Immunoglobulins play a vital role in host immune response and in the pathogenesis of conditions like asthma. Therapeutic agents such as monoclonal antibodies target specific elements of the asthmatic inflammatory cascade. Decisions to utilize these medications are often based on systemic inflammatory profiling without direct insight into the airway inflammatory profile. We sought to investigate the relationship between immunoglobulin and cytokine profiles in the airway and systemic immune compartments of adult asthmatics. Blood sampling and bronchoscopy with bronchoalveolar lavage (BAL) were performed in 76 well-defined adult asthmatics. Antibody and cytokine profiles were measured in both BAL and serum using ELISA and quantibody arrays. There was no relationship between BAL and serum levels of IgE. This is of significance in an asthma population. For some analytes, correlation analysis was significant (P < 0.05) indicating representativeness of our cohort and experimental setup in those cases. Nevertheless, the predictive power (r2) of the BAL-to-serum comparisons was mostly low except for TNF-α (r2 = 0.73) when assuming a simple (linear) relationship. This study highlights the importance of sample site when investigating the roles of immunoglobulins and cytokines in disease pathogenesis and suggests that both localized and systemic immune responses are at play. The prescription of asthma monoclonal therapy is generally based on systemic evaluation of cytokine and immunoglobulin levels. Our research suggests that this approach may not fully reflect the pathophysiology of the disease and may provide insight into why some patients respond to these targeted therapies while others do not.

BackgroundTracheostomies in children are associated with significant morbidity, poor quality of life, excess healthcare costs and excess mortality. The underlying mechanisms facilitating adverse respiratory outcomes in tracheostomised children are poorly understood. We aimed to characterise airway host defence in tracheostomised children using serial molecular analyses. MethodsTracheal aspirates, tracheal cytology brushings and nasal swabs were prospectively collected from children with a tracheostomy and controls. Transcriptomic, proteomic and metabolomic methods were applied to characterise the impact of tracheostomy on host immune response and the airway microbiome. ResultsChildren followed up serially from the time of tracheostomy up to 3 months postprocedure (n=9) were studied. A further cohort of children with a long-term tracheostomy were also enrolled (n=24). Controls (n=13) comprised children without a tracheostomy undergoing bronchoscopy. Long-term tracheostomy was associated with airway neutrophilic inflammation, superoxide production and evidence of proteolysis when compared with controls. Reduced airway microbial diversity was established pre-tracheostomy and sustained thereafter. ConclusionsLong-term childhood tracheostomy is associated with a inflammatory tracheal phenotype characterised by neutrophilic inflammation and the ongoing presence of potential respiratory pathogens. These findings suggest neutrophil recruitment and activation as potential exploratory targets in seeking to prevent recurrent airway complications in this vulnerable group of patients.

"The Potential Role of Gastric Microbiology in Respiratory Disease." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp.

When initiated the human microbiome project did not include the lungs and airways in its sampling sites, indicating an under appreciation of the role of the human lung microbiome in health and disease. This paradigm has recently changed through the use of culture independent methods to characterise the human lung microbiome. The original thinking, that the normal lung was essentially sterile, had previously been challenged by findings of microaspiration in normal volunteers and in patients with decreased levels of consciousness. The sterile lung was also questioned by findings of clinically occult infection markers in lung allograft recipients. What is arguably a “rediscovery” of the importance of the human lung microbiome may still underappreciate physiological and patho-physiological inter-relationships between organ systems, studied in separate research disciplines. In particular, microaspiration may be an important, direct mechanism through which the lung microbiome is modulated. As well as aspiration related to gastro-oesophageal reflux and microaspiration the authors feel that the importance of dysphagia in chronic lung disease, will be increasingly recognised in frailty related microbiome exchange between the oropharynx into the lung. This review therefore discusses interconnections in the human microbiome, with a focus on the potential for aerodigestive pathophysiology and microaspiration. Potential connections with human lung disease are discussed and contextualised within a developing literature. This review therefore highlights much needed new targets for translational intervention in lung pathophysiology and underlies the importance of a mixed disciplinary team approach for the future.

Introduction: The purpose of this study is to explore IPF patients' eating and drinking experiences, and the impact of any changes subsequent to their diagnosis and any coping mechanisms. Methods: This qualitative study used purposive sampling to recruit IPF patients from IPF support groups and clinics. Semi-structured, in-depth interviews explored patients' experiences of their eating and drinking, using an evidence-informed topic guide. Interviews were recorded via video or telephone call, transcribed, and data coded and analysed using a reflexive thematic analysis. Results: Fifteen IPF patients (9 M, 6 F), median age 71, range (54-92) years were interviewed via telephone call (n: 5), video call (n: 10). Three main themes were identified, along with several sub-themes. These were 1) 'Eating, as such, is no longer a pleasure'. This theme focused on the physical and sensory changes in eating and drinking, and the subsequent emotional and social impact. 2) 'It is something that happens naturally and just try and get on with it'. This theme centred on the self-employed strategies used to manage changes to eating and drinking. 3) 'What is normal and the new normal'. This theme focuses on patients' information seeking to understand eating and drinking changes and what are their expectations about these changes. Conclusion: To our knowledge, this is the first study to report on IPF patients' lived experience of swallowing changes due to their diagnosis Findings demonstrate that some patients have substantial struggles and challenges with eating and drinking, affecting them physically, emotionally and socially. There is a need to provide better patient information for this area.

Introduction: Poly (I:C) is a synthetic double-stranded RNA, a TLR3 ligand and mimic of viral infection. Epithelial cells exposed to poly (I:C) release inflammatory cytokines (e.g. IL-6) and display delayed wound healing. miR-149-5p may regulate inflammation and targets transcription factor p63, a key regulator of airway epithelium wound repair. Aims: To determine the role of miR-149-5p in inflammatory responses and regulation of p63 in bronchial and alveolar epithelial cells exposed with poly (I:C). Methods: Bronchial (BEAS-2B) or alveolar (A549) epithelial cell lines were maintained as submerged culture in BEBM supplemented with insulin-transferrin-sodium selenite, linoleic-BSA (ITS+1: 1%) or F-12K with ITS+1 (1%) during stimulation with poly (I:C, 0.5 µg/ml) for 48h, respectively. Cell viability was determined by lactate dehydrogenase release. miR-149-5p expression was assessed using TaqMan assay. IL-6 release and p63 expression were determined by ELISA and immunoblotting techniques, respectively. Results: Poly (I:C) stimulation caused a significant BEAS-2B cell death after 24 and 48h stimulation but showed no effect on A549 cell viability. BEAS-2B cells exposed to poly (I:C) expressed lower level of miR-149-5p expression, which was correlated with increased IL-6 release and p63 expression and after 24 and 48h, respectively. Ectopic expression of miR-149-5p mimic in BEAS-2B cells, suppressed p63 after 48h. Although miR-149-5p expression was supressed in A549 cells stimulated with poly (I:C) after 48h, the IL-6 release and p63 expression remained undetected. Conclusion: miR-149-5p may regulate IL-6 and p63 expressions. BEAS-2B cells respond differently to poly(I:C) compared with A549 cells.

Infection remains a significant contributor to morbidity and mortality in patients with myeloma. This guideline was developed by a multidisciplinary group of clinicians who specialise in the management of patients with myeloma and infection from the medical and scientific advisory group from Myeloma Australia and the National Centre for Infections in Cancer. In addition to summarising the current epidemiology and risk factors for infection in patients with myeloma, this guideline provides recommendations that address three key areas in the prevention of infection: screening for latent infection, use of antimicrobial prophylaxis and immunoglobulin replacement and vaccination against leading respiratory infections (severe acute respiratory syndrome coronavirus 2, influenza and Streptococcus pneumoniae) and other preventable infections. This guideline provides a practical approach to the prevention of infection in patients with myeloma and harmonises the clinical approach to screening for infection, use of prophylaxis and vaccination to prevent infectious complications.

Introduction: Aim: to describe IPF patients' perceptions of swallowing dysfunction, laryngopharyngeal reflux and laryngeal hypersensitivity symptoms. Methods: A cross sectional observational study. Recruitment was undertaken through UK pulmonary fibrosis support groups and the Newcastle Interstitial Lung Diseases clinic, between (January 2021- November 2021). Three validated questionnaires were used (1) The Eating Assessment Tool-10 (EAT-10), (normal <3), (2) The Reflux Symptoms Index (RSI), (normal ≤13) and (3) The Newcastle Laryngeal Hypersensitivity (LHQ) Questionnaire. Results: Forty IPF patients were recruited (26 M: 14 F, age median 71, range 52-92 years) 30% used oxygen. EAT-10 mean score was 5.9 (±6.5), range 0 to 20, (23/40) 57% of patients had swallowing difficulty evident by a total EAT-10 score >3. The most common symptoms reported were coughing on eating and food sticking in the throat. The RSI mean score was 15.6 (±8.3), range from 2 to 31, (24/40) 60% had an RSI score >13, suggesting the presence of extra-oesophageal reflux in some patients. The Newcastle LHQ scores indicated that 25 patients reported alterations to laryngeal sensation, and had a total LHQ scores that were below the lower level of 95% CI of published normal LHQ score (17.8). Conclusion: In this study, a majority of IPF patients reported swallowing difficulty, reflux and laryngeal sensation symptoms. Further work is indicated to explore these symptoms fully in this vulnerable group.

Primary epithelial cell models to provide insights into subglottic stenosis. We read with interest the recent study by Sharif et al. Using computational analyses of a previous scRNA-seq from seven idiopathic subglottic stenosis (iSGS) patients and three matched controls, the authors described a central role for a dysregulated epithelium in the pathophysiology of SGS. This study of genetic risk in SGS showed that TGFβ response pathways and epithelial–mesenchymal transition (EMT) were prominently implicated. The findings compliment and extend previous work from the group, and the North American Airway Collaborative (NoAAC), to provide collaborative new translational research into SGS. We thank Sharif et al. for referencing our work investigating the role of EMT in lung allograft dysfunction and collaborative research with Australian investigators in smoking-related pathophysiology, which provided context for their work in SGS. We agree that EMT-related airways remodeling in SGS might be a key area to research and have previously developed primary and cell-line-based models to study pathways of epithelial injury in SGS. We think our results may be complimentary to the computational data presented by Sharif et al. Gastroesophageal reflux disease has been implicated in patients with iSGS and we found that bile acids were a potent stimulus for EMT in primary epithelial cells grown from patient subglottic brushings. Related work indicated that having a role in airways remodeling EMT is an important mechanism in malignancy and may be a mechanistic link for the increased risk associated with airflow obstruction per se, over and above risk associated with smoking. We suggest that primary airway culture systems may have a key role in further research. Patientderived cell models are amenable to studying differences in disease pathophysiology related to age and sex, for example, and allow the screening of potential new treatment approaches. We suggest that this has the potential to compliment the elegant computational methods of Sharif et al. and work of others implicating EMT in iSGS. ADIL ALDHAHRANI, PhD Department of Clinical Laboratory Sciences, Turabah University College, Taif University, Taif, Saudi Arabia

Background Highly variable responses to immune checkpoint inhibitors (ICI) among NSCLC patients underscore the need for predictive biomarkers to guide therapy selection. In this study, RNA-seq data were used to classify patients into our previously reported TME subtypes, which are prognostic for survival and predictive for immunotherapy response across multiple cancers, including NSCLC.1 Here, we further validated these TME subtypes in ICI-treated NSCLC patients Methods NSCLC patients from three independent cohorts, GSE135222 (n = 27), SU2C-MARK (n = 152), and a novel retrospective cohort from St. Luke's Cancer Institute (n = 161), were screened for the following criteria: (1) advanced lung adenocarcinoma (LUAD) or advanced squamous cell carcinoma (LUSC); (2) ICI-naive at the time of biopsy; (3) no EGFR or ALK alterations, resulting in 258 qualified samples. As described by Bagaev et al.1 transcriptomic-based TME subtyping was applied to classify each sample as fibrotic (F), immune-enriched/fibrotic (IE/F), immune-enriched/non-fibrotic (IE), or desert (D). TME cell types were characterized using Kassandra cell deconvolution.2 Logrank tests were used for Kaplan-Meier and Cox proportional hazards models. Results Overall (OS, p = 0.002) and progression-free (PFS, p = 0.0002) survival were calculated for each TME subtype using ICI-treated NSCLC cohorts (table 1). IE samples had the best prognosis (median OS 38.7 mos., p = 0.009; median PFS 9.0 mos., p = 0.001), and the D samples exhibited statistically significant lower OS (median 14.1 mos, p = 0.002) and PFS (median 3.3 mos., p = 0.002). TME subtypes were further grouped into immune-enriched 'hot' (IE + IE/F, median OS 29.6 and PFS 8.9 mos.) and 'cold' (D + F, median OS 13.3 and PFS 4.1 mos.) groups, which further improved patient separation by PFS (p = 0.0002) and OS (p = 1e-05). Independent of PD1 status, CD8 T cell infiltration was qualitatively and quantitatively associated with better OS (HR 0.23, p < 0.005) and PFS (HR 0.25, p < 0.005) in LUAD but not in LUSC. Conclusions Here, transcriptomic-based TME subtypes and cell type deconvolution were applied to an unselected cohort of NSCLC patients receiving ICIs. The TME subtypes and CD8 T cell infiltration were associated with OS and PFS. When samples were grouped into 'hot' (IE + IE/F) and 'cold' (D + F) TME subtypes, sample stratification further improved. Our results were consistent with previous findings,1 but further investigation is required to validate these TME subtypes as predictive biomarkers. References A. Bagaev et al. 'Conserved pan-cancer microenvironment subtypes predict response to immunotherapy,' Cancer Cell, 2021; 39(6): 845–865.e7, Jun. 2021, doi: 10.1016/j.ccell.2021.04.014. A. Zaitsev et al. 'Precise reconstruction of the TME using bulk RNA-seq and a machine learning algorithm trained on artificial transcriptomes,' Cancer Cell, Aug. 2022; 40(8): 879–894.e16, , doi: 10.1016/j.ccell.2022.07.006. Ethics Approval The human data analyzed here was collected under IRB protocol at St. Luke's Cancer Institute.

Background Unfortunately, many COPD patients continue to exacerbate despite good adherence to GOLD Class D recommended therapy. Acute exacerbations lead to an increase in symptoms, decline in lung function and increased mortality rate. The purpose of this review is to do a literature search for any prophylactic anti-microbial treatment trials in GOLD class D patients who ‘failed’ recommended therapy and discuss the role of COPD phenotypes, lung and gut microbiota and co-morbidities in developing a tailored approach to anti-microbial therapies for high frequency exacerbators. Main text There is a paucity of large, well-conducted studies in the published literature to date. Factors such as single-centre, study design, lack of well-defined controls, insufficient patient numbers enrolled and short follow-up periods were significant limiting factors in numerous studies. One placebo-controlled study involving more than 1000 patients, who had 2 or more moderate exacerbations in the previous year, demonstrated a non-significant reduction in exacerbations of 19% with 5 day course of moxifloxacillin repeated at 8 week intervals. In Pseudomonas aeruginosa ( Pa ) colonised COPD patients, inhaled antimicrobial therapy using tobramycin, colistin and gentamicin resulted in significant reductions in exacerbation frequency. Viruses were found to frequently cause acute exacerbations in COPD (AECOPD), either as the primary infecting agent or as a co-factor. However, other, than the influenza vaccination, there were no trials of anti-viral therapies that resulted in a positive effect on reducing AECOPD. Identifying clinical phenotypes and co-existing conditions that impact on exacerbation frequency and severity is essential to provide individualised treatment with targeted therapies. The role of the lung and gut microbiome is increasingly recognised and identification of pathogenic bacteria will likely play an important role in personalised antimicrobial therapies. Conclusion Antimicrobial therapeutic options in patients who continue to exacerbate despite adherence to guidelines-directed therapy are limited. Phenotyping patients, identification of co-existing conditions and assessment of the microbiome is key to individualising antimicrobial therapy. Given the impact of viruses on AECOPD, anti-viral therapeutic agents and targeted anti-viral vaccinations should be the focus of future research studies.

Introduction: Bronchial epithelial cells exposed to poly(I:C) release inflammatory cytokines (e.g. IL-6). microRNAs (e.g. miR-149-5p) have emerged as regulators of inflammation. There are however different reports on poly(I:C) dosage and culture conditions. Aims: To establish a dose-dependent study of poly(I:C) in different culture conditions to assess the 1) epithelial cell viability, 2) release of IL-6, and 3) miR-149-5p expression. Methods: BEAS-2B cells were maintained as submerged cultures in 3 conditions including BEGM, BEBM supplemented with insulin-transferrin-sodium selenite, linoleic-BSA (ITS+1: 1%) during stimulation, or 24h prior and during the stimulation. Cells were incubated with poly(I:C) for 24h. Cell viability was assessed by lactate dehydrogenase release and IL-6 level was determined using ELISA assay. miR-149-5p expression was assessed at 6-24h after incubation with poly(I:C), using TaqMan assay. We used PRISM software for statistical analysis. Results: Poly(I:C) (0.1-50ug/ml) caused no significant cell death in cells maintained in BEGM but significantly reduced viability for cells cultured in serum free conditions. Poly(I:C) (0.5-5ug/ml) and (0.5ug/ml) resulted in significant release of IL-6 in cells maintained in BEGM and serum free conditions, respectively. IL-6 release was about 7 folds greater from cells in serum free conditions compared with BEGM after incubation with poly(I:C) (0.5ug/ml). miR-149-5p expression decreased over time which was statistically significant after 24h incubation of cells with poly(I:C) (0.5ug/ml) in serum free media. Conclusion: Different culture conditions affect cell viability and IL-6 release in response to poly(I:C). miR-149-5p may regulate IL-6 expression.