ERJ Open Research

Chronic Pseudomonas aeruginosa lung infections are becoming harder to treat due to global escalation of antimicrobial resistance (AMR). Bacteriophage (phage) therapy has emerged as a promising adjunct to conventional antibiotics, especially in chronic lung infections such as those seen in cystic fibrosis (CF). However, phage monotherapy may be limited by the emergence of phage-resistant bacterial populations and there remains limited preclinical evidence evaluating both antimicrobial efficacy and host safety in physiologically relevant human airway models. Here, we evaluated the safety and antimicrobial activity of Kara-mokiny 3, a myovirus bacteriophage, alone and in combination with subinhibitory concentrations of tobramycin using fully differentiated paediatric primary airway epithelial cells (pAECs) cultured at the air-liquid interface (ALI). Kara-mokiny 3 rapidly reduced P. aeruginosa viability and exhibited synergistic activity with tobramycin, resulting in significantly greater bacterial killing than either treatment alone. Importantly, phage treatment replicated efficiently in the presence of its bacterial host while preserving epithelial morphology, mucin production and epithelial barrier architecture., without inducing cytotoxicity or excessive IL-6 and IL-8 inflammatory responses. These findings demonstrate that phage-antibiotic combination therapy can enhance antimicrobial activity while maintaining epithelial safety in a physiologically relevant human airway model. This study represents one of the first comprehensive evaluations of phage-antibiotic combination therapy in differentiated primary airway epithelial cultures, providing important preclinical evidence supporting the development of personalised phage-based therapies for the treatment of MDR pulmonary infections. ImportanceThe rise of MDR P. aeruginosa has created an urgent need for alternative treatment strategies for chronic lung infections. Although phage therapy is receiving increasing clinical attention, there is limited evidence evaluating its safety and efficacy in physiologically relevant human airway models. Using differentiated primary airway epithelial cultures, we demonstrate that a phage-antibiotic combination reduces bacterial burden without compromising epithelial integrity and toxicity or excessive inflammatory responses. These findings provide translational evidence supporting phage-antibiotic combination therapy and highlight the value of primary airway epithelial models for the preclinical assessment of emerging antimicrobial interventions, supporting the translation of personalised phage therapies.

In 1992, inhaled NO (iNO) at low doses entered the practice of medicine for cardiopulmonary indications. Recently, iNO at higher doses has been tested in diverse pulmonary infections. However, nothing is known about the ability of exogenous NO gas to kill Mycobacterium tuberculosis (Mtb), the leading cause of death from infection between major viral pandemics. Here we mimicked exposure conditions used in recent human studies of high-dose iNO to explore the effects of NO gas against Mtb in vitro and in mice. We saw a profound bactericidal effect of NO gas in vitro against Mtb incubated in shallow, mildly acidic fluid. Mtb-infected mice tolerated inhaled NO well, except for developing more methemoglobinemia than humans at the same level of exposure. In Mtb-infected mice with poorly aerated pulmonary infiltrates, inhaled NO had an anti-inflammatory effect but did not reduce the bacterial burden. These results may help inform the decision whether to test inhaled NO as an adjunctive treatment for tuberculosis, and if so, in what settings and with what goals.

Background Exposure to environmental tobacco smoke (ETS) negatively affects children`s health, but few studies examined parental smoking behaviour in families of children with respiratory diseases. We studied parental smoking prevalence, characteristics, and changes over one year among families in the Swiss Paediatric Airway Cohort (SPAC). Methods We included children aged 0-17 years referred to paediatric respiratory outpatient clinics in Switzerland from 2017 to 2024. Parents answered a questionnaire at the initial clinic visit and again after one year. We used multivariable logistic regression to explore the characteristics of mothers and fathers who smoked and assessed changes in smoking behavior over one year. Results Among 4,199 children (median age 9 years [IQR 5-12]), 31% were exposed to parental smoking at baseline (paternal smoking: 16%; maternal smoking: 6%; both parents smoking: 9%). Mothers were more likely to smoke if they had a lower education level (OR 2.0, 95%CI 1.6-2.5 for compulsory education vs university education), did not have Swiss nationality (OR 1.3, 1.0-1.6) and lived in a socially disadvantaged neighborhood (OR 1.3, 1.0-1.7). Similar associations were observed for fathers. In addition, fathers were more likely to smoke if they were unemployed (OR 2.0, 1.3-3.2 vs having a full-time job. The strongest predictor of smoking was having a partner who smoked, with ORs above 6 for both mothers and fathers. Parents of 2,338 children completed the one-year follow-up questionnaire. Data from 2226 mothers and 1895 fathers showed that among baseline smokers with follow-up data, 225 (78%) mothers and 382 (81%) of fathers continued smoking, and only 63 (22%) of mothers and 90 (19%) of fathers quit. Among baseline non-smokers, 47 (2%) mothers and 54 (3%) fathers started smoking. Conclusions One-third of children consulting respiratory specialists in Switzerland are exposed to parental smoking. ETS exposure was strongly associated with socio-economic factors. Even after visiting a specialized clinic, most parents continued to smoke. This highlights the urgent need for stronger national smoking policies and targeted support to help these parents quit and stay smoke-free.

Background: The systemic immune responses associated with persistent respiratory symptoms (PRS) after exposure to airborne environmental pollutants remain poorly understood. Objective: To identify immune disturbances associated with PRS, defined as persistent wheeze, cough, or breathlessness, we examined systemic immune responses and airway function in a cross-sectional cohort with detailed histories of airborne pollutant exposure. Methods: Never-smoking post-deployment Veterans with PRS (n=16) or without PRS (n=24) underwent chest computed tomography, pulmonary function testing, and oscillometry to assess structural and functional airway abnormalities. Peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3/CD28 antibodies, lipopolysaccharide, or {beta}-glucan, and cytokine production was measured. Correlation analyses evaluated associations between cytokine responses and physiological measures of airway function. Results: Oscillometry, but not conventional pulmonary function testing or chest computed tomography, detected small-airway abnormalities in participants with PRS, including significantly greater frequency dependence of resistance and higher resonant frequency. Baseline PBMC cytokine concentrations were similar between groups. After stimulation, however, PBMCs from participants with PRS showed increased IL-17A production consistent with a type 17 (T17) response; innate stimulation also increased the type 2 (T2) cytokines IL-33 and IL-4. T2/T17 cytokine responses correlated positively with oscillometric measures of small-airway dysfunction. Conclusion: Individuals with PRS exhibited a stimulus-dependent systemic T2/T17 immune signature that was associated with early small-airway dysfunction. Clinical Implication: Stimulus-dependent systemic immune profiling, combined with oscillometry, may help identify early respiratory abnormalities in pollutant-exposed individuals whose conventional pulmonary tests remain normal.

Research on Pseudomonas aeruginosa adaptation in cystic fibrosis (CF) has historically relied on comparing chronic isolates to laboratory reference strains, or evolving reference strains in environments simulating chronic CF. This work has established a small set of genes, including lasR, mucA, and mexZ, as canonical markers of CF patho-adaptation. However, without broad non-CF comparators, it remains unclear how specific these signatures are to CF. We used a structured literature review to define 20 historically emphasized "canonical CF genes", then evaluated their mutational patterns across 4,475 genetically distinct P. aeruginosa genomes from seven defined clinical and environmental contexts. We tested four competing hypotheses: (1) enrichment in adult CF alone, (2) in adult and pediatric CF combined, (3) in chronic lung infections broadly (including non-CF bronchiectasis), or (4) no strong environment-specific enrichment. We found little evidence that canonical gene mutations were specifically enriched in adult CF or CF more broadly. Instead, loss-of-function and individual mutations in genes including mucA, mexB, and mexZ were enriched across chronic lung infections, while most canonical genes (including lasR) showed no strong environment-specific enrichment. These results demonstrate that a canon of genes believed to drive patho-adaptation in CF instead largely reflects the narrow comparative framework of past studies rather than CF-exclusive selection. Our findings emphasize shared evolutionary pressures between CF and non-CF bronchiectasis, highlighting opportunities to exchange research and therapeutic insights across chronic infection clinical contexts.

Rationale: Pulmonary hypertension is defined by pulmonary hemodynamics, but diagnostic and prognostic biomarkers remain limited. Nuclear magnetic resonance (NMR) spectroscopy provides detailed insights, particularly in the lipid metabolism. Objectives: To explore circulating NMR-derived metabolites and lipoprotein-related parameters for their association with pulmonary hemodynamics and to analyse their prognostic properties in pulmonary arterial hypertension (PAH). Methods: Retrospective analysis of a PAH cohort with complete diagnostic workup including right heart catheterization and baseline serum samples, from the prospective GRaz Pulmonary Hypertension-Metabolism (GRAPH-M) registry. Measurements: NMR-derived metabolites and lipoprotein-related parameters were analyzed for their association with clinically relevant parameters of PAH. We defined PHIHDL, a score derived from high-density lipoprotein (HDL) related measures based on their strong association with pulmonary hemodynamics, and evaluated its prognostic value. Results: We included 100 patients with PAH treated at the PH clinic of LKH University Hospital, Medical University of Graz, between 2011 and 2021. Age was 61{+/-}15 years, female/male ratio 2.5, BMI 26 {+/-}7 kg/m2, mPAP 41{+/-}16 mmHg, PAWP 8.8{+/-}3.2 mmHg, PVR 8.0{+/-}4.9 WU, and median survival was 8.0 years. During follow-up, 46 patients died. We identified a cluster of 12 HDL-related measures that showed significant inverse association to pulmonary hemodynamics and derived PHIHDL from the reversed scaled average of these particles. PHIHDL was associated with all-cause mortality after adjustment for age and sex (HR 2.96, 95% CI 1.52-5.70), independent of the clinical risk scores COMPERA 2.0 and REVEAL Lite2. Conclusion: PHIHDL, a pulmonary hemodynamics-based metabolomic score, provides independent prognostic information beyond established risk scores in PAH.

Background Elexacaftor-tezacaftor-ivacaftor (ETI) has been demonstrated to improve lung function in clinical trials; however, evidence describing effects on trajectories and whether long-term improvements are sustained (>1-year) is lacking. We estimated within-person lung clearance index (LCI) trajectories before and after ETI initiation, assessing changes in level and rate of change, alongside acute LCI change, up to three years after ETI initiation. Methods Prospective observational study of children at a tertiary hospital. Children aged 3-17 years with [&ge;]2 LCI testing occasions (i) before and (ii) after starting ETI were used to describe lung function trajectories. Children with [&ge;]1 pre-ETI and [&ge;]1 post-ETI LCI occasion(s) were used to describe acute LCI change after ETI initiation. Age-adjusted LCI trajectories for time periods (i) before and (ii) after ETI initiation were estimated using linear mixed-effects models, and pre- and post-ETI LCIs were compared using paired Wilcoxon tests. Results Mean pre-ETI and post-ETI longitudinal changes in LCI were -0.007 (95% CI: -0.28, 0.27; n=35) and 0.12 (95% CI: -0.17, 0.41; n=20) turnovers per year, respectively. Before ETI initiation, 57% (30/53) of patients had an LCI[&ge;]7.1 turnovers (indicating impaired lung function), compared to 26% (14/53) post-ETI, with a median LCI difference of -0.70 (95% CI -0.84, -0.46; p<0.001) turnovers. Within-individual variability in LCI decreased post-ETI. Conclusions Our real-world data within a unique longitudinal study provide a comprehensive picture of ETI benefit by outlining not only acute improvement in LCI but maintained stability in LCI trajectories and improved LCI stability sustained up to three years post-initiation.

Inadequate autophagy promotes smoking-induced cellular senescence involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome axis. For the first time, we investigated the therapeutic potential of pemafibrate, a putative TFEB inducer. COPD lung epithelial cells showed reduced TFEB expression. Pemafibrate enhanced autophagy/mitophagy flux and restored lysosomal acidification observed during cigarette smoke (CS) extract exposure in human bronchial epithelial cells, resulting in reduced cellular senescence. TFEB knockdown demonstrated involvement of pemafibrate-induced TFEB in these effects. Pemafibrate induced TFEB expression, mitigated alveolar enlargement and airflow obstruction, and attenuated the CS-induced increase in static lung compliance in a long-term CS-exposed mouse model. It reduced the CS exposure-induced cellular senescence, possibly through autophagy/mitophagy, as suggested by bulk RNA sequencing of mouse lungs. A retrospective cohort study showed that patients given pemafibrate displayed attenuated FEV1.0 decline compared with those given bezafibrate or fenofibrate. In conclusion, pemafibrate is a promising therapeutic agent for COPD, potentially exerting its effects through the regulation of the TFEB-autophagy/mitophagy-lysosome axis.

Background: The prevalence of cannabis use is increasing at an alarming rate owing to its legalization and decriminalization in some countries. Epidemiological evidence on the association between cannabis use and cancer is inconsistent and conflicting. Herein, we performed two-sample Mendelian randomization (MR) to investigate whether cannabis use is causally associated with site-specific cancers in individuals of European ancestry. Methods: We identified 22 independent genetic variants strongly associated with cannabis use (p-value < 5 x 10-8) in a large meta-analysis of genome-wide association studies of individuals of European ancestry. Genome-wide association summary-level data on site-specific cancers were obtained from individuals of European ancestry in FinnGen, Finland. MR analyses were performed using the inverse-variance weighted (IVW) and multivariable method. Sensitivity analyses were performed using the simple median, weighted median, MR-Egger, and MR pleiotropy residual sum and outlier methods. Results: Our multivariable IVW analyses adjusted for cigarette smoking found that genetic liability to cannabis use was causally associated with esophageal cancer (odds ratio [OR] =1.74, 95% confidence interval [CI] =1.29-2.15, p-value =0.013) and lung cancer (OR=1.35, 95% CI = 1.13-1.58, p-value =0.009). However, genetic liability to cannabis use exerted a protective effect against pancreatic cancer (OR=0.77, 95% CI =0.57-0.91, pvalue=0.032) in individuals of European ancestry in the FinnGen. Our sensitivity analyses found no evidence of horizontal pleiotropy between cannabis use and site-specific cancers. Conclusion: We found that genetic liability to cannabis use was associated with esophageal, lung, and pancreatic cancers in individuals of European ancestry.

Introduction: Idiopathic inflammatory myopathy-associated interstitial lung disease (IIM-ILD) is a major cause of morbidity and mortality. We tested whether quantitative myositis-specific autoantibodies and proteomic profiling capture biological heterogeneity and prognosis beyond categorical serology. Methods: Myositis-specific autoantibodies were quantified using the luciferase immunoprecipitation systems assay, and 184 serum proteins were measured in 226 IIM patients; 199 with higher-ILD-risk autoantibodies (Jo-1/MDA5/PL-7/PL-12/EJ), 27 with lower-ILD-risk autoantibodies (Mi-2/NXP2/TIF1{gamma}) and 35 healthy controls. We identified shared and subgroup-specific differences by comparing each subgroup with controls, then correlated quantitative autoantibody and protein levels within higher-risk subgroups. Additional analyses included pathway enrichment, unsupervised clustering, longitudinal lung-function change, and mortality. Results: Higher-ILD-risk subgroups shared interferon-responsive CXCR3 chemokine, IL-6/JAK/STAT3, and apoptosis signaling. Dominant autoantibody subgroup profiles differed: interferon/CXCR3 chemokine signaling with T-cell activation and monocyte recruitment in anti-Jo-1; proteostasis/antigen-processing and vascular/cellular stress signals in anti-MDA5; IL-6/macrophage and profibrotic signals in anti-PL-12; and apoptotic and innate immune activation with metabolic/redox-stress signals in anti-PL-7. Within higher-ILD-risk subgroups, autoantibody levels correlated with interferon-response, profibrotic, and metabolic/vascular proteins (r=0.40-0.74; nominal p<0.05). Unsupervised clustering identified four proteomic endotypes beyond autoantibody type, including an injury-stress endotype associated with worse lung function and poorer survival, and a chemokine/checkpoint-high endotype with relatively preserved lung function. Across 203 participants with 38 deaths, a weighted 10-protein score was associated with all-cause mortality (HR, 3.28; 95% CI, 2.12-5.08; p<0.001). Conclusions: Integrated quantitative autoantibodies and proteomic profiling revealed shared inflammatory biology, autoantibody-associated signatures, and an injury-stress endotype associated with poor survival in IIM-ILD, supporting risk stratification beyond categorical serology.

Background: Olfactory dysfunction is a recognised but poorly characterised comorbidity of Primary Ciliary Dyskinesia (PCD). No prior systematic review has synthesised its prevalence or clinical correlates. Methodology: A PRISMA compliant systematic review and meta-analysis was conducted. Five databases were searched to February 2026. Observational studies reporting olfactory function in confirmed PCD were included. Risk of Bias was assessed using the Newcastle-Ottawa Scale. A random-effects meta-analysis using the Freeman-Tukey double arcsine transformation was performed to calculate pooled prevalence with 95% confidence intervals (CI) and prediction intervals (PI). Results: Twelve studies (n=865) were included. Overall pooled prevalence of olfactory dysfunction was 43.4% (95% CI 25.2-62.5%; 95% PI 0.1-99.0%). Objective psychophysical testing yielded a significantly higher pooled prevalence of 66.1% (95% CI 55.5-76.0%; 95% PI 38.4-88.9%) compared to patient-reported outcome measures (30.5%; 95% CI 11.4-54.0%). Older age, greater sinonasal disease burden, and specific ciliary ultrastructural defects were associated with worse olfactory function. A striking discordance between objective dysfunction and subjective awareness was observed across multiple studies. Conclusions: Olfactory dysfunction is highly prevalent in PCD and substantially under-recognised by patients. Routine objective olfactory screening should be integrated into standard multidisciplinary PCD care.

BackgroundModerate to severe tricuspid regurgitation (TR) affects approximately 1.6 million Americans, yet more than 90% of patients with significant TR remain untreated. Women exhibit higher TR prevalence and more rapid disease progression than men, but the valve-intrinsic mechanisms underlying these sex disparities remain unclear. We hypothesized that sex and circulating testosterone influence tricuspid leaflet remodeling during right-sided pressure overload. MethodsFemale, castrated male (C-Male), and non-castrated male (NC-Male) adult Dorset sheep (n = 45) underwent pulmonary artery banding (PAB) and were followed for 13 {+/-} 1.5 weeks. Tricuspid leaflets were evaluated using morphometry, 3D profilometry, biaxial mechanical testing, histology, and bulk RNA sequencing. Sex-stratified differential gene expression was performed, and pathway enrichment of key biological processes were compared between sexes. ResultsPAB produced a uniform hemodynamic stimulus and equivalent moderate-to-severe TR across sex groups. Despite similar TR burden, leaflet remodeling diverged substantially by sex and castration status. C-Males developed the broadest remodeling phenotype, characterized by diffuse multi-leaflet growth, thickening, increased nuclei count, and low-strain stiffening. Females demonstrated more restricted leaflet and region-specific structural and cellular changes, along with circumferential low-strain stiffening. NC-Males exhibited preferential septal remodeling characterized by growth, thickening, increased nuclei count, and radial high-strain stiffening. Transcriptomic analysis revealed that females upregulated a focused matricellular remodeling program enriched for extracellular space organization (67 DEGs; FDR=0.025), whereas C-Males activated coordinated extracellular matrix and apoptosis-regulatory programs (388 DEGs; FDR=0.009). In contrast, NC-Males exhibited broad transcriptional response (406 DEGs) without significant pathway enrichment. ConclusionsTricuspid leaflet maladaptation during pressure overload is sex-dependent and testosterone-sensitive, involving distinct structural, mechanical, and transcriptional remodeling programs. These findings identify sex and testosterone status as previously under-recognized modulators of tricuspid valve remodeling and may help explain clinical sex disparities in TR progression. NOVELTY AND SIGNIFICANCE What is known?O_LIPulmonary hypertension and right ventricular pressure overload are linked to tricuspid leaflet remodeling through leaflet thickening, enlargement, and altered mechanical properties. C_LIO_LISex and sex-steroid hormones regulate fibrosis and extracellular matrix remodeling in cardiovascular tissues, but their role in tricuspid leaflet remodeling remains poorly understood. C_LI What new information does this article contribute?O_LISex and circulating testosterone status influence the magnitude, spatial distribution, biomechanical behavior, and transcriptional organization of tricuspid leaflet remodeling during pressure overload. C_LIO_LIFemales, castrated males, and non-castrated males develop distinct remodeling programs characterized by focused matricellular remodeling, coordinated extracellular matrix/apoptosis signaling, and diffuse transcriptional activation, respectively. C_LIO_LIThese findings identify sex and hormonal status as biological regulators of tricuspid valve maladaptation during functional tricuspid regurgitation. C_LI SummarySex differences in tricuspid regurgitation progression are recognized clinically, yet the mechanobiological basis underlying these disparities remains poorly understood. Using a controlled ovine model of pressure overload-induced secondary tricuspid regurgitation, we demonstrated that tricuspid leaflet maladaptation is a sex-specific and testosterone-sensitive process spanning structural, mechanical, and transcriptional scales. Under comparable hemodynamic overload, all animals developed significant tricuspid regurgitation, but leaflet remodeling patterns diverged substantially across sexes. Castrated male sheep exhibited the broadest maladaptive phenotype, characterized by diffuse multi-leaflet growth and thickening, increased low-stretch stiffness, and coordinated extracellular matrix and apoptosis-regulatory transcriptional programs. Female sheep developed more spatially restricted remodeling accompanied by a focused matricellular and extracellular matrix secretory response, whereas non-castrated male sheep demonstrated selective leaflet remodeling with broad, but less coordinated, transcriptional activation. Different remodeling patterns emerged in females and castrated males despite comparable testosterone levels, suggesting that testosterone depletion alone does not fully explain these tricuspid valve remodeling phenotypes. These findings establish sex and testosterone status as previously underrecognized biological regulators of tricuspid leaflet maladaptation and support the emerging view that valve leaflets are active, mechanobiologically responsive, participants in functional tricuspid regurgitation progression.

Abstract Importance: Socially vulnerable patients have a high burden of obstructive sleep apnea, but the stage of the referral pathway at which access barriers arise is uncertain. Objective: To determine whether area-level social deprivation was associated with appointment scheduling, wait time, cancellations, or no-shows among adults referred for specialist obstructive sleep apnea care. Design: This was a cross-sectional study evaluating patients referred from December 1, 2016 through November 30, 2019. Data were analyzed from January 30, 2026 to May 16, 2026. Setting: Foothills Medical Centre Sleep Centre in Calgary, Canada. Participants: Adults referred to a tertiary academic sleep centre in Calgary, Alberta, Canada. Eligible patients had valid provincial health insurance and either a scheduled clinic appointment or home sleep apnea test data available. Exposures: Quintiles of the four Canadian Index of Multiple Deprivation domains: residential instability, economic dependency, ethnocultural composition, and situational vulnerability. Main Outcomes and Measures: The primary outcome was receipt of a scheduled specialist appointment. Secondary outcomes were time from referral to the first attended appointment and number of appointment cancellations or no-shows. Results: Among 3111 patients (mean [SD] age, 53.7 [14.3] years; 40.7% female), 1766 (56.7%) were scheduled and 1647 (52.9%) attended an appointment. Each quintile increase in situational vulnerability was associated with lower odds of scheduling (adjusted odds ratio [95% confidence interval] 0.86 [0.81-0.92]), whereas each quintile increase in ethnocultural composition was associated with higher odds (adjusted odds ratio [95% confidence interval] 1.32 [1.22-1.43]). Residential instability and economic dependency were not associated with scheduling. No deprivation domain was associated with time to the first attended appointment, cancellations, or no-shows. Conclusions and Relevance: In this cohort, area-level deprivation was associated with whether patients were scheduled for an appointment but not with wait time or missed visits after scheduling. These findings suggest that equity interventions should focus on completion of referral and scheduling processes.

Background: Higher Body Mass Index (BMI) is an established risk factor of sleep disturbance. It is not known if the effect is homogeneous across the lifecourse or if there is a particular time point in life that might be best to target. Methods: Two-sample Mendelian randomization (MR) was used to investigated the effect of childhood adiposity (adjusting on adulthood adiposity and obstructive sleep apnea (OSA)) on insomnia, morning chronotype, sleep duration, daytime sleepiness and daytime napping. Similarly, total, and direct effect of adulthood adiposity on these outcomes was explored. We used summary statistics from a genome-wide association study (GWAS) of UK Biobank for childhood and adulthood adiposity (n=453,169) and large-scale consortia of OSA (Million Veteran Program) (n=410,268), insomnia, and chronotype (23andMe) (n=1,978,022 and n=248,1000, respectively). Results: Two-sample univariable MR analysis provided no evidence of an effect of genetically predicted childhood adiposity on later life insomnia (Odds ratio (OR)= 0.94, 95% Confidence interval (CI)= 0.87, 1.03). Whereas, multivariable MR (adjusted for adulthood adiposity) analysis provide strong evidence of direct protective effect of genetically predicted childhood adiposity on later life insomnia (OR= 0.70, CI= 0.64, 0.77). Further, both in univariable and multivariable MR, a strong positive effect of increased childhood body size on morning chronotype was observed (OR= 1.16, CI= 1.01, 1.33 and OR= 1.36, CI= 1.15, 1.62, respectively) after accounting for adulthood body size. In both analysis the estimate did not change considerably after aditionally adjusting for OSA. However, childhood and adulthood adiposity found to be associated with OSA and OSA with insomnia. In both univariable and multivariable analysis, increased body size in adulthood increased the risk of having insomnia and a morning chronotype. Conclusions: The findings suggest that higher body size in childhood is not a risk factor for later life insomnia, whereas higher body size in adulthood was. Further, if healthy body size is maintained in adulthood, high childhood adiposity may decrease the risk of insomnia and increase the risk of being a morning person in later life. Keywords: childhood, adulthood, obesity, insomnia, morning chronotype, medelian randomization

Air-liquid interface (ALI) cultures consisting of well-differentiated primary respiratory epithelial cells (PRECs) provide a versatile in vitro model for pharmacological studies and to investigate host-pathogen interactions. Proliferation and differentiation of PRECs require complex media containing several growth factors, hormones, and nutrients. Usually, some of these essential components are provided by the addition of fetal calf serum (FCS). However, several disadvantages of FCS and, most importantly, ethical concerns regarding the method of serum collection have encouraged researchers to find alternatives. Human platelet lysate (hPL) has emerged as a promising alternative to FCS for supporting cell expansion in vitro. In the present study, we investigated the effects of different concentrations of hPL on the proliferation of porcine PRECs and their subsequent differentiation under ALI conditions. Cell morphology was assessed by phase-contrast microscopy, while cell proliferation was evaluated using the ClickTech EdU Cell Proliferation Kit and visualization of proliferating cells by fluorescence microscopy. Differentiation under ALI conditions was monitored by immunofluorescence staining of ciliated cells and the establishment of an intact epithelial barrier was confirmed by measuring transepithelial electrical resistance (TEER). We found that 5% hPL supported efficient cell growth and the subsequent formation of a functional, well-differentiated airway epithelium comparable to or even better than 10% FCS. Thus, hPL offers a reproducible, ethically sound, and scalable alternative to FCS for complex cell culture models in respiratory research, drug development, and host-pathogen interaction studies. LO_SCPLOWAYC_SCPLOW SO_SCPLOWUMMARYC_SCPLOWRespiratory epithelial cells from the lungs of slaughtered animals, such as pigs, can be used for cell culture models to study respiratory diseases and drug development. Air-liquid interface (ALI) cultures closely mimic the natural environment of the airways by exposing the cells to air, making them a valuable alternative to animal experiments. To grow and mature properly, these cells require nutrients and growth factors that are commonly supplied by serum from unborn calves (FCS). However, for ethical and scientific reasons, the use of FCS should be avoided. Therefore, we evaluated whether human platelet lysate (hPL) derived from expired blood donations could replace FCS in ALI cultures. We found that adding 5% hPL to the medium supported efficient cell growth and the development of a well-differentiated airway epithelium. This approach enables the use of an improved and ethically superior model of the (porcine) respiratory tract in accordance with the 3Rs principle.

In chronic pulmonary infection airways are colonized by genetically and phenotypically diverse Pseudomonas aeruginosa populations, yet almost everything known about P. aeruginosa pathogenesis has been learned from single clinical isolates or laboratory reference strains studied in isolation. Whether a diverse population behaves as the sum of its members remains unclear. We infected differentiated primary cystic fibrosis (CF) airway epithelial cells (CF-pAECs) at air-liquid interface with whole P. aeruginosa populations collected from the sputum of three adults with CF, and, in parallel, with genetic variants retrieved from the same populations. We found that host responses to whole populations were not predicted by responses to their derivative variants, and mixed populations did not exhibit the average expected response of the members. Variation in host response was dominated by the tissue-remodeling mediators vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9), which differed distinctly between individual variants, whereas core pro-inflammatory cytokines (IL-6, IL-8, TNF-, IFN-{gamma}) varied comparatively little. Bacterial transcriptomes recorded during infection showed the same asymmetry. Clinical populations shared a transcriptional state distinct from PAO1, and mixed populations maintained stable expression of core regulatory, secretion, and DNA-repair loci (including hfq, xcpT, and ssb), while the derivative variants grown alone exhibited differential transcriptional profiles. Interactions among co-existing lineages therefore shape both bacterial physiology and host response, suggesting that the diverse population, not the single clone, is the appropriate unit of study in chronic infection. ImportanceIn chronic respiratory infections, lungs are not colonized by a clonal population but by diverse co-evolving Pseudomonas aeruginosa lineages that have often evolved together for many years. By comparing primary human cystic fibrosis (CF) airway epithelial cells responses to whole P. aeruginosa populations taken from sputum against their isolated sub-lineages, we demonstrate that host inflammatory responses to the population could not be predicted from the responses to their derivative individual variants, and the bacteria themselves behaved differently in a population than they did alone. These findings highlight that working on single isolates risk misrepresenting chronic infection biology and emphasize that effective therapeutic strategies must target population-level dynamics rather than single isolated strains.

Background Idiopathic Pulmonary Fibrosis (IPF) is a fatal chronic lung disease with limited therapeutic options. While alveolar epithelial injury and fibroblast activation are well-studied, endothelial-mesenchymal transition (EndoMT) is emerging as a critical pathogenic mechanism. The regulatory role of exosomal miRNAs in pulmonary fibrosis remains unclear. This study investigates serum exosomal miRNAs, particularly let-7a-5p, in modulating EndoMT during the onset of pulmonary fibrosis. Methods Clinical cohorts of IPF patients and healthy controls were enrolled. Serum exosomal miRNAs were profiled, followed by differential expression and functional enrichment analyses. In vitro experiments involved human pulmonary artery endothelial cells (HPAECs) transfected with let-7a-5p mimic or inhibitor. Dual-luciferase reporter assays confirmed the binding between let-7a-5p and TGFBR1. HPAECs were co-cultured with lung epithelial cells to examine paracrine signaling. In vivo studies used a bleomycin-induced mouse model with let-7a-5p agomir administration. Assessments included histopathological staining, hydroxyproline content, Western blot, qPCR, micro-CT, and pulmonary function tests. Results Let-7a-5p was significantly downregulated in serum exosomes from IPF patients, correlating with clinical indicators. Mechanistically, let-7a-5p directly bound the TGFBR1 3'UTR to inhibit its expression. Inhibition of let-7a-5p upregulated -SMA, FN1, smad2/3 phosphorylation, and collagen I, while downregulating CD31 and VE-cadherin. Therapeutically, let-7a-5p mimic reversed bleomycin-induced EndoMT and suppressed epithelial-mesenchymal transition (EMT) via paracrine signaling. Mice administered agomir showed reduced fibrosis, improved lung function, and suppressed TGF-{beta}/Smad signaling. Conclusion Serum exosomal let-7a-5p suppresses pulmonary fibrosis by targeting TGFBR1 to inhibit EndoMT. Its downregulation in IPF patients correlates with disease progression, highlighting its biomarker potential.

Background To characterize Coronavirus disease 2019 (COVID-19) symptom severity, and recovery from pre-infection through one month, overall and by risk groups. Methods Symptomatic adults aged [&ge;]18 years with test-confirmed COVID-19 were enrolled from ambulatory care clinics within a national U.S. retail pharmacy network between 10/24/2024 and 08/29/2025 (NCT05160636). Adjusted mixed models for repeated measures estimated least-squares mean changes (LSE) and standard errors (SE) from pre-infection and on Days 1-7, 10, 14, and Week 4 from enrollment in composite symptom scores (sum of severity ratings (0-3) across 14 symptoms), counts of mild-to-severe, moderate-to-severe, and severe symptoms, overall and by age and clinical risk status. Effect sizes (ES) were defined as small (0.2-<0.5), medium ([&ge;]0.5), and large ([&ge;]0.8). Results The analysis included 608 adults. On Day 1, symptom severity rose sharply from pre-infection for the composite symptom score (LSE 14.2 [SE 0.3]; ES 2.22), mild-to-severe (7.6 [0.1]; 2.72), moderate-to-severe (5.0 [0.2]; 1.77); and severe (1.8 [0.1]; 0.92) (all p<0.001). By Week 4, composite score (0.7 [0.2]; 0.26), mild-to-severe (0.5 [0.1]; 0.23); moderate-to-severe symptoms (0.1 [0.1]; 0.17) and severe symptoms (0.2 [0.1]; 0.5) remained slightly above baseline (all p[&le;]0.025). Elevated severe symptom durations varied: high-risk adults (through Day 3), adults <50 years (through Day 7), and adults [&ge;]50 years (through Day 7). Conclusions COVID-19 was associated with notable acute symptoms in outpatients, followed by gradual improvement over time, although symptoms still persisted at four weeks. Improvement in severe symptoms varied by individual risk profile, reinforcing the importance risk-based follow-up and ongoing monitoring.

The role of bioaerosols in the transmission of pathogens and antimicrobial resistance (AMR) is of increasing clinical importance, particularly in settings housing vulnerable populations. Air filtration (e.g. HEPA filtration) and ventilation (e.g. minimum air changes per hour) measures are designed to restrict the airborne transmission of microorganisms. Despite these measures, airborne transmission remains a persistent issue in hospitals, workplaces, aged care, and schools, and is not typically assessed in routine surveillance for infection prevention. Here, we evaluated the efficacy of a high-volume air sampling approach to capture the indoor 'aerobiome', and investigated the potential for bioaerosols to mediate disease and AMR transmission in workplace and hospital settings. Our sampling approach demonstrates the benefits of simple decontamination procedures and personal protective equipment on the ability to distinguish genuine low biomass signals in air samples from blank controls, enabling reliable and sensitive microbial detection down to a limit of 69 bacterial cells/m3 of air. In a workplace bathroom setting, increased airborne biomass was strongly associated with human activity. This diminished significantly after a few hours of no activity, yet persisted in the indoor environment, with viable identical bacterial strains recovered from bioaerosols and bathroom surfaces across months of sampling. Applying our approach in a hospital ward, air samples from occupied patient rooms were not distinguishable from blank controls and contained negligible fungal and bacterial content, with only trace contributions from human occupancy. Our findings indicate that air filtration measures in this ward are effective at minimising airborne risks, but periodic testing of high-risk areas may be valuable in indoor settings with greater human traffic and may contribute key information to outbreak investigations.

Introduction Individuals with Gastroesophageal Reflux Disease (GERD) often experience airway inflammation and bronchoconstriction as a result of reflux aspiration and/or vagally mediated reflexes. The Impulse Oscillometry System (IOS) is a sensitive, non-invasive tool that can detect subtle changes in airway resistance. While there are few studies exploring airway resistance in GERD globally, no studies have been conducted in Sri Lanka. Therefore, we aim to compare the airway resistance using IOS in medical undergraduates with and without symptomatic GERD. Methods A cross-sectional study was conducted among 811 medical undergraduates (31.1% male; mean age 22.9 years) at the Faculty of Medicine, Rajarata University of Sri Lanka. Symptomatic GERD was screened using the validated GerdQ, and a cutoff of[&ge;]8 was used to diagnose those with GERD symptoms. Of the 242 (29.8%) with GERD symptoms, 188 with chronic respiratory diseases or recent respiratory symptoms were excluded, and 50 with GERD symptoms and 50 healthy, age- and sex-matched controls were recruited. Lung function was assessed using IOS and spirometry, according to American Thoracic Society (ATS) and European Respiratory Society (ERS) guidelines. Results Prevalence of symptomatic GERD among medical undergraduates was 29.8% (242/811). The common symptoms among GERD were heartburn (89.6%, 217/242) and regurgitation (85.5%, 207/242). Oscillometry parameters including, R5-R20 Hz (15.29% vs 9.69%, p=0.002), Fres (14.95 1/s vs 13.37 1/s, p = 0.04), and AX (0.66 vs 0.48, p = 0.02) were significantly higher in students with symptomatic GERD (mean = 15.29%) than in healthy controls (mean = 9.69%; p = 0.002). However, spirometry parameters including FEV1, FVC, and PERF did not differ between the GERD-positive and control groups. Conclusion Individuals with symptomatic GERD demonstrated a higher peripheral airway resistance compared to controls, whereas no significant difference was observed in upper airway resistance. This could be due to the gastric acid stimulation of vagal nerve terminations in the lower part of the esophageal wall, leading to increased resistance in the peripheral airways through vagally mediated bronchoconstriction.