise the leading causes of lung disease-related mortality worldwide. Exposure to tobacco smoke is a mutual aetiology underlying the two diseases, accounting for almost 90% of cases. There is accumulating evidence supporting the role of immune dysfunction, the lung microbiome, extracellular vesicles and underlying genetic susceptibility in the development of COPD and lung cancer. Further, epigenetic factors, involving DNA methylation and microRNA expression, have been implicated in both diseases. Chronic inflammation is a key feature of COPD and could be a potential driver of lung cancer development. Using next generation technologies, further studies investigating the genomics, epigenetics and gene-environment interaction in key molecular pathways will continue to elucidate the pathogenic mechanisms underlying the development of COPD and lung cancer, and contribute to the development of novel diagnostic and prognostic tools for early intervention and personalised therapeutic strategies.

Potential clinical utility of multiple target quantitative polymerase chain reaction (qPCR) array to detect microbial pathogens in patients with chronic obstructive pulmonary disease (COPD).

BACKGROUND: Culture-independent methods such as quantitative polymerase chain reaction (qPCR) are more sensitive for detecting pathogens than conventional culture. This study aimed to test the clinical potential of a multiple target qPCR array in identifying sputum pathogens, compared to traditional culture. METHODS: Forty chronic obstructive pulmonary disease (COPD) patients provided spontaneous sputum and blood samples during an exacerbation event (n=25 patients) and in stable state (n=15 patients). Sputum was processed and analysed by microscopy, culture and sensitivity testing (MCS) to identify living microbial isolates, and multiple target qPCR (44 targets for bacterial and fungal pathogens and antibiotic resistance genes), and 16S rRNA gene sequencing. RESULTS: Six microbial isolates (5 bacterial, 1 fungal) were cultured from 20 exacerbation and 10 stable patient sputum samples. Four of these microbial isolates had their presence in patient sputum confirmed by qPCR. All bacterial targets detected by qPCR were further confirmed by 16S rRNA gene sequencing at a genus level. qPCR identified significantly more bacterial pathogens than culture (P<0.001). The most prevalent bacterial species identified by qPCR were Streptococcus pneumoniae (72% of patients), Pseudomonas aeruginosa (40%), Prevotella oris (32%) and Haemophilus influenzae (17%). Microbial species diversity and richness were not significantly different between samples obtained from exacerbating and clinically stable cases. 16S rRNA gene sequencing identified Pseudomonas 4408227 (P=0.022, FDR =0.043 AUC =0.72) as a significantly different bacterial OTU (operational taxonomic units) in exacerbation sputum samples compared to stable state samples. CONCLUSIONS: Multiple target qPCR was more sensitive for detection of sputum pathogens in COPD patients than conventional culture. 16S rRNA gene sequencing confirmed the identity at a genus level of all bacterial targets detected by qPCR, as well as identifying bacterial OTUs that could potentially be used to distinguish between exacerbation and stable COPD disease states. Multiple target qPCR pathogen detection in the sputum of COPD patients warrants further investigation to determine how it may influence COPD clinical management.

The contribution of respiratory microbiome analysis to a treatable traits model of care.

The composition of the airway microbiome in patients with chronic airway diseases, such as severe asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis and cystic fibrosis (CF), has the potential to inform a precision model of clinical care. Patients with these conditions share overlapping disease characteristics, including airway inflammation and airflow limitation. The clinical management of chronic respiratory conditions is increasingly moving away from a one-size-fits-all model based on primary diagnosis, towards care targeting individual disease traits, and is particularly useful for subgroups of patients who respond poorly to conventional therapies. Respiratory microbiome analysis is an important potential contributor to such a 'treatable traits' approach, providing insight into both microbial drivers of airways disease, and the selective characteristics of the changing lower airway environment. We explore the potential to integrate respiratory microbiome analysis into a treatable traits model of clinical care and provide a practical guide to the application and clinical interpretation of respiratory microbiome analysis.