RESUMO
BACKGROUND: Matrix metalloproteinase-9 (MMP-9) and its inhibitor tissue inhibitor of metalloproteinase-1 (TIMP-1) are involved in the pathogenesis of airway inflammation in patients with chronic obstructive pulmonary disease (COPD). However, no study so far has addressed their value as noninvasive biomarkers of airways inflammation. OBJECTIVE: To evaluate MMP-9 and TIMP-1 concentrations in the exhaled breath condensate (EBC) of patients with stable COPD and also during the exacerbation episode. METHODS: EBC and serum samples were collected in 17 stable-phase COPD patients who were current smokers as well as during their first exacerbation episode, and in 22 asymptomatic smokers. EBC and serum levels of MMP-9 and TIMP-1 were measured with ELISA kit. RESULTS: Mean EBC MMP-9 and TIMP-1 levels were higher in patients with stable COPD than in asymptomatic smokers. Exacerbation of COPD increased 2-fold the exhalation of MMP-9 (18.5 ± 10.1 ng/ml vs. 8.9 ± 6.2 ng/ml, p = 0.01) and TIMP-1 (to 41.1 ± 20.4 ng/ml vs. 16.4 ± 6.8 ng/ml, p < 0.001). Both, MMP-9 and TIMP-1 in EBC correlated negatively with FEV(1) (% predicted) at baseline (r = -0.78, p < 0.001 and r = -0.73, p < 0.001) and during the exacerbation episode (r = -0.57, p = 0.02 and r = -0.65, p = 0.005). Similar negative correlations were noted with FVC (% predicted), except for MMP-9 in EBC at exacerbation. Exhaled MMP-9 and TIMP-1 did not correlate with serum concentrations in COPD patients, either at baseline or during exacerbation. CONCLUSION: Exhaled MMP-9 and TIMP-1 increased during COPD exacerbation and was negatively correlated with spirometric variables, which suggests the usefulness of their measurement in EBC for the monitoring of airways inflammation. However, to better assess their diagnostic or prognostic value larger studies are necessary.
Assuntos
Expiração , Metaloproteinase 9 da Matriz/análise , Doença Pulmonar Obstrutiva Crônica/enzimologia , Inibidor Tecidual de Metaloproteinase-1/análise , Idoso , Biomarcadores/metabolismo , Testes Respiratórios , Progressão da Doença , Feminino , Humanos , Masculino , Estudos Prospectivos , Índice de Gravidade de Doença , EspirometriaRESUMO
Chronic obstructive pulmonary disease (COPD) is predominantly the result of years of cigarette smoking. Increased oxidative stress in COPD derives from the increased burden of inhaled oxidants (cigarette smoke), air pollution and the increase in reactive oxygen and nitrogen species (ROS and RNS), generated by some inflammatory, immune, and structural airways cells. In view of the lack of therapy that might inhibit the progress of the disease, there is an urgent need for a successful therapeutic approach. Apocynin is a molecule inhibiting activation of NADPH oxidase - enzyme generating ROS and RNS precursor. Thus, our aim was to analyze apocynin influence on hydrogen peroxide and nitrite concentrations in EBC of COPD patients. Apocynin reduced concentration of H(2)O(2) in COPD patients 60 and 120 min after apocynin inhalation, in comparison to placebo (0.43 µM vs. 0.59 µM, and 0.4 µM vs. 0.59 µM respectively, p < 0.05). Moreover, apocynin decreased NO(2)(-) ions concentration in airways of COPD patients after apocynin nebulization (3.97 µM vs. 4.48 µM after 30 min, 3.82 µM vs. 4.48 µM after 60 min, and 3.76 µM vs. 4.48 µM after 30 min respectively, p < 0.05). No adverse effects have been observed. The results suggest that apocynin might be considered as anti-inflammatory agent, and, possibly used in therapy of COPD.
Assuntos
Testes Respiratórios , Peróxido de Hidrogênio/metabolismo , Nitritos/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Acetofenonas/farmacologia , Adulto , Idoso , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , OxirreduçãoRESUMO
AIMS: Sleep is divided into two major stages, non-rapid eye movement (NREM) and rapid eye movement (REM), which are distinct in various neuroendocrine respects. NREM/REM cycles influence insulin and glucagon secretion; however, glucose concentrations in REM compared with NREM have not been directly explored. The aim was to investigate the differences in glucose concentrations in interstitial fluid (IGC) between NREM/REM cycles using a continuous glucose monitoring system (CGMS). METHODS: Thirteen subjects were eligible for analysis out of the 28 enrolled. All underwent standard polysomnography for the assessment of sleep stages and the exclusion of sleep apnoea syndrome with CGMS and subsequent morning oral glucose tolerance test (exclusion of glucose intolerance or diabetes). RESULTS: The IGC in REM fell in 12 out of the 13 subjects, whereas the IGC in NREM increased in eight out of the 13 subjects. Therefore, the mean change of IGC differed in direction between sleep stages: -0.028 (-0.045 to -0.011) for REM vs. 0.005 (-0.012 to 0.017) for NREM [median (QR), P = 0.007, n = 13], with the mean difference of 0.038 mmol/l x 5 min(-1) (95% confidence interval 0.012, 0.064). The mean glucose concentration in REM sleep was lower than in NREM: 4.29 +/- 1.00 vs. 4.53 +/- 0.90 mmol/l (mean +/- sd, P = 0.003, n = 13). CONCLUSIONS: The decrease in IGC in REM compared with NREM sleep, with lower absolute values, may arise from different physiological events observed in these sleep stages. The REM-related decline in glucose concentrations may be a risk factor for nighttime hypoglycaemia.
Assuntos
Glicemia/metabolismo , Encéfalo/metabolismo , Hipoglicemia/metabolismo , Adulto , Encéfalo/fisiopatologia , Teste de Tolerância a Glucose , Humanos , Hipoglicemia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Polissonografia , Sono/fisiologia , Sono REM/fisiologia , Adulto JovemRESUMO
BACKGROUND: This study was designed to investigate the effect of cigarette smoking on hydrogen peroxide (H2O2) and thiobarbituric reactive substances (TBARs) concentrations in exhaled breath condensate (EBC) in patients with community acquired pneumonia (CAP). METHODS: H2O2 and TBARs concentrations in EBC were determined with spectrofluorimetrical assays. RESULTS: Non-smoking CAP patients (n = 24) exhaled 1.4, 1.8 and 1.7 times more H2O2 than the smoking patients with CAP (n = 19) as assessed one (0.73 +/- 0.32 microM v. 0.51 +/- 0.36 microM), three (0.84 +/- 0.31 microM v. 0.47 +/- 0.24 microM) and five (0.66 +/- 0.28 microM v. 0.40 +/- 0.35 microM) days after admission (p < 0.05 in each case). Over 10 days of hospital treatment, mean level of exhaled H2O2 0.45 +/- 0.22 microM in CAP patients with smoking history was decreased if compared with 0.71 +/- 0.19 microM exhaled H2O2 in CAP group (p = 0.005). On the contrary, TBARs concentration evaluated over entire study period was increased in smoking CAP patients (median 0.02 microM, range 0-0.32 microM) compared with non-smoking group (median 0.01 microM, range 0-0.21 microM, p < 0.05). Concurrent, active smoking status was related with the decreased levels of H2O2 exhaled in breath condensate within the course of CAP but it appeared to increase levels of TBARs. CONCLUSIONS: The differential alternations of oxidative parameters in EBC with respect to the smoking status might provide evidence of increased H2O2 decomposition and enhanced generation of reactive species in airways of CAP patients.
Assuntos
Peróxido de Hidrogênio/metabolismo , Pneumonia Bacteriana/metabolismo , Fumar/efeitos adversos , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Idoso , Antibacterianos/uso terapêutico , Testes Respiratórios , Infecções Comunitárias Adquiridas/metabolismo , Interpretação Estatística de Dados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Pneumonia Bacteriana/diagnóstico , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/fisiopatologia , Espécies Reativas de Oxigênio , Testes de Função Respiratória , Fumar/metabolismo , Espectrometria de Fluorescência , Espirometria , Fatores de TempoRESUMO
The clinical efficacy of inhalatory furosemide (Fu) has been extensively studied in bronchial asthma patients but there are only a few studies addressing its action on cells participating in the underlying inflammatory process. Therefore, we investigated the effect of Fu on human peripheral blood polymorphonuclear leukocytes (PMNL) at concentrations that can be achieved in the bronchial lining fluid by inhalation, i.e. 10(-5), 10(-4) and 10(-3) M. The influence of Fu on the following PMNL parameters was investigated: intracellular calcium changes ([Ca2+]i) as a part of signal transduction and luminol dependent chemiluminescence (LCL) as an indirect measure of NADPH-oxidase activation upon n-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation; chemotaxis to fMLP, phagocytosis and intracellular killing of Staphylococcus aureus. Incubation with Fu resulted in a concentration dependent reduction of Ca2+ influx and Fu (10(-3) M) decreased the main Ca2+ parameters to one half of the control values and to the level obtained in calcium-free buffer. In contrast, Fu had no effect if preincubated with the cells and then removed by washing. The LCL signal was reduced by Fu (10(-3) M) from 2000 +/- 870 to 550 +/- 440 arbitrary units [aU] (p<0.05). In contrast to the [Ca2+]i measurements, a slightly diminished LCL was also observed following preincubation with Fu and washing. No effect of Fu was found on phagocytosis and intracellular killing of St. aureus. Fu diminished chemotaxis to fMLP but at 10(-3) M it also displayed weak chemoattractant properties. The differential action of Fu on human PMNL may add to the understanding of its topical and restricted efficacy in bronchial asthma.
Assuntos
Diuréticos/farmacologia , Furosemida/farmacologia , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inibidores , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Cálcio/análise , Cálcio/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Fura-2 , Humanos , Técnicas In Vitro , Indicadores e Reagentes , Medições Luminescentes , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/imunologia , Fagocitose/efeitos dos fármacos , Staphylococcus aureus/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina/farmacologiaRESUMO
Enhanced exhalation of H2O2 and TBARs have been reported in various inflammatory lung diseases. This may reflect activated phagocytes influx and free radical generation in the airways. However, to apply these compounds as markers of oxidative stress it is necessary to understand factors influencing their exhalation in healthy subjects. We investigated the concentration of H2O2 and TBARs in expired breath condensate (EBC) of 58 healthy volunteers. EBC was collected seven times every 4 h during 24 h and three times every 7 d during 2 consecutive weeks. The H2O2 exhalation revealed diurnal variation with two-peak values 0.45 +/- 0.29 microM and 0.43 +/- 0.22 microM at 12:00 and 24:00 h. The lowest concentrations, 0.26 +/- 0.13 microM and 0.25 +/- 0.26 microM, were found at 20:00 and 8:00 h. Cigarette smokers exhaled about 2.4 times more H(2)O(2) than never smoked subjects. Moreover, in contrast to nonsmokers, cigarette smokers' H2O2 exhalation was stable over 2 week observation. The mean H2O2 concentration estimated over the whole 2 week period was higher in subjects above 40 years regardless of smoking habit, and it positively correlated with age in never smoked subjects (p <.004). Smoking of one cigarette caused 1.8-fold rise in H2O2 exhalation (p <.01). The baseline H2O2 levels correlated with cumulative cigarette consumption (p <.05) and MEF 25% of predicted (p <.05). Neither moderate exercise nor one puff of salbutamol nor ipratropium influenced significantly the concentration of H2O2 and TBARs in EBC. Only 4 of 120 EBC specimens from never smoked subjects revealed detectable levels of TBARs. Cigarette smokers exhaled more TBARs (p <.05) than never smoked volunteers. Our results indicate that healthy never smoked subjects exhale H2O2 with diurnal variation and significant changes over 2 week observation. Cigarette smoking enhanced H2O2 generation in the airways. These results could be useful for planning studies with exhaled H2O2 as a marker of airway inflammation. Occasional detection of TBARs in EBC of never smoked persons may be a result of sufficient antioxidant activity in the airways that protects tissues from peroxidative damage.
Assuntos
Peróxido de Hidrogênio/metabolismo , Respiração , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Adulto , Fatores Etários , Albuterol/administração & dosagem , Albuterol/farmacologia , Índice de Massa Corporal , Broncodilatadores/farmacologia , Ritmo Circadiano/efeitos dos fármacos , Exercício Físico/fisiologia , Feminino , Humanos , Ipratrópio/administração & dosagem , Ipratrópio/farmacologia , Masculino , Ventilação Pulmonar/efeitos dos fármacos , Análise de Regressão , Respiração/efeitos dos fármacos , Fatores Sexuais , Fumar/efeitos adversos , EspirometriaRESUMO
The influence of Cefodizime (CDZ) on in vitro activity of polymorphonuclear leukocytes (PMNL) from healthy subjects was assessed. Preincubation with CDZ enhanced phagocytosis and intracellular killing of Staphylococcus aureus by PMNL. Contrary to numerous clinical reports, no significant effect of CDZ preincubation on PMNL response to n-formyl-methionyl-leucyl-phenylalanine was found with respect to intracellular calcium changes, degranulation, hydrogen peroxide production, and chemiluminescence. These results suggest that augmented microbicidal activity of PMNL is not related to the enhanced production of reactive oxygen species in healthy subjects.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antibacterianos/farmacologia , Cefotaxima/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fagocitose/efeitos dos fármacos , Staphylococcus aureus/imunologia , Adulto , Cálcio/metabolismo , Cefotaxima/farmacologia , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Feminino , Humanos , Peróxido de Hidrogênio/metabolismo , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Medições Luminescentes , Masculino , Fagocitose/imunologia , Staphylococcus aureus/genéticaRESUMO
UNLABELLED: We have investigated whether pretreatment with N-acetylcysteine (NAC) and/or ambroxol (Amb), drugs known as reactive oxygen species (ROS) scavengers, would minimize lipopolysaccharide (LPS)-induced leucocyte accumulation in rat lung microvasculature and protect lungs from damage and the effect of these drugs on chemotactic peptide (fMLP)-induced chemiluminescence of human polymorphonuclear leukocytes (PMNs). Animals were injected ip with NAC (27.6 mg/kg, n=8), ambroxol (70 mg/kg, n=8), combination NAC+ambroxol (n=8), or 1 ml buffer alone (n=8), once a day for 3 consecutive days. Then animals were injected with LPS (17 mg/kg), and killed 3 h later. In each of another four groups eight rats were used as a control, and received the same drug treatment but LPS was replaced with 0.9% NaCl. PMNs and macrophages (Ms) were counted in histologic slides of lung tissue. Using computer image analysis we measured the area of alveolar profiles. Luminol-enhanced chemiluminescence was measured in PMNs suspensions obtained from healthy volunteers. Chemiluminescence intensity was measured in resting and fMLP-stimulated cells, and compared between cells incubated with Amb, NAC or distilled water. We observed significant differences in the number of PMNs and Ms, alveolar profile area between control and LPS-treated animals (P<0.01). PMNs and Ms were numerous in lungs of LPS-administered animals (PMNs: Median (M)=137.5 per 6 high power fields range (r)=54.0; Ms: M=123.0 r=11.0), less numerous in ambroxol-treated group (PMNs: M=101.5 r=32.0 and Ms:53.5 r=36.0), not abundant in NAC (PMNs:M=56.0 r=28.0 and Ms:M=20.5 r=13.0) and in NAC+ambroxol treated rats (PMNs:M=53.5 r=21.0 and Ms:M=29.0 r=9.0), and rare in LPS+drugs-untreated control group (PMNs:M=40.5 r=19.0 and Ms:M=18.5 r=15.0). Chemiluminescence assay revealed that 100 micro;M ambroxol stimulated fMLP-induced PMNs chemiluminescence and NAC of the same concentration had no significant effect. CONCLUSION: In our experiment we showed that pretreatment with NAC and ambroxol may inhibit phagocyte influx to rat lung and may protect it from damage. We also revealed that NAC at dose 27.6 mg/kg has stronger protective properties than ambroxol at dose 70 mg/kg and this may result from enhancing effect of ambroxol on fMLP-provoked PMNs chemiluminescence.
Assuntos
Acetilcisteína/farmacologia , Ambroxol/farmacologia , Endotoxinas/farmacologia , Expectorantes/farmacologia , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Fagócitos/efeitos dos fármacos , Animais , Escherichia coli/metabolismo , Humanos , Medições Luminescentes , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Masculino , Neutrófilos/efeitos dos fármacos , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The imbalance between oxidants and antioxidants is known to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoking is the most frequent factor responsible for development of COPD by leading to oxidant overload in the lower airways, due to presence of its own oxidants and to recruitment and activation of pulmonary phagocytes. We aimed to determine whether (1) patients with stable COPD have higher thiobarbituric acid-reactive substances (TBARs, an end-product of lipid peroxidation) and H2O2 levels in expired breath condensate than healthy subjects who have never smoked; (2) COPD subjects who are current smokers exhale more TBARs and H2O2 than COPD ex-smokers and those who have never smoked; and (3) concentration of TBARs correlates with H2O2 levels in the breath condensate of COPD patients. The TBAR and H2O2 content in expired breath condensate of 17 healthy nonsmoking subjects and 44 patients (11 current smokers, 20 ex-smokers and 13 who had never smoked) with stable COPD [forced expiratory volume in 1 s (FEV1) 63.3 +/- 16.3% and FEV1 reversibility 5.2 +/- 4.3% predicted value] was measured spectrofluorimetrically by the thiobarbituric acid and homovanillic acid methods, respectively. The mean concentrations of TBARs and H2O2 in the expired breath condensate of COPD subjects were 12 (0.48-0.86 microM vs. 0.04 +/- 0.14 microM; P < 0.05) and 10 times (0.48 +/- 0.67 microM vs. 0.05 +/- 0.07 microM; P < 0.005) higher than in healthy controls. Current smokers with COPD did not exhale more H2O2 than COPD ex-smokers and those who had never smoked. TBARs levels shared only a tendency to be higher in the breath condensate of smoking COPD subjects than in that of ex-smokers (0.92 +/- 1.49 microM vs. 0.35 +/- 0.44 microM) and of COPD subjects who had never smoked (0.92 +/- 1.49 microM vs. 0.30 +/- 0.53 microM). No correlation was found between TBAR and H2O2 levels in the whole COPD group. These variables did not correlate with cigarette smoking status and the time from smoking cessation. Subjects with stable COPD exhibit increased lipid peroxidation and H2O2 generation in the airways. Current cigarette smoking does not distinguish COPD subjects with respect to TBARs and H2O2 exhalation.
Assuntos
Antioxidantes/efeitos adversos , Peróxido de Hidrogênio/metabolismo , Pneumopatias Obstrutivas/metabolismo , Fumar/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Antioxidantes/metabolismo , Biomarcadores/análise , Testes Respiratórios , Feminino , Volume Expiratório Forçado , Humanos , Pneumopatias Obstrutivas/etiologia , Masculino , Pessoa de Meia-Idade , Fumar/efeitos adversosRESUMO
We have found an increased H2O2 level in expired air of asthmatic patients. Neutrophils from these subjects generated higher amounts of superoxide radicals after challenge with phorbol esters than those from healthy subjects which may result from an increased activity of NADPH-oxidase. The enhanced Ca2+ mobilisation in neutrophils from asthmatics could be responsible for increased production and subsequent elevated H2O2 concentration in expired breath condensate. In this study we wished to determine whether neutrophils of asthmatic patients have enhanced [Ca2+]i response after N-formyl-methionyl-leucyl-phenylalanine--fMLP challenge as compared with cells from healthy donors, and if so, does it correlate with H2O2 levels in expired air. We examined 21 patients, 10 healthy individuals as a control group (mean age 34.3 +/- 5.5, 6 males and 4 females) and 11 asthmatic subjects (mean age 38.2 +/- 7.2, 7 males and 4 females). The rise of [Ca2+]i as an early event of neutrophil activation, was measured spectrofluorimetically with Fura-2-AM. The mean H2O2 level, measured spectrofluorimetrically in the expired breath of asthmatics, was 20-fold higher than that in healthy control (0.18 +/- 0.20 vs. 0.01 +/- 0.04 microM, p < 0.05). [Ca2+]i increase after challenge by fMLP (delta [Ca2+]i) was much higher in asthmatics than in control group (205.0 +/- 44 vs. 113.0 +/- 22 nM, p < 0.05, respectively). A strong correlation was observed between H2O2 and delta [Ca2+]i and maximal velocity of increase in [Ca2+]i in asthmatics (r = 0.87, p < 0.01 and r = 0.64, p < 0.05). We conclude that elevated H2O2 level in the expired breath condensate of asthmatics can be generated by activated neutrophils in the course of mucosal inflammation observed in bronchial asthma.
Assuntos
Asma/imunologia , Asma/metabolismo , Peróxido de Hidrogênio/metabolismo , Ativação de Neutrófilo/imunologia , Adulto , Asma/sangue , Testes Respiratórios , Cálcio/metabolismo , Feminino , Humanos , Peróxido de Hidrogênio/análise , Masculino , Pessoa de Meia-Idade , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Alvéolos Pulmonares/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cigarette smoking is the most common factor responsible for the development of chronic obstructive pulmonary disease (COPD) leading to oxidant overload in the lower airways because of the presence of oxidants in cigarette smoke and recruitment and activation of pulmonary phagocytes. In this study we intended to determine whether: 1) patients with stable COPD have higher H2O2 levels in expired breath condensate than healthy nonsmoking subjects and 2) whether cigarette smoking increases H2O2 exhalation in patients with stable COPD. The H2O2 content of the expired breath condensate of 17 healthy nonsmoking subjects and 38 patients (10 current smokers, 17 exsmokers and 11 who have never smoked) with stable COPD (forced expiratory volume in one second (FEV1) 63.3 +/- 15.5% of predicted value) was measured spectrofluorimetrically (homovanillic acid method). The mean H2O2 concentration in the expired breath condensate of COPD subjects was 10-times higher than that found in healthy controls (0.55 +/- 0.69 microM versus 0.05 +/- 0.07 microM, p < 0.005). There were no significant differences between H2O2 levels found in current smokers with COPD (0.44 +/- 0.56 microM) and COPD subjects who have never smoked (0.49 +/- 0.70 microM). No correlation was found between expired H2O2 and daily cigarette consumption or cumulative cigarette consumption in current smokers or exsmokers with COPD. These findings demonstrate that subjects with stable chronic obstructive pulmonary disease exhibit increased H2O2 generation in the airways and that cigarette smoking does not increase H2O2 production.
Assuntos
Peróxido de Hidrogênio/metabolismo , Pneumopatias Obstrutivas/metabolismo , Oxidantes/metabolismo , Fumar/efeitos adversos , Testes Respiratórios , Estudos de Casos e Controles , Feminino , Humanos , Pneumopatias Obstrutivas/complicações , Pneumopatias Obstrutivas/diagnóstico , Masculino , Pessoa de Meia-IdadeRESUMO
The respiratory burst of polymorphonuclear leukocytes depends on activation of many enzymes and generation of variety of second messengers. One of important links leading to polymorphonuclear leukocytes (PMNL) activation is a transient rise in intracellular free calcium concentration ([Ca2+]i). Serine proteinase inhibitors such as alpha-1-proteinase inhibitor (alpha1PI), phenylmethylsulphonylfluoride (PMSF) and soybean trypsin inhibitor (SBTI) were reported to inhibit human PMNL respiratory burst. In this study we tested the hypothesis whether these inhibitors can inhibit the rise in [Ca2+]i after stimulation of human PMNL with 10(-7) M n-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) or platelet activating factor (PAF). [Ca2+]i was measured with use of a fluorescent probe Fura-2AM under conditions of 100 nM and 1 mM extracellular Ca2+ concentration. Preincubation of PML with 600 mg/dl of alpha1PI or SBTI for 30 min at 37 degrees C had no influence on Ca2+ response to all agonists in comparison with cells treated with the same concentration of human serum albumin. However, 0.5 mM PMSF enhanced 1.7-fold (p < 0.002) [Ca2+]i rise after challenge with FMLP while did not affect significantly Ca2+ response to PAF and LTB4. The stimulatory effect of PMSF after addition of FMLP was dependent on increased Ca2+ influx from extracellular space. Our results suggest that suppression of PMNL respiratory burst by serine proteinase inhibitors is not mediated via Ca2+ pathway and that some proteases take part in Ca2+ response to FMLP.
Assuntos
Cálcio/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Inibidores de Serina Proteinase/farmacologia , Adulto , Feminino , Humanos , Técnicas In Vitro , Líquido Intracelular/metabolismo , Leucotrieno B4/farmacologia , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fluoreto de Fenilmetilsulfonil/farmacologia , Fator de Ativação de Plaquetas/farmacologia , Explosão Respiratória/efeitos dos fármacos , Inibidores da Tripsina/farmacologiaRESUMO
Staphylococcal serine proteinase (SSP) can influence various functions of human polymorphonuclear leukocytes (PMNL) including chemotaxis and phagocytosis. Since the rise in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation, we tested the ability of SSP to increase the intracellular free calcium concentration in human PMNL using the fluorescent calcium indicator Fura-2AM. PMNL isolated from healthy donors responded to SSP in the concentration range of 10 to 100 micrograms/ml. The highest Ca2+ rise (104 +/- 47 nM) was observed for 10 micrograms/ml SSP. It was mainly dependent (81 +/- 11%) on extracellular calcium influx, however, SSP mobilized 68 +/- 7% of Ca2+ from intracellular calcium stores. Boiling of SSP or preincubation with phenylmethylsulphonylfluoride (an serine proteinase inhibitor) did not change its ability to increase intracellular free calcium concentration in PMNL. It suggests that active center of SSP is not responsible for Ca2+ mobilization. Finally, PMNL responded to each of three consecutive stimulations with SSP independently of the presence of high or low extracellular Ca2+ concentration. This may be an additional mechanism responsible for activation of human PMNL and degradation of alveolar walls during the staphylococcal infection in the lower airways.
Assuntos
Proteínas de Bactérias/farmacologia , Cálcio/metabolismo , Neutrófilos/efeitos dos fármacos , Serina Endopeptidases/farmacologia , Adulto , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Compartimento Celular , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Indóis/farmacologia , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Transporte de Íons/efeitos dos fármacos , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismoRESUMO
Platelet activating factor (PAF), a potent lipid mediator, has been implicated in the pathogenesis of airways inflammation in bronchial asthma. Binding of PAF to its receptor (Nakamura et al., 1991) leads to changes of intracellular Ca2+ concentration ([Ca2+]i) that is crucial to cell activation. Therefore, the aim of our study was to investigate whether PMNL of asthmatic patients stimulated with PAF (10(-7) M) differ in relation to changes of [Ca2+]i from cells of healthy subjects. PMNL from asthmatic patients revealed attenuated first response to PAF stimulation--increase in [Ca2+]i (delta[Ca2+]i) was 1.3-fold lower in cells of asthmatics (P < 0.05) versus PMNL of healthy subjects. As determined in experiments with low extracellular calcium concentration, Ca2+ release from internal stores tended to be increased in asthmatics and hence the difference in total Ca2+ response was related to decrease in Ca2+ influx. Thus the contribution of Ca2+ from internal stores to the total first Ca2+ response upon PAF stimulation was two-fold higher (58 +/- 18 vs. 29 +/- 8%, P < 0.001) in PMNL of asthmatics compared with healthy subjects. Two subsequent Ca2+ responses evoked by stimulations with the agonist in 1 mM Ca2+ buffer did not differ between study groups. In low Ca2+ buffer PMNL of 50% of asthmatics responded to the second stimulation while cells of healthy subjects remained unresponsive. The altered Ca2+ responses in PMNL of asthmatic subjects may reflect previous contact with mediator(s) that occur in vivo which may be at least partially explained by the phenomenon of down regulation reported for PAF receptor upon cell stimulation.
Assuntos
Asma/sangue , Cálcio/metabolismo , Neutrófilos/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adulto , Feminino , Humanos , Técnicas In Vitro , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacosRESUMO
We describe the case of a 22 yr old male patient with cystic fibrosis, who, after long-term antibiotic treatment of pulmonary infection, developed a haemorrhagic diathesis with severe bleeding from the mucus membrane of the mouth, and haematuria. Rapid recovery was observed after infusion of vitamin K. During 8 months of follow-up, no evidence of recurrence of the clotting disturbances and anaemia were noted. The combination of impaired absorption of vitamin K due to underlying disease with the antibiotic-induced suppression of vitamin K synthesis by intestinal bacteria could be a possible explanation for this disorder.
Assuntos
Cefuroxima/efeitos adversos , Cefalosporinas/efeitos adversos , Fibrose Cística/complicações , Gentamicinas/efeitos adversos , Transtornos Hemorrágicos/induzido quimicamente , Netilmicina/efeitos adversos , Pneumonia/tratamento farmacológico , Adulto , Cefuroxima/uso terapêutico , Cefalosporinas/uso terapêutico , Fibrose Cística/tratamento farmacológico , Seguimentos , Gentamicinas/uso terapêutico , Transtornos Hemorrágicos/tratamento farmacológico , Transtornos Hemorrágicos/fisiopatologia , Humanos , Masculino , Netilmicina/uso terapêutico , Pneumonia/complicações , Pneumonia/microbiologia , Fatores de Tempo , Vitamina K/uso terapêuticoRESUMO
Polymorphonuclear leukocytes isolated from peripheral blood of asthmatics appear to be primed to release more reactive oxygen species than cells of healthy subjects. The enhanced agonist-induced rise in the intracellular free calcium concentration may be responsible for this increased respiratory burst. To test this hypothesis we studied the N-formyl-methionyl-leucyl-phenylalanine- and cyclopiazonic acid--(an inhibitor of Ca(2+)-ATPase of intracellular calcium stores) induced calcium increase in the polymorphonuclear leukocytes of 28 subjects (16 with moderate asthma, 69.6% +/- 8.3% predicted normal peak expiratory flow and 12 normal controls) using a fluorescent probe Fura-2AM at 100 nM and 1 mM extracellular calcium concentrations. In 1 mN calcium, the N-formyl-methionyl-leucyl-phenylalanine-induced calcium increase was 1.7-fold higher in asthmatics than in healthy subjects. Similarly, the contribution of calcium from intracellular stores to the calcium response to N-formyl-methionyl-leucyl-phenylalanine was higher in asthmatics (55% +/- 14% vs. 39% +/- 14%, P < 0.01). The pool of calcium released from intracellular stores by N-formyl-methinoyl-leucyl-phenylalanine and cyclopiazonic acid was 2.3- and 2.2-fold larger than in control cells. There was a correlation between maximal intracellular calcium concentration related to N-formyl-methionyl-leucyl-phenylalanine-induced calcium release from intracellular stores and forced expiratory volume in 1 s expressed as percentage predicted and reversibility in asthmatics (r = 0.63, r = -0.53, P < 0.05). In conclusion, polymorphonuclear leukocytes of asthmatics exhibit an altered calcium response that is mainly dependent on increased calcium release from intracellular stores.
Assuntos
Asma/sangue , Cálcio/sangue , Neutrófilos/metabolismo , Adulto , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Estudos de Casos e Controles , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Técnicas In Vitro , Indóis/farmacologia , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Masculino , Pessoa de Meia-Idade , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacosRESUMO
Cigarette smoking causes an influx of mononuclear phagocytes and polymorphonuclear leucocytes into the lower airways. These cells have altered oxygen metabolism and release more H2O2 than phagocytes from nonsmokers. In this study, we intended to determine whether asymptomatic cigarette smokers exhale more H2O2 than healthy nonsmokers. The content of H2O2 in the expired condensate of 27 nonsmokers and 33 cigarette smokers was measured spectrofluorimetrically (homovanillic acid method). The mean H2O2 level in the expired breath condensate of all cigarette smokers was about fivefold higher than that found in the whole nonsmoker group (0.24 +/- 0.32 versus 0.05 +/- 0.11 nM). However, only 16 smokers (49%) and 6 nonsmokers (22%) had detectable levels of H2O2 in expired breath that reached values 0.49 +/- 0.28 and 0.23 +/- 0.10 nM, respectively. Although the cigarette smoking status was similar for both male and female smokers, females expired 2.5 fold less H2O2 than males (0.15 +/- 0.24 (n = 21) versus 0.38 +/- 0.39 (n = 12) nM. No correlation was found between expired H2O2 levels and cigarette smoking status expressed as the daily cigarette consumption, cumulative cigarette consumption and urinary cotinine concentration. It is suggested that in some smokers, expressed H2O2 can be a noninvasive marker of oxidant overload in the lower airways related to cigarette smoking.
Assuntos
Testes Respiratórios , Peróxido de Hidrogênio/análise , Fumar/metabolismo , Adulto , Cotinina/análise , Cotinina/urina , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Changes of [Ca2+]i in human polymorphonuclear leukocytes (PMNL) were studied. PMNL suspension was activated three times every 5 min with 10(-7) M PAF and fMLP. Both PAF and fMLP, induced three consecutive [Ca2+]i transients in PMNL suspended in medium with 1 mM Ca2+. The first Ca2+ response was a result of Ca2+ release from internal stores and the extracellular Ca2+ influx, while the second and third responses were completely dependent on Ca2+ influx from extracellular space. The contribution of Ca2+ from intracellular stores to the first PAF-induced Ca2+ response was about 1.4-fold lower in comparison with the first fMLP induced Ca2+ response (27 +/- 1 vs 37 +/- 6% (p < 0.05). Previous addition of PAF enhanced 3-fold (p < 0.001) the PMNL response to fMLP while cells pretreated with fMLP failed to increase their [Ca2+]i after challenge with PAF. PMNL from 40% of donors did not respond to PAF in the presence of 100 nM Ca2+. However, the cells responding to PAF as the cells treated with fMLP or cyclopiazonic acid released almost the entire Ca2+ from intracellular stores after challenge. Subtraction of mean [Ca2+]i transients in the presence of 100 nM Ca2+ from that obtained in medium with 1 mM Ca2+ showed that, in PMNL stimulated with PAF in contrast to the cells treated with fMLP, the onset of Ca2+ influx from extracellular space precedes Ca2+ release from intracellular stores. These results suggest that PAF-induced Ca2+ influx from extracellular space is at least partly independent of Ca2+ release from intracellular stores.
Assuntos
Fatores de Coagulação Sanguínea/farmacologia , Cálcio/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fator de Ativação de Plaquetas , Cálcio/análise , Espaço Extracelular/metabolismo , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Humanos , Líquido Intracelular/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
The increase in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation. The undecapeptide substance P can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, phagocytosis, and respiratory burst. In this study we investigated the ability of low-concentration (that can occur in vivo) substance P (10(-7) M) and its precursor alpha-protachykinin (3 x 10(-7) M) to increase the intracellular free calcium concentration in human polymorphonuclear leukocytes. Cells isolated from ten healthy donors were incubated with substance P or alpha-protachykinin in 1 mM calcium medium for 5 min and the intracellular free calcium concentration was monitored using the fluorescent calcium indicator Fura-2am. Polymorphonuclear leukocytes from 40% of donors responded to both agonists. The substance P- and alpha-protachykinin-induced increase in intracellular free calcium concentration was 59 +/- 13 nM and 58 +/- 12 nM and the extracellular calcium influx contributed to 87 +/- 8% and 54 +/- 8% of the calcium response, respectively. alpha-Protachykinin released almost all the calcium from intracellular stores, while substance P mobilized only 24 +/- 5% of this calcium pool. Finally, cells that responded to a single challenge with substance P and alpha-protachykinin were able to increase their intracellular free calcium concentration in response to each of three consecutive stimulations with these agonists. This may be an additional mechanism by which substance P and its precursor modify the function of human polymorphonuclear leukocytes.
Assuntos
Cálcio/sangue , Neutrófilos/efeitos dos fármacos , Precursores de Proteínas/farmacologia , Substância P/farmacologia , Taquicininas/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Proteínas Recombinantes/farmacologia , Valores de ReferênciaRESUMO
A rapid transient rise in the intracellular free calcium concentration ( Ca2+]i) is an important step in human polymorphonuclear leukocytes (PMNL) activation. This can be caused by many inflammatory mediators and has been implicated in the regulation of various cellular reactions. In this study we investigated the changes of [Ca2+]i in human PMNL activated three times with 10(-7)M n-formyl-methionyl-leucyl-phenylalanine (FMLP). PMNL in the presence of 1 mM Ca2+ were able to respond to three consecutive stimulations with FMLP. The first Ca2+ response was the highest one and was a result of Ca2+ release from internal stores (which was responsible for about 30% of maximal increment in [Ca2+]i) and the extracellular Ca2+ influx. Experiments with PMNL suspended in a medium containing 100 nM Ca2+ and pretreated with 1 nM Ni2+ (an inorganic calcium channel blocker) revealed that the second and third response is completely dependent on the extracellular Ca2+ influx. Changes of the time interval between stimulations had no influence on the occurrence of extracellular Ca2+ influx related to second addition of FMLP. Elongation of the time interval up to 30 min did not restore the release of Ca2+ from internal stores. It indicates the occurrence of dissociation of Ca2+ release from intracellular stores and extracellular Ca2+ influx during the second and third PMNL response to FMLP.