The elastase inhibitory capacity and the alpha 1-proteinase inhibitor and bronchial inhibitor content of bronchoalveolar lavage fluids from healthy subjects

Pulmonary emphysema is currently thought to be due to an elastase-antielastase imbalance with resultant destruction of alveolar structures. The present study was aimed at testing whether alpha 1-proteinase inhibitor (alpha 1 PI) is the major component of the antielastase screen of the lower respiratory tract of healthy subjects. Bronchoalveolar lavage was performed in 8 nonsmokers (27.8 +/- 3.8 years) and 9 smokers (25 +/- 0.96 years). The lavage fluids were tested for leukocyte and pancreatic elastase inhibitory capacity (LEIC and PEIC) and immunoreactive alpha 1 PI and bronchial inhibitor (brI) content. The mean +/- s.e.m. levels of LEIC, PEIC, alpha 1 PI and brI were 0.16 +/- 0.039, 0.042 +/- 0.006, 0.09 +/- 0.007 and 0.013 +/- 0.002 mol/mol albumin, respectively. Thus, on the average, the molar concentration of brI was about 14% that of alpha 1 PI. The difference between LEIC and alpha 1 PI did not reach statistical significance (P = 0.0503). The PEIC was however significantly lower than the alpha 1 PI levels (P less than 0.05), indicating that the lavage fluids contained both active and inactive alpha 1 PI. Nonsmokers and smokers did not differ in their LEIC, PEIC, alpha 1 PI and brI levels. When the data were examined on an individual basis, the subjects could be divided into 2 groups: group I (n = 9; 3 nonsmokers, 6 smokers) whose LEIC/alpha 1 PI molar ratios were higher than unity and group II (n = 8; 5 nonsmokers, 3 smokers) whose LEIC/alpha 1 PI molar ratios were equal or lower than unity. Group I subjects had significantly higher LEIC values (0.26 +/- 0.05 mol elastase inhibited/mol albumin) than group II individuals (0.055 +/- 0.006; P less than 0.001) but the two groups had similar levels of immunoreactive alpha 1 PI (0.09 and 0.08 mol alpha 1 PI/mol albumin for group I and II, respectively), functionally active alpha 1 PI (percentage of active alpha 1 PI: 53% and 37% for group I and II, respectively) and immunoreactive brI (0.016 and 0.010 mol brI/mol albumin for group I and II, respectively). These results suggested that the lavage fluids from group I contained significant amounts of undefined leukocyte elastase inhibitor(s). Gel filtration of a lavage fluid from group I showed that the undefined elastase inhibitor(s) co-eluted with brI. Most of the lavage fluids were still able to inhibit leukocyte elastase following removal of alpha 1 PI by perchloric acid precipitation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Qualitative studies of lung lavage alpha 1-proteinase inhibitor

A method is described which enables identification of the molecular size of alpha 1-proteinase inhibitor (alpha 1-PI) in biological fluids. This technique when applied to bronchoalveolar lavage fluids clearly demonstrates alpha 1-PI in three molecular forms; the native molecule (Mr approximately equal to ++54 000), a partially proteolysed form (Mr approximately equal to 49 000) and in a form suggestive of a complex with enzyme (Mr approximately equal to 82 000). Samples showing the presence of native alpha 1-PI inhibited more porcine pancreatic elastase than samples where no native alpha 1-PI was seen or where the predominant form was partially proteolysed alpha 1-PI (p less than 0.01). Although the predominant band of alpha 1-PI was more frequently the partially proteolysed form in current smokers (p less than 0.01), there was no clear difference in the inhibitory function of alpha 1-PI between current smokers and non-smokers and those with and without airflow obstruction.     

Smoking and COPD increase sputum levels of extracellular superoxide dismutase

Extracellular superoxide dismutase (ECSOD) is the major superoxide-scavenging enzyme in the lung. Certain ECSOD polymorphisms are protective against COPD. We postulated that smokers and COPD subjects would have altered levels of ECSOD in the lung, airway secretions, and/or plasma. Lung tissue ECSOD was evaluated from nonsmokers, smokers, and subjects with mild to very severe COPD by Western blot, immunohistochemistry, and ELISA. ECSOD levels in plasma, bronchoalveolar lavage fluid (BALF), and induced-sputum supernatants were analyzed by ELISA and correlated with smoking history and disease status. Immunohistochemistry identified ECSOD in extracellular matrix around bronchioles, arteries, and alveolar walls, with decreases seen in the interstitium and vessels of severe COPD subjects using digital image analysis. Plasma ECSOD did not differ between COPD subjects and controls nor based on smoking status. ECSOD levels in induced sputum supernatants were elevated in current smokers and especially in COPD subjects compared to nonsmokers, whereas corresponding changes could not be seen in the BALF. ECSOD expression was reduced around vessels and bronchioles in COPD lungs. Substantial increases in sputum ECSOD in smokers and COPD is interpreted as an adaptive response to increased oxidative stress and may be a useful biomarker of disease activity in COPD.     

Cigarette smoking depletes cells spontaneously secreting Th(1) cytokines in the human airway

Cigarette smoking may modify the immune balance in the airway since it alters the course of diseases in which immune system has an important role. This study examined whether cigarette smoking could affect the distribution of cells secreting Th(1) or Th(2) cytokines in the human airway. We utilized cytokine ELISPOT assay to detect and quantitate the frequencies of cells spontaneously secreting cytokines in bronchoalveolar lavage fluid (BALF). BALF was collected from six non-smokers and four heavy cigarette smokers without clinical airway symptoms. Cytokine ELISPOT assay was performed to quantitate cells secreting interleukin (IL-)2, IL-4 and interferon (IFN-)gamma with or without phorbor 12-myristate 13-acetate (PMA) stimulation. There were no cells spontaneously secreting IL-2 detected in all samples from smokers whereas most of non-smokers had detectable IL-2-secreting cells. The number of IFN-gamma-secreting cells was also extremely decreased in smokers. Mitogen-stimulated Th(1) cytokine-secreting cells were again significantly decreased in smokers' airways. The frequency of IL-2-secreting cells and CD4/CD8 ratio in BALF had a weak positive correlation. IL-4-secreting cells were not detected in any samples from both groups. These results show that cigarette smoking depletes Th(1) cytokine-secreting cells in the human airway. It may explain the susceptibility of smokers to certain airway disease conditions such as viral or mycobacterial infections and allergic diseases.     

Bronchoalveolar lavage fluid obtained from smokers exhibits increased monocyte chemokinetic activity

Alveolar macrophages, which are cells derived from blood monocytes, accumulate within the lower respiratory tract of cigarette smokers. One mechanism to account for this accumulation of alveolar macrophages may be an increase in the migration of blood monocytes into the lungs of smokers. To evaluate this hypothesis, bronchoalveolar lavage fluid (BALF) was obtained from 15 smokers and 16 nonsmokers. The smokers' BALF possessed a significantly increased capacity to attract normal blood monocytes when evaluated using a blind-well chamber technique (26.2 +/- 7.6 vs 14.8 +/- 6.9 cells/high-power field, P less than 0.01). Checkerboard analysis of the activity revealed that it was predominantly chemokinetic. Partial characterization of the activity in smokers' BALF revealed that it was lipid soluble but only partially sensitive to trypsin and heat. The chemokinetic activity correlated with alveolar macrophage numbers in the BALF (r = 0.4391, P = 0.009). Furthermore, both the chemokinetic activity and alveolar macrophage number correlated with alterations of respiratory function (forced expiratory volume in 1 s, diffusing capacity for carbon monoxide, and forced expiratory flow at 75% of the vital capacity). These results suggest that the increase in alveolar macrophage number present in the BALF of cigarette smokers may be due, at least in part, to an increased amount of chemokinetic factor(s) in the smokers' BALF, and these factor(s) may participate in the decline of respiratory function associated with cigarette smoking, probably by recruiting monocytes into lung.     

Flow cytometric evaluation of lymphocyte subpopulations in BALF of healthy smokers and nonsmokers

The purpose of this work was to evaluate the normal lymphocyte phenotype in the bronchoalveolar lavage fluid (BALF). BAL was carried out in 12 untreated healthy nonsmoking volunteers and in 9 cigarette smokers. For the analysis of lymphocyte subsets by two-color flow cytometry, the monoclonal antibodies used were directed anti: CD3, CD4, CD8, CD16, CD19, D25, CD45, CD56 and anti HLA-DR. An increase in the total number of cells in BALF of smoking persons and increased proportion of macrophages was observed. The percentage of CD8+ lymphocytes was 1.7 times higher, whereas the proportions of CD4+ cells, and a CD4+/CD8+ ratio were lower 1.5 and 2.6 times, respectively, in the BALF of cigarette smoking persons when compared with nonsmoking volunteers. The changes did not depend on the age of the person. In conclusion, we suggest that the decreased CD4/CD8 ratio and the elevated CD8 T cell subset may be regarded as a potential risk factor associated with clinically asymptomatic lung cancer. Moreover, in the interpretation of BALF from patients with pulmonary diseases cell proportions of nonsmoking and of smoking persons should be compared with the respective controls.     

Systemic and pulmonary oxidative stress in idiopathic pulmonary fibrosis.

An oxidant/antioxidant imbalance has been proposed in patients with idiopathic pulmonary fibrosis (IPF). We tested this hypothesis by measuring various parameters of the oxidant/antioxidant balance in the plasma of 12 patients with IPF (7 nonsmokers and 5 smokers); in the bronchoalveolar lavage fluid (BALF) of 24 patients with IPF (17 nonsmokers and 7 smokers) and 31 healthy subjects (23 nonsmokers and 8 smokers). The trolox equivalent antioxidant capacity (TEAC) in plasma and BALF was lower in nonsmoking patients with IPF (plasma 0.55+/-0.1 mM, p<.001; BALF 4.8+/-1.2 microM, mean +/-SEM, p<.01), compared with healthy nonsmokers (plasma 1.33+/-0.03 mM; BALF 10+/-2 microM). Similar trends in plasma and BALF TEAC were observed in smoking patients with IPF in comparison with healthy smokers. The decrease in BALF TEAC was concomitant with a decrease in BALF protein thiol levels, but the decrease TEAC levels in plasma in IPF patients was not accompanied by a decrease in protein thiol levels. Reduced glutathione (GSH) was lower in BALF in nonsmoking patients with IPF (1.0+/-0.1 microM) compared with healthy nonsmokers (2.3+/-0.2 microM, p<.001). In contrast, GSH levels were higher in smoking patients with IPF (5.2+/-1.1 microM, p<.001) than in nonsmoking patients. GSSG levels were not different in any of the groups. The levels of products of lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS) in plasma and BALF were significantly increased in both smoking (plasma 2.2+/-0.5 microM, p<.01; BALF 0.18+/-0.04 microM, p<.001), and nonsmoking (plasma 2.1+/-0.3 microM, p<.01; BALF 0.22+/-0.05 microM, p<.001) IPF patients, compared with healthy nonsmokers (plasma 1.4+/-0.3 microM; BALF 0.05+/-0.004 microM). These data show evidence of oxidant/antioxidant imbalance in the lungs of patients with IPF, which is also reflected as systemic oxidant stress.     

Effects of fluticasone propionate inhalation on levels of arachidonic acid metabolites in patients with chronic obstructive pulmonary disease

BACKGROUND: In smoking COPD patients the bronchoalveolar lavage (BAL) fluid contains high numbers of inflammatory cells. These cells might produce arachidonic acid (AA) metabolites, which contribute to inflammation and an increased bronchomotor tone. AIMS: To investigate levels of AA metabolites in BAL fluid, before and after inhaled glucocorticoid therapy: fluticasone propionate (FP) 1 mg per day, or placebo. METHODS: A double-blind placebo controlled trial lasting six months. COPD patients were selected by clinical criteria and the presence of bronchial hyper-responsiveness (BHR). Lung function was recorded and in BAL fluid we counted cell numbers and measured LTB4, LTC4/D4/E4, PGE2, 6kPGF1alpha, PGF2alpha and TxB2. A control group consisted of asymptomatic smokers (n=6). RESULTS: Paired data were obtained from 9 FP treated and 11 placebo patients. BAL cells were almost exclusively alveolar macrophages. In patients and controls both cellularity and levels of AA metabolites were equal Cell numbers did not change after treatment. Statistically significant decreases after FP therapy were noticed for PGE2 (30%), 6kPGF1alpha (41%) and PGF2alpha (54%). CONCLUSIONS: In COPD, the capability of inflammatory cells to produce certain AA metabolites was decreased after inhaled FP treatment. This result is discussed in its relation to clinical effects, the influence of smoking, and the results of an earlier, similar study in asthma patients.     

Functional activity of phagocytes of bronchoalveolar lavage in patients with fibro-cavernous and infiltrative pulmonary tuberculosis

Functional activity of the bronchoalveolar lavage fluid (BALF) phagocytes was studied in 33 and 16 patients with fibro-cavernous and infiltrative pulmonary tuberculosis (FCPT and IPT, respectively). Complex examination of BALF, alveolar macrophages and neutrophils sedimented from BALF has shown interrelationship between functional activity of the cells and the form of pulmonary tuberculosis. Higher neopterin content and activity of elastase mainly secreted into BALF by activated alveolar macrophages and neutrophils, respectively, reflect higher secretory activity of both types of cells in FCPT. In FCPT this is combined with higher bactericidal activity of neutrophils, which significantly correlates with their adenosine deaminase (ADA) activity. Comparison of changes of the biochemical parameters studied in BALF (neopterin, elastase, ADA and its isoenzymes, 2-deoxy-ADA) and bactericidal activity of the sedimented cells obviously reflects different sides of BALF phagocytes functioning. Taking into consideration modern concepts on the mechanisms of regulation of phagocyte cells one may suggest the existence of differences in intercellular interactions in various forms of pulmonary tuberculosis.     

白介素-18在被动吸烟诱导的慢性阻塞性肺疾病大鼠中的表达

目的 研究白介紊-18(Interleukin-18,IL-18)在被动吸烟诱导的慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)大鼠中的表达变化.方法 将20只大鼠随机分为2组,即正常对照组和COPD模型组.应用单纯被动吸烟法建立大鼠COPD模型,香烟烟雾暴露时间为6个月.利用酶联免疫吸附法测定2组大鼠血清和支气管肺泡灌洗液(bronchoal veolar lavage fluid,BALF)中的IL-18浓度,用实时定量RT-PCR法测定BALF中IL-18 mRNA的表达水平,用HE染色法观察肺组织形态学改变,用免疫组织化学染色法检测IL-18在肺组织中的表达.结果 1.COPD模型组血清和BALF中的IL-18浓度较正常对照组显著增加(P＜0.01);2.COPD模型组BALF中IL-18 mRNA的表达水平较正常对照组显著增高(P＜0.01);3.COPD模型组肺组织中IL-18的表达较正常对照组显著增加(P＜0.01).结论 被动吸烟诱导的COPD大鼠外周血和肺部均高表达IL-18,提示IL-18在吸烟所致的COPD发病机制中可能起重要作用.     

Clara cell protein (CC16) in serum and bronchoalveolar lavage fluid of subjects exposed to asbestos

The Clara cell protein (CC16) is a small and readily diffusible protein of 16kDa secreted by bronchiolar Clara cells in the distal airspaces. These epithelial cells are altered in several pulmonary pathological processes induced by various lung toxicants. In the search for a new biomarker of asbestos-induced lung impairment, we used a sensitive immunoassay to determine the levels of CC16 in bronchoalveolar fluid (BALF) and serum of subjects exposed to asbestos compared with a group of healthy controls. In the BALF of asbestos-exposed subjects there was an insignificant trend towards CC16 elevation compared with controls, with a (mean ±SD of 0.81 ±0.65mg l^-1 for asbestos-exposed subjects (n = 23) versus 0.39 ±0.19mg l^-1 for controls (n = 11) (p = 0.09). In serum, CC16 concentration was significantly increased among asbestos-exposed subjects, with values of 27.2 ±24.0 µg l^-1 for asbestos-exposed subjects (n = 34) versus 16.1 ±7.6 µg l^-1 for controls (n = 34) (p = 0.01). Regarding the effects of smoking, there were significant differences between generally lower CC16 levels in serum and BALF (p = 0.05 and 0.001, respectively) of smokers compared with the higher levels in non-smokers. Serum CC16 levels positively correlated with those in BALF, which is consistent with a diffusional transfer of CC16 from the bronchoalveolar space into the serum. No association, however, emerged between the levels of CC16 in serum or BALF and either the duration of asbestos exposure or the severity of the lung impairment as assessed by chest X-ray. These findings suggest that exposure to asbestos elicits early changes in the local and, importantly, also the systemic levels of CC16. This pneumoprotein therefore appears as a promising non-invasive biomarker of asbestos-induced lung injury and occupational disease in both smoking and non-smoking exposed subjects.     

Oxidation of alpha 1-protease inhibitor: role of lipid peroxidation products

Previously we demonstrated that in vivo exposure of humans to NO2 resulted in significant inactivation of alpha 1-protease inhibitor (alpha 1-PI) in the bronchoalveolar lavage fluid. However, alpha 1-PI retains its elastase inhibitory activity in vitro when exposed to 10 times the concentration of NO2 used in vivo. We suggested exogenous oxidants such as O2 and NO2 exert their effect in vivo in part through lipid peroxidation. We investigated the mechanism of inactivation of alpha 1-PI in the presence or absence of lipids under oxidant atmosphere. alpha 1-PI in solutions containing phosphate buffer (control), 0.1 mM stearic acid (saturated fatty acid, 18:0), or 0.1 mM linoleic acid (polyunsaturated fatty acid, 18:2) was exposed to either N2 or NO2 (50 ppm for 4 h). Elastase inhibitory capacity of alpha 1-PI was significantly diminished in the presence of 0.1 mM linoleic acid and under NO2 atmosphere (75 +/- 8% of control, P less than 0.01), whereas there was no change in elastase inhibitory capacity of alpha 1-PI in the presence or absence (buffer only) of 0.1 mM stearic acid under a similar condition (109 +/- 11 and 94 +/- 6%, respectively). The inactivated alpha 1-PI as the result of peroxidized lipid could be reactivated by dithiothreitol and methionine sulfoxide peptide reductase, suggesting oxidation of methionine residue at the elastase inhibitory site. Furthermore the inhibitory effect of peroxidized lipid on alpha 1-PI could be prevented by glutathione and glutathione peroxidase and to some extent by alpha-tocopherol.     

Altered airway surfactant phospholipid composition and reduced lung function in asthma.

Pulmonary surfactant in bronchoalveolar lavage fluid (BALF) and induced sputum from adults with stable asthma (n = 36) and healthy controls (n = 12) was analyzed for phospholipid and protein compositions and function. Asthmatic subjects were graded as mild, moderate, or severe. Phospholipid compositions of BALF and sputum from control subjects were similar and characteristic of surfactant. For asthmatic subjects, the proportion of dipalmitoyl phosphatidylcholine (16:0/16:0PC), the major phospholipid in surfactant, decreased in sputum (P < 0.05) but not in BALF. In BALF, mole percent 16:0/16:0PC correlated with surfactant function measured in a capillary surfactometer, and sputum mole percent 16:0/16:0PC correlated with lung function (forced expiratory volume in 1 s). Neither surfactant protein A nor total protein concentration in either BALF or sputum was altered in asthma. These results suggest altered phospholipid composition and function of airway (sputum) but not alveolar (BALF) surfactant in stable asthma. Such underlying surfactant dysfunction may predispose asthmatic subjects to further surfactant inhibition by proteins or aeroallergens in acute asthma episodes and contribute to airway closure in asthma. Consequently, administration of an appropriate therapeutic surfactant could provide clinical benefit in asthma.     

Differences in acute lung response to elastase instillation in two rodent species may determine differences in severity of emphysema development

Elastase intratracheal instillation induces early emphysema in rodents. However, Syrian Golden hamsters develop more severe emphysema than Sprague-Dawley rats. We have reported species differences in oxidant/antioxidant balance modulating antiprotease function early after instillation. We now hypothesize that other components of the initial lung response to elastase might also be species-dependent. Sprague-Dawley rats and Syrian Golden hamsters received a single dose of pancreatic elastase (0.55 U/100 g body wt) to study acute lung injury biomarkers. Using serum, lung, and bronchoalveolar lavage fluid (BALF) samples, we evaluated changes in alveolar-capillary permeability, alpha 1-antitrypsin (α(1)-AT) concentration and activity, glutathione content, and proinflammatory cytokines. Rats showed a large increase in alveolar-capillary permeability and few hemorrhagic changes, whereas hamsters exhibited large hemorrhagic changes (P < 0.01) and mild transendothelial passage of proteins. Western blots showed a 30-fold increase in BALF α(1)-AT concentration in rats and only a 7-fold increase in hamsters (P < 0.001), with [α(1)-AT-elastase] complexes only in rats, suggesting differences in antiprotease function. This was confirmed by the α(1)-AT bioassay showing 20-fold increase in α(1)-AT activity in rats and only twofold increase in hamsters (P < 0.001). In rats, results were preceded by a 3-, 60-, and 20-fold increase in IL-6, IL-1β, and TNF-α respectively (P < 0.001). In hamsters, only IL-1β and TNF-α showed mild increases. All parameters studied were back to baseline by 4 days. In conclusion, several components of the initial lung response showed species differences. Cytokine release pattern and functional inhibition of α(1)-AT were the most significant components differing among species and could account for differences in susceptibility to elastase.     

Proteomic analysis of pulmonary edema fluid and plasma in patients with acute lung injury

Proteomics is the large-scale analysis of protein profiles. This approach has not yet been reported in the study of acute lung injury (ALI). This study details protein profiles in plasma and pulmonary edema fluid (EF) from 16 ALI patients and plasma and bronchoalveolar lavage fluid (BALF) from 12 normal subjects. More than 300 distinct protein spots were evident in the EF and BALF of both normal subjects and ALI patients. Of these, 158 were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the plasma and EF protein profile of ALI patients, there were multiple qualitative changes. For instance, in all normal subjects, but in only one of the ALI patients, seven distinct surfactant protein A isoforms were evident. Nearly all ALI patients also had protein spots that indicate truncation or other posttranslational modifications. Several of these novel changes could serve as new biomarkers of lung injury.     

General and local reactions in the proteolytic system during the development of experimental pneumonia

Proteolytic enzymes and their inhibitors were studied in the serum and bronchoalveolar lavage of rats with acute and chronic experimental pulmonary inflammation. An increase in the inhibitory potential of the blood serum and bronchoalveolar lavage at the acute stage of inflammation was shown. At the chronic stage of pulmonary inflammation the level of the main inhibitors decreased to normal or zero values in the blood and lavage fluid, respectively. It was observed that alpha 1-proteinase inhibitor serum concentration correlated with the level of bronchoalveolar antitrypsin.     

O3-induced change in bronchial reactivity to methacholine and airway inflammation in humans

The increase in airway responsiveness induced by O3 exposure in dogs is associated with airway epithelial inflammation, as evidenced by an increase in the number of neutrophils (polymorphonuclear leukocytes) found in epithelial biopsies and in bronchoalveolar lavage fluid. We investigated in 10 healthy, human subjects whether O3-induced hyperresponsiveness was similarly associated with airway inflammation by examining changes in the types of cells recovered in bronchoalveolar lavage fluid obtained after exposure to air or to O3 (0.4 or 0.6 ppm). We also measured the concentrations of cyclooxygenase and lipoxygenase metabolites of arachidonic acid in lavage fluid. We measured airway responsiveness to inhaled methacholine aerosol before and after each exposure and performed bronchoalveolar lavage 3 h later. We found more neutrophils in the lavage fluid from O3-exposed subjects, especially in those in whom O3 exposure produced an increase in airway responsiveness. We also found significant increases in the concentrations of prostaglandins E2, F2 alpha, and thromboxane B2 in lavage fluid from O3-exposed subjects. These results show that in human subjects O3-induced hyperresponsiveness to methacholine is associated with an influx of neutrophils into the airways and with changes in the levels of some cyclooxygenase metabolites of arachidonic acid.     

Lipid peroxidation and antielastase activity in the lung under oxidant stress: role of antioxidant defenses

The role of lipid peroxidation in the inactivation of alpha 1-protease inhibitor (alpha 1-PI) in the alveolar lining fluid of human subjects has been examined under oxidant stress. Exposure to nitrogen dioxide (NO2) at 4 ppm for 3 h resulted in a significant increase in the amount of lipid peroxidation products in the alveolar lining fluid, with conjugated dienes the predominant species. Four-week supplementation with vitamins C and E before NO2 exposure markedly decreased the levels of conjugated dienes (control 804 +/- 103 pmol/micrograms total phospholipids vs. vitamin-supplemented 369 +/- 58, P = 0.003). Malondialdehydes, although detectable in the lavage fluid, contributed little to the total amount of lipid peroxidation products, and the levels were comparable in both groups. NO2 exposure in the absence of vitamin supplementation caused a significant decrease in the elastase inhibitory capacity (EIC) of the alveolar lining fluid in the control group but not in the vitamin-supplemented group [control 3.67 +/- 0.32 micrograms alpha 1-PI/micrograms porcine pancreatic elastase (PPE) vs. vitamin-supplemented 2.75 +/- 0.17, P less than 0.03]. The vitamin-supplemented group had a lower level of conjugated dienes and a higher EIC. Conversely, the control group had higher levels of conjugated dienes and a lower EIC in their lavage fluid. These studies demonstrate that lipid peroxidation occurs as an early event during oxidant exposure in the lungs of normal subjects. The appearance of lipid peroxidation products in the lavage fluid is associated with a decrease in the EIC of the alveolar lining fluid. Vitamins C and E diminish lipid peroxidation and preserve the EIC of the lower respiratory tract fluid during oxidant stress.     

Metabolomic analysis of bronchoalveolar lavage fluid from cystic fibrosis patients

Metabolite profiles of bronchoalveolar lavage fluid (BALF) from pediatric patients with cystic fibrosis (CF) were correlated to the degree of airway inflammation using nuclear magnetic resonance spectroscopy-based metabolomics. BALF was collected from 11 children with CF during clinically indicated bronchoscopy. The spectra from BALF with high levels of neutrophilic airway inflammation displayed signals from numerous metabolites, whereas the spectra from subjects with low levels of inflammation were very sparse. The metabolites identified in samples taken from subjects with high inflammation include known markers of inflammation such as amino acids and lactate, as well as many novel signals. Statistical analysis highlighted the most important metabolites that distinguished the high- from the low-inflammation groups. This first demonstration of metabolomics of human BALF shows that clear distinctions in the metabolic profiles can be observed between subjects experiencing high versus low inflammation and is a first step toward the goal of discovering novel biomarkers of airway inflammation.     

Acid-stable low molecular mass proteinase inhibitors in human lung lavage

An acid-stable, low molecular mass proteinase inhibitor, bronchial mucus proteinase inhibitor (BMPI), has been isolated from sputum and partially characterised. A single band with a modal molecular mass of 18 700 was observed following SDS-polyacrylamide gel electrophoresis. BMPI inhibited human leukocyte elastase, cathepsin G, trypsin and chymotrypsin, but not porcine pancreatic elastase. Although BMPI had a molecular mass close to the similarly isolated inhibitor of Girard et al. (Girard, F., Tournier, J.M., Polu, J.M. & Sadoul, P. (1980), Bull. Eur. Physiopathol. Respir. 16 (Suppl.) 237-245), and although it showed immunological cross reactivity to the low molecular mass inhibitor of Kramps et al. (Kramps, J.A., Franken, C., Meyer, C.J.L.M. & Dijkman, J.H. (1981) J. Histochem. Cytochem. 29, 712-719), it was found to have an amino-acid profile different to any previously described inhibitor. BMPI was detectable in bronchoalveolar lavage fluid collected from healthy and diseased human lungs. The median molar ratio of BMPI/alpha 1-proteinase inhibitor (alpha 1 PI) observed in these lavage samples was 0.7, which is generally higher than those derived from the data of other authors. This suggests that BMPI is a different protein to those previously described, although its exact relationship to other low molecular mass proteinase inhibitors remains to be determined.