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    Asthma-associated differences in microbial composition of induced sputum. Marri Pradeep Reddy,Stern Debra A,Wright Anne L,Billheimer Dean,Martinez Fernando D The Journal of allergy and clinical immunology BACKGROUND:It is increasingly evident that microbial colonization of the respiratory tract might have a role in the pathogenesis of asthma. OBJECTIVE:We sought to characterize and compare the microbiome of induced sputum in asthmatic and nonasthmatic adults. METHODS:Induced sputum samples were obtained from 10 nonasthmatic subjects and 10 patients with mild active asthma (8/10 were not using inhaled corticosteroids). Total DNA was extracted from sputum supernatants and amplified by using primers specific for the V6 hypervariable region of bacterial 16s rRNA. Samples were barcoded, and equimolar concentrations of 20 samples were pooled and sequenced with the 454 GS FLX sequencer. Sequences were assigned to bacterial taxa by comparing them with 16s rRNA sequences in the Ribosomal Database Project. RESULTS:All sputum samples contained 5 major bacterial phyla: Firmicutes, Proteobacteria, Actinobacteria, Fusobacterium, and Bacteroidetes, with the first 3 phyla accounting for more than 90% of the total sequences. Proteobacteria were present in higher proportions in asthmatic patients (37% vs 15%, P < .001). In contrast, Firmicutes (47% vs 63%, P = .17) and Actinobacteria (10% vs 14%, P = .36) were found more frequently in samples from nonasthmatic subjects, although this was not statistically significant. Hierarchical clustering produced 2 significant clusters: one contained primarily asthmatic samples and the second contained primarily nonasthmatic samples. In addition, samples from asthmatic patients had greater bacterial diversity compared with samples from nonasthmatic subjects. CONCLUSION:Patients with mild asthma have an altered microbial composition in the respiratory tract that is similar to that observed in patients with more severe asthma. 10.1016/j.jaci.2012.11.013
    Induced sputum proteome in healthy subjects and asthmatic patients. Gharib Sina A,Nguyen Elizabeth V,Lai Ying,Plampin Jessica D,Goodlett David R,Hallstrand Teal S The Journal of allergy and clinical immunology BACKGROUND:Asthma is a heterogeneous disease characterized by abnormal airway pathophysiology and susceptibility to different stimuli, as exemplified by a subset of patients with exercise-induced bronchoconstriction. Induced sputum provides a noninvasive method to sample airway biofluids that are enriched in proteins. OBJECTIVE:We hypothesized that novel mechanisms in the pathogenesis of asthma might be revealed by studying the patterns of protein expression in induced sputum. METHODS:We used shotgun proteomics to analyze induced sputum from 5 healthy subjects and 10 asthmatic patients, including 5 with exercise-induced bronchoconstriction. Differential protein expression among asthmatic patients, asthma subphenotypes, and control subjects was determined by using spectral counting and computational methods. RESULTS:Using Gene Ontology analysis, we defined the functional landscape of the induced sputum proteome and applied network analysis to construct a protein interaction map for this airway compartment. Shotgun proteomics analysis identified a number of proteins the differential enrichment or depletion of which robustly distinguished asthmatic patients from healthy control subjects and captured the effects of exercise on induced sputum proteome. Functional and network analysis identified key processes, including proteolytic activity, that are known contributors to airway remodeling. Importantly, this approach highlighted previously unrecognized roles for differentially expressed proteins in pathways implicated in asthma, such as modulation of phospholipase A(2) by secretoglobin, a putative role for S100A8/9 in human asthma, and selective upregulation of complement component 3a in response to exercise in asthmatic patients. CONCLUSION:Computationally intensive analysis of induced sputum proteome is a powerful approach to understanding the pathophysiology of asthma and a promising methodology to investigating other diseases of the airways. 10.1016/j.jaci.2011.07.053
    Type 2-high asthma is associated with a specific indoor mycobiome and microbiome. Vandenborght Louise-Eva,Enaud Raphaël,Urien Charlotte,Coron Noémie,Girodet Pierre-Olivier,Ferreira Stéphanie,Berger Patrick,Delhaes Laurence The Journal of allergy and clinical immunology BACKGROUND:The links between microbial environmental exposures and asthma are well documented, but no study has combined deep sequencing results from pulmonary and indoor microbiomes of patients with asthma with spirometry, clinical, and endotype parameters. OBJECTIVE:The goal of this study was to investigate the links between indoor microbial exposures and pulmonary microbial communities and to document the role of microbial exposures on inflammatory and clinical outcomes of patients with severe asthma (SA). METHODS:A total of 55 patients with SA from the national Cohort of Bronchial Obstruction and Asthma cohort were enrolled for analyzing their indoor microbial flora through the use of electrostatic dust collectors (EDCs). Among these patients, 22 were able to produce sputum during "stable" or pulmonary "exacerbation" periods and had complete pairs of EDC and sputum samples, both collected and analyzed. We used amplicon targeted metagenomics to compare microbial communities from EDC and sputum samples of patients according to type 2 (T2)-asthma endotypes. RESULTS:Compared with patients with T2-low SA, patients with T2-high SA exhibited an increase in bacterial α-diversity and a decrease in fungal α-diversity of their indoor microbial florae, the latter being significantly correlated with fraction of exhaled nitric oxide levels. The β-diversity of the EDC mycobiome clustered significantly according to T2 endotypes. Moreover, the proportion of fungal taxa in common between the sputum and EDC samples was significantly higher when patients exhibited acute exacerbation. CONCLUSION:These results illustrated, for the first time, a potential association between the indoor mycobiome and clinical features of patients with SA, which should renew interest in deciphering the interactions between indoor environment, fungi, and host in asthma. 10.1016/j.jaci.2020.08.035
    Microbiota abnormalities in inflammatory airway diseases - Potential for therapy. Gollwitzer Eva S,Marsland Benjamin J Pharmacology & therapeutics Increasingly the development of novel therapeutic strategies is taking into consideration the contribution of the intestinal microbiota to health and disease. Dysbiosis of the microbial communities colonizing the human intestinal tract has been described for a variety of chronic diseases, such as inflammatory bowel disease, obesity and asthma. In particular, reduction of several so-called probiotic species including Lactobacilli and Bifidobacteria that are generally considered to be beneficial, as well as an outgrowth of potentially pathogenic bacteria is often reported. Thus a tempting therapeutic approach is to shape the constituents of the microbiota in an attempt to restore the microbial balance towards the growth of 'health-promoting' bacterial species. A twist to this scenario is the recent discovery that the respiratory tract also harbors a microbiota under steady-state conditions. Investigators have shown that the microbial composition of the airway flora is different between healthy lungs and those with chronic lung diseases, such as asthma, chronic obstructive pulmonary disease as well as cystic fibrosis. This is an emerging field, and thus far there is very limited data showing a direct contribution of the airway microbiota to the onset and progression of disease. However, should future studies provide such evidence, the airway microbiota might soon join the intestinal microbiota as a target for therapeutic intervention. In this review, we highlight the major advances that have been made describing the microbiota in chronic lung disease and discuss current and future approaches concerning manipulation of the microbiota for the treatment and prevention of disease. 10.1016/j.pharmthera.2013.08.002
    Bacterial biogeography of adult airways in atopic asthma. Durack Juliana,Huang Yvonne J,Nariya Snehal,Christian Laura S,Ansel K Mark,Beigelman Avraham,Castro Mario,Dyer Anne-Marie,Israel Elliot,Kraft Monica,Martin Richard J,Mauger David T,Rosenberg Sharon R,King Tonya S,White Steven R,Denlinger Loren C,Holguin Fernando,Lazarus Stephen C,Lugogo Njira,Peters Stephen P,Smith Lewis J,Wechsler Michael E,Lynch Susan V,Boushey Homer A, Microbiome BACKGROUND:Perturbations to the composition and function of bronchial bacterial communities appear to contribute to the pathophysiology of asthma. Unraveling the nature and mechanisms of these complex associations will require large longitudinal studies, for which bronchoscopy is poorly suited. Studies of samples obtained by sputum induction and nasopharyngeal brushing or lavage have also reported asthma-associated microbiota characteristics. It remains unknown, however, whether the microbiota detected in these less-invasive sample types reflect the composition of bronchial microbiota in asthma. RESULTS:Bacterial microbiota in paired protected bronchial brushings (BB; n = 45), induced sputum (IS; n = 45), oral wash (OW; n = 45), and nasal brushings (NB; n = 27) from adults with mild atopic asthma (AA), atopy without asthma (ANA), and healthy controls (HC) were profiled using 16S rRNA gene sequencing. Though microbiota composition varied with sample type (p < 0.001), compositional similarity was greatest for BB-IS, particularly in AAs and ANAs. The abundance of genera detected in BB correlated with those detected in IS and OW (r median [IQR] 0.869 [0.748-0.942] and 0.822 [0.687-0.909] respectively), but not with those in NB (r = 0.004 [- 0.003-0.011]). The number of taxa shared between IS-BB and NB-BB was greater in AAs than in HCs (p < 0.05) and included taxa previously associated with asthma. Of the genera abundant in NB, only Moraxella correlated positively with abundance in BB; specific members of this genus were shared between the two compartments only in AAs. Relative abundance of Moraxella in NB of AAs correlated negatively with that of Corynebacterium but positively with markers of eosinophilic inflammation in the blood and BAL fluid. The genus, Corynebacterium, trended to dominate all NB samples of HCs but only half of AAs (p = 0.07), in whom abundance of this genus was negatively associated with markers of eosinophilic inflammation. CONCLUSIONS:Induced sputum is superior to nasal brush or oral wash for assessing bronchial microbiota composition in asthmatic adults. Although compositionally similar to the bronchial microbiota, the microbiota in induced sputum are distinct, reflecting enrichment of oral bacteria. Specific bacterial genera are shared between the nasal and the bronchial mucosa which are associated with markers of systemic and bronchial inflammation. 10.1186/s40168-018-0487-3
    Microbial diversity within the airway microbiome in chronic pediatric lung diseases. Hahn Andrea,Warnken Stephanie,Pérez-Losada Marcos,Freishtat Robert J,Crandall Keith A Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases The study of the airway microbiome in children is an area of emerging research, especially in relation to the role microbial diversity may play in acute and chronic inflammation. Three such pediatric airway diseases include cystic fibrosis, asthma, and chronic lung disease of prematurity. In cystic fibrosis, the presence of Pseudomonas spp. is associated with decreased microbial diversity. Decreasing microbial diversity is also associated with poor lung function. In asthma, early viral infections appear to drive changes in bacterial diversity which may be associated with asthma risk. Premature infants with Ureaplasma spp. are at higher risk for chronic lung disease due to inflammation. Microbiome changes due to prematurity also appear to affect the inflammatory response to viral infections post-natally. Importantly, microbial diversity can be measured using metataxonomic (e.g., 16S rRNA sequencing) and metagenomic (e.g., shotgun sequencing) approaches. A metagenomics approach may be preferable as it can provide further granularity of the sample composition, identifying the bacterial species or strain, information on additional microbial components, including fungal and viral components, information about functional genomics of the microbiome, and information about antimicrobial resistance mutations. Future studies of pediatric airway diseases incorporating these techniques may provide evidence for new treatment approaches for these vulnerable patient populations. 10.1016/j.meegid.2017.12.006
    Sputum proteomics in inflammatory and suppurative respiratory diseases. Gray Robert D,MacGregor Gordon,Noble Donald,Imrie Margaret,Dewar Maria,Boyd A Christopher,Innes J Alastair,Porteous David J,Greening Andrew P American journal of respiratory and critical care medicine RATIONALE:Markers of inflammatory activity are important for assessment and management of many respiratory diseases. Markers that are currently unrecognized may be more valuable than those presently believed to be useful. OBJECTIVES:To identify potential biomarkers of suppurative and inflammatory lung disease in induced sputum samples. METHODS:Induced sputum was collected from 20 healthy control subjects, 24 patients with asthma, 24 with chronic obstructive pulmonary disease, 28 with cystic fibrosis (CF), and 19 with bronchiectasis. Twelve patients with CF had sputum sampled before and after antibiotic therapy for an infective exacerbation. The fluid phase of induced sputum was analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy on three protein array surfaces. Some protein markers were selected for identification, and relevant ELISA assays sought. For 12 patients with CF, both SELDI-TOF and ELISA monitored changes in inflammatory responses during infective exacerbations. MEASUREMENTS AND MAIN RESULTS:SELDI-TOF identified potential biomarkers that differentiated each of the disease groups from healthy control subjects: at a significance of P < 0.01, there were 105 for asthma, 113 for chronic obstructive pulmonary disease, 381 for CF, and 377 for bronchiectasis. Peaks selected for protein identification yielded calgranulin A, calgranulin B, calgranulin C, Clara cell secretory protein, lysosyme c, proline rich salivary peptide, cystatin s, and hemoglobin alpha. On treatment of an infective CF exacerbation, SELDI-TOF determined falls in levels of calgranulin A and calgranulin B that were mirrored by ELISA-measured falls in calprotectin (heterodimer of calgranulins A and B). CONCLUSIONS:Proteomic screening of sputum yields potential biomarkers of inflammation. The early development of a clinically relevant assay from such data is demonstrated. 10.1164/rccm.200703-409OC
    Induced sputum: a window to lung pathology. Nicholas Ben,Djukanović Ratko Biochemical Society transactions Sputum is recognized as a sampling method for the monitoring and assessment of chronic lung diseases such as asthma, COPD (chronic obstructive pulmonary disease) and cystic fibrosis. Sputum samples the central airways and its protein components (e.g. mucins and cytokines), cellular components (e.g. eosinophils and neutrophils) and microbiological components (e.g. viruses and bacteria) can be used as markers of disease severity, exacerbation, susceptibility or progression. This paper describes the basic constituents of induced sputum and how these influence the quantification and identification of novel biomarkers of chronic lung diseases using techniques such as proteomics. 10.1042/BST0370868