European Respiratory Journal

BackgroundTBX4 variants are a recognised cause of paediatric pulmonary hypertension (PH), often associated with interstitial lung disease (ILD). Evidence for ILD-directed therapy in this group is lacking. MethodsWe conducted a retrospective study of children ([≤]18 years) with TBX4-associated PH at a national centre (2001-2025). ILD was defined using ChILD-EU criteria. Patients treated with pulsed intravenous methylprednisolone were assessed for response using ChILD-EU categories. Secondary outcomes included respiratory severity score (RSS), functional class (FC), echocardiographic measures, and NT-proBNP. ResultsOf 21 children, 11 (52%) had ILD; 9 received corticosteroids. Median age at treatment was 0.8 years. A clear or best response occurred in 7/9 (78%). RSS improved in 6/9 (p=0.02), with all children on respiratory support showing partial or complete weaning. Functional class improved in all with FC III/IV at baseline (p=0.02). Right ventricular function improved (TAPSE z-score +1.65, p=0.04), and elevated NT-proBNP normalised. Key clinical milestones included ECMO weaning, transplant delisting, and discontinuation of prostacyclin therapy. No significant adverse effects were observed. Untreated children showed no early improvement. ConclusionsCorticosteroids were associated with meaningful improvements in respiratory and PH outcomes in TBX4-associated PH with ILD. Prospective evaluation is warranted.

Background: Idiopathic pulmonary fibrosis (IPF) is a rare disease with a poor prognosis. Disease risk involves rare and common genetic variants. However, an inverse association have been described between them. Accordingly, IPF patients with a higher polygenic risk score (PRS) for IPF are less likely to carry rare deleterious variants and vice versa. Here, we evaluate weather PRS of IPF could serve as an additional criterion to patient prioritisation for rare variant discovery. Methods: We identified carriers based on the presence of rare qualifying variants (QVs) in genes linked to monogenic forms of pulmonary fibrosis in 888 IPF patients from the Pulmonary Fibrosis Foundation Patient Registry (PFF-PR). Genome-wide association study (GWAS) summary statistics from independent cohorts were used to construct a whole-genome PRS (WG-PRS) using a clumping and thresholding method (C+T) and a Bayesian method (SBayesRC). PRS were also derived from 19 known common sentinel IPF variants (Sentinel-PRS). Logistic regression models were used to evaluate associations between PRS and carrier status. Discriminatory performance was evaluated using area under the curve (AUC) analysis, and comparisons were made with DeLong test. Validation was performed in 472 IPF individuals from the UK PROFILE cohort. Results: IPF-PRS were strongly associated with the QVs carrier status: Odds Ratio [OR] 0.65 (95% Confidence Interval [CI] 0.53-0.79) for WG-PRSC+T, OR 0.71 (95% CI 0.59-0.86) for WG-PRSSBayesRC, and OR 0.77 (95% CI 0.63-0.94) for Sentinel-PRS. Adding WG-PRS to the patient personal clinical history improved the prediction of QVs carriers: AUC=0.62 for the clinical model, AUC=0.68 for WG-PRSC+T (DeLong test, p=9.54x10-4) and AUC=0.66 for WG-PRSSBayesRC (DeLong test, p=0.02). Adding of IPF-PRS to clinical variables correctly reclassified 22.8% of carriers when using WG-PRSC+T, 20.8% when using Sentinel-PRS, and 16.7% for WG-PRSSBayesRC. WG-PRSSBayesRC and the Sentinel-PRS also demonstrated improved prediction of QVs carriers in telomere-related genes in PROFILE. Conclusions: Incorporating IPF-PRS into a model based on the patient clinical history improves the identification of QVs carriers. Although the overall discriminatory power was moderate, these findings raise de the possibility of using WG-PRS as useful criterion for rare variant discovery in patients with IPF and enhance decision-making.

Genome-wide association studies of idiopathic pulmonary fibrosis (IPF) have identified 35 common genetic risk loci associated with IPF susceptibility. In this study, we evaluated the effects of the reported variants in clinically curated non-European individuals. Despite limited sample sizes, we observed partial replication, limited transferability of some variants and evidence of ancestry-specific effects. The MUC5B promoter variant rs35705950 emerged as the dominant and most consistent signal across ancestries. Our findings highlight the need for larger, well-characterised studies in understudied populations to support robust discovery and translation.

Background Acute respiratory distress syndrome (ARDS) is characterised by substantial physiological heterogeneity, which contribute to a very variable clinical outcomes and therefore inconsistent responses to ventilatory strategies. We aimed to externally validate physiological ARDS subphenotypes previously identified using routine ventilatory and gas-exchange variables, assess their prognostic relevance across independent cohorts, and examine heterogeneity of treatment effect according to PEEP strategy. Methods Unsupervised Gaussian Mixture Modelling was used to identify physiological subphenotypes based on ventilatory mechanics and gas-exchange parameters. Labels were subsequently used to train and validate supervised classifiers using XGBoost. Prognostic relevance was assessed across three independent cohorts, including two randomised controlled trials (ALVEOLI and LOVS). Predictive enrichment for PEEP strategy was evaluated using individual patient data from ALVEOLI and LOVS (n = 1,532) using intention-to-treat analyses, applying both one-stage and two-stage fixed-effects IPD meta-analytic approaches to test for interaction between physiological subphenotype and PEEP strategy. Results Two distinct physiological subphenotypes, termed Efficient and Restrictive, were replicated across independent cohorts. Across each cohort, patients classified as Restrictive consistently exhibited higher all-cause 28-day mortality compared to Efficient patients. When pooled across studies, the Restrictive subphenotype was associated with a significantly increased risk of death (pooled odds ratio 1.75, 95% CI 1.36-2.24), with no evidence of between-study heterogeneity. Predictive analyses showed a statistically significant interaction between physiological subphenotype and PEEP strategy in the one-stage IPD model (p for interaction = 0.037), with concordant findings in the two-stage fixed-effects IPD meta-analysis (interaction OR 1.91, 95% CI 1.00-3.66; I2 = 0%). Higher PEEP was associated with increased mortality in Efficient patients and reduced mortality in Restrictive patients, indicating effect modification by physiological subphenotype. Interpretation Physiological ARDS subphenotypes derived from routinely collected bedside data provide robust and externally validated prognostic stratification across observational and randomised trial cohorts. The observed interaction with PEEP strategy suggests that underlying physiological profiles may influence treatment response, supporting the concept that physiology-based be a starting point for personalized medicine and therefore better ventilatory strategies in future clinical trials.

Background Recent guidelines differ in how fractional exhaled nitric oxide (FeNO) is used to diagnose school-age asthma, either as one of several tests with a cut-off at 25 ppb or as a single rule-in test at 35 ppb. Evidence on its diagnostic performance and clinical utility in subgroups remain limited. Methods We analysed data from 1,979 school-age children in the Swiss Paediatric Airway Cohort referred for suspected asthma. We investigated FeNO performance with diagnosis by paediatric pulmonologists as reference standard using receiver operating characteristics curves, selected cut-offs and simulated predictive values across different prevalence. Subgroup analyses considered allergic sensitisation with allergic rhinitis and current inhaled corticosteroid (ICS) use. Results In the overall cohort (asthma diagnosis 70%), FeNO showed poor discrimination for asthma (AUC 0.66; 95% CI 0.64-0.68) with an optimal cut-off at 22 ppb. At 25 and 35 ppb, sensitivity was low (43%, 95% CI 40-46; 31%, 95% CI 29-34) and specificity moderate to high (84%, 95% CI 77-84; 90%, 95% CI 87-92). Positive predictive value at 35 ppb was 88% and was 57% when simulated at a prevalence of 30%. FeNO had no diagnostic value in non-sensitised children and lower performance in sensitised children with allergic rhinitis than in those without (AUC 0.59 vs 0.68). Current ICS use did not influence performance. Conclusion FeNO has limited diagnostic performance as a stand-alone test for school-age asthma, and underlying asthma prevalence and allergic characteristics should be considered in the interpretation.

Equine asthma (EA) is a highly prevalent, chronic, inflammatory disease of the lower airways ranging from mild-to-moderate to severe clinical presentations. Diagnosis currently relies on bronchoalveolar lavage fluid (BALF) cytology, an invasive method associated with interobserver variability, which highlights the need for more reproducible approaches. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional gene regulation. They are stable and readily detectable in body fluids and have shown promising results as biomarkers in human asthma. The aim of this study was to characterize miRNA abundance profiles in BALF and serum from horses with distinct EA endotypes to evaluate their biomarker potential and explore their involvement in disease pathogenesis. A total of 43 horses were included and classified as either EA (n=32) or controls (n=11), based on clinical examination and BALF cytology. The EA horses were further divided into three endotypes based on BALF inflammatory cell composition: neutrophilic asthma (n=10), mastocytic asthma (n=15), and mixed asthma (n=7). RNA was isolated from both serum and BALF samples and analyzed by quantitative real-time PCR (qPCR) targeting 103 miRNAs linked to asthma and pulmonary inflammation in humans. Differential miRNA abundance was analyzed across EA endotypes. The most significantly differentially abundant miRNAs were used for in silico target prediction and pathway enrichment analyses. Horses with mixed EA had significantly lower levels of eca-miR-125a-3p and eca-miR-125b-5p in BALF compared to controls. Additionally, eca-miR-146a-5p abundance was significantly increased in BALF from horses with neutrophilic EA compared to mastocytic EA. Target and pathway enrichment analyses for eca-miR-146a-5p identified immune-relevant pathways, such as MAPK and T-cell receptor signaling, supporting its involvement in inflammatory processes associated with asthma. This study identified three promising candidates, eca-miR-125a-3p, eca-miR-125b-5p, and eca-miR-146a-5p, as potential biomarkers associated with different EA endotypes. These miRNAs are interesting candidates for further investigation in an independent cohort.

BackgroundPleuroparenchymal fibroelastosis (PPFE) is a rare, fibrotic lung disease with poor prognosis, usually affecting adults which most commonly occurs idiopathically. Biallelic pathogenic variants in DGUOK cause mitochondrial DNA (mtDNA) depletion syndrome, predominantly affecting infants with severe hepatic and neurological symptoms. Detailed description of pulmonary manifestations with late-onset presentation have not been reported. MethodsWe describe nine patients with PPFE and DGUOK-associated mitochondriopathy. Clinical, radiological, histopathological, and genetic data were systematically collected from all patients. Functional studies, single nucleus RNA sequencing (snRNAseq), immunofluorescence staining, transmission electron microscopy and respiratory chain enzyme activity assays were conducted on patient-derived fibroblasts, muscle or lung tissues. mtDNA content quantification was performed on whole genome sequencing (WGS) data. ResultsAll patients (ages 5-36) presented with progressive dyspnea, weight loss and some with spontaneous pneumothoraces. Chest computed tomography and lung biopsies showed features of PPFE. Biallelic pathogenic DGUOK variants were identified in all patients, seven of them carry an unreported intronic variant leading to mtDNA depletion. snRNAseq of lung tissue from four pediatric patients identified Aberrant Basaloid cells and intermediate cells as their precursor localized at the fibrotic edge. Mitochondrial alterations were identified by electron microscopy. ConclusionPPFE in children and young adults is associated with DGUOK-related mitochondriopathy. For the first time, we demonstrate Aberrant Basaloid cells in pediatric fibrotic lung tissue. Since pulmonary involvement may be underrecognized or misinterpreted and the clinical presentation may not always be typical of a mitochondriopathy, we recommend genetic testing in all patients with PPFE of unknown origin.

Lung fibroblasts are key regulators of tissue homeostasis and extracellular matrix (ECM) remodeling, and their aberrant activation drives the progressive parenchymal scarring characteristic of idiopathic pulmonary fibrosis (IPF), a fatal disease with limited therapeutic options. Despite their central pathogenic role, lung fibroblasts are difficult to isolate due to their embedded position within the ECM, and standard in vitro culture conditions may lead to the loss of their native functional and transcriptional characteristics, hampering the study of fibroblast behavior in disease. The transcriptional heterogeneity of lung fibroblast subtypes and the extent to which culture-induced alterations diverge from native tissue signatures remain poorly understood. Here, we integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics of lung tissue from IPF patients and age-matched healthy donors with transcriptomic profiling of cultured fibroblasts collected at passages 1 and 6 after isolation using three optimized protocols: whole lung cell suspension (WLCS), negative fraction enrichment, and outgrowth. Tissue-based analysis identified six transcriptionally distinct mesenchymal subtypes: alveolar, adventitial, inflammatory, peribronchial, CTHRC1+ and smooth muscle cell (SMC). The fibroblast subtype CTHRC1+ represented the most transcriptionally activated pro-fibrotic subtype, showing the greatest upregulation of ECM biosynthesis genes, a prominent role in intercellular communication, and preferential enrichment within fibroblastic foci in IPF lung tissue. Pseudotime trajectory analysis supported a directional transcriptional continuum from alveolar and inflammatory fibroblasts toward the CTHRC1+ state, driven by coordinated activation of pro-fibrotic transcription factors, including RUNX2, CREB3L1, and SCX. In vitro culture progressively reshaped fibroblast transcriptional identity relative to native tissue, with increased collagen and matrix metalloproteinase (MMP) expression during passaging, loss of distinct CTHRC1+ fibroblasts, and gain of alveolar fibroblasts displaying pro-fibrotic activation across all isolation protocols. These findings provide a high-resolution transcriptional map of lung fibroblast heterogeneity in IPF and highlight critical limitations of standard in vitro culture systems for recapitulating native fibroblast diversity, with important implications for the development and evaluation of fibroblast-targeted therapeutic strategies in IPF.

Aminoglycoside drugs, amikacin, streptomycin, and amikacin liposome inhalation suspension are crucial for treating refractory Mycobacterium avium-intracellulare complex pulmonary disease. In Mycobacterium tuberculosis, cross-resistance occurs between amikacin and kanamycin, but not between amikacin and streptomycin in genetic drug susceptibility testing. However, the occurrence of cross-resistance among aminoglycosides remains unclear in M. avium-intracellulare complex. We aimed to evaluate cross-resistance among aminoglycosides to determine whether streptomycin or kanamycin remains effective after the development of amikacin resistance. This single-center retrospective study included 20 patients with amikacin-resistant M. avium-intracellulare complex harboring rrs mutations. Paired analyses of streptomycin and kanamycin minimum inhibitory concentration values before and after amikacin resistance development were performed. In addition, streptomycin- and kanamycin-resistant strains were generated in vitro and resistance-associated mutations were identified using whole-genome sequencing. No significant increase was observed in streptomycin minimum inhibitory concentration values following amikacin resistance. In contrast, kanamycin values uniformly increased to >256 g/mL after the acquisition of amikacin resistance. Furthermore, amikacin- and kanamycin-resistant isolates shared mutations at position 1408 in the rrs gene, whereas streptomycin-resistant isolates exhibited mutations at position 20 in the rrs gene. These results suggest that amikacin and kanamycin exhibit cross-resistance in M. avium-intracellulare complex, whereas amikacin and streptomycin may not. Two cases in our cohort in which streptomycin treatment was effective after the acquisition of amikacin resistance further support these findings. In conclusion, streptomycin may be a potential therapeutic alternative for amikacin-resistant M. avium-intracellulare complex pulmonary disease. Future studies correlating streptomycin minimum inhibitory concentration values with clinical outcomes are required.

BackgroundChronic obstructive pulmonary disease (COPD) is a chronic lung condition characterised by accelerated lung aging. Extracellular vesicles (EVs), which can be categorised into large EVs (LEVs) and small EVs (SEVs), may play a critical role in intercellular communication. They contribute to the pathogenesis of COPD by transporting and transferring microRNAs (miRNAs). This study profiles cells and EV-associated miRNAs from both healthy and COPD small airway (SA)-epithelial cells and SA-fibroblasts and identifies the biological pathways associated with these miRNAs. MethodsEVs were isolated from conditioned media of healthy and COPD SA-epithelial cells and SA-fibroblasts, both at baseline and following H2O2 exposure. MiRNAs were extracted from cells and EVs and analysed by small RNA (smRNA) sequencing. ResultsSmRNA sequencing of COPD SA-epithelial cells and EVs revealed that four miRNAs were upregulated and fourteen were downregulated in the cells compared to healthy controls. COPD LEVs displayed nine upregulated and ten downregulated miRNAs, while SEVs showed ten upregulated and eleven downregulated miRNAs. Only one miRNA consistently upregulated in COPD SA-epithelial cells, LEVs, and SEVs. The various differentially expressed miRNAs were primarily associated with cellular senescence pathways. In SA-fibroblasts 39 miRNAs were upregulated in COPD compared to healthy cells. 14 miRNAs were upregulated in COPD LEVs and 11 downregulated, whereas SEVs exhibited twenty upregulated and eleven downregulated miRNAs. Overlap was limited, with only three miRNAs consistently upregulated in SA-fibroblasts and EVs. These miRNAs were linked to pathways related to fibrosis and cellular senescence. Furthermore, oxidative stress alters the miRNA profiles detected in cells and EVs differently between cells from healthy individuals and COPD patients. ConclusionsCOPD alters miRNA signatures in cells and their EVs, with limited overlap between compartments. These COPD-associated miRNAs are enriched in pathways driving cellular senescence and fibrosis, suggesting a potential role in disease progression.

Introduction: Although temporal variation is the hallmark of asthma, recommended diagnostic approaches largely rely on single clinic-based measurements. Ambulatory monitoring captures diurnal and day-to-day variability and may therefore enhance diagnostic accuracy. We evaluated the clinical feasibility and potential utility of home spirometry and fractional exhaled nitric oxide (FeNO) monitoring in asthma diagnosis. Methods: Symptomatic, untreated adults with GP-suspected asthma underwent diagnostic tests including bronchodilator reversibility, in-clinic FeNO, blood eosinophil counts and bronchial challenge. Participants measured spirometry and FeNO four times daily over one week; during the second week spirometry were measured twice daily. The reference standard was provided (asthma/not-asthma) by an expert panel of at least two asthma specialists based on clinical history and the results of all in-clinic testing; home spirometry (except for peak expiratory flow) and FeNO measurements were blinded to the panel. Results: Of 67 eligible participants, 51(76%) were recruited, and 38 had asthma confirmed or excluded by the panel. 1058 home spirometry measurements were obtained from 37(73%) participants; 848 home FeNO readings were obtained from 39(76%) participants. Among those completing at least one home measurement, median (IQR) adherence was 66.7(58.6-97.6)% for spirometry and 78.5(51.8-103.6)% for FeNO. Collection of health impact data for economic evaluation was feasible. In participants with a confirmed diagnostic outcome who completed home measurements (FeNO: n=32; spirometry: n=28), the putative home-testing metrics demonstrated high sensitivities at [≥]90% specificity, and outperformed peak expiratory flow diurnal variability. Incorporating home testing into the BTS/NICE/SIGN 2024 diagnostic pathway had the potential to reduce reliance on bronchial challenge testing by 57%. Conclusions: Home spirometry and FeNO testing and the prospective collection of health-economic data in the diagnostic setting were feasible. Home-based testing strategy showed early potential to improve asthma diagnosis and pathway efficiency. These findings support further evaluation through an adequately powered diagnostic accuracy study and health-economic assessment.

Background: Our group previously reported that lung cancer (LC) screening history results and subsequent timing of diagnosis are associated with significant differences in survival outcomes. As a follow-up study, we sought to develop novel personalized risk models that considered screening history for incidence cancers, interval LCs, and prevalence LCs. Methods: Using data from the CT-arm of the NLST, four independent case-control analyses were conducted to develop parsimonious risk models. Controls (n=26,038) were those never diagnosed with LC. The four LC case groups were 270 prevalence LCs, 44 interval LCs, 206 screen-detected LCs (SDLCs) that had a baseline positive screen, and 164 SDLCs that had a baseline negative screen. For each case-control analysis, univariable analyses identified statistically significant covariates from 48 variables and then significant covariates were included into a stepwise backward selection approach to identify a model with the most informative covariates. Results: For prevalence LCs, the model (AUC=0.711) included age, pack-years smoked, BMI, smoking status, smoking onset age, personal history of cancer, family history of LC, alcohol consumption, and milling occupation. For interval LCs, the model (AUC=0.734) included age, smoking status, smoking onset age, cigar smoking, marital status, and asbestos occupation. For baseline positive SDLCs, the model (AUC=0.685) included age, pack-years smoked, BMI, emphysema, chemicals/plastics exposure, and milling occupation. For baseline negative SDLCs, the model (AUC=0.701) included age, pack-years smoked, BMI, smoking status, emphysema, sarcoidosis, and sandblasting occupation. Conclusions: Besides smoking and age, which are inclusion criteria for screening, these models identified other important risk factors which could be used to provide personalized LC risk assessment and screening management.

BackgroundTuberculosis (TB) is the second-leading cause of deaths from infectious agents and remains a global health threat. Ethionamide (ETH) is a prodrug used in regimens for multidrug-resistant TB, and, partly due to side effects that can lead to low treatment adhesion, resistance arises. Changes in EthA, the monooxygenase that activates ETH, are the main mechanism of resistance. Yet, of hundreds of EthA substitutions found in resistant isolates, only a handful have been annotated as resistance determinants. ResultsAn in silico analysis was carried out on a previously described panel of Mycobacterium tuberculosis clinical isolates for which genomes and ETH susceptibility testing results were available. EthA substitutions were mapped, revealing the existence of hotspots in its sequence. Visualization of the hotspots in the EthA structural model shows that they cluster in three regions, including ligand binding pockets. Models were built of twenty-three variants found in resistant isolates and changes in local configuration was mapped to identify investigate impact on ETH activation. Information from these models contributed to establishing five criteria for scoring whether substitutions are most likely to lead to resistance. Using these criteria, EthA D58G was selected and its expression is shown to increase growth in high ETH concentrations. ConclusionFunctionally relevant regions of EthA are revealed and point out priority substitutions for functional studies, enhancing identification and detection of substitutions not been previously associated with resistance.

BackgroundLong COVID is characterised by persistent systemic inflammation and endothelial dysfunction, with increasing evidence implicating thromboinflammatory mechanisms. Platelet-monocyte aggregates (PMA) represent a sensitive marker of platelet activation and immune-vascular interactions, but their role in Long COVID remains incompletely defined. MethodsThis study quantified circulating PMA in 20 Long COVID patients and 20 healthy controls using a two-colour imaging flow cytometry assay targeting CD14 (a monocyte receptor for pathogen-associated molecular patterns, PAMPs) and CD62P (P-selectin). PMA were expressed as a percentage of total monocytes, and platelet attachment patterns were classified into single versus multiple platelet binding. Statistical analyses included Shapiro-Wilk normality testing, unpaired t-tests, Mann-Whitney U tests or two-way ANOVA as appropriate, and linear regression for correlation analysis. ResultsCirculating PMA were significantly elevated in Long COVID patients compared with controls (29.19 [20.02-37.26] vs 4.59 [2.67-7.16], p < 0.0001). Long COVID samples showed a reduced proportion of monocytes with single platelet attachment and a corresponding increase in multiple platelet binding (p < 0.0001). In controls, %PMA increased with age (p < 0.01), whereas no age association was observed in Long COVID, indicating an elevated baseline independent of age. ConclusionsLong COVID is associated with markedly increased platelet-monocyte aggregation and altered platelet attachment dynamics, consistent with sustained thromboinflammatory activity. PMA represent a sensitive cellular marker of platelet-driven immune activation and may have utility as an accessible biomarker for stratifying thromboinflammatory burden in Long COVID.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by aberrantly activated, apoptosis-resistant profibrotic lung (myo)fibroblasts. Prior research has demonstrated that lung fibroblasts from patients with IPF exhibit resistance to DNA damage, suggesting that this behavior contributes to their persistent survival and continuous proliferation. We propose that elevated levels of the DNA damage repair protein RAD51 regulate myofibroblast activation and apoptosis and provide a potential therapeutic target to impede fibrosis progression. MethodsHuman lung fibroblasts were transfected with siRNA against RAD51 or treated with RAD51-specific inhibitor B02 and markers of fibrosis, DNA damage, apoptosis, metabolic reprogramming, and mitochondrial dynamics were assessed. The preclinical efficacy of B02 was evaluated in human precision cut lung slices (PCLS) and in a mouse model of pulmonary fibrosis. FindingsRAD51 expression was significantly upregulated in the lungs and lung fibroblasts of IPF patients. Knockdown or inhibition of RAD51 in fibroblasts reduced profibrotic marker expression, suppressed mTORC1 signaling and mitochondrial function, and increased apoptosis susceptibility. Pharmacological inhibition of RAD51 shifted the profibrotic phenotype towards a fibrosis-resolving state in human and mouse PCLS, and in a bleomycin-induced mouse model of lung fibrosis. InterpretationThe inhibition of RAD51 exerts therapeutic benefits in lung fibrosis by promoting apoptosis. Our findings identify that inhibiting RAD51 with B02 in fibroblasts impairs DNA repair and induces metabolic reprogramming, making it a potential therapeutic target. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPulmonary fibrosis (PF) is characterized by excessive fibroblast activation and subsequent deposition of extracellular matrix (ECM) proteins, which ultimately disrupt normal lung architecture. A significant contributing factor to the pathogenesis of pulmonary fibrosis is the presence of fibroblasts that are resistant to apoptosis, preventing normal wound healing. Recent studies highlight the DNA repair protein RAD51 as effective in protecting fibroblasts from death induced by chemotherapy and ionizing radiation. These finding suggested that RAD51 could have a role in fibroblast activation and apoptosis resistance in pulmonary fibrosis. Added value of this studyWe demonstrated that RAD51 is important for maintaining apoptosis-resistant fibrotic fibroblasts and their metabolic abnormalities. Our findings indicated that TGF{beta}-mediated upregulation of RAD51 reduces DNA damage, activates multiple pathways related to fibroblast activation and proliferation, and induces metabolic reprogramming, ultimately regulating apoptosis. Mechanistically, RAD51 inhibition enhanced p53 acetylation at lysine 120 and upregulated the expression proapoptotic proteins PUMA/BAK in mitochondria, promoting apoptosis. Pharmacological inhibition of RAD51 using the specific inhibitor B02 during the fibrotic phase of experimental lung disease effectively ameliorated pulmonary fibrosis. Implications of all the available evidenceOur findings establish that RAD51 plays an important role in the survival of apoptosis-resistant fibrotic fibroblasts. We propose that reducing RAD51 expression leads to the metabolic reprogramming of activated fibroblasts, resulting in decreased mitochondrial respiration, reduced ATP levels, and diminished glycolysis or glutaminolysis. These observations suggest that targeting energy metabolism through RAD51 inhibition could be a viable strategy to enhance apoptosis, thereby creating a therapeutically targetable pathway in fibrotic cells. These findings highlight the potential of RAD51 as a therapeutic target for the treatment of IPF.

BackgroundIn patients requiring respiratory support, clinicians rely on physical exam, radiologic, laboratory, and ventilator-derived measures for the provision of sufficient support while minimizing ventilator and "work of breathing" induced lung injury. Point of care lung ultrasound (LUS) is a widely available tool in hospital and clinic environments. To date, LUS has not been used to evaluate lung strain. MethodsWe collected LUS images in four anesthetized, neuromuscularly blocked, and mechanically ventilated pigs being used for another experiment. A feature tracking tool was developed which tracked echo-bright lung structures in ten second clips obtained in triplicate of the right and left, upper and lower lung fields using tidal volumes of 4, 6, 8, 10, and 12 mL/kg. Pleural lines were manually drawn and a program for quantifying lung strain developed with assistance from Anthropic Claude Artificial Intelligence tool. Structures were identified in inspiratory and expiratory frames and tracked bidirectionally with median strain per frame used for calculations. ResultsTriplicate measures of lung ultrasound images in four pigs had a median coefficients of variation of 35% (23-47% IQR) and linear modeling of strain with tidal volumes of 4-12 mL/kg showed positive correlation with R2 value ranging from 0.89 to 0.97. Strain measurements were similar after bronchial administration of 1.5M hydrochloric acid. ConclusionsRegional lung strain quantification using LUS is a viable and potentially useful tool for respiratory support management.

Rationale: Sputum-based testing using Xpert MTB/RIF Ultra (Xpert) is the most common molecular testing method for diagnosing tuberculosis (TB). Objectives: To evaluate whether sputum quality influences Xpert positivity and diagnostic accuracy. Methods: We screened consecutive people for presumptive TB in India, the Philippines, Vietnam, Nigeria, South Africa, Uganda, and Zambia as part of the R2D2 TB Network and ADAPT studies. Participants provided 2-3 sputum samples for Xpert and culture reference testing. The quality of the first sputum sample was graded following standardized procedures by trained research staff and used for Xpert testing. We performed logistic regression to evaluate whether sputum grade was independently associated with Xpert positivity, and calculated sensitivity and specificity of Xpert against a culture-based microbiological reference standard (MRS). Measurements and Main Results: Among 1,855 participants, 798 (43%) were female, 348 (19%) were living with HIV (PLHIV), and 1795 (97%) had a cough of [&ge;]2 weeks. Overall, 313 (17%) had a positive Xpert result. Most sputum samples were salivary (83%). Xpert positivity was lowest among salivary samples (16.1%) and highest among purulent samples (31.2%). After adjusting for demographic and clinical variables, there was no significant association between any sputum grade and Xpert positivity. Xpert sensitivity (salivary: 89%, mucoid: 91%, mucopurulent: 87%, purulent: 100%) and specificity (>98%) were high across sputum grades. Conclusions: Sputum quality was not independently associated with Xpert positivity and Xpert sensitivity was high across all sputum grades. These findings support molecular testing of all sputum samples for TB diagnosis regardless of macroscopic appearance.

Background: Extreme-phenotype comparisons allowed the discovery of novel asthma genetic risk loci. However, this approach remains unexplored in epigenome-wide association studies (EWAS). We aimed to identify bulk and cell-specific methylation markers of asthma with severe exacerbations across diverse ancestry groups. Methods: We conducted a meta-EWAS of 739,543 CpGs in whole blood among 1,192 African American and Latino pediatric populations, comparing non-asthmatics and asthma exacerbators. Genome-wide CpGs were followed up for replication in a meta-analysis across 1,516 ethnically diverse participants and in a cross-tissue evaluation of 393 nasal samples. We conducted differentially methylated region (DMRs), cell-type-deconvoluted, and quantitative trait loci analyses (whole-genome sequencing n=1,668; RNA-seq n=1,209). We examined enrichment in traits, pathways, and druggable genes, and analyzed DNAm predictors of plasma proteins and aging. Results: DNAm at 505 CpGs and 119 DMRs in whole blood were associated with asthma exacerbations (p<9x10-8, {lambda}=1.05). We replicated 25 CpGs in blood cells, cross-validated 7 in nasal samples, and detected 42 cell-specific DNAm markers mainly driven by T cells. DNAm at 134 CpGs was associated with gene expression in whole blood, including 118 associations with T-cell receptor genes, and 446 CpGs were regulated by [&ge;]1 genetic variant. We found enrichment for previous associations with environmental exposures, immune disorders, immune and inflammatory pathways, and druggable genes by developmental drugs. 21 methylation-predicted plasma proteins, involved in host defense, and one lung aging clock were associated with asthma exacerbations. Conclusions: The first meta-EWAS of extreme asthma phenotypes identified hundreds of novel DNAm markers, suggesting novel methylation biomarkers and candidate drugs for asthma and supporting the role of T cells.

Pulmonary fibrosis (PF) involves excessive collagen accumulation, yet mechanisms shifting the balance of synthesis and degradation toward net deposition remain unclear. Myeloperoxidase (MPO) inversely correlates with survival in PF. Using the bleomycin model, we found MPO knockout (MPOko) mice were protected from fibrosis, and pharmacological MPO inhibition after peak inflammation (day 7) recapitulated this protection. MPO persisted in lung tissue 21 days post-injury despite neutrophil efflux, linking acute inflammation to sustained remodeling. Mechanistically, we identified that MPO inhibits Cathepsin K (CatK), a potent collagenolytic enzyme involved in fibrosis resolution. Notably, CatK gene expression (CTSK) is elevated in PF, suggesting post-translational inhibition of CatK. MPOko and inhibitor-treated mice exhibited elevated CatK activity after bleomycin; exogenous addition of pathophysiologic concentrations of MPO reduced CatK activity in mouse precision-cut lung slices and human fibroblasts. Biochemically, MPO reduced CatK activity to 33% of control. In two distinct cohorts of PF patients, we observed significantly increased MPO protein levels in platelet poor plasma and in lung tissue. In PF patients, 62% had MPO levels in platelet poor plasma exceeding healthy controls, while lung tissue from other PF patients showed significantly elevated MPO staining. Plasma levels were inversely correlated with decreased survival, FVC, and DLCO. These findings establish MPO as a post-translational inhibitor of CatK-mediated collagenolysis, revealing a mechanism linking acute inflammation to sustained fibrosis and suggest a patient subpopulation that may benefit from MPO-targeted therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/713467v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@d8fc5eorg.highwire.dtl.DTLVardef@1a088fcorg.highwire.dtl.DTLVardef@818b7dorg.highwire.dtl.DTLVardef@ecdca0_HPS_FORMAT_FIGEXP M_FIG C_FIG Myeloperoxidase persists in lung tissue after injury and inhibits cathepsin K activity, impairing collagen degradation and promoting extracellular matrix accumulation during pulmonary fibrosis.

Tuberculosis (TB) is a significant cause of morbidity and mortality in children and adolescents, causing 172,000 deaths in 2024 in children and adolescents worldwide. Diagnostic challenges are pronounced in pediatrics, in which collecting respiratory specimens is challenging and TB is often paucibacillary, leading to delayed diagnosis and increased mortality. We describe the protocol and methodology of the Pediatric TB Diagnostic (PDTBDx) cohort, a study with the primary aim of evaluating non-sputum-based TB diagnostics for diagnosis and treatment response in children. This is a prospective observational cohort study of >400 children recruited from inpatient and outpatient clinical sites in Nairobi, Kenya. Children <15 years presenting to study clinical sites with TB symptoms will be considered for enrollment as symptomatic participants. Enrolled participants will undergo rigorous clinical assessment and longitudinal follow-up to ensure appropriate diagnostic classification by NIH consensus statement guidelines for pediatric TB. Baseline evaluation includes symptom assessment, chest x-ray, HIV testing, respiratory TB culture and GeneXpert Ultra, and urine LAM. Subsequent visits occur at week 2, months 1, 2, 4, 6,12 and 24. Blood and urine specimens will be collected at baseline and at follow-up visits for storage for evaluation of novel diagnostic assays, including exosome-based and CRISPR-based TB biomarkers. This large, prospective cohort of pediatric participants with and without TB follows a consistent and rigorous protocol for diagnosing childhood TB, in concordance with internationally recognized guidelines. Assays evaluated in PDTBDx will guide improved diagnostic strategies for pediatric TB.