Transient increases in albumin and hyaluronan in bronchoalveolar lavage fluid after quitting smoking: possible signs of reparative mechanisms.

Inhalation of tobacco smoke results in an accumulation of cells in the lower respiratory tract. The inflammatory response in the alveoli and lung interstitium may also be reflected by increased bronchoalveolar lavage (BAL) fluid concentrations of extracellular matrix components. The present study investigated the influence of smoking on the BAL fluid concentrations of albumin (ALB), hyaluronan (HA) and fibronectin (FN). Lavage fluids from 18 smokers were analysed before and at 1, 3, 6, 9 and 15 months after smoking cessation. Never-smokers (n = 112) served as a reference group. The total cell concentration and the concentrations of macrophages, lymphocytes, neutrophils and eosinophils were higher (P < 0.001-0.01) in smokers' BAL fluid than in never-smokers', but the values returned to normal within 9 months of smoking cessation. The HA concentration was higher (P < 0.001) in smokers' than in never-smokers' BAL fluid, but FN and ALB did not differ. Transient increases in the concentrations of ALB and HA (P < 0.01 for both) was observed within 6 months of smoking cessation. These findings indicate a temporary heightened alveolar-capillary permeability and an increased production and/or degradation of HA, being enhanced following smoking cessation. The findings probably reflect an initiation of a reparative process.

Stereoselective high-performance liquid chromatographic assay of (+/-)-delmopinol in plasma using solid-phase extraction, a chiral derivatizing agent and electrochemical detection.

An enantioselective HPLC bioanalytical method for (+/-)-delmopinol was established in order to elucidate the pharmacokinetic behaviour of this chiral drug. (+/-)-Delmopinol and (+/-)-M1652, a structurally related compound used as internal standard, were extracted from plasma by a solid-phase extraction procedure using CN cartridges. The enantiomers were derivatized with a chiral derivatizing agent (R,R)-O,O'-di-p-toluoyl tartaric acid anhydride yielding diastereomeric derivatives which were separated on a reversed-phase column with acetonitrile-0.1 M ammonium acetate buffer (65:35, v/v) pH 5.7 as mobile phase. The resolution values of the diastereomeric derivatives of (-)- and (+)-M1652 and of the derivatives of (-)- and (+)-delmopinol were 1.03 and 1.46, respectively. The limit of quantitation was approximately 3 pmol (1 ng)/enantiomer per 0.5 ml plasma using electrochemical detection (+0.75 V versus Pd/PdO reference electrode). The effectiveness of the derivatization was > 98% and the total recovery of (+/-)-delmopinol and of (+/-)-M1652 from plasma or serum was found to be approximately 50%. The assay was applied to enantioselective pharmacokinetic investigations in humans, rats and dogs but showing here only one concentration time curve of the (+)- and (-)-delmopinol in a human subject after administering (+/-)-delmopinol in form of an aqueous mouth wash solution for 60 s.

Long term effects of alumina on components of bronchoalveolar lavage fluid from rats.

Significant differences in several components of bronchoalveolar lavage fluid (BAL fluid) have previously been reported in aluminium potroom workers compared with controls. The present paper describes the long term effects in rats of one time exposure to potroom aluminium oxide without fluorides (primary alumina (PA)) or with adsorbed fluorides (secondary alumina (SA)) on components of BAL fluid. Alumina dust (40 mg) suspended in saline was instilled intratracheally; controls received saline. Bronchoalveolar lavage (BAL) was performed one, four, and 12 months after exposure. The number of cells in BAL fluid was increased significantly (p < 0.05) by SA but not PA. The increase was mainly macrophages, but the concentrations of neutrophils also increased about 10-fold one and 12 months after exposure. Although albumin and hyaluronan concentrations did not differ from those of controls, fibronectin concentrations were significantly (p < 0.001) increased one year after exposure both in PA exposed and SA exposed rats. The results indicate that SA, possibly because of adhered fluorides, induces early changes in alveolar cell populations including persistent neutrophilia. These cellular changes may have a destructive effect. The late pronounced increase of fibronectin in both PA and SA exposed rats indicates a delayed effect of alumina on the extracellular matrix.

Extracellular matrix components in bronchoalveolar lavage fluid in quartz exposed rats.

To investigate the long term effects of quartz, bronchoalveolar lavage (BAL) and analysis of lung silica were performed in rats (n = 20) one, four, and 12 months after exposure to intratracheally instilled crystalline silica. Total and relative concentrations of silica in the lungs were highest one month after exposure. At this time BAL fluid concentrations of total cells, macrophages, and lymphocytes increased five to 10-fold compared with saline instilled controls (n = 19). The number of polymorphonuclear cells (PMNs) increased about 200-fold. The increased number of PMNs persisted during the year. Furthermore, albumin and fibronectin concentrations increased continually during the year, about two to fivefold the values of controls. Hyaluronan, by contrast, increased during the four month period (about eightfold) but decreased after one year to the one month concentration. Phospholipids in BAL fluid, raised already after one month, remained high at one year. The findings suggest progressive damage of the alveolar and interstitial tissues. Moreover, the increases in components of the extracellular matrix capable of building fibrotic networks are in agreement with the microscopical findings of fibrosis. Because only total cells, macrophages, and albumin concentrations correlated weakly with the silica contents of the lung, it is unlikely that the relation between quartz burden and the reaction in the lung is simple.

Bronchoalveolar lavage fibronectin in patients with sarcoidosis: correlation to hyaluronan and disease activity.

Bronchoalveolar lavage was performed in 51 patients with sarcoidosis and in 21 healthy nonsmokers. The concentration of fibronectin was significantly higher (p less than 0.001) in lavage fluid from sarcoid patients (median 267 micrograms.l-1) than in that of controls (46 micrograms.l-1). Furthermore, a significantly higher concentration of fibronectin was found in patients with active disease than in those in whom the disease was inactive (p less than 0.001). In a six month follow-up perspective, patients with a progressive disease course had significantly higher levels of fibronectin than those who had a stable or regressive disorder (p less than 0.01). Correspondingly, lavage hyaluronan was higher (p less than 0.001) in sarcoid patients (55 micrograms.l-1) than in controls (9 micrograms.l-1) and higher (p less than 0.01) in those with active than in those with inactive disease. Patients with progressive disease had higher (p less than 0.01) concentrations of hyaluronan than those in whom the disease was stable. A significant correlation was found between lavage fibronectin levels and hyaluronan (r = 0.81, p less than 0.001). The percentage of mast cells was also higher in patients with active than in those with inactive disease (p less than 0.01) and higher in progressive than in stable sarcoidosis (p less than 0.001). Ten out of 10 patients with progressive disease had mast cells greater than or equal to 0.5%, hyaluronan greater than or equal to 50 micrograms.l-1 and fibronectin greater than or equal to 350 micrograms.l-1 compared to eight out of 41 patients with stable or regressive disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of fibronectin and its degradation products in bronchoalveolar lavage fluid.

In order to estimate, in sarcoidosis, the extent of degradation and the availability of functionally active fibronectin (FN) in the alveolar space, native and total (= native + degraded) FN were determined in the bronchoalveolar lavage (BAL) fluid of 28 non-smoking patients with sarcoidosis, 12 healthy non-smokers and 24 asymptomatic smokers. The FN was measured in unconcentrated BAL fluid in the presence of the protease inhibitor aprotinin: total FN was assayed by a double-antibody sandwich enzyme-linked immunoadsorbent assay (ELISA) and native FN by a gelatin-antibody sandwich ELISA. Both in controls and in sarcoid patients, concentrations of native FN were lower than those of total FN, indicating that FN is degraded in the alveolar space. Compared to non-smoking controls, the sarcoid patients had significantly increased concentrations of both native and total FN (p less than 0.001 for both) in the BAL fluid. Native FN in percentage of total FN was similar in non-smoking sarcoids and controls, representing about 80% and 70%, respectively. In contrast, native FN corresponded to only 30% in the group of control smokers, indicating higher degradation in smokers. Thus, in smokers, less intact collagen-binding FN is available for forming an extracellular matrix, which may be necessary for repair. On the other hand, the elevation of FN, especially in its native form, in sarcoidosis implies that more of it may be available for repair but, if in excess, also for the build-up of fibrotic tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of an inhaled corticosteroid, budesonide, on alveolar macrophage function in smokers.

Selected functions of alveolar macrophages obtained by bronchoalveolar lavage of 12 healthy smokers were examined before and after eight weeks' treatment with an inhaled glucocorticosteroid, budesonide (400 micrograms twice daily). After budesonide treatment spontaneous as well as opsonised zymosan triggered prostaglandin E2 (PGE2) secretion from harvested cells was reduced; no such reduction in opsonised zymosan triggered leukotriene B4 (LTB4) production was observed. Neither the capacity to phagocytose opsonised yeast particles nor the superoxide radical generation triggered by the calcium ionophore A23187, 4 beta-phorbol 12-myristate 13-acetate (PMA), or opsonised zymosan ex vivo were more than marginally affected by the glucocorticosteroid treatment in vivo. Lavage fluid concentrations of angiotensin converting enzyme (ACE), however, after treatment were twice those before treatment and concentrations of fibronectin were reduced to half. Albumin concentrations in lavage fluid were not affected by the glucocorticosteroid treatment. In separate experiments treatment of alveolar macrophages with 10(-7) or 10(-6) M budesonide overnight in vitro did not affect their superoxide radical or PGE2 generation but significantly blocked LTB4 release. These data indicate that inhaled gluco-corticosteroid treatment may affect synthesis or release (or both) of ACE and fibronectin by alveolar macrophages from healthy smokers whereas other functions of these cells, such as the generation of reactive oxygen derived products ex vivo, are only marginally affected.

Extracellular matrix components in bronchoalveolar lavage fluid in sarcoidosis and their relationship to signs of alveolitis.

In lung sarcoidosis, the mutual relationships of three components of the extracellular matrix, fibronectin (FN), hyaluronan (HA), and type III procollagen peptide (PCP), were investigated in the bronchoalveolar lavage (BAL) fluid. Furthermore, their relation to signs of alveolitis and of fibrosis was examined. Sarcoidosis patients (n = 74) had significantly (p less than 0.001) increased BAL fluid concentrations of FN, HA, and PCP, as well as albumin and lymphocytes, compared to controls (n = 57). The increases were significantly higher in clinically active than in inactive sarcoidosis. FN, HA, and PCP were significantly correlated to markers of alveolitis, such as albumin (r approximately 0.6-0.7; p less than 0.001) and lymphocytes (r approximately 0.4-0.5; p less than 0.001 for FN and HA; p less than 0.05 for PCP), indicating that an alveolar inflammatory process may be a prerequisite for the increased production of the three components. Since correlations between FN and HA and functional parameters (VC, TLC, FEV1.0, and DLCO) were low (r approximately 0.2-0.3, p less than 0.05-0.01 for FN and HA), the increased levels of the extracellular markers do not seem to reflect developed fibrosis. The three markers of extracellular matrix showed significant (p less than 0.001) mutual correlations in the sarcoid patients (r approximately 0.7). FN and HA were correlated even in controls (r = 0.5; p less than 0.01). The findings are in agreement with our hypothesis that these compounds may participate in the buildup of an extracellular network that supports the healing process but in excess may eventually lead to fibrosis.

Characteristics of alveolar cells and soluble components in bronchoalveolar lavage fluid from non-smoking aluminium potroom workers.

Aluminium potroom workers have been reported to develop severe pneumoconiosis and bronchial hyperreactivity. The influence of inhalation of aluminium oxide and fluorides on the alveolar milieu was studied by bronchoalveolar lavage (BAL) in 14 male non-smoking potroom workers; 28 non-smoking healthy volunteers served as controls. The total numbers, concentrations, and proportions of various alveolar cells did not differ between the groups. The concentrations of albumin and fibronectin in BAL fluid were significantly higher (p less than 0.01 for both) in the exposed workers, reflecting an increased alveolar capillary permeability and an activation of alveolar macrophages (AMs). The concentration of angiotensin converting enzyme, another AM marker, was, however, decreased (p less than 0.01) in the workers. The concentration of hyaluronan, a fibroblast marker, did not differ between the groups. AMs from workers had a decreased capacity (p less than 0.05) to interact with yeast C3b particles but not to ingest them. The expression of HLA-DR and OKM1 on the cell surfaces of AMs were equal in the two groups. The BAL findings were not accompanied by restrictive lung disease in the workers. The fact that only a discrete alveolitis was found in the potroom workers may be due to a low grade of exposure to alumina and fluorides and to frequent use of respiratory protection equipment.

100 cases of external cephalic version, with special reference to fetomaternal transfusion.

The incidence of fetomaternal transfusion (FMT) and localization of the placenta was studied in 100 attempted external versions (ECV). In only one case could a significant FMT be detected with the Kleihauer-Betke test. No major complications occurred. Sixty per cent of the attempted versions were successful. Multiparity and either dorsal or fundal placental localization were found to be favorable factors. The determination of AFP was of no use for the detection of FMT.

99mTc-DTPA clearance measured by a dual head gamma camera in healthy subjects and patients with sarcoidosis. Studies of reproducibility and relation to bronchoalveolar lavage findings.

99mTc-DTPA clearance was studied in ten healthy non smokers, five asymptomatic smokers and nine non smoking patients with sarcoidosis in the supine position with a dual head gamma camera allowing simultaneous information of regional clearance rates in frontal and dorsal projections. In the patients with sarcoidosis, a bronchoalveolar lavage was performed prior to the clearance study. DTPA clearance rate was measured during 60-90 min and data were corrected for recirculating radioactivity. The coefficients of variation for measurements on 2 consecutive days in the 10 healthy non smokers were 9%-11% for the right and left lung, anterior and posterior projections. The T1/2 calculated from total lung projections were 90-92 min for the anterior view and 84-85 min for the posterior view. Regional measurements did not add further information. No apico-basal difference was found but there was a significant fronto-dorsal gradient in 99mTc-DTPA clearance in the supine position. Smokers had significantly (P less than 0.01) faster clearance rates (T1/2 28 +/- 10 min) than healthy controls. In the sarcoidosis group clearance rates were increased in four patients and no relationships were found between DTPA clearance rates and inflammatory markers (lymphocytes, albumin, ACE) in the bronchoalveolar lavage fluid.

Relationship between serum neopterin and lymphocytic alveolitis in sarcoidosis.

We have previously found elevated levels of serum (S-) neopterin in patients with active sarcoidosis. In the present study we therefore investigated the relationship between elevated S-neopterin and sarcoid alveolitis as reflected in bronchoalveolar (BAL) findings. Patients with elevated percentages of BAL-lymphocytes (n = 19) had significantly higher (p less than 0.01) S-neopterin levels (median 12.1 nmol/L) than those (n = 11) with normal percentages (median 8.5 nmol/L). Of 19 patients with elevated lymphocyte percentage 18 had increased S-neopterin. There was a significant correlation between S-neopterin levels and the concentration (p less than 0.01) as well as the percentage (p less than 0.05) of BAL-lymphocytes. On the other hand, no correlation was found between S-neopterin and the BAL-albumin/S-albumin ratios, the concentration of macrophages or the concentration of angiotensin converting enzyme in the BAL fluid. The results indicate a close relationship between S-neopterin and lymphocytic alveolitis in sarcoidosis. Furthermore, a possible relationship between elevated S-neopterin and the ratios of T helper to T cytotoxic-suppressor cells (TH/TC-S) in blood, assumed to reflect changes of the ratios in alveolitis, was examined. However, 34 of 37 sarcoid patients had TH/TC-S ratios within the normal range. The ratios of patients with normal and elevated S-neopterin did not differ.

Exposure to microorganisms, airway inflammatory changes and immune reactions in asymptomatic dairy farmers. Bronchoalveolar lavage evidence of macrophage activation and permeability changes in the airways.

Lung function, bronchial reactivity, serum antibody levels, bronchoalveolar lavage (BAL) fluid and the magnitude of exposure to airborne microorganisms during farm work were studied in 10 healthy farmers with no respiratory symptoms. Lung function and bronchial reactivity were not altered. An increase in the amount of antibodies against several microorganisms could be demonstrated but precipitating antibodies were observed only in two subjects. In the BAL fluid the nonsmoking farmers had elevated proportion of lymphocytes and elevated levels of albumin, fibronectin and angiotensin-converting enzyme while the levels of hyaluronan and procollagen III N-terminal peptide were within normal limits. The lung function, bronchial reactivity, serum antibody levels and BAL findings were not correlated with the exposure to airborne microorganisms. We conclude that healthy farmers, exposed to mold dust, may exhibit signs of alveolitis including albumin leakage, accumulation of inflammatory cells and alveolar macrophage activation.

Human alveolar macrophages from smokers have an impaired capacity to secrete LTB4 but not other chemotactic factors.

The function of alveolar macrophages (AM phi s) was studied in terms of the secretion of various chemotactic factors. Human AM phi s were obtained by BAL from healthy non-smokers and smokers. One of the chemotactic factors was LTB4, an arachidonic acid metabolite of the lipoxygenase pathway. The amount of LTB4 was determined in culture medium, in cell homogenate and in BAL-fluid. The total chemotactic activity for neutrophils was measured in culture medium and in BAL-fluid. AM phi s from smokers showed an impaired secretion of LTB4. The spontaneous secretion in vitro was inhibited by 90% (p less than 0.05) and the stimulated one was blocked by 84% (p less than 0.05). This impairment was not followed by a decrease in total chemotactic activity, indicating the existence of other chemotactic factors than LTB4. Preliminary characterization of the chemotactic activity by gel filtration demonstrated at least four different chemotactic factors. Budesonide inhibited both the release of LTB4 and the total chemotactic activity in medium from stimulated AM phi s.

Hyaluronate in bronchoalveolar lavage fluid in sarcoidosis and its relationship to alveolar cell populations.

Hyaluronate is a potential marker of activated pulmonary fibroblasts and appears in increased amounts in bronchoalveolar lavage fluid from patients with sarcoidosis. This study was performed to investigate a possible link between the local immune response and pulmonary fibroblast proliferation. The median hyaluronate concentration in the lavage fluid from 23 sarcoid patients was 12.0 (interquartile range 7.5-28.5) micrograms/l. The hyaluronate concentration was positively correlated to the concentration and proportion of lymphocytes (p less than 0.001 and p less than 0.01, respectively) as well as to the concentrations of T lymphocyte subsets (OKT4+ p less than 0.01, OKT8+ p less than 0.05). No correlation was found between the hyaluronate concentration and the OKT4+/OKT8+ ratio. Furthermore, a significant correlation was observed in the lavage fluid between hyaluronate and angiotensin-converting enzyme, a marker of monocyte/macrophage activity (p less than 0.01). Thus, the intensity of the sarcoid alveolitis was associated with biochemical signs of pulmonary fibroblast proliferation/activation in sarcoidosis.

Subcellular localization of angiotensin-converting enzyme in the human alveolar macrophage.

The present investigation was performed in order to elucidate the subcellular localization of angiotensin-converting enzyme (ACE) in human alveolar macrophages. A pure population of alveolar macrophages was obtained by centrifugal elutriation of bronchoalveolar lavage (BAL) fluid from seven sarcoid patients. The cells were homogenized by sonication and the postnuclear supernatant was fractionated on a discontinuous sucrose gradient. Fractions of particulate material were collected and characterized by marker enzymes. The distribution pattern of ACE closely resembled that of NADPH-cytochrome-c-reductase and sialyltransferase, markers of the endoplasmic reticulum and the Golgi complex, respectively, indicating a common localization. This localization is compatible with synthesis taking place in the alveolar macrophage.

Relationship between changed alveolar-capillary permeability and angiotensin converting enzyme activity in serum in sarcoidosis.

The effect of altered alveolar-capillary permeability on angiotensin converting enzyme (ACE) activity in serum (SACE) was studied in 45 patients with sarcoidosis and 21 healthy controls. In sarcoidosis increased albumin concentrations in the bronchoalveolar lavage fluid (L albumin) and increased ratios of L albumin to albumin in serum (S albumin) indicated an increased permeability of the alveolar-capillary membrane. ACE activity in the lavage fluid (LACE) was correlated with the number of alveolar macrophages in controls, indicating that it may come from these cells. LACE was high in active sarcoidosis while in inactive disease it was similar to that in controls. SACE in sarcoidosis was significantly increased. Ninety per cent of patients with increased L albumin had increased SACE. SACE activity was significantly correlated with concentrations of L albumin and with LACE activity. The relationships between signs of increased membrane permeability and SACE and between LACE and SACE suggest that excess SACE in sarcoidosis may, at least partly, originate in the alveolar space.

Sarcoidosis in an apparently healthy volunteer.

As previously reported in this journal, alveolitis may occur in asymptomatic individuals exposed to antigens known as causative agents in hypersensitivity pneumonitis. We found an alveolitis, probably of sarcoid origin, in an apparently healthy volunteer.