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Rationale: IL-33 is a proinflammatory cytokine thought to play a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). A recent clinical trial using an anti-IL-33 antibody showed a reduction in exacerbation and improved lung function in ex-smokers but not current smokers with COPD. Objectives: This study aimed to understand the effects of smoking status on IL-33. Methods: We investigated the association of smoking status with the level of gene expression of IL-33 in the airways in eight independent transcriptomic studies of lung airways. Additionally, we performed Western blot analysis and immunohistochemistry for IL-33 in lung tissue to assess protein levels. Measurements and Main Results: Across the bulk RNA-sequencing datasets, IL-33 gene expression and its signaling pathway were significantly lower in current versus former or never-smokers and increased upon smoking cessation (P < 0.05). Single-cell sequencing showed that IL-33 is predominantly expressed in resting basal epithelial cells and decreases during the differentiation process triggered by smoke exposure. We also found a higher transitioning of this cellular subpopulation into a more differentiated cell type during chronic smoking, potentially driving the reduction of IL-33. Protein analysis demonstrated lower IL-33 levels in lung tissue from current versus former smokers with COPD and a lower proportion of IL-33-positive basal cells in current versus ex-smoking controls. Conclusions: We provide strong evidence that cigarette smoke leads to an overall reduction in IL-33 expression in transcriptomic and protein level, and this may be due to the decrease in resting basal cells. Together, these findings may explain the clinical observation that a recent antibody-based anti-IL-33 treatment is more effective in former than current smokers with COPD.
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Doença Pulmonar Obstrutiva Crônica , Fumantes , Humanos , Interleucina-33/genética , Fumar/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Perfilação da Expressão GênicaRESUMO
There are no methods sensitive enough to detect enzymes within cells, without the use of analyte labeling. Here we show that it is possible to detect protein ion signals of three different H2S-synthesizing enzymes inside microglia after pretreatment with silver nanowires (AgNW) using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Protein fragment ions, including the fragment of amino acid (C4H8N+ = 70 amu), fragments of the sulfur-producing cystathionine-containing enzymes, and the Ag+ ion signal could be detected without the use of any labels; the cells were mapped using the C4H8N+ amino acid fragment. Scanning electron microscopy imaging and energy-dispersive X-ray chemical analysis showed that the AgNWs were inside the same cells imaged by TOF-SIMS and transformed chemically into crystalline Ag2S within cells in which the sulfur-producing proteins were detected. The presence of these sulfur-producing cystathionine-containing enzymes within the cells was confirmed by Western blots and confocal microscopy images of fluorescently labeled antibodies against the sulfur-producing enzymes. Label-free TOF-SIMS is very promising for the label-free identification of H2S-contributing enzymes and their cellular localization in biological systems. The technique could in the future be used to identify which of these enzymes are most contributory.
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Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Microglia/enzimologia , Prata/farmacologia , Enxofre/química , Sulfurtransferases/metabolismo , Animais , Transporte Biológico , Linhagem Celular Transformada , Camundongos , Microglia/efeitos dos fármacos , Microglia/ultraestrutura , Microscopia Eletrônica de Varredura , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Nanofios/química , Prata/química , Espectrometria de Massa de Íon Secundário , Enxofre/metabolismoRESUMO
BACKGROUND: Severe asthma is a heterogeneous condition, as shown by independent cluster analyses based on demographic, clinical, and inflammatory characteristics. A next step is to identify molecularly driven phenotypes using "omics" technologies. Molecular fingerprints of exhaled breath are associated with inflammation and can qualify as noninvasive assessment of severe asthma phenotypes. OBJECTIVES: We aimed (1) to identify severe asthma phenotypes using exhaled metabolomic fingerprints obtained from a composite of electronic noses (eNoses) and (2) to assess the stability of eNose-derived phenotypes in relation to within-patient clinical and inflammatory changes. METHODS: In this longitudinal multicenter study exhaled breath samples were taken from an unselected subset of adults with severe asthma from the U-BIOPRED cohort. Exhaled metabolites were analyzed centrally by using an assembly of eNoses. Unsupervised Ward clustering enhanced by similarity profile analysis together with K-means clustering was performed. For internal validation, partitioning around medoids and topological data analysis were applied. Samples at 12 to 18 months of prospective follow-up were used to assess longitudinal within-patient stability. RESULTS: Data were available for 78 subjects (age, 55 years [interquartile range, 45-64 years]; 41% male). Three eNose-driven clusters (n = 26/33/19) were revealed, showing differences in circulating eosinophil (P = .045) and neutrophil (P = .017) percentages and ratios of patients using oral corticosteroids (P = .035). Longitudinal within-patient cluster stability was associated with changes in sputum eosinophil percentages (P = .045). CONCLUSIONS: We have identified and followed up exhaled molecular phenotypes of severe asthma, which were associated with changing inflammatory profile and oral steroid use. This suggests that breath analysis can contribute to the management of severe asthma.
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Asma/diagnóstico , Nariz Eletrônico , Eosinófilos/patologia , Inflamação/diagnóstico , Neutrófilos/patologia , Adulto , Testes Respiratórios , Análise por Conglomerados , Estudos de Coortes , Progressão da Doença , Expiração , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Índice de Gravidade de DoençaRESUMO
Oxidative stress is believed to be a major driver of inflammation in smoking asthmatics. The U-BIOPRED project recruited a cohort of Severe Asthma smokers/ex-smokers (SAs/ex) and non-smokers (SAn) with extensive clinical and biomarker information enabling characterization of these subjects. We investigated oxidative stress in severe asthma subjects by analysing urinary 8-iso-PGF2α and the mRNA-expression of the main pro-oxidant (NOX2; NOSs) and anti-oxidant (SODs; CAT; GPX1) enzymes in the airways of SAs/ex and SAn. All the severe asthma U-BIOPRED subjects were further divided into current smokers with severe asthma (CSA), ex-smokers with severe asthma (ESA) and non-smokers with severe asthma (NSA) to deepen the effect of active smoking. Clinical data, urine and sputum were obtained from severe asthma subjects. A bronchoscopy to obtain bronchial biopsy and brushing was performed in a subset of subjects. The main clinical data were analysed for each subset of subjects (urine-8-iso-PGF2α; IS-transcriptomics; BB-transcriptomics; BBr-transcriptomics). Urinary 8-iso-PGF2α was quantified using mass spectrometry. Sputum, bronchial biopsy and bronchial brushing were processed for mRNA expression microarray analysis. Urinary 8-iso-PGF2α was increased in SAs/ex, median (IQR) = 31.7 (24.5-44.7) ng/mmol creatinine, compared to SAn, median (IQR) = 26.6 (19.6-36.6) ng/mmol creatinine (p< 0.001), and in CSA, median (IQR) = 34.25 (24.4-47.7), vs. ESA, median (IQR) = 29.4 (22.3-40.5), and NSA, median (IQR) = 26.5 (19.6-16.6) ng/mmol creatinine (p = 0.004). Sputum mRNA expression of NOX2 was increased in SAs/ex compared to SAn (probe sets 203922_PM_s_at fold-change = 1.05 p = 0.006; 203923_PM_s_at fold-change = 1.06, p = 0.003; 233538_PM_s_at fold-change = 1.06, p = 0.014). The mRNA expression of antioxidant enzymes were similar between the two severe asthma cohorts in all airway samples. NOS2 mRNA expression was decreased in bronchial brushing of SAs/ex compared to SAn (fold-change = -1.10; p = 0.029). NOS2 mRNA expression in bronchial brushing correlated with FeNO (Kendal's Tau = 0.535; p< 0.001). From clinical and inflammatory analysis, FeNO was lower in CSA than in ESA in all the analysed subject subsets (p< 0.01) indicating an effect of active smoking. Results about FeNO suggest its clinical limitation, as inflammation biomarker, in severe asthma active smokers. These data provide evidence of greater systemic oxidative stress in severe asthma smokers as reflected by a significant changes of NOX2 mRNA expression in the airways, together with elevated urinary 8-iso-PGF2α in the smokers/ex-smokers group. Trial registration ClinicalTrials.gov-Identifier: NCT01976767.
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Asma/metabolismo , Estresse Oxidativo/fisiologia , Fumar Tabaco/efeitos adversos , Adulto , Asma/patologia , Biomarcadores/metabolismo , Broncoscopia , Estudos de Coortes , Feminino , Humanos , Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Fumar/metabolismo , Escarro/metabolismo , Fumar Tabaco/metabolismoRESUMO
Here we examine the organ level toxicology of both carbon black (CB) and silver nanoparticles (AgNP). We aim to determine metal-specific effects to respiratory function, inflammation and potential interactions with lung lining fluid (LLF). C57Bl6/J male mice were intratracheally instilled with saline (control), low (0.05 µg/g) or high (0.5 µg/g) doses of either AgNP or CB 15 nm nanospheres. Lung histology, cytology, surfactant composition and function, inflammatory gene expression, and pulmonary function were measured at 1, 3, and 7 days post-exposure. Acutely, high dose CB resulted in an inflammatory response, increased neutrophilia and cytokine production, without alteration in surfactant composition or respiratory mechanics. Low dose CB had no effect. Neither low nor high dose AgNPs resulted in an acute inflammatory response, but there was an increase in work of breathing. Three days post-exposure with CB, a persistent neutrophilia was noted. High dose AgNP resulted in an elevated number of macrophages and invasion of lymphocytes. Additionally, AgNP treated mice displayed increased expression of IL1B, IL6, CCL2, and IL10. However, there were no significant changes in respiratory mechanics. At day 7, inflammation had resolved in AgNP-treated mice, but tissue stiffness and resistance were significantly decreased, which was accompanied by an increase in surfactant protein D (SP-D) content. These data demonstrate that the presence of metal alters the response of the lung to nanoparticle exposure. AgNP-surfactant interactions may alter respiratory function and result in a delayed immune response, potentially due to modified airway epithelial cell function.
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PURPOSE OF REVIEW: Asthma is a heterogeneous disease consisting of different phenotypes that are driven by different mechanistic pathways. The purpose of this review is to emphasize the important role of precision medicine in asthma management. RECENT FINDINGS: Despite asthma heterogeneity, the approach to management has been on the basis of disease severity, with the most severe patients reserved for the maximum treatments with corticosteroids and bronchodilators. At the severe end, the recent availability of biologic therapies in the form of anti-IgE (omalizumab) and anti-IL5 therapies (mepolizumab and reslizumab) has driven the adaptation of precision medicine. These therapies are reserved for severe asthma with defined either allergic or eosinophilic background, respectively. SUMMARY: Unbiased definition of phenotypes or endotypes (which are phenotypes defined by mechanisms) is an important step towards the use of precision medicine in asthma. Although T2-high asthma has been defined with targets becoming available for treating allergic or eosinophilic asthma, the definition of non-T2 phenotypes remains a priority. Precision medicine is also dependent on the definition of biomarkers that can help differentiate between these phenotypes and pinpoint patients suitable for specific-targeted therapies. Thus, precision medicine links phenotypes (endotypes) to targeted treatments for better outcomes.
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Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Asma/fisiopatologia , Terapia de Alvo Molecular , Fenótipo , Medicina de Precisão , Anticorpos Anti-Idiotípicos/uso terapêutico , Asma/genética , Biomarcadores/análise , Humanos , Índice de Gravidade de DoençaRESUMO
RATIONALE: Stratification of asthma at the molecular level, especially using accessible biospecimens, could greatly enable patient selection for targeted therapy. OBJECTIVES: To determine the value of blood analysis to identify transcriptional differences between clinically defined asthma and nonasthma groups, identify potential patient subgroups based on gene expression, and explore biological pathways associated with identified differences. METHODS: Transcriptomic profiles were generated by microarray analysis of blood from 610 patients with asthma and control participants in the U-BIOPRED (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes) study. Differentially expressed genes (DEGs) were identified by analysis of variance, including covariates for RNA quality, sex, and clinical site, and Ingenuity Pathway Analysis was applied. Patient subgroups based on DEGs were created by hierarchical clustering and topological data analysis. MEASUREMENTS AND MAIN RESULTS: A total of 1,693 genes were differentially expressed between patients with severe asthma and participants without asthma. The differences from participants without asthma in the nonsmoking severe asthma and mild/moderate asthma subgroups were significantly related (r = 0.76), with a larger effect size in the severe asthma group. The majority of, but not all, differences were explained by differences in circulating immune cell populations. Pathway analysis showed an increase in chemotaxis, migration, and myeloid cell trafficking in patients with severe asthma, decreased B-lymphocyte development and hematopoietic progenitor cells, and lymphoid organ hypoplasia. Cluster analysis of DEGs led to the creation of subgroups among the patients with severe asthma who differed in molecular responses to oral corticosteroids. CONCLUSIONS: Blood gene expression differences between clinically defined subgroups of patients with asthma and individuals without asthma, as well as subgroups of patients with severe asthma defined by transcript profiles, show the value of blood analysis in stratifying patients with asthma and identifying molecular pathways for further study. Clinical trial registered with www.clinicaltrials.gov (NCT01982162).
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Corticosteroides/uso terapêutico , Asma/sangue , Asma/tratamento farmacológico , Perfilação da Expressão Gênica/métodos , Corticosteroides/sangue , Adulto , Análise por Conglomerados , Estudos de Coortes , Europa (Continente) , Feminino , Humanos , Masculino , Análise em Microsséries/estatística & dados numéricos , Pessoa de Meia-Idade , Estudos Prospectivos , Índice de Gravidade de Doença , Transcriptoma/efeitos dos fármacosRESUMO
We investigated changes in gene expression that occur in chronic obstructive pulmonary disease (COPD) after corticosteroid treatment and sought to identify the mechanisms that regulate these changes. Biopsy samples were taken from patients with COPD (Global Initiative for Chronic Obstructive Lung Disease stage I to II) before and after treatment with fluticasone propionate (FP)/salmeterol (SM) (50/500, 4 wk). Gene expression was measured by microarray and was confirmed by real-time reverse transcription-quantitative PCR (RT-qPCR). The effect of FP on IgG expression and B-cell proliferation in the presence of oxidative stress was also studied. FP/SM significantly increased the expression of 180 genes while repressing 343 genes. The top 5 down-regulated genes were associated with immunoglobulin production, whereas the immunomodulatory FK506 binding protein (FK506BP) was up-regulated. Genes including IL6, IL8, and TBET-encoding TBX21 were unaffected. FP reduced IgG protein and mRNA expression and proliferation of human B cells through the dephosphorylation of ERK-1/2 via increased DUSP1 (dual-specificity protein phosphatase 1) expression. Consistent with in vivo data, oxidative stress did not prevent FP-induced suppression of IgG expression in human B cells in vitro Changes in expression were validated by RT-qPCR and by gene set enrichment analysis in distinct COPD cohorts. FP may reduce the adaptive immune response in COPD and may be more effective in patients with an increased B-cell/antibody response indicated by high autoantibody titers.-Lee, J., Machin, M., Russell, K. E., Pavlidis, S., Zhu, J., Barnes, P. J., Chung, K. F., Adcock, I. M., Durham, A. L. Corticosteroid modulation of immunoglobulin expression and B-cell function in COPD.
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Linfócitos B/fisiologia , Combinação Fluticasona-Salmeterol/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulinas/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Adulto , Idoso , Linfócitos B/efeitos dos fármacos , Broncodilatadores/administração & dosagem , Broncodilatadores/uso terapêutico , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Feminino , Combinação Fluticasona-Salmeterol/administração & dosagem , Glucocorticoides/administração & dosagem , Glucocorticoides/uso terapêutico , Humanos , Imunoglobulinas/genética , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Projetos Piloto , TranscriptomaRESUMO
Asthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases of the airway, although the drivers and site of the inflammation differ between diseases. Asthmatics with a neutrophilic airway inflammation are associated with a poor response to corticosteroids, whereas asthmatics with eosinophilic inflammation respond better to corticosteroids. Biologicals targeting the Th2-eosinophil nexus such as anti-interleukin (IL)-4, anti-IL-5, and anti-IL-13 are ineffective in asthma as a whole but are more effective if patients are selected using cellular (eg, eosinophils) or molecular (eg, periostin) biomarkers. This highlights the key role of individual inflammatory mediators in driving the inflammatory response and for accurate disease phenotyping to allow greater understanding of disease and development of patient-oriented antiasthma therapies. In contrast to asthmatic patients, corticosteroids are relatively ineffective in COPD patients. Despite stratification of COPD patients, the results of targeted therapy have proved disappointing with the exception of recent studies using CXC chemokine receptor (CXCR)2 antagonists. Currently, several other novel mediator-targeted drugs are undergoing clinical trials. As with asthma specifically targeted treatments may be of most benefit in specific COPD patient endotypes. The use of novel inflammatory mediator-targeted therapeutic agents in selected patients with asthma or COPD and the detection of markers of responsiveness or nonresponsiveness will allow a link between clinical phenotypes and pathophysiological mechanisms to be delineated reaching the goal of endotyping patients.
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Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Inflamação/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , HumanosRESUMO
The UK Refractory Asthma Stratification Programme (RASP-UK) will explore novel biomarker stratification strategies in severe asthma to improve clinical management and accelerate development of new therapies. Prior asthma mechanistic studies have not stratified on inflammatory phenotype and the understanding of pathophysiological mechanisms in asthma without Type 2 cytokine inflammation is limited. RASP-UK will objectively assess adherence to corticosteroids (CS) and examine a novel composite biomarker strategy to optimise CS dose; this will also address what proportion of patients with severe asthma have persistent symptoms without eosinophilic airways inflammation after progressive CS withdrawal. There will be interactive partnership with the pharmaceutical industry to facilitate access to stratified populations for novel therapeutic studies.
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Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Asma/epidemiologia , Pesquisa Biomédica/métodos , Gerenciamento Clínico , Cooperação do Paciente , Medição de Risco , Humanos , Reino UnidoRESUMO
In chronic obstructive pulmonary disease (COPD), oxidative stress regulates the inflammatory response of bronchial epithelium and monocytes/macrophages through kinase modulation and has been linked to glucocorticoid unresponsiveness. Glycogen synthase-3ß (GSK3ß) inactivation plays a key role in mediating signaling processes upon reactive oxygen species (ROS) exposure. We hypothesized that GSK3ß is involved in oxidative stress-induced glucocorticoid insensitivity in COPD. We studied levels of phospho-GSK3ß-Ser9, a marker of GSK3ß inactivation, in lung sections and cultured monocytes and bronchial epithelial cells of COPD patients, control smokers, and nonsmokers. We observed increased levels of phospho-GSK3ß-Ser9 in monocytes, alveolar macrophages, and bronchial epithelial cells from COPD patients and control smokers compared with nonsmokers. Pharmacological inactivation of GSK3ß did not affect CXCL8 or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression but resulted in glucocorticoid insensitivity in vitro in both inflammatory and structural cells. Further mechanistic studies in monocyte and bronchial epithelial cell lines showed that GSK3ß inactivation is a common effector of oxidative stress-induced activation of the MEK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways leading to glucocorticoid unresponsiveness. In primary monocytes, the mechanism involved modulation of histone deacetylase 2 (HDAC2) activity in response to GSK3ß inactivation. In conclusion, we demonstrate for the first time that ROS-induced glucocorticoid unresponsiveness in COPD is mediated through GSK3ß, acting as a ROS-sensitive hub.
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Dexametasona/farmacologia , Glucocorticoides/farmacologia , Quinase 3 da Glicogênio Sintase/fisiologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Idoso , Células Cultivadas , Dexametasona/uso terapêutico , Feminino , Expressão Gênica/efeitos dos fármacos , Glucocorticoides/uso terapêutico , Glicogênio Sintase Quinase 3 beta , Histona Desacetilase 2/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Macrófagos Alveolares/enzimologia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/enzimologia , Transdução de SinaisRESUMO
Oxidative stress enhances inflammation and reduces the effectiveness of corticosteroids, but the inflammatory signalling pathways induced by oxidants remain ill-defined. Phosphorylation of histone 3 at serine 10 (H3-Pser10) marks out a subset of inflammatory genes for transcription, several of which are induced in oxidant-associated inflammation. However, the influence of oxidants or of corticosteroids on this modification remains unknown. We assessed the regulation of H3-Pser10 by oxidants and lipopolysaccharide (LPS) in human blood monocytes and lung macrophages and the effectiveness of its abolition in controlling inflammatory gene expression in cells from asthmatic subjects compared to corticosteroids alone. Both oxidants and LPS promoted the induction of H3-Pser10 which was unaffected by corticosteroids. The induction of H3-Pser10 was mediated through p38α mitogen-activated protein kinase (MAPK) and IκB kinase 2 (IKK-2) signalling. Consequently, inhibitors of p38α MAPK or IKK-2 used in combination with dexamethasone were more effective at controlling inflammatory gene expression from monocytes and lung macrophages from asthmatic patients than the corticosteroid alone. Therefore, reduction of H3-Pser10 by inhibition of p38α MAPK or of IKK-2 may provide greater anti-inflammatory control than corticosteroids alone in oxidant-associated inflammation such as severe asthma.
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Corticosteroides/farmacologia , Histonas/metabolismo , Monócitos/efeitos dos fármacos , Oxidantes/farmacologia , Serina/metabolismo , Adulto , Feminino , Histonas/química , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Hydrogen sulfide (H2S), a gas characterized by the odor of rotten eggs, is produced by many cells in the airways and lungs, and may regulate physiologic and pathophysiologic processes. It plays a role in cellular signaling, and represents the third gasotransmitter after nitric oxide and carbon monoxide. Endogenous and exogenous H2S have anti-inflammatory and anti-proliferative effects, with inhibitory effects in models of lung inflammation and fibrosis. Under certain conditions, H2S may also be proinflammatory. It is generally a vasodilator and relaxant of airway and vascular smooth muscle cells. It acts as a reducing agent, being able to scavenge superoxide and peroxynitrite. H2S is detectable in serum and in sputum supernatants with raised levels observed in asthmatics. The sputum levels correlated inversely with lung function. H2S may play a role in the pathogenesis of asthma.
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Asma/diagnóstico , Asma/metabolismo , Sulfeto de Hidrogênio/metabolismo , Asma/fisiopatologia , Biomarcadores/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Pulmão/metabolismo , Pulmão/fisiopatologia , Fenótipo , Escarro/metabolismoRESUMO
Ozone is an oxidizing environmental pollutant that contributes significantly to respiratory health. Exposure to increased levels of ozone has been associated with worsening of symptoms of patients with asthma and COPD (chronic obstructive pulmonary disease). In the present study, we investigated the acute and chronic effects of ozone exposure-induced oxidative stress-related inflammation mechanics in mouse lung. In particular, we investigated the oxidative stress-induced effects on HDAC2 (histone deacetylase 2) modification and activation of the Nrf2 (nuclear factor erythroid-related factor 2) and HIF-1α (hypoxia-inducible factor-1α) signalling pathways. Male C57BL/6 mice were exposed to ozone (3 p.p.m.) for 3 h a day, twice a week for a period of 1, 3 or 6 weeks. Control mice were exposed to normal air. After the last exposure, mice were killed for BAL (bronchoalveolar lavage) fluid and lung tissue collection. BAL total cell counts were elevated at all of the time points studied. This was associated with increased levels of chemokines and cytokines in all ozone-exposed groups, indicating the presence of a persistent inflammatory environment in the lung. Increased inflammation and Lm (mean linear intercept) scores were observed in chronic exposed mice, indicating emphysematous changes were present in lungs of chronic exposed mice. The antioxidative stress response was active (indicated by increased Nrf2 activity and protein) after 1 week of ozone exposure, but this ability was lost after 3 and 6 weeks of ozone exposure. The transcription factor HIF-1α was elevated in 3- and 6-week ozone-exposed mice and this was associated with increased gene expression levels of several HIF-1α target genes including Hdac2 (histone deacetylase 2), Vegf (vascular endothelial growth factor), Keap1 (kelch-like ECH-associated protein 1) and Mif (macrophage migration inhibitory factor). HDAC2 protein was found to be phosphorylated and carbonylated in nuclear and cytoplasm fractions, respectively, and was associated with a decrease in DNA-binding activity and protein expression of HDAC2. Decreased HDAC2 activity, most likely a direct result of protein modification, in combination with the loss of the antioxidative stress response and activation of the HIF-1α pathway, contribute to the inflammatory response and emphysema observed in ozone-exposed mice.
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Poluentes Atmosféricos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ozônio/farmacologia , Pneumonia/induzido quimicamente , Idoso , Animais , Antioxidantes/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Células Cultivadas , Citocinas/biossíntese , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 2/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/metabolismo , Oxidantes Fotoquímicos/administração & dosagem , Oxidantes Fotoquímicos/farmacologia , Ozônio/administração & dosagem , Fosforilação/efeitos dos fármacos , Pneumonia/genética , Pneumonia/patologia , Pneumonia/fisiopatologia , Enfisema Pulmonar/induzido quimicamente , RNA Mensageiro/genética , Superóxido Dismutase/metabolismoRESUMO
PURPOSE OF REVIEW: Severe asthma remains a condition in search of deeper understanding and of newer effective therapies. Risk factors for developing severe asthma, phenotyping of disease, characterizing the inflammatory response and understanding of poor therapeutic responses to corticosteroids are important areas of research. The development of biomarkers for phenotypic diagnosis and prognostic reasons is important. RECENT FINDINGS: Severe asthma has been defined as asthma that does not respond adequately to asthma medications, particularly corticosteroids, with continuing loss of control of asthma and future risk of exacerbations and side effects from corticosteroid therapy. This is a heterogeneous condition and cluster analyses have yielded phenotypes on the basis of age of onset of disease, sex, lung function, atopy and questionnaire data. Use of sputum eosinophil counts has further defined a group with persistent eosinophilic inflammation despite corticosteroid therapy, and a noneosinophilic group with uncontrolled asthma. SUMMARY: Use of a single biomarker provides value in phenotyping and in predicting response to treatment but many more biomarkers such as those derived from -omic approaches remain to be used in the analysis. A systems biology approach to finding novel biomarkers is the way forward to stratify severe asthma so that appropriate and distinct therapies can be selected for each subtype.
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Asma/metabolismo , Eosinofilia/metabolismo , Óxido Nítrico/metabolismo , Escarro/metabolismo , Asma/diagnóstico , Asma/imunologia , Biomarcadores/metabolismo , Análise por Conglomerados , Progressão da Doença , Eosinofilia/diagnóstico , Eosinofilia/imunologia , Humanos , Fenótipo , Prognóstico , Fatores de Risco , Índice de Gravidade de Doença , Inquéritos e QuestionáriosRESUMO
RATIONALE: There is increasing evidence for the presence of autoantibodies in chronic obstructive pulmonary disease (COPD). Chronic oxidative stress is an essential component in COPD pathogenesis and can lead to increased levels of highly reactive carbonyls in the lung, which could result in the formation of highly immunogenic carbonyl adducts on "self" proteins. OBJECTIVES: To determine the presence of autoantibodies to carbonyl-modified protein in patients with COPD and in a murine model of chronic ozone exposure. To assess the extent of activated immune responses toward carbonyl-modified proteins. METHODS: Blood and peripheral lung were taken from patients with COPD, age-matched smokers, and nonsmokers with normal lung function, as well as patients with severe persistent asthma. Mice were exposed to ambient air or ozone for 6 weeks. Antibody titers were measured by ELISA, activated compliment deposition by immunohistochemistry, and cellular activation by ELISA and fluorescence-activated cell sorter. MEASUREMENTS AND MAIN RESULTS: Antibody titer against carbonyl-modified self-protein was significantly increased in patients with Global Initiative for Chronic Obstructive Lung Disease stage III COPD compared with control subjects. Antibody levels inversely correlated with disease severity and showed a prevalence toward an IgG1 isotype. Deposition of activated complement in the vessels of COPD lung as well as autoantibodies against endothelial cells were also observed. Ozone-exposed mice similarly exhibited increased antibody titers to carbonyl-modified protein, as well as activated antigen-presenting cells in lung tissue and splenocytes sensitized to activation by carbonyl-modified protein. CONCLUSIONS: Carbonyl-modified proteins, arising as a result of oxidative stress, promote antibody production, providing a link by which oxidative stress could drive an autoimmune response in COPD.
Assuntos
Autoanticorpos/metabolismo , Estresse Oxidativo/imunologia , Carbonilação Proteica/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Idoso , Animais , Asma/imunologia , Autoanticorpos/sangue , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Análise por Pareamento , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Ozônio , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/patologia , Índice de Gravidade de Doença , Fumar/efeitos adversosRESUMO
BACKGROUND AND OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN or CD147) induces the production of matrix metalloproteinases (MMP) such as MMP-9, which plays an important role in COPD. We determined its cellular origin and role in MMP-9 production in COPD. METHODS: Bronchial biopsies, alveolar macrophages (AM) and blood monocytes (BM) from patients with COPD, healthy smokers and non-smokers, and bronchial epithelial cells (EC) from surgically resected airways from patients with COPD were stimulated with LPS or CRP in the presence and absence of an anti-EMMPRIN blocking antibody. EMMPRIN in BAL, plasma, conditioned media and cell lysates was quantified and immunohistochemical localization of EMMPRIN was determined in bronchial biopsies. MMP-9 activity and mRNA was also determined. RESULTS: EMMPRIN levels in BAL fluid were higher in patients with COPD compared with non-smokers and smokers. There was greater EMMPRIN expression in EC from patients with COPD compared with smokers and non-smokers. EC secreted and expressed more EMMPRIN protein than BM and AM. Blocking EMMPRIN decreased MMP-9 activity in supernatant of EC, but not in those from AM and BM, and decreased MMP-9 mRNA expression in EC. CONCLUSIONS: The increased EMMPRIN expression in COPD is reflected by an increased release from bronchial EC, which are one of the main source of EMMPRIN. EMMPRIN regulates MMP-9 expression in COPD.
Assuntos
Basigina/metabolismo , Brônquios/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Doença Pulmonar Obstrutiva Crônica/metabolismo , Adolescente , Adulto , Anticorpos Bloqueadores/farmacologia , Biópsia , Proteína C-Reativa/farmacologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/metabolismo , Masculino , Doença Pulmonar Obstrutiva Crônica/cirurgia , Fumar/metabolismo , Adulto JovemRESUMO
Indacaterol is a novel once-daily long-acting ß2-agonist (LABA) for the treatment of chronic obstructive pulmonary disease (COPD) that is currently completing Phase II and Phase III trials. It represents the first of a group of drugs now referred to as the 'ultra-LABAs'. It has recently gained approval in Europe, and is pending regulatory review in the USA. Indacaterol produces a rapid and sustained bronchodilation that lasts for at least 24 h in patients with COPD. To date, 1-year studies with indacaterol indicate that it can be taken once daily with good overall safety and tolerability profiles. The therapeutic potential for indacaterol is supported by data on patient-reported outcomes with an improvement in symptoms such as dyspnea, exercise capacity and quality of life, and a reduction in exacerbations. It is likely that indacaterol could be used in conjunction with other agents such as inhaled corticosteroids and anticholinergics for the treatment of COPD as a single once-daily combination treatment.