BACKGROUND: The human respiratory tract harbours diverse microbial communities crucial for health, but their dynamics during environmental perturbations like smoking remain poorly understood. While smoking is a major risk factor for various diseases, its compartment-specific effects on the respiratory microbiome and potential recovery following cessation have not been fully elucidated. Here, we present a longitudinal, multi-site study of respiratory microbiome dynamics in smokers undergoing cessation, benchmarked against healthy never-smokers. METHODS: Using standardized sampling of the anterior nares, oropharynx, and bronchoalveolar lavage (BAL), combined with 16S rRNA gene amplicon sequencing and rigorous contamination controls, we characterized community composition, diversity, inter-individual variability, and microbial interactions across airway compartments. RESULTS: Smokers exhibited pronounced microbiome alterations: nasal richness increased, while lung richness and core taxa were decreased. Smoking-induced changes were compartment-specific and most pronounced in nose and lung. The degree of individual-specific differences in community structure was elevated in smokers and correlated with smoking history. Short-term cessation (6 weeks) led to minor shifts in genus abundance but increased similarity between oropharyngeal and lung communities, whereas long-term cessation (1 year) resulted in partial restoration, particularly in lung and nasal microbiomes. Some genera, including Haemophilus and Prevotella_7, showed persistent alterations, suggesting lasting smoking effects. Network analyses revealed that smoking disrupted microbial co-occurrence and reduced community connectivity, whereas cessation partially restored interaction networks, with central taxa remaining altered and dynamics differing between oropharynx and lung, reflecting different underlying ecological assembly processes. Recovery trajectories were highly individualized, with lung microbiomes influenced by deterministic processes and upper airway microbiomes shaped by stochastic factors, explaining site-specific responses and the persistence of personalized microbial signatures. CONCLUSION: These results provide the first time-resolved, multi-compartment characterization of respiratory microbiome recovery after smoking cessation, revealing that smoking leaves lasting, site-specific imprints on airway microbial communities and interactions. Our findings underscore the need for individual and compartment-specific approaches when designing microbiome-based interventions to support respiratory health.

Background Pathogenic mycobacteria, such as Mycobacterium tuberculosis complex (Mtbc), and non-tuberculous mycobacteria (NTMs) can cause severe chronic pulmonary infections. However, not all infected patients develop active disease, and it remains unclear whether key lung microbiome taxa play a role in the pathogenesis of tuberculosis (TB) and NTM lung diseases (LD). Here, we aim to further define the lung microbiome composition in TB, and NTM-LD prior to the initiation of therapy. Study design We employed 16S rRNA amplicon sequencing to characterize the baseline microbiome in bronchoalveolar lavage fluid (BALF) from patients diagnosed with TB (n = 23), NTM-LD (n = 19), or non-infectious inflammatory disease (n = 4). We applied depletion of human cells, removal of extracellular DNA, implementation of a decontamination strategy, and exploratory whole-metagenome sequencing (WMS) of selected specimens. Results Genera Serratia and unclassified Yersiniaceae dominated the lung microbiome of most patients with a mean relative abundance of >15% and >70%, respectively. However, at the sub-genus level, as determined by amplicon sequence variants (ASVs), TB-patients exhibited increased community diversity, and distinct signatures of ASV_7, ASV_21 abundances which resulted in a significant association with disease state. Exploratory WMS, and ASV similarity analyses suggested the presence of Serratia liquefaciens , Serratia grimesii , Serratia myotis and/or Serratia quinivorans in TB and NTM-LD patients. Conclusions The lung microbiome of TB-patients harbored a distinct, and heterogenous structure, with specific occurrences of certain Serratia traits. Some of these traits may play a role in understanding the microbial interactions in the lung microbiome of patients infected with Mtbc.

Sudden aggravations of chronic inflammatory airway diseases are difficult-to-foresee life-threatening episodes for which advanced prognosis-systems are highly desirable. Here we present an experimental chip-based fluidic system designed for the rapid and sensitive measurement of biomarkers prognostic for potentially imminent asthma or COPD exacerbations. As model biomarkers we chose three cytokines (interleukin-6, interleukin-8, tumor necrosis factor alpha), the bacterial infection marker C-reactive protein and the bacterial pathogen Streptococcus pneumoniae-all relevant factors in exacerbation episodes. Assay protocols established in laboratory environments were adapted to 3D-printed fluidic devices with emphasis on short processing times, low reagent consumption and a low limit of detection in order to enable the fluidic system to be used in point-of-care settings. The final device demonstrator was validated with patient sample material for its capability to detect endogenous as well as exogenous biomarkers in parallel.

Population-based biobanking is an essential element of medical research that has grown substantially over the last two decades, and many countries are currently pursuing large national biobanking initiatives. The rise of individual biobanks is paralleled by various networking activities in the field at both the national and international level, such as BBMRI-ERIC in the EU. A significant contribution to population-based biobanking comes from large cohort studies and national repositories, including the United Kingdom Biobank (UKBB), the CONSTANCES project in France, the German National Cohort (NAKO), LifeLines in the Netherlands, FinnGen in Finland, and the All of Us project in the U.S. At the same time, hospital-based biobanking has also gained importance in medical research. We describe some of the scientific questions that can be addressed particularly well by the use of population-based biobanks, including the discovery and calibration of biomarkers and the identification of molecular correlates of health parameters and disease states. Despite the tremendous progress made so far, some major challenges to population-based biobanking still remain, including the need to develop strategies for the long-term sustainability of biobanks, the handling of incidental findings, and the linkage of sample-related and sample-derived data to other relevant resources.

Tuberculosis (TB) and sarcoidosis are both granulomatous diseases. Here, we compared the immunological microenvironments of granulomas from TB and sarcoidosis patients using in situ sequencing (ISS) transcriptomic analysis and multiplexed immunolabeling of tissue sections. TB lesions consisted of large necrotic and cellular granulomas, whereas “multifocal” granulomas with macrophages or epitheloid cell core and a T-cell rim were observed in sarcoidosis samples. The necrotic core in TB lesions was surrounded by macrophages and encircled by a dense T-cell layer. Within the T-cell layer, compact B-cell aggregates were observed in most TB samples. These B-cell clusters were vascularized and could contain defined B-/T-cell and macrophage-rich areas. The ISS of 40–60 immune transcripts revealed the enriched expression of transcripts involved in homing or migration to lymph nodes, which formed networks at single-cell distances in lymphoid areas of the TB lesions. Instead, myeloid-annotated regions were enriched in CD68, CD14, ITGAM, ITGAX, and CD4 mRNA. CXCL8 and IL1B mRNA were observed in granulocytic areas in which M. tuberculosis was also detected. In line with ISS data indicating tertiary lymphoid structures, immune labeling of TB sections expressed markers of high endothelial venules, follicular dendritic cells, follicular helper T cells, and lymph-node homing receptors on T cells. Neither ISS nor immunolabeling showed evidence of tertiary lymphoid aggregates in sarcoidosis samples. Together, our finding suggests that despite their heterogeneity, the formation of tertiary immune structures is a common feature in granulomas from TB patients.

Biobanking plays a pivotal role in biomedical research by providing standardized processing, precise storing, and management of biological sample collections along with the associated data. Effective data management is a prerequisite to ensure the integrity, quality, and accessibility of these resources. This review provides a current landscape of data management in biobanking, discussing key challenges, existing strategies, and potential future directions. We explore multiple aspects of data management, including data collection, storage, curation, sharing, and ethical considerations. By examining the evolving technologies and methodologies in biobanking, we aim to provide insights into addressing the complexities and maximizing the utility of biobank data for research and clinical applications.

BACKGROUND:Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8+ T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells.METHODS:We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasome in vitro, ex vivo and in vivo using RNA and Western blot analyses. CD8+ T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8+ T-cell numbers were determined in patients' peripheral blood using tetramer technology.RESULTS:Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cells in vitro, ex vivo and in vivo. In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8+ T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8+ T-cells compared to healthy controls and asthmatics.CONCLUSION:Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8+ T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections.

IntroductionSarcoidosis is a highly variable disease in terms of organ involvement, type of onset and course. Associations of genetic polymorphisms with sarcoidosis phenotypes have been observed and suggest genetic signatures.MethodsAfter obtaining a positive vote of the competent ethics committee we genotyped 1909 patients of the deeply phenotyped Genetic-Phenotype Relationship in Sarcoidosis (GenPhenReSa) cohort of 31 European centers in 12 countries with 116 potentially disease-relevant single-nucleotide polymorphisms (SNPs). Using a meta-analysis, we investigated the association of relevant phenotypes (acute vs. sub-acute onset, phenotypes of organ involvement, specific organ involvements, and specific symptoms) with genetic markers. Subgroups were built on the basis of geographical, clinical and hospital provision considerations.ResultsIn the meta-analysis of the full cohort, there was no significant genetic association with any considered phenotype after correcting for multiple testing. In the largest sub-cohort (Serbia), we confirmed the known association of acute onset with TNF and reported a new association of acute onset an HLA polymorphism. Multi-locus models with sets of three SNPs in different genes showed strong associations with the acute onset phenotype in Serbia and Lublin (Poland) demonstrating potential region-specific genetic links with clinical features, including recently described phenotypes of organ involvement.DiscussionThe observed associations between genetic variants and sarcoidosis phenotypes in subgroups suggest that gene–environment-interactions may influence the clinical phenotype. In addition, we show that two different sets of genetic variants are permissive for the same phenotype of acute disease only in two geographic subcohorts pointing to interactions of genetic signatures with different local environmental factors. Our results represent an important step towards understanding the genetic architecture of sarcoidosis.

Respiratory infections by Gram-negative bacteria are a major cause of global morbidity and mortality. Alveolar macrophages (AMs) play a central role in maintaining lung immune homeostasis and host defense by sensing pathogens via pattern recognition receptors (PRR).

In patients with severe #COVID19, increased levels of autoantibodies against PAR1 were found. These might serve as allosteric agonists of PAR1 on endothelial cells and platelets, and thus might contribute to the pathogenesis of microthrombosis in COVID-19. https://bit.ly/3pqM9Vv.

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼102 colony forming units (CFUs) and ∼103 CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity.

BACKGROUND:Comprehensive studies investigated the role of T-cells in asthma which led to personalised treatment options targeting severe eosinophilic asthma. However, little is known about the contribution of B-cells to this chronic inflammatory disease. In this study we investigated the contribution of various B-cell populations to specific clinical features in asthma. METHODS:In the All Age Asthma Cohort (ALLIANCE), a subgroup of 154 adult asthma patients and 28 healthy controls were included for B-cell characterisation by flow cytometry. Questionnaires, lung function measurements, blood differential counts and allergy testing of participants were analysed together with comprehensive data on B-cells using association studies and multivariate linear models. RESULTS:Patients with severe asthma showed decreased immature B-cell populations while memory B-cells were significantly increased compared with both mild-moderate asthma patients and healthy controls. Furthermore, increased frequencies of IgA+ memory B-cells were associated with impaired lung function and specifically with parameters indicative for augmented resistance in the peripheral airways. Accordingly, asthma patients with small airway dysfunction (SAD) defined by impulse oscillometry showed increased frequencies of IgA+ memory B-cells, particularly in patients with mild-moderate asthma. Additionally, IgA+ memory B-cells significantly correlated with clinical features of SAD such as exacerbations. CONCLUSIONS:With this study we demonstrate for the first time a significant association of increased IgA+ memory B-cells with asthma and SAD, pointing towards future options for B-cell-directed strategies in preventing and treating asthma.

ABSTRACT Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of a well-characterized cohort of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen (HLA) region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a highly pleiotropic ∼0.9-Mb inversion polymorphism and characterized the potential effects of the inversion in detail. Our data, together with the 5 th release of summary statistics from the COVID-19 Host Genetics Initiative, also identified a new locus at 19q13.33, including NAPSA , a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.

The nuclear long non-coding RNA LUCAT1 has previously been identified as a negative feedback regulator of type I interferon and inflammatory cytokine expression in human myeloid cells. Here, we define the mechanistic basis for the suppression of inflammatory gene expression by LUCAT1. Using comprehensive identification of RNA-binding proteins by mass spectrometry as well as RNA immunoprecipitation, we identified proteins important in processing and alternative splicing of mRNAs as LUCAT1-binding proteins. These included heterogeneous nuclear ribonucleoprotein C, M, and A2B1. Consistent with this finding, cells lacking LUCAT1 have altered splicing of selected immune genes. In particular, upon lipopolysaccharide stimulation, the splicing of the nuclear receptor 4A2 (NR4A2) gene was particularly affected. As a consequence, expression of NR4A2 was reduced and delayed in cells lacking LUCAT1. NR4A2-deficient cells had elevated expression of immune genes. These observations suggest that LUCAT1 is induced to control the splicing and stability of NR4A2, which is in part responsible for the anti-inflammatory effect of LUCAT1. Furthermore, we analyzed a large cohort of patients with inflammatory bowel disease as well as asthma and chronic obstructive pulmonary disease. In these patients, LUCAT1 levels were elevated and in both diseases, positively correlated with disease severity. Collectively, these studies define a key molecular mechanism of LUCAT1-dependent immune regulation through post-transcriptional regulation of mRNAs highlighting its role in the regulation of inflammatory disease.

Due to the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), deepening the host genetic contribution to severe COVID-19 may further improve our understanding about underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany, as well as hypothesis-driven targeted analysis of the human leukocyte antigen (HLA) region and chromosome Y haplotypes. We include detailed stratified analyses based on age, sex and disease severity. In addition to already established risk loci, our data identify and replicate two genome-wide significant loci at 17q21.31 and 19q13.33 associated with severe COVID-19 with respiratory failure. These associations implicate a highly pleiotropic ~0.9-Mb 17q21.31 inversion polymorphism, which affects lung function and immune and blood cell counts, and the NAPSA gene, involved in lung surfactant protein production, in COVID-19 pathogenesis.

To examine the role of smoking on the bacterial community composition of the upper and the lower respiratory tract, a monocentric, controlled prospective study was performed, including healthy smokers, ex-smokers and never-smokers. Smokers were further grouped according to their smoking history. Bacterial diversity was analysed using a molecular barcoding approach based on directly extracted DNA. Our study shows for the first time distinct bacterial response patterns in the upper and lower respiratory tract to cigarette smoking leading to a higher abundance of opportunistic pathogens. The clinical significance of these dysbioses for health needs to be further explored.

The interplay of particles with pulmonary surfactant, a lipid‐protein material pivotal for lung function, is hypothesized as a key factor that has not been routinely considered in the current in vitro models when determining the fate of inhaled nanomaterials. To explain its influence on cellular uptake and protective effects, nanoparticles are studied on two models of alveolar cells, in the absence or presence of pulmonary surfactant. Composition and interfacial performance of native human and porcine surfactants, a commercially available bovine surfactant (Alveofact), and an artificial lung lining fluid are characterized using shotgun lipidomics and biophysical approaches (i.e., Langmuir surface balances and captive bubble surfactometry). Plain and aminofunctionalized silica nanoparticles and a novel antimycobacterial nanoformulated benzothiazinone (BTZ043) are selected as examples of neutral, positively charged and therapeutically relevant nanoparticles, respectively. They are deposited onto monocultures of human alveolar epithelial and phagocytic cell lines in the presence or absence of the surfactant preparations, modeling the alveolar milieu. Only surfactant preparations with high interfacial activity and distinctive composition mitigated the toxicity of aerosolized particles, along with a tendency of aerosolized particles to aggregate. Key requirements of surfactant preparations needed when studying interactions of nanomaterials with the pulmonary air‐blood barrier in vitro are identified.

[This corrects the article DOI: 10.1371/journal.pone.0238619.].

Purpose Long-term non-invasive ventilation (NIV) is recommended for patients with stable chronic obstructive lung disease (COPD) and chronic hypercapnia. High inspiratory pressure NIV (hiNIV) and a significant reduction of arterial pCO(2)have been shown to prolong survival. Often, patients on hiNIV describe severe respiratory distress, known as "deventilation syndrome", after removal of the NIV mask in the morning. Mechanical pursed lips breathing ventilation (PLBV) is a new non-invasive ventilation mode that mimics the pressure-curve of pursed lips breathing during expiration. The clinical impact of switching patients from standard NIV to PLBV has not been studied so far. Patients and methods In this hypothesis generating study, we retrospectively analysed the effects of switching COPD patients (stage GOLD III-IV) from conventional NIV to PLBV. Medical records of all patients who had an established NIV and were switched to PLBV between March 2016 and October 2017 were screened. Patients were included if they complained of shortness of breath on mask removal, used their conventional NIV regularly, and had a documented complete diagnostic workup including lung function testing, blood gas analysis and 6-minute walk test (6MWT) before and after 3-7 days of PLBV. Results Six male and 10 female patients (median age 65.4 years; IQR 64.0-71.3) with a previous NIV treatment duration of 38 months (median; IQR 20-42) were analysed. After PLVB initiation, the median inspiratory ventilation pressure needed to maintain the capillary pre-switch pCO(2)level was reduced from 19.5 mbar (IQR 16.0-26.0) to 13.8 mbar (IQR 12.5-14.9; p<0.001). The median 6MWT distance increased from 200m (IQR 153.8-266.3) to 270m (IQR 211.3-323.8; p<0.001). Median forced vital capacity (FVC) increased from 49.5% to 53.0% of the predicted value (p = 0.04), while changes in FEV(1)and residual volume (RV) were non-significant. Conclusion Based on this small retrospective analysis, we hypothesise that switching patients with COPD GOLD III-IV and chronic hypercapnia from conventional NIV to PLBV may increase exercise tolerance and FVC in the short term.

The significance of human biorepositories for modern medical research, particularly for comprehensive population-based genetic analyses, is constantly growing. While large and centralized institutions are usually considered best suited to meet the increasing demand for high-quality “biobanks,” most medical research institutions still host rather heterogeneous and fragmented biobanking activities, undertaken by clinical departments with oftentimes rather different scientific scope. Undoubtedly, most clinicians and medical researchers would appreciate infrastructural support in terms of the storage and handling of their biosamples, but they are also likely to expect access to their samples avoiding extensive formal requirements. We report on the establishment of the PopGen 2.0 Network (P2N), an overarching alliance of initially seven biobanks from Northern Germany which adopted a joint but lean governance structure and use-and-access policy for their samples and data. In addition, the members of P2N have pursued an intense collaboration on ethical, legal and social issues and maintain a common IT infrastructure. The implementation of P2N has substantially improved the prospects of biobank-based research at the participating institutions. The network may thus serve as a role model for similar initiatives geared at linking pre-existing biorepositories for the benefit of research quality, efficiency, and transparency.