RESUMO
BACKGROUND: The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported. METHODS: To investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90ß respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and ß-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting. RESULTS: The epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and ß-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90ß that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells. CONCLUSIONS: The eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.
Assuntos
Antiasmáticos/farmacologia , Brônquios/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/farmacologia , Cadeias Leves de Miosina/metabolismo , Pyroglyphidae/imunologia , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Antígenos CD , Brônquios/enzimologia , Brônquios/imunologia , Caderinas/metabolismo , Linhagem Celular , Dextranos/metabolismo , Impedância Elétrica , Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Permeabilidade , Fosforilação , Interferência de RNA , Fatores de Tempo , Transfecção , beta Catenina/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Previous studies have shown that matrix metalloproteinase-9 (MMP-9) and its cognate inhibitor TIMP-1, inflammatory cytokine TNF-α, and the OPG/RANK/RANKL system may each play individual roles in the pathogenesis of osteoporosis in patients with COPD. In the present study, we investigated the interrelationships of these factors in male COPD patients with and without osteoporosis. The serum levels of MMP-9, MMP-9/TIMP-1 ratio, TNF-α, RANKL, OPG, and the RANKL/OPG ratio were higher in COPD patients with osteoporosis than in individuals with normal or low bone mineral density (BMD) (N = 30, all P < 0.05 or < 0.01). The lung function FEV1%Pre and the BMD of the lumbar spine and femoral neck were found to be negatively correlated with MMP-9 serum level (r = -0.36, P < 0.05, r = -0.58, P < 0.001, and r = -0.62, P < 0.01, respectively), RANKL serum level (r = -0.21, P < 0.05, and r = -0.25, P < 0.05, and r = -0.26, P < 0.05, respectively), and RANKL/OPG ratio (r = -0.23, P < 0.05, r = -0.33, P < 0.05, and r = -0.38, P < 0.05, respectively). However, they had no correlation with TIMP-1, TNF-α, OPG, or RANK. The MMP-9 serum level was found to be positively correlated with TNF-α level (r = 0.35, P < 0.05) and RANKL/OPG ratio (r = 0.27, P < 0.05) but not associated with RANKL. These results suggest that MMP-9, TNF-α, and the OPG/RANK/RANKL system may be closely interrelated and may play interactive roles in pathogenesis of osteoporosis in COPD.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Osteoporose/metabolismo , Osteoprotegerina/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Idoso , Índice de Massa Corporal , Densidade Óssea , Estudos de Casos e Controles , Humanos , Masculino , Pessoa de Meia-Idade , Osteoporose/complicações , Doença Pulmonar Obstrutiva Crônica/complicações , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
OBJECTIVE: To explore the risk factors for hospitalization case fatality of patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). METHODS: A retrospective review of medical records was performed for 182 hospitalized AECOPD patients at Nanfang Hospital from January 2010 to August 2012. Their general information, condition in stable stage, the results of spirometry, blood routine test, blood gas analysis and C-reactive protein (CRP) were collected and analyzed. The risk factors for mortality were analyzed by multivariable Logistic regression. RESULTS: Among them, 42 died during hospitalization. Univariate analysis revealed that 8 factors had significant differences between two groups (all P < 0.05): high exacerbation risk (death vs improvement group, 90.4% vs 70.0%) , low peripheral absolute lymphocyte count (73.8% vs 47.1%), high CRP (50.0% vs 17.1%), concurrent anemia (50.0% vs 27.1%), hypoproteinemia (71.4% vs 46.4%), hypercapnia (64.3% vs 30.7%), chronic pulmonary heart disease (76.1% vs 40.7%) and ischemic heart disease (19.0% vs 7.0%). By multiple Logistic regression analysis, high CRP (OR = 3.226, P = 0.009), hypercapnia (OR = 2.928, P = 0.013), chronic pulmonary heart disease (OR = 2.510, P = 0.045), low peripheral absolute lymphocyte count (OR = 2.488, P = 0.045) were the independent risk factors for hospitalization case fatality of AECOPD patients. CONCLUSION: Low peripheral absolute lymphocyte count, high CRP, hypercapnia and chronic pulmonary heart disease were the independent risk factors for mortality in hospitalized AECOPD patients.
Assuntos
Mortalidade Hospitalar , Doença Pulmonar Obstrutiva Crônica/mortalidade , Idoso , Feminino , Humanos , Modelos Logísticos , Masculino , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: To explore the post-therapeutic change of cathelicidin LL-37 in asthmatics of different inflammatory phenotypes. METHODS: Thirty-four patients with initially diagnosed asthma (asthma group) and 14 normal subjects (control group) were recruited at Nanfang Hospital from August 2009 to August 2010 for this prospective study. Sputum and venous blood samples were collected and analyzed for cell differential. Eosinophilic asthma was defined as the count of sputum eosinophils ≥ 3%. The LL-37 concentrations in plasma and sputum supernatant were measured by enzyme-linked immunosorbent assay (ELISA) kit. The subjects were treated with budesonide/formoterol (160/4.5 µg) one inhalation twice daily and re-examined after 1 month. RESULTS: Prior to treatment, there were no differences between the asthma and control groups in the levels of LL-37 in plasma and sputum supernatant (P = 0.427,0.427). The plasma concentrations of LL-37 in asthma group were negatively correlated with baseline forced expiratory volume in one second (FEV(1), r = -0.470, P = 0.005), percent predicted of FEV(1) (FEV(1)%pred, r = -0.421, P = 0.013) and forced vital capacity (FVC, r = -0.367, P = 0.033). After treatment, the plasma and sputum supernatant concentrations of LL-37 (M (Q(R))) in the asthma group (5.6 (16.2), 65.6 (184.0) µg/L) were significantly higher than those baseline concentrations (5.03 (9.21), 28.40(109.76) µg/L, P = 0.005, 0.015). In the eosinophilic asthma subgroup, the plasma and sputum supernatant concentrations of LL-37 (M (Q(R))) after treatment (5.3 (19.3), 65.6 (185.2) µg/L) were significantly higher than those baseline concentrations (6.7 (8.9) L, 35.3 (102.0) µg/L, P = 0.021,0.014). And in the non-eosinophilic asthma subgroup, the changes of plasma and sputum supernatant concentrations of LL-37 showed no significant differences (P = 0.139, 0.386). In the asthma group, the correlations between plasma concentrations of LL-37 and FEV(1), FEV(1)%pred, FVC were not statistically significant (P = 0.283, 0.706,0.272) after treatment. CONCLUSIONS: LL-37 may participate in the aggravation of asthma. The elevated concentrations of LL-37 in eosinophilic asthma is probably due to the resolved suppression of LL-37 expression by eosinophilic inflammation. But its mechanism needs further researches.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Asma/metabolismo , Asma/terapia , Adulto , Asma/patologia , Estudos de Casos e Controles , Feminino , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , CatelicidinasRESUMO
Respiratory syncytial virus (RSV) infection is the main cause of lower respiratory tract infection in children. However, there is no effective treatment for RSV infection. Here, we aimed to identify potential biomarkers to aid in the treatment of RSV infection. Children in the acute and convalescence phases of RSV infection were recruited and proteomic analysis was performed to identify differentially expressed proteins (DEPs). Subsequently, promising candidate proteins were determined by functional enrichment and protein-protein interaction network analysis, and underwent further validation by western blot both in clinical and mouse model samples. Among the 79 DEPs identified in RSV patient samples, 4 proteins (BPGM, TPI1, PRDX2, and CFL1) were confirmed to be significantly upregulated during RSV infection. Functional analysis showed that BPGM and TPI1 were mainly involved in glycolysis, indicating an association between RSV infection and the glycolysis metabolic pathway. Our findings provide insights into the proteomic profile during RSV infection and indicated that BPGM, TPI1, PRDX2, and CFL1 may be potential therapeutic biomarkers or targets for the treatment of RSV infection.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Biomarcadores , Criança , Humanos , ProteômicaRESUMO
OBJECTIVE: To investigate the role of epidermal growth factor receptor (EGFR) signaling pathway in bronchial epithelial actin stress fiber (F-actin) rearrangement induced by house dust mite (HDM). METHODS: Normal human bronchial epithelial cells (16HBE) were stimulated with HDM with or without pretreatment with AG-1478, an EGFR inhibitor. The levels of phospho(p)-EGFR, F-actin, E-cadherin and ß-catenin in the cell cultures were detected with Western blotting. The localizations of F-actin, E-cadherin and ß-catenin in the bronchial epithelial cells were determined with immunofluorescence assay, and the transmembrane electrical resistance (TER) and FITC-dextran flux (FITC-DX) in the cells were measured to assess the barrier function of the bronchial epithelia. RESULTS: HDM stimulation of the cells for 10 min resulted in significantly increased p-EGFR expression (P<0.05) without causing obvious changes in the expression of E-cadherin (P>0.05) or ß-catenin (P>0.05). Immunofluorescence assay revealed delocalization of E-cadherin and ß-catenin in HDM-treated 16HBE cells, shown by their diffusion from the cell membrane to the cytoplasm. In HDM-treated cells, the TER was significantly decreased to (70.00∓4.33)% and the FITC-DX was significantly increased to (115.98∓4.34)%; Inhibition of EGFR reversed the delocalization of E-cadherin and ß-catenin, improved the TER to (90.00∓3.75)% and lowered the FITC-DX to (101.10∓2.10)%. HDM induced increased expression and rearrangement of F-actin, which was obviously inhibited by pretreatment of the cells with AG-1478 (P<0.05). CONCLUSION: EGFR signaling pathway mediates HDM-induced F-actin rearrangement in human bronchial epithelial cells to contribute to epithelial barrier dysfunction.
Assuntos
Células Epiteliais/citologia , Receptores ErbB/metabolismo , Pyroglyphidae , Mucosa Respiratória/fisiopatologia , Transdução de Sinais , Actinas/metabolismo , Animais , Antígenos CD/metabolismo , Brônquios/citologia , Caderinas/metabolismo , Células Cultivadas , Humanos , beta Catenina/metabolismoRESUMO
OBJECTIVE: To explore the role of the receptor for advanced glycation end products (RAGE) in regulating the expression of MUC5AC and mucus production in a mouse model of toluene diisocyanate (TDI)?induced asthma. METHODS: BALB/c mice were randomly divided into control group, vehicle (AOO) group, TDI?induced asthma group and RAGE inhibitor (FPS?ZM1) group. PAS staining, Western blotting, and immunohistochemistry were used to analyze the changes in mucus production and MUC5AC expression in the airway of the mice, and the expression of p?ERK was detected with Western blotting. In vitro cultured human bronchial epithelial cell line 16HBE was transfected with lentiviral vector carrying short hairpin RNA targeting RAGE (shRNA?RAGE) and subsequently challenged with a TDI?human serum albumin (TDI-HSA) conjugate, and the changes in cellular MUC5AC mRNA expression as detected using RT-PCR; the protein expressions of ERK and p?ERK in the cells were examined with Western blotting. The effect of ERK inhibitor U0126 pretreatment on MUC5AC mRNA expression was also analyzed in the cells. RESULTS: Compared with the control mice, TDI-induced asthmatic mice showed significantly higher rates of PAS positivity and increased MUC5AC and p?ERK expressions in the airway (P<0.05). Treatment with FPS?ZM1 significantly decreased PAS positivity and lowered MUC5AC and p?ERK expressions in the airway of the asthmatic mice (P<0.05). Exposure of 16HBE cells to TDI?HSA caused a significant increase in MUC5AC mRNA expression and p?ERK protein expression (P<0.05), while RAGE knockdown obviously suppressed TDI?HSA-induced upregulation of p-ERK and MUC5AC mRNA (P<0.05). Treatment with the ERK inhibitor U0126 also lowered TDI?HSA?induced up?regulation of MUC5AC mRNA in the cells (P<0.05). CONCLUSION: RAGE signaling induces MUC5AC expression via extracellular signal-regulated kinase pathway to promote mucus overproduction in mice with TDI-induced asthma.
Assuntos
Asma/metabolismo , Mucina-5AC/metabolismo , Muco/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Asma/induzido quimicamente , Benzamidas/farmacologia , Butadienos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Nitrilas/farmacologia , Distribuição Aleatória , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Tolueno 2,4-Di-IsocianatoRESUMO
OBJECTIVE: To evaluate fractional exhaled nitric oxide (FENO) level in patients with subacute cough and its value in predicting the patients' response to inhaled corticosteroids (ICS) treatment. METHODS: A total of 100 patients with persistent cough lasting more than 3 weeks were enrolled, including 52 patients with subacute cough and 48 with chronic cough. FENO, spirometry, and responses to ICS therapy of the patients were evaluated. RESULTS: The recruited patients had a median (inter-quartile ranges) FENO level of 19 ppb (12-30 ppb). Patients with chronic cough had a significantly higher median FENO level than those with subacute cough (20.5 vs 16 ppb; Z=-2.245, P=0.025). A FENO level ≥25 ppb was recorded in 15 (28.8%) patients with subacute cough, as compared with 20 (41.6%) in patients with chronic cough (χ(2)=1.801, P=0.179). With a FENO ≥25 ppb as the critical value to justify ICS treatment, 15 patients with subacute cough received ICS and 14 (93.3%) of them showed obvious relief of cough after 2 weeks of therapy, a response rate similar to that of 85.0% (17/20) in patients with chronic cough receiving the treatment (χ(2)=0.588, P=0.443). In patients with subacute cough, those with cough variant asthma (CVA) or eosinophilic bronchitis (EB) had a significantly higher median FENO level than those with postinfectious cough [(16 (11-31) ppb vs 11 (8-19) ppb, P<0.01]. In the etiological analysis, CVA or EB was identified in 23 (44.2%) of the patients with subacute cough, as compared 21 (43.8%) in patients with chronic cough (χ(2)=0.002, P=0.961). CONCLUSION: FENO may be an important indicator for etiological diagnosis of subacute cough and for predicting the response to ICS treatment.
Assuntos
Corticosteroides/uso terapêutico , Tosse/tratamento farmacológico , Óxido Nítrico/análise , Testes Respiratórios , Doença Crônica , Tosse/diagnóstico , Expiração , Feminino , Humanos , MasculinoRESUMO
OBJECTIVE: To establish a rapid and economic method for isolating human peripheral blood eosinophils with high viability for patch-clamp studies and investigate the electrophysiological properties of Ca(2+)-activated K(+) channel of the isolated cells. METHODS: Peripheral blood eosinophils were isolated by modified discontinuous Percoll density gradient centrifugation, and the electric currents in the single Ca(2+)-activated K(+) channels of the cells were recorded using patch-clamp technique with cell-attached configuration. RESULTS: The purity of the eosinophils from healthy donors reached (90.5+/-1.6)%, with a viability rate over 99% and recovery rate of (48.2+/-6.9)%. The isolated cells were morphologically intact, from which Ca(2+)-activated K(+) channel activity could be detected. CONCLUSION: The peripheral blood eosinophils isolated using this rapid, simple and highly efficient method are characterized by high purity and viability without obvious cellular injuries, which are ideal for patch-clamp studies.
Assuntos
Eosinófilos/citologia , Separação Celular , Humanos , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/análiseRESUMO
OBJECTIVE: To study the effects of doxofylline on calcium-activated potassium (K(Ca)) channels of human peripheral blood eosinophils in asthma. METHODS: Peripheral blood eosinophils from patients with asthma were isolated and divided equally into two groups, a control group and a doxofylline incubated group. The data were recorded using cell-attached configuration of patch-clamp technique and the kinetic changes of K(Ca) channels activated by 0.2 micromol/L platelet activating factor (PAF) were compared. RESULTS: As compared with the control group, the open probability of the K(Ca) channels decreased from 0.135 +/- 0.021 to 0.044 +/- 0.018, the open time from (5.75 +/- 0.40) ms to (2.39 +/- 0.13) ms, while the close time increased from (2.17 +/- 0.50) ms to (23.73 +/- 2.50) ms in the doxofylline incubated group. The differences were significant between the two groups (all P < 0.05). CONCLUSION: Doxofylline could decrease the open probability of the channels as a result of both the shortening of open period and the prolongation of close time.
Assuntos
Asma/metabolismo , Eosinófilos/metabolismo , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Teofilina/análogos & derivados , Adolescente , Adulto , Asma/sangue , Feminino , Humanos , Masculino , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/metabolismo , Teofilina/farmacologia , Teofilina/uso terapêutico , Adulto JovemRESUMO
OBJECTIVE: To study genes that differentially expressed in peripheral blood eosinophils of patient with asthmatic. METHODS: Total RNA extracted from eosinophils of patient at the time of exacerbation was taken as the tester and the total RNA from eosinophils of the same patient after improvement as driver. The ds cDNA was synthesized by SMART PCR cDNA Synthesis technology. The differentially expressed genes were obtained by suppression subtractive hybridization (SSH) technology and were further identified by Southern blot. RESULTS: Twelve differentially expressed genes including transformation growth factor beta activated kinase like (TAK1L) protein, cGMP gated channel protein were obtained. CONCLUSION: Increased expression of these genes were involved in the regulation of proinflammatory response, signal transduction, energy metabolism and cell apoptosis, which may help to clarify the molecular mechanism of eosinophils in asthma and may provide the theoretical base for finding out the new medicines towards eosinophils.
Assuntos
Asma/genética , DNA Complementar/análise , Eosinófilos/metabolismo , Asma/sangue , Asma/patologia , Southern Blotting , Clonagem Molecular , Humanos , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase , Transdução de SinaisRESUMO
The strong association between bcl-2-like 11 (BIM) triggered apoptosis and the presence of epidermal growth factor receptor (EGFR) mutations has been proven in nonsmall cell lung cancer (NSCLC). However, the relationship between EGFR-tyrosine kinase inhibitor's (TKI's) efficacy and BIM polymorphism in NSCLC EGFR is still unclear.Electronic databases were searched for eligible literatures. Data on objective response rates (ORRs), disease control rates (DCRs), and progression-free survival (PFS) stratified by BIM polymorphism status were extracted and synthesized based on random-effect model. Subgroup and sensitivity analyses were conducted.A total of 6 studies that involved a total of 773 EGFR mutant advanced NSCLC patients after EGFR-TKI treatment were included. In overall, non-BIM polymorphism patients were associated with significant prolonged PFS (hazard ratio 0.63, 0.47-0.83, Pâ=â0.001) compared to patients with BIM polymorphism. However, only marginal improvements without statistical significance in ORR (odds ratio [OR] 1.71, 0.91-3.24, Pâ=â0.097) and DCR (OR 1.56, 0.85-2.89, Pâ=â0.153) were observed. Subgroup analyses showed that the benefits of PFS in non-BIM polymorphism group were predominantly presented in pooled results of studies involving chemotherapy-naive and the others, and retrospective studies. Additionally, we failed to observe any significant benefit from patients without BIM polymorphism in every subgroup for ORR and DCR.For advanced NSCLC EGFR mutant patients, non-BIM polymorphism ones are associated with longer PFS than those with BIM polymorphism after EGFR-TKIs treatment. BIM polymorphism status should be considered an essential factor in studies regarding EGFR-targeted agents toward EGFR mutant patients.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas , Genes erbB-1/genética , Proteínas de Membrana/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Humanos , Mutação , Polimorfismo Genético , Resultado do TratamentoRESUMO
BACKGROUND: Evidence describing the association between pulmonary function and carotid atherosclerosis has been inconclusive and the role of smoking in this association is unclear. We therefore examined this association in the Guangzhou Biobank Cohort Study-CVD Subcohort. METHODS: Common carotid artery (CCA) intima-media thickness (IMT) and carotid plaques were measured by B-mode ultrasonography and lung function by spirometry using a turbine flowmeter. Chronic obstructive pulmonary disease (COPD) was defined as the ratio of forced expiratory volume in 1 s (FEV1) to forced vital capacity (FVC) of less than 0.70. Predicted FEV1 and FVC were derived using equations for Chinese. RESULTS: Of 1625 participants aged 50 + years, 382 (23.5%) had evidence of carotid plaque. The mean CCA-IMT was higher in those with COPD than those without (0.82 ± 0.29 mm versus 0.76 ± 0.31 mm, P = 0.02). We found no evidence that the association of pulmonary function with CCA-IMT varied by smoking status (P values interaction: 0.23-0.83). After adjustment for a wide range of potential confounders, the increased risks of thickened CCA-IMT (CCA-IMT ≥1.0 mm) in those with COPD became marginally nonsignificant (adjusted odds ratio (OR) 1.45, 95% confidence interval (CI) 0.91-2.29; P = 0.12). Compared to those in the highest tertile, participants in the lowest tertile of FEV1 observed to predicted ratio had increased risk of thickened CCA-IMT (adjusted OR 2.18, 95% CI 1.42-3.34) and carotid plaque (adjusted OR 1.50, 95% CI 1.08-2.09), while participants in the lowest tertile of FVC observed to predicted ratio had increased risk of thickened CCA-IMT (adjusted OR 2.29, 95% CI 1.46-3.58), but the adjusted OR for carotid plaque was marginally nonsignificant (adjusted OR 1.29, 95% CI 0.93-1.80; P = 0.13). CONCLUSION: Independent of smoking status, poor pulmonary function was dose-dependently associated with carotid atherosclerosis in older Chinese. (281 words).
Assuntos
Doenças das Artérias Carótidas/fisiopatologia , Pulmão/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fatores Etários , Idoso , Bancos de Espécimes Biológicos , Doenças das Artérias Carótidas/diagnóstico , Doenças das Artérias Carótidas/epidemiologia , Artéria Carótida Primitiva/diagnóstico por imagem , Espessura Intima-Media Carotídea , Distribuição de Qui-Quadrado , China/epidemiologia , Estudos de Coortes , Estudos Transversais , Inglaterra , Feminino , Volume Expiratório Forçado , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Valor Preditivo dos Testes , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Medição de Risco , Fatores de Risco , Espirometria , Capacidade VitalRESUMO
OBJECTIVE: To amplify double-strand cDNA from small amount of total RNA of eosinophils from asthma patients by Super SMART cDNA synthesis technique. METHODS: The eosinophils were purified from the peripheral blood of asthma patients before and after treatment by Percoll gradient centrifugation, from which the total RNA was extracted using TRIzol kit. First-strand cDNA synthesis and double-strand cDNA amplification were performed using Super SMART cDNA synthesis technique. The quality of the obtained cDNA was evaluated by gradient cDNA electrophoresis, and the amplification efficiency determined by cDNA quantification. RESULT: From 20 ng total RNA, 7.155 microg and 6.568 microg of the tester and driver double- strand cDNAs respectively were obtained successfully, and the result of electrophoresis indicated high quality and purity of the cDNA acquired. CONCLUSION: Super SMART cDNA synthesis technique can effectively amplify high-quality double-strand DNA from a very small amount of total RNA, which may facilitate the exploration of the mechanism of asthma from the genetic level.
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Asma/genética , DNA Complementar/biossíntese , Eosinófilos/metabolismo , Perfilação da Expressão Gênica , Reação em Cadeia da Polimerase/métodos , Asma/sangue , DNA Complementar/genética , RNA Mensageiro/genética , Taq PolimeraseRESUMO
OBJECTIVE: To explore the molecular mechanism of eosinophils for its role in bronchial asthma. METHODS: The total RNA extracted from the eosinophils of patients during the onset of asthma was used as the tester and the total RNA obtained after treatment served as the driver. cDNA suppression subtractive hybridization (SSH) was performed using the protocols described in the Clontech SMART PCR cDNA Syn thesis Kit and PCR-Select cDNA Subtraction Kit. The subtracted cDNA was then inserted into T vector to generate subtracted cDNA library. Clones of the subtracted cDNA library were screened by hybridization and the insert sequence of the positive clones was compared with the sequence in the GenBank. RESULTS: A total of 400 clones selected from the subtracted cDNA library were amplified by PCR and about 85% of these clones contained inserts. Six differential cDNA fragments were acquired after two differential screening. These genes were involved in the regulation of proinflammatory response, signal transduction, energy metabolism and cell apoptosis. CONCLUSION: Differentially expressed genes of the eosinophils during the onset and the remission stage of bronchial asthma can be effectively cloned by SSH, which provides a solid foundation for clarifying the molecular mechanism of eosinophils in asthma and a theoretical base for clinical treatment and prevention of asthma.
Assuntos
Asma/sangue , Eosinófilos/metabolismo , Hibridização de Ácido Nucleico/métodos , Southern Blotting , Clonagem Molecular , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: To observe the effect of 25-hydroxyvitamin D3 on the permeability and ZO-1 expression in normal human airway epithelial cells. METHODS: MTT assay was used to assess the viability of human airway epithelial cell line 16HBE following a 24-hour exposure to different concentrations of 25-hydroxy vitamin D3, and the transepithelial electrical resistance (TER) of the cell monolayer was measured using a Millicell-ERS voltohmmeter. Real-time quantitative RT-PCR was employed to determine the changes of ZO-1 mRNA expression in the cells following the exposures. RESULTS: Exposure to 25-hydroxyvitamin D3 resulted in significantly increased permeability of 16HBE cells, but the exspression of ZO-1 showed no obvious changes. 25-hydroxyvitamin D3 at 4×10(-9) mol/L showed the strongest effect in increasing the permeability of cell monolayer. CONCLUSION: 25-hydroxyvitamin D3 increases the permeability of normal bronchial airway epithelial cell monolayer in vitro, but this effect is not mediated by upregulation of ZO-1 expression.
Assuntos
Brônquios/citologia , Calcifediol/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Brônquios/metabolismo , Calcifediol/farmacocinética , Linhagem Celular , Células Epiteliais/citologia , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
OBJECTIVE: To investigate the level of the patients perceived control of asthma (PCA) in South China and analyze the risk factors contributing to inadequate PCA. METHODS: A total of 150 asthmatic out-patients consisting of 86 males and 64 females aged 19-65 (38.6∓11.7) years were enrolled in this investigation. The patients were asked to complete questionnaires of the demographic data, perceived control of asthma (PCAQ-6) scales, asthma control test (ACT) scales and Standard asthma-specific quality of life [AQLQ(S)] scale. The data of spirometric measurements, blood cell count and induced sputum cell count were also collected. RESULTS: All the 150 asthmatic out-patients recruited completed the questionnaires and examinations. The PCAQ-6 scores ranged from 10 to 26 (18.75∓3.42) in these patients (18.6∓3.28 in male and 18.95∓3.6 in female patients), significantly lower than those reported in other countries (P<1). PCA was positively correlated to the level of asthma control (r(p)=0.377, P=0.000) and AQLQ(S) scores (r(p)=0.675, P=0.000). Multiple linear regression showed that PCA was positively correlated to FEV1% and blood neutrophil counts, and inversely to asthma duration. CONCLUSION: The level of the PCA appears inadequate in South China. The PCA can affect the level of asthma control and asthma-specific quality of life. The factors contributing to inadequate PCA include primarily asthma duration, lung function and blood neutrophil counts.
Assuntos
Asma/prevenção & controle , Asma/psicologia , Conhecimentos, Atitudes e Prática em Saúde , Inquéritos e Questionários , Adulto , Idoso , Asma/sangue , China , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos , Qualidade de Vida , Adulto JovemRESUMO
OBJECTIVE: To investigate the effect of toluene diisocyanate (TDI) on the production of reactive oxygen species (ROS) and the permeability of human bronchial epithelial (HBE) cells. METHODS: TDI-human serum albumin (TDI-HSA) conjugate was prepared using a modified Son's method. MTT assay was used to assess HBE cell viability after exposure to different concentrations of TDI-HSA. The level of intracellular ROS of HBE cells was detected by flow cytometry with an oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) uploading, and the permeability of cell monolayer was assessed by detecting the transepithelial electrical resistance (TEER). RESULTS: The exposure to 120 µg/ml TDI-HSA did not obviously affect the cell viability. Compared with the control group, the intracellular fluorescent intensity increased significantly in the cells exposed to 20, 60, and 100 µg/ml TDI-HSA (P<0.05). The intracellular ROS production increased significantly after 100 µg/ml TDI-HSA treatment (P<0.05), but the increment in ROS production was significantly suppressed by pretreatment of the cells with N-acetylcysteine (NAC) (P<0.05), which also enhanced the TEER decreased by TDI-HSA treatment (P<0.05). CONCLUSIONS: TDI enhances the permeability of HBE cell monolayer partially through a ROS-mediated pathway, suggesting the importance of oxidative stress in TDI-induced pulmonary diseases.
Assuntos
Brônquios/citologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tolueno 2,4-Di-Isocianato/farmacologia , Linhagem Celular , Células Epiteliais/citologia , Humanos , Estresse Oxidativo/efeitos dos fármacos , Albumina Sérica/farmacologiaRESUMO
OBJECTIVE: Eosinophils play a pivotal role in asthmatic airway inflammation. We previously found a significantly high expression of Slingshot-1L (SSH-1L) in peripheral eosinophils in acute exacerbations of asthma. Objective To investigate the expression and localization patterns of SSH-1L in peripheral blood eosinophils of asthmatic patients and their changes after treatment with inhaled corticosteroids. METHODS: We recruited 4 outpatients with acute exacerbations of asthma who received no previous corticosteroid treatment and 1 healthy volunteer. From all the subjects 30 ml peripheral venous blood samples were collected before and after a 3-month treatment with inhaled fluticasone. The eosinophils were isolated, purified and counted, and the expressions of SSH-1L in the eosinophils were examined by RT-PCR and Western blotting. The localization of SSH-1L phosphatases in the peripheral eosinophils was detected by immunofluorescence assay in one patient. RESULTS: SSH-1L phosphatases distributed diffusely in the cytoplasm, especially dense near the membrane of the peripheral eosinophils. Glucocorticoids treatment resulted in a significant reduction in both the SSH-1L mRNA expression (0.7403∓0.1124 vs 0.4101∓0.0363, P=0.001) and SSH-1L protein expression (0.3410∓0.1337 vs 0.1543∓0.0551, P=0.039). CONCLUSION: A high expression of SSH-1L in peripheral eosinophils in acute exacerbations of asthma may play a role in the activation and migration of eosinophils. The efficacy of inhaled corticosteroids in asthma control might be partly attributed to a down-regulated expression of SSH-1L.
Assuntos
Asma/sangue , Eosinófilos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Adulto , Idoso , Asma/tratamento farmacológico , Feminino , Glucocorticoides/uso terapêutico , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To investigate the effect of dimethylsulfoxide on the expression of thymic stromal lymphopoietin (TSLP) in human bronchial epithelial cell (HBE). METHODS: 16HBE cells were incubated in the presence of dimethylsulfoxide at different concentrations, and the cell proliferation changes were observed. The expressions of TSLP mRNA and protein in the cells were detected by real-time quantitative PCR and ELISA, respectively. RESULTS: Dimethylsulfoxide induced significantly increased TSLP mRNA expression in HBE cells (P<0.01) in a concentration-dependent manner. The level of TSLP protein in the supernatant was also increased after dimethylsulfoxide treatment, but high concentration of dimethylsulfoxide resulted in e inhibited cell proliferation. CONCLUSION: Dimethylsulfoxide may affect the immunomodulatory function of HBE cells.