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1.
Chest ; 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34801592

RESUMO

BACKGROUND: Improved understanding of the pathways associated with airway pathophysiology in chronic obstructive pulmonary disease (COPD) will identify new predictive biomarkers and novel therapeutic targets. RESEARCH QUESTION: Which physiologic pathways are altered in the airways of subjects with COPD and predict exacerbations? STUDY DESIGN AND METHODS: We applied a mass spectrometric panel of metabolomic biomarkers related to mucus hydration and inflammation to sputa from the SPIROMICS multi-center COPD study. Biomarkers elevated in sputa from subjects with COPD were evaluated for relationships to measures of COPD disease severity and their ability to predict future exacerbations. RESULTS: Sputum supernatants from 980 subjects (77 healthy non-smokers [NS], 341 smokers with preserved spirometry [SPS], and 562 COPD subjects [178 GOLD 1, 303 GOLD 2, and 81 GOLD 3]) were analyzed. Biomarkers from multiple pathways were elevated in COPD and correlated with sputum neutrophil counts. Among the most significant analytes (at FDR 0.1) were sialic acid, hypoxanthine, xanthine, methylthioadenosine, adenine, and glutathione. Sialic acid and hypoxanthine were strongly associated with measures of disease severity, and elevation of these biomarkers was associated with shorter time to exacerbation and improved prediction models of future exacerbations. INTERPRETATION: Biomarker evaluation implicated pathways involved in mucus hydration, adenosine metabolism, methionine salvage, and oxidative stress in COPD airway pathophysiology. Therapies that target these pathways may be of benefit in COPD, and a simple model adding sputum soluble phase biomarkers improves prediction of pulmonary exacerbations.

2.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34548396

RESUMO

Elevated levels of MUC5AC, one of the major gel-forming mucins in the lungs, are closely associated with chronic obstructive lung diseases such as chronic bronchitis and asthma. It is not known, however, how the structure and/or gel-making properties of MUC5AC contribute to innate lung defense in health and drive the formation of stagnant mucus in disease. To understand this, here we studied the biophysical properties and macromolecular assembly of MUC5AC compared to MUC5B. To study each native mucin, we used Calu3 monomucin cultures that produced MUC5AC or MUC5B. To understand the macromolecular assembly of MUC5AC through N-terminal oligomerization, we expressed a recombinant whole N-terminal domain (5ACNT). Scanning electron microscopy and atomic force microscopy imaging indicated that the two mucins formed distinct networks on epithelial and experimental surfaces; MUC5B formed linear, infrequently branched multimers, whereas MUC5AC formed tightly organized networks with a high degree of branching. Quartz crystal microbalance-dissipation monitoring experiments indicated that MUC5AC bound significantly more to hydrophobic surfaces and was stiffer and more viscoelastic as compared to MUC5B. Light scattering analysis determined that 5ACNT primarily forms disulfide-linked covalent dimers and higher-order oligomers (i.e., trimers and tetramers). Selective proteolytic digestion of the central glycosylated region of the full-length molecule confirmed that MUC5AC forms dimers and higher-order oligomers through its N terminus. Collectively, the distinct N-terminal organization of MUC5AC may explain the more adhesive and unique viscoelastic properties of branched, highly networked MUC5AC gels. These properties may generate insight into why/how MUC5AC forms a static, "tethered" mucus layer in chronic muco-obstructive lung diseases.


Assuntos
Células Epiteliais/metabolismo , Mucina-5AC/química , Mucina-5AC/metabolismo , Mucina-5B/química , Mucina-5B/metabolismo , Mucosa Respiratória/metabolismo , Células Cultivadas , Células Epiteliais/citologia , Humanos , Mucosa Respiratória/citologia
3.
Eur Respir J ; 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172469

RESUMO

QUESTION: Cystic fibrosis (CF) is characterised by the accumulation of viscous, adherent mucus in the lungs. While several hypotheses invoke a direct relationship with CFTR dysfunction (i.e., acidic airway surface liquid (ASL) pH, low [HCO3 -], airway dehydration), the dominant biochemical alteration of CF mucus remains unknown. MATERIALS/METHODS: We characterised a novel cell line (CFTR-KO Calu3 cells) and the responses of human bronchial epithelial (HBE) cells from subjects with G551D or F508del mutations to Ivacaftor and Elexacaftor-Tezacaftor-Ivacaftor (ETI). A spectrum of assays such as short-circuit currents (Isc), qPCR, ASL pH, western blotting (WB), light scattering/refractometry (SEC-MALS), scanning electron microscopy (SEM), % solids, and particle tracking were performed to determine the impact of CFTR function on mucus properties. RESULTS: Loss of CFTR function in Calu3 cells resulted in ASL pH acidification and mucus hyperconcentration (dehydration). Modulation of CFTR in CF HBE cells did not affect ASL pH or mucin mRNA expression, but decreased mucus concentration, relaxed mucus network ultrastructure, and improved mucus transport. In contrast with modulator-treated cells, a large fraction of airway mucins remained attached to naïve CF cells following short apical washes, as revealed by the use of reducing agents to remove residual mucus from the cell surfaces. Extended hydration, but not buffers alkalised with NaOH or HCO3 -, normalised mucus recovery to modulator-treated cell levels. CONCLUSION: These results indicate that airway dehydration, not acidic pH and/or low [HCO3 -], is responsible for abnormal mucus properties in CF airways and CFTR modulation predominantly restores normal mucin entanglement.

4.
Lancet Respir Med ; 9(11): 1241-1254, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34058148

RESUMO

BACKGROUND: We previously described the contributions of increased total airway mucin concentrations to the pathogenesis and diagnosis of the chronic bronchitic component of chronic obstructive pulmonary disease (COPD). Here, we investigated the relative contribution of each of the major airway gel-forming mucins, MUC5AC and MUC5B, to the initiation, progression, and early diagnosis of airways disease in COPD. METHODS: SPIROMICS was a multicentre, observational study in patients aged 40-80 years recruited from six clinical sites and additional subsites in the USA. In this analysis, MUC5AC and MUC5B were quantitated by stable isotope-labelled mass spectrometry in induced sputum samples from healthy never-smokers, ever-smokers at risk for COPD, and ever-smokers with COPD. Participants were extensively characterised using results from questionnaires, such as the COPD assessment test (CAT) and St George's Respiratory Questionnaire; quantitative CT, such as residual volume/total lung capacity ratio (RV/TLC) and parametric response mapping-functional small airway disease (PRM-fSAD); and pulmonary function tests, such as FEV1, forced vital capacity (FVC), and forced expiratory flow, midexpiratory phase (FEF25-75%). Absolute concentrations of both MUC5AC and MUC5B were related to cross-sectional (baseline, initial visit) and 3-year follow-up longitudinal data, including lung function, small airways obstruction, prospective acute exacerbations, and smoking status as primary outcomes. This study is registered with ClinicalTrials.gov (NCT01969344). FINDINGS: This analysis included 331 participants (mean age 63 years [SEM 9·40]), of whom 40 were healthy never-smokers, 90 were at-risk ever-smokers, and 201 were ever-smokers with COPD. Increased MUC5AC concentrations were more reliably associated with manifestations of COPD than were MUC5B concentrations, including decreased FEV1 and FEF25-75%, and increased prospective exacerbation frequency, RV/TLC, PRM-fSAD, and COPD assessment scores. MUC5AC concentrations were more reactive to cigarette smoke exposure than were MUC5B concentrations. Longitudinal data from 3-year follow-up visits generated a multivariate-adjusted odds ratio for two or more exacerbations of 1·24 (95% CI 1·04-1·47, p=0·015) for individuals with high baseline MUC5AC concentration. Increased MUC5AC, but not MUC5B, concentration at baseline was a significant predictor of FEV1, FEV1/FVC, FEF25-75%, and CAT score decline during the 3-year follow-up. Moreover, current smokers in the at-risk group showed raised MUC5AC concentrations at initial visits and decreased lung function over 3 years. By contrast, former smokers in the at-risk group showed normal MUC5AC concentrations at the initial visit and preserved lung function over 3 years. INTERPRETATION: These data indicate that increased MUC5AC concentration in the airways might contribute to COPD initiation, progression, exacerbation risk, and overall pathogenesis. Compared with MUC5B, greater relative changes in MUC5AC concentrations were observed as a function of COPD severity, and MUC5AC concentration seems to be an objective biomarker to detect disease in at-risk and pre-COPD individuals. These data suggest that MUC5AC-producing pathways could be potential targets for future therapeutic strategies. Thus, MUC5AC could be a novel biomarker for COPD prognosis and for testing the efficacy of therapeutic agents. FUNDING: National Institutes of Health; National Heart, Lung, and Blood Institute.

5.
Respir Res ; 22(1): 127, 2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33906653

RESUMO

BACKGROUND: Soluble receptor for advanced glycation end products (sRAGE) is a proposed emphysema and airflow obstruction biomarker; however, previous publications have shown inconsistent associations and only one study has investigate the association between sRAGE and emphysema. No cohorts have examined the association between sRAGE and progressive decline of lung function. There have also been no evaluation of assay compatibility, receiver operating characteristics, and little examination of the effect of genetic variability in non-white population. This manuscript addresses these deficiencies and introduces novel data from Pittsburgh COPD SCCOR and as well as novel work on airflow obstruction. A meta-analysis is used to quantify sRAGE associations with clinical phenotypes. METHODS: sRAGE was measured in four independent longitudinal cohorts on different analytic assays: COPDGene (n = 1443); SPIROMICS (n = 1623); ECLIPSE (n = 2349); Pittsburgh COPD SCCOR (n = 399). We constructed adjusted linear mixed models to determine associations of sRAGE with baseline and follow up forced expiratory volume at one second (FEV1) and emphysema by quantitative high-resolution CT lung density at the 15th percentile (adjusted for total lung capacity). RESULTS: Lower plasma or serum sRAGE values were associated with a COPD diagnosis (P < 0.001), reduced FEV1 (P < 0.001), and emphysema severity (P < 0.001). In an inverse-variance weighted meta-analysis, one SD lower log10-transformed sRAGE was associated with 105 ± 22 mL lower FEV1 and 4.14 ± 0.55 g/L lower adjusted lung density. After adjusting for covariates, lower sRAGE at baseline was associated with greater FEV1 decline and emphysema progression only in the ECLIPSE cohort. Non-Hispanic white subjects carrying the rs2070600 minor allele (A) and non-Hispanic African Americans carrying the rs2071288 minor allele (A) had lower sRAGE measurements compare to those with the major allele, but their emphysema-sRAGE regression slopes were similar. CONCLUSIONS: Lower blood sRAGE is associated with more severe airflow obstruction and emphysema, but associations with progression are inconsistent in the cohorts analyzed. In these cohorts, genotype influenced sRAGE measurements and strengthened variance modelling. Thus, genotype should be included in sRAGE evaluations.


Assuntos
Pulmão/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/sangue , Enfisema Pulmonar/sangue , Receptor para Produtos Finais de Glicação Avançada/sangue , Idoso , Biomarcadores/sangue , Feminino , Volume Expiratório Forçado , Humanos , Estudos Longitudinais , Pulmão/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Fenótipo , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/diagnóstico , Enfisema Pulmonar/fisiopatologia , Índice de Gravidade de Doença , Espirometria , Tomografia Computadorizada por Raios X , Capacidade Vital
6.
J Cyst Fibros ; 20(5): 851-856, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33674211

RESUMO

BACKGROUND: The CFTR modulator ivacaftor has been variably effective in treating individuals with cystic fibrosis (CF) who harbor CFTR gating variants such as G551D, as well as other classes of CFTR variants when used with other modulators. Because CFTR genotype does not fully explain this variability, defining genetic modifiers of response to modulator therapy is of particular interest to the field of individualized CF drug therapy. Previous studies have proposed that a variant in SLC26A9 (rs7512462) is associated with lung disease severity and with response to treatment with ivacaftor in individuals with CF who carry G551D or gating variants. METHODS: Given the implications for CF treatment, we re-examined the reported associations in three cohorts; patients enrolled in the Twin and Siblings study at Johns Hopkins University, the CF modifier study at the University of North Carolina at Chapel Hill, and the prospective G551D Observational (GOAL) study. The GOAL study was specifically designed to measure lung function response to ivacaftor. RESULTS: We find no association between SLC26A9 (rs7512462) genotype and lung disease severity (n = 272) or change in lung function at one-, three-, and six-month intervals following ivacaftor treatment(n = 141) in individuals with CF who carry at least one G551D variant. CONCLUSIONS: Our inability to replicate this association indicates that rs7512462 genotype should not be used in treatment decisions.

7.
J Allergy Clin Immunol ; 147(5): 1594-1601, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33667479

RESUMO

Severe asthma accounts for almost half the cost associated with asthma. Severe asthma is driven by heterogeneous molecular mechanisms. Conventional clinical trial design often lacks the power and efficiency to target subgroups with specific pathobiological mechanisms. Furthermore, the validation and approval of new asthma therapies is a lengthy process. A large proportion of that time is taken by clinical trials to validate asthma interventions. The National Institutes of Health Precision Medicine in Severe and/or Exacerbation Prone Asthma (PrecISE) program was established with the goal of designing and executing a trial that uses adaptive design techniques to rapidly evaluate novel interventions in biomarker-defined subgroups of severe asthma, while seeking to refine these biomarker subgroups, and to identify early markers of response to therapy. The novel trial design is an adaptive platform trial conducted under a single master protocol that incorporates precision medicine components. Furthermore, it includes innovative applications of futility analysis, cross-over design with use of shared placebo groups, and early futility analysis to permit more rapid identification of effective interventions. The development and rationale behind the study design are described. The interventions chosen for the initial investigation and the criteria used to identify these interventions are enumerated. The biomarker-based adaptive design and analytic scheme are detailed as well as special considerations involved in the final trial design.


Assuntos
Asma , Biomarcadores , Medicina de Precisão , Ensaios Clínicos Controlados Aleatórios como Assunto , Humanos , Projetos de Pesquisa
8.
NPJ Biofilms Microbiomes ; 7(1): 14, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547327

RESUMO

Chronic obstructive pulmonary disease (COPD) is heterogeneous in development, progression, and phenotypes. Little is known about the lung microbiome, sampled by bronchoscopy, in milder COPD and its relationships to clinical features that reflect disease heterogeneity (lung function, symptom burden, and functional impairment). Using bronchoalveolar lavage fluid collected from 181 never-smokers and ever-smokers with or without COPD (GOLD 0-2) enrolled in the SubPopulations and InteRmediate Outcome Measures In COPD Study (SPIROMICS), we find that lung bacterial composition associates with several clinical features, in particular bronchodilator responsiveness, peak expiratory flow rate, and forced expiratory flow rate between 25 and 75% of FVC (FEF25-75). Measures of symptom burden (COPD Assessment Test) and functional impairment (six-minute walk distance) also associate with disparate lung microbiota composition. Drivers of these relationships include members of the Streptococcus, Prevotella, Veillonella, Staphylococcus, and Pseudomonas genera. Thus, lung microbiota differences may contribute to airway dysfunction and airway disease in milder COPD.


Assuntos
Bactérias/classificação , Pulmão/microbiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , RNA Ribossômico 16S/genética , Análise de Sequência de RNA/métodos , Adulto , Idoso , Bactérias/isolamento & purificação , Líquido da Lavagem Broncoalveolar/microbiologia , Feminino , Volume Expiratório Forçado , Humanos , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/microbiologia , Espirometria
9.
Chest ; 160(1): 94-103, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33539837

RESUMO

BACKGROUND: Mild expiratory flow limitation may not be recognized using traditional spirometric criteria based on the ratio of FEV1/FVC. RESEARCH QUESTION: Does slow vital capacity (SVC) instead of FVC increase the sensitivity of spirometry to identify patients with early or mild obstructive lung disease? STUDY DESIGN AND METHODS: We included 854 current and former smokers from the Subpopulations and Intermediate Outcome Measures in COPD Study cohort with a postbronchodilator FEV1/FVC ≥ 0.7 and FEV1 % predicted of ≥ 80% at enrollment. We compared baseline characteristics, chest CT scan features, exacerbations, and progression to COPD (postbronchodilator FEV1/FVC, < 0.7) during the follow-up period between 734 participants with postbronchodilator FEV1/SVC of ≥ 0.7 and 120 with postbronchodilator FEV1/SVC < 0.7 at the enrollment. We performed multivariate linear and logistic regression models and negative binomial and interval-censored proportion hazards regression models adjusted for demographics and smoking exposure to examine the association of FEV1/SVC < 0.7 with those characteristics and outcomes. RESULTS: Participants with FEV1/SVC < 0.7 were older and had lower FEV1 and more emphysema than those with FEV1/SVC ≥ 0.7. In adjusted analysis, individuals with postbronchodilator FEV1/SVC < 0.7 showed a greater percentage of emphysema by 0.45% (95% CI, 0.09%-0.82%), percentage of gas trapping by 2.52% (95% CI, 0.59%-4.44%), and percentage of functional small airways disease based on parametric response mapping by 2.78% (95% CI, 0.72%-4.83%) at baseline than those with FEV1/SVC ≥ 0.7. During a median follow-up time of 1,500 days, an FEV1/SVC < 0.7 was not associated with total exacerbations (incident rate ratio [IRR], 1.61; 95% CI, 0.97-2.64), but was associated with severe exacerbations (IRR, 2.60; 95% CI, 1.04-4.89). An FEV1/SVC < 0.7 was associated with progression to COPD during a 3-year follow-up even after adjustment for demographics and smoking exposure (hazard ratio, 3.93; 95% CI, 2.71-5.72). We found similar results when we examined the association of prebronchodilator FEV1/SVC < 0.7 or FEV1/SVC less than the lower limit of normal with chest CT scan features and progression to COPD. INTERPRETATION: Low FEV1 to SVC in current and former smokers with normal spirometry results can identify individuals with CT scan features of COPD who are at risk for severe exacerbations and is associated with progression to COPD in the future. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01969344T4; URL: www.clinicaltrials.gov.


Assuntos
Volume Expiratório Forçado/fisiologia , Pulmão/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumantes , Tomografia Computadorizada por Raios X/métodos , Capacidade Vital/fisiologia , Progressão da Doença , Seguimentos , Humanos , Pulmão/diagnóstico por imagem , Estudos Prospectivos , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Espirometria/métodos
10.
Am J Respir Crit Care Med ; 203(8): 957-968, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33180550

RESUMO

Rationale: The relative roles of mucus plugs and emphysema in mechanisms of airflow limitation and hypoxemia in smokers with chronic obstructive pulmonary disease (COPD) are uncertain.Objectives: To relate image-based measures of mucus plugs and emphysema to measures of airflow obstruction and oxygenation in patients with COPD.Methods: We analyzed computed tomographic (CT) lung images and lung function in participants in the Subpopulations and Intermediate Outcome Measures in COPD Study. Radiologists scored mucus plugs on CT lung images, and imaging software automatically quantified emphysema percentage. Unadjusted and adjusted relationships between mucus plug score, emphysema percentage, and lung function were determined using regression.Measurements and Main Results: Among 400 smokers, 229 (57%) had mucus plugs and 207 (52%) had emphysema, and subgroups could be identified with mucus-dominant and emphysema-dominant disease. Only 33% of smokers with high mucus plug scores had mucus symptoms. Mucus plug score and emphysema percentage were independently associated with lower values for FEV1 and peripheral oxygen saturation (P < 0.001). The relationships between mucus plug score and lung function outcomes were strongest in smokers with limited emphysema (P < 0.001). Compared with smokers with low mucus plug scores, those with high scores had worse COPD Assessment Test scores (17.4 ± 7.7 vs. 14.4 ± 13.3), more frequent annual exacerbations (0.75 ± 1.1 vs. 0.43 ± 0.85), and shorter 6-minute-walk distance (329 ± 115 vs. 392 ± 117 m) (P < 0.001).Conclusions: Symptomatically silent mucus plugs are highly prevalent in smokers and independently associate with lung function outcomes. These data provide rationale for targeting patients with mucus-high/emphysema-low COPD in clinical trials of mucoactive treatments.Clinical trial registered with www.clinicaltrials.gov (NCT01969344).


Assuntos
Hipóxia/induzido quimicamente , Hipóxia/fisiopatologia , Muco , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/fisiopatologia , Fumar/efeitos adversos , Idoso , Feminino , Volume Expiratório Forçado , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Função Respiratória , Fumantes , Capacidade Vital
11.
JCI Insight ; 6(1)2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33232302

RESUMO

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Animais , Sistemas CRISPR-Cas , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Feminino , Trato Gastrointestinal/patologia , Trato Gastrointestinal/fisiopatologia , Técnicas de Inativação de Genes , Humanos , Masculino , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Sistema Respiratório/patologia , Sistema Respiratório/fisiopatologia , Distribuição Tecidual , Transcriptoma
12.
Am J Respir Crit Care Med ; 203(10): 1275-1289, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33321047

RESUMO

Rationale: Identification of the specific cell types expressing CFTR (cystic fibrosis [CF] transmembrane conductance regulator) is required for precision medicine therapies for CF. However, a full characterization of CFTR expression in normal human airway epithelia is missing. Objectives: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single-cell RNA (scRNA) sequencing (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. scRNA in situ hybridization (ISH) and single-cell qRT-PCR were performed for validation. In vitro culture systems correlated CFTR function with cell types. Lentiviruses were used for cell type-specific transduction of wild-type CFTR in CF cells. Measurements and Main Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and, particularly, small airway superficial epithelia, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, whereas the expression in ciliated cells was infrequent and low. scRNA ISH and single-cell qRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to those of normal control subjects. CFTR mediated Cl- secretion in cultures tracked secretory cell, but not ionocyte, densities. Furthermore, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells and not with ionocytes. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not in ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Células Epiteliais/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Estudos de Casos e Controles , Técnicas de Cultura de Células , Humanos
13.
PLoS One ; 15(11): e0239189, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33253230

RESUMO

Genome wide association studies (GWAS) have identified several genomic loci with candidate modifiers of cystic fibrosis (CF) lung disease, but only a small proportion of the expected genetic contribution is accounted for at these loci. We leveraged expression data from CF cohorts, and Genotype-Tissue Expression (GTEx) reference data sets from multiple human tissues to generate predictive models, which were used to impute transcriptional regulation from genetic variance in our GWAS population. The imputed gene expression was tested for association with CF lung disease severity. By comparing and combining results from alternative approaches, we identified 379 candidate modifier genes. We delved into 52 modifier candidates that showed consensus between approaches, and 28 of them were near known GWAS loci. A number of these genes are implicated in the pathophysiology of CF lung disease (e.g., immunity, infection, inflammation, HLA pathways, glycosylation, and mucociliary clearance) and the CFTR protein biology (e.g., cytoskeleton, microtubule, mitochondrial function, lipid metabolism, endoplasmic reticulum/Golgi, and ubiquitination). Gene set enrichment results are consistent with current knowledge of CF lung disease pathogenesis. HLA Class II genes on chr6, and CEP72, EXOC3, and TPPP near the GWAS peak on chr5 are most consistently associated with CF lung disease severity across the tissues tested. The results help to prioritize genes in the GWAS regions, predict direction of gene expression regulation, and identify new candidate modifiers throughout the genome for potential therapeutic development.


Assuntos
Fibrose Cística/genética , Expressão Gênica/genética , Genes Modificadores/genética , Locos de Características Quantitativas/genética , Estudos de Coortes , Feminino , Regulação da Expressão Gênica/genética , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Masculino
14.
Sci Rep ; 10(1): 20133, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208859

RESUMO

COPD, chronic bronchitis (CB) and active smoking have all been associated with goblet cell hyperplasia (GCH) in small studies. Active smoking is strongly associated with CB, but there is a disconnect between CB clinical symptoms and pathology. Chronic cough and sputum production poorly correlate with the presence of GCH or COPD. We hypothesized that the primary determinant of GCH in ever smokers with or without airflow obstruction is active smoking. Goblet Cell Density (GCD) was measured in 71 current or former smokers [32 subjects without COPD and 39 COPD subjects]. Endobronchial mucosal biopsies were stained with Periodic Acid Schiff-Alcian Blue, and GCD was measured as number of goblet cells/mm basement membrane. GCD was divided into tertiles based on log10 transformed values. Log10GCD was greater in current smokers compared to former smokers. Those with classically defined CB or SGRQ defined CB had a greater log10 GCD compared to those without CB. Current smoking was independently associated with tertile 3 (high log10GCD) whereas CB was not in multivariable regression when adjusting for lung function and demographics. These results suggest that GCH is induced by active smoke exposure and does not necessarily correlate with the clinical symptoms of CB.


Assuntos
Bronquite Crônica/patologia , Células Caliciformes/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Fumar/patologia , Idoso , Feminino , Humanos , Hiperplasia/patologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fumantes
16.
Sci Rep ; 10(1): 10562, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601308

RESUMO

Levels of iron and iron-related proteins including ferritin are higher in the lung tissue and lavage fluid of individuals with chronic obstructive pulmonary disease (COPD), when compared to healthy controls. Whether more iron in the extracellular milieu of the lung associates with distinct clinical phenotypes of COPD, including increased exacerbation susceptibility, is unknown. We measured iron and ferritin levels in the bronchoalveolar lavage fluid (BALF) of participants enrolled in the SubPopulations and InteRmediate Outcome Measures In COPD (SPIROMICS) bronchoscopy sub-study (n = 195). BALF Iron parameters were compared to systemic markers of iron availability and tested for association with FEV1 % predicted and exacerbation frequency. Exacerbations were modelled using a zero-inflated negative binomial model using age, sex, smoking, and FEV1 % predicted as clinical covariates. BALF iron and ferritin were higher in participants with COPD and in smokers without COPD when compared to non-smoker control participants but did not correlate with systemic iron markers. BALF ferritin and iron were elevated in participants who had COPD exacerbations, with a 2-fold increase in BALF ferritin and iron conveying a 24% and 2-fold increase in exacerbation risk, respectively. Similar associations were not observed with plasma ferritin. Increased airway iron levels may be representative of a distinct pathobiological phenomenon that results in more frequent COPD exacerbation events, contributing to disease progression in these individuals.


Assuntos
Proteínas de Ligação ao Ferro/metabolismo , Ferro/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Líquido da Lavagem Broncoalveolar/química , Progressão da Doença , Feminino , Ferritinas/metabolismo , Volume Expiratório Forçado , Humanos , Ferro/fisiologia , Proteínas de Ligação ao Ferro/fisiologia , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/metabolismo , Testes de Função Respiratória , Fatores de Risco , Índice de Gravidade de Doença
17.
Am J Respir Cell Mol Biol ; 63(4): 464-477, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32543909

RESUMO

By enhancing tissue repair and modulating immune responses, Foxp3+ regulatory T cells (Tregs) play essential roles in resolution from lung injury. The current study investigated the effects that Tregs exert directly or indirectly on the transcriptional profiles of type 2 alveolar epithelial (AT2) cells during resolution in an experimental model of acute lung injury. Purified AT2 cells were isolated from uninjured mice or mice recovering from LPS-induced lung injury, either in the presence of Tregs or in Treg-depleted mice, and transcriptome profiling identified differentially expressed genes. Depletion of Tregs resulted in altered expression of 49 genes within AT2 cells during resolution, suggesting that Tregs present in this microenvironment influence AT2-cell function. Biological processes from Gene Ontology enriched in the absence of Tregs included those describing responses to IFN. Neutralizing IFN-γ in Treg-depleted mice reversed the effect of Treg depletion on inflammatory macrophages and B cells by preventing the increase in inflammatory macrophages and the decrease in B cells. Our results provide insight into the effects of Tregs on AT2 cells. Tregs directly or indirectly impact many AT2-cell functions, including IFN type I and II-mediated signaling pathways. Inhibition of IFN-γ expression and/or function may be one mechanism through which Tregs accelerate resolution after acute lung injury.


Assuntos
Lesão Pulmonar Aguda/imunologia , Células Epiteliais Alveolares/imunologia , Interferon gama/imunologia , Pulmão/imunologia , Linfócitos T Reguladores/imunologia , Transcriptoma/imunologia , Animais , Linfócitos B/imunologia , Feminino , Fatores de Transcrição Forkhead/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia
18.
Cell ; 182(2): 429-446.e14, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32526206

RESUMO

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Sistema Respiratório/virologia , Genética Reversa/métodos , Idoso , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , COVID-19 , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Fibrose Cística/patologia , DNA Recombinante , Feminino , Furina/metabolismo , Humanos , Imunização Passiva , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Mucosa Nasal/virologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Sistema Respiratório/patologia , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Células Vero , Virulência , Replicação Viral
19.
Chest ; 158(6): 2333-2345, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32450244

RESUMO

BACKGROUND: Little is known about the concordance of atopy with asthma COPD overlap. Among individuals with COPD, a better understanding of the phenotypes characterized by asthma overlap and atopy is needed to better target therapies. RESEARCH QUESTION: What is the overlap between atopy and asthma status among individuals with COPD, and how are categories defined by the presence of atopy and asthma status associated with clinical and radiologic phenotypes and outcomes in the Genetic Epidemiology of COPD Study (COPDGene) and Subpopulation and Intermediate Outcome Measures in COPD Study (SPIROMICS)? STUDY DESIGN AND METHODS: Four hundred three individuals with COPD from SPIROMICS and 696 individuals from COPDGene with data about specific IgEs to 10 common allergens and mixes (simultaneous assessment of combination of allergens in similar category) were included. Comparison groups were defined by atopic and asthma status (neither, atopy alone, atopic asthma, nonatopic asthma, with atopy defined as any positive specific IgE (≥0.35 KU/L) to any of the 10 allergens or mixes and asthma defined as self-report of doctor-diagnosed current asthma). Multivariable regression analyses (linear, logistic, and zero inflated negative binomial where appropriate) adjusted for age, sex, race, lung function, smoking status, pack-years smoked, and use of inhaled corticosteroids were used to determine characteristics of groups and relationship with outcomes (exacerbations, clinical outcomes, CT metrics) separately in COPDGene and SPIROMICS, and then adjusted results were combined using meta-analysis. RESULTS: The prevalence of atopy was 35% and 36% in COPD subjects from SPIROMICS and COPDGene, respectively, and less than 50% overlap was seen between atopic status with asthma in both cohorts. In meta-analysis, individuals with nonatopic asthma had the most impaired symptom scores (effect size for St. George's Respiratory Questionnaire total score, 4.2; 95% CI, 0.4-7.9; effect size for COPD Assessment Test score, 2.8; 95% CI, 0.089-5.4), highest risk for exacerbations (incidence rate ratio, 1.41; 95% CI, 1.05-1.88) compared with the group without atopy or asthma. Those with atopy and atopic asthma were not at increased risk for adverse outcomes. INTERPRETATION: Asthma and atopy had incomplete overlap among former and current smokers with COPD in COPDGene and SPIROMICS. Nonatopic asthma was associated with adverse outcomes and exacerbation risk in COPD, whereas groups having atopy alone and atopic asthma had less risk.


Assuntos
Síndrome de Sobreposição da Doença Pulmonar Obstrutiva Crônica e Asma , Hipersensibilidade Imediata , Imunoglobulina E , Doença Pulmonar Obstrutiva Crônica , Estado Asmático , Síndrome de Sobreposição da Doença Pulmonar Obstrutiva Crônica e Asma/epidemiologia , Síndrome de Sobreposição da Doença Pulmonar Obstrutiva Crônica e Asma/imunologia , Síndrome de Sobreposição da Doença Pulmonar Obstrutiva Crônica e Asma/fisiopatologia , Síndrome de Sobreposição da Doença Pulmonar Obstrutiva Crônica e Asma/terapia , Variação Biológica da População , Gerenciamento Clínico , Feminino , Humanos , Hipersensibilidade Imediata/epidemiologia , Hipersensibilidade Imediata/fisiopatologia , Imunoglobulina E/análise , Imunoglobulina E/classificação , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Prevalência , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/terapia , Fatores de Risco , Fumar/epidemiologia , Estado Asmático/epidemiologia , Estado Asmático/imunologia
20.
Proteomics ; 20(12): e1900278, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32386347

RESUMO

Novel proteomics platforms, such as the aptamer-based SOMAscan platform, can quantify large numbers of proteins efficiently and cost-effectively and are rapidly growing in popularity. However, comparisons to conventional immunoassays remain underexplored, leaving investigators unsure when cross-assay comparisons are appropriate. The correlation of results from immunoassays with relative protein quantification is explored by SOMAscan. For 63 proteins assessed in two chronic obstructive pulmonary disease (COPD) cohorts, subpopulations and intermediate outcome measures in COPD Study (SPIROMICS), and COPDGene, using myriad rules based medicine multiplex immunoassays and SOMAscan, Spearman correlation coefficients range from -0.13 to 0.97, with a median correlation coefficient of ≈0.5 and consistent results across cohorts. A similar range is observed for immunoassays in the population-based Multi-Ethnic Study of Atherosclerosis and for other assays in COPDGene and SPIROMICS. Comparisons of relative quantification from the antibody-based Olink platform and SOMAscan in a small cohort of myocardial infarction patients also show a wide correlation range. Finally, cis pQTL data, mass spectrometry aptamer confirmation, and other publicly available data are integrated to assess relationships with observed correlations. Correlation between proteomics assays shows a wide range and should be carefully considered when comparing and meta-analyzing proteomics data across assays and studies.


Assuntos
Infarto do Miocárdio/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumantes/estatística & dados numéricos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Imunoensaio/métodos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Doença Pulmonar Obstrutiva Crônica/sangue
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