Induced sputum, bronchoalveolar lavage and blood from mild asthmatics: inflammatory cells, lymphocyte subsets and soluble markers compared

Airway inflammation in asthma can be measured directly by invasive bronchoalveolar lavage (BAL), directly and relatively noninvasively by induced sputum and indirectly from peripheral blood. We compared cellular and fluid phase indices of inflammation in induced sputum, BAL and blood from 11 adults with mild stable asthma. On one day, induced sputum selected from saliva was collected and on the next, blood and BAL. Median results of sputum compared with BAL showed a higher number of nonsquamous cells (53 versus 0.8 x 10(6) cells x mL(-1), p=0.003), more neutrophils (34.3 versus 1.0%, p<0.001), CD4+ and CD19+ T-cells (76.5 versus 54.7%, p=0.01 and 5.2 versus 1.1%, p=0.03, respectively), fewer macrophages (603 versus 95.0%, p=0.002) and markedly higher levels of eosinophil cationic protein (ECP) (264 versus 2.0 microg x L(-1), p<0.001), tryptase (17.6 versus 2.2 UI x L(-1), p<0.001) and fibrinogen (1,400 versus 150 microg x L(-1), p=0.001). Sputum and BAL neutrophils and CD4+ T-cells were strongly correlated. Sputum and BAL differed from blood by having higher proportions of T-cells (94.9 and 98.9% versus 87.7%, p=0.002) and lower proportions of CD19+ T-lymphocytes (p=0.04 and 0.006). Sputum also differed from blood by having higher proportions of CD4+ T-cells (76.5 versus 51.4%, p=0.001), lower proportions of CD8+ cells (24.0 versus 403%, p=0.04) and a higher CD4+/CD8+ ratio (3.3 versus 1.4, p=0.01). We conclude that in mild asthmatics, sputum, bronchoalveolar lavage and blood measure different compartments of inflammation. Induced selected sputum has the advantage over bronchoalveolar lavage of higher density of cell recovery and stronger signal for fluid-phase markers.     

Oleamide potentiates benzodiazepine-sensitive gamma-aminobutyric acid receptor activity but does not alter minimum alveolar anesthetic concentration

Short-term exposure to ozone at peak ambient levels induces neutrophil influx and impairs lung function in healthy humans. In order to investigate the mechanisms contributing to neutrophil recruitment and to examine the role of T-cells in the acute inflammatory response, we exposed 12 healthy humans to 0.2 parts per million (ppm) of ozone and filtered air on two separate occasions for 2 h with intermittent periods of rest and exercise (minute ventilation = 30 L x min(-1)). Fibreoptic bronchoscopy was performed 6 h after the end of exposures. Total protein, tryptase, histamine, myeloperoxidase, interleukin (IL)-8 and growth-related oncogene-alpha (Gro-alpha) were measured and total and differential cell counts were performed in bronchoalveolar lavage (BAL) fluid. Flow cytometry was performed on BAL cells to study total T-cells, T-cell receptors (alphabeta and gammadelta), T-cell subsets (CD4+ and CD8+ cells) and activated T-cell subsets (CD25+). Using immunohistochemistry, neutrophils, mast cells, total T-cell numbers, T-cell subsets, CD25+ T-cells and leukocyte endothelial adhesion molecules including P-selectin, E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 were quantified in the bronchial biopsies. Paired samples were available from nine subjects. Following ozone exposure there was a threefold increase in the proportion of polymorphonuclear neutrophils (PMNs) (p=0.07) and epithelial cells (p=0.05) in BAL fluid. This was accompanied by increased concentrations of IL-8 (p=0.01), Gro-alpha (p=0.05) and total protein (p=0.058). A significant positive correlation was demonstrated between the two chemokines and proportion of PMNs in BAL fluid. After ozone exposure there was a significant decrease in the CD4/CD8 ratio (p=0.05) and the proportion of activated CD4+ (p=0.01) and CD8+ T-cells (p=0.04). However, no significant changes were demonstrable in any of the inflammatory markers studied in the biopsies. Short-term exposure of healthy humans to 0.2 ppm ozone induced a neutrophil influx in peripheral airways at 6 h post exposure, but no apparent inflammatory response in proximal airways. This response seems to be mediated at least in part by interleukin-8 and growth-related oncogene-alpha.     

Comparable efficacy of hydrogenated versus nonhydrogenated plant sterol esters on circulating cholesterol levels in humans

To study the role of T lymphocytes in asthma and its relationship with clinical severity, atopic status and bronchial hyperreactivity (BHR), we have examined T cell populations in peripheral blood of 32 stable asthmatic (18 non atopic, 14 atopic) and 10 non atopic-non asthmatic person using flow cytometry. BHR was determined with histamine challenge test. The percentages of CD4+, CD8+ T lymphocytes, CD4/CD8 ratios, T cell activation markers (IL-2R, CD25; HLA-DR) were not different from control (p > 0.05). The percentage of CD16+ CD56+ cells were higher in peripheral blood of asthmatics (15.5% versus 9.3% p < 0.05). In the asthmatic group, there was inverse correlation between BHR and CD4/CD8 ratio (p < 0.05). The percentages of CD5+ T lymphocytes bearing activation marker CD25 significantly higher in severely asthmatics when compared with mild asthmatics (13.2 +/- 2.0 versus 7.8 +/- 1.1% p < 0.05). These results suggest that natural killer activity is increased in the asthmatics, severely asthmatics have more T lymphocytes bearing activation marker CD25 and BHR would be related with CD4/CD8 ratio rather than the other T lymphocytes subpopulations in peripheral blood.     

Estimation of relative economic value for herd life of dairy cattle from profile equations

The phagocytic capability afforded by neutrophil influx into the lungs is essential to ward off invading bacteria. The objective of this study was to evaluate the effect of prior neutrophil recruitment induced by alveolar instillation of endotoxin (LPS, 200 micrograms/kg) 16 h before a pulmonary infection caused by instillation of live Pseudomonas aeruginosa ([PYO]: 1.5 x 10(8) colony-forming units [cfu]/kg) in rats. A first series of experiments showed that lipopolysaccharide (LPS) instillation induced recruitment of alveolar neutrophils that were capable, ex vivo, of elastase exocytosis, reactive oxygen species secretion, and PYO killing. In a second set of experiments, LPS followed by PYO was compared with PYO alone (n = 11 surviving rats in each group). Parameters were studied 24 h after the bacterial challenge. As compared with PYO alone, pretreatment with LPS followed by PYO was associated with decreased mortality (0% versus 54%, p < 0.05), decreased protein leakage into bronchoalveolar lavage (BAL) fluid (1.8 +/- 0.4 versus 13.5 +/- 2.2 mg/ml, p < 0.001), and improved bacterial clearance from BAL (4.0 +/- 1.4 x 10(2) versus 1.2 +/- 0.5 x 10(4) cfu/ml, p < 0.05) and from pulmonary parenchyma (8.5 +/- 6.4 x 10(5) versus 1.9 +/- 0.8 x 10(7) cfu/ml, p < 0.05). We conclude that prior alveolar endotoxin instillation induces local recruitment of functionally active neutrophils, and that this is associated with resistance to subsequent experimental pneumonia.     

IFN-gamma and IL-12 are increased in active compared with inactive tuberculosis

Cytokine-mediated immune responses to Mycobacterium tuberculosis infection are important determinants of M. tuberculosis disease development and pathology. However, the distinction between changes in cytokine profile attributable to M. tuberculosis infection and those associated with active pulmonary tuberculosis is unclear. We have compared T cells and their subsets, macrophages, and cytokine messenger RNA (mRNA) profile in the bronchoalveolar lavage (BAL) of patients with active pulmonary tuberculosis with inactive tuberculosis subjects. Ten patients with microbiologically confirmed active pulmonary tuberculosis and 25 subjects with inactive tuberculosis were recruited. Bronchoscopy with BAL was undertaken in all cases and BAL cytospins were examined using the techniques of immunocytochemistry and in situ hybridization. There was a significant increase in the percentage of BAL cells that were CD8+ T cells in active tuberculosis compared with inactive tuberculosis (mean +/- SEM: 7.2 +/- 0.9 versus 2.1 +/- 0.4, p < 0.001), but not CD3+ or CD4+ T cells nor macrophages. There were significant increases in the percentage of BAL cells expressing mRNA for interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) in active versus inactive pulmonary tuberculosis subjects (8.0 +/- 0.6 versus 3.7 +/- 0.4 and 28.4 +/- 2.3 versus 10.2 +/- 1.0, p < 0.001, respectively). There were no significant differences between the active and inactive groups in the number of cells expressing mRNA for IL-2, tumor necrosis factor-alpha (TNF-alpha), IL-4, and IL-5. In conclusion, active pulmonary tuberculosis is associated with increased numbers of CD8+ cells and marked increases in the expression of IL-12 and IFN-gamma mRNA in the BAL, both of which may be useful markers of disease activity.     

The immunopathology and clinical relevance of lymphocyte cultures in liver transplantation

Lymphocytes infiltrating human liver allograft biopsies were expanded in vitro for 3 to 5 days in recombinant IL-2 and then uniformly quantified and phenotypically characterized. The extent of proliferation was correlated with the degree and pattern of lymphocyte infiltration of the source biopsy as well as with the subsequent clinical outcome. Each of the 117 cases was assigned to one of three primary clinical outcome groups based on a retrospective evaluation of the clinical course before and after biopsy. The groups consisted of cases involving viral infection (n = 21), rejection (n = 40), or nonrejection-related allograft dysfunction (n = 56). The rejection group showed significantly greater in vitro expansion of lymphocytes (4201 +/- 685) compared to the nonrejection group (2720 +/- 408, P < 0.05). However, cases from the viral infection group showed the highest overall average lymphocyte growth (6655 +/- 2595, P < 0.05). Immunohistologic evaluation of the source liver transplant biopsy demonstrated increased T-cell infiltration of portal triads primarily by CD8+ T-cells in rejection compared to nonrejection cases (semiquantitative grade 1.3 +/- 0.1 versus 1.0 +/- 0.1, P < 0.05). The viral infection group demonstrated more significant T-cell infiltration (again predominantly CD8+) of the lobules compared to cases without viral infection (1.9 +/- 0.3 versus 1.3 +/- 0.1, P < 0.05). Immunohistologic evaluation of the cultured lymphocytes from the biopsies demonstrated a marked predominance (75% of cultures) of CD8+ T-cells compared to CD4+ T-cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of neutrophils in the pathogenesis of idiopathic pulmonary fibrosis

STUDY OBJECTIVES: The prognostic value of the neutrophil count in BAL fluid (BALF) has been controversial. The role of neutrophils in this inflammatory lung disease, therefore, was evaluated in this study by additional measures. MATERIALS AND METHODS: We performed BAL in 22 patients with idiopathic pulmonary fibrosis (IPF) diagnosed by open lung biopsy specimen. Percent polymorphonuclear leukocyte (PMN) in BALF and absolute neutrophil counts were compared with those of normal nonsmokers. Elastase complexed to alpha-1-proteinase inhibitor (alpha1-PI) in plasma and BALF was measured as a marker of elastase burden, and neutrophil distribution in 22 lung tissues was observed by immunohistochemistry using antineutrophil elastase antibody. RESULTS: Percent PMN and absolute neutrophil counts in BALF did not increase in patients with IPF as compared with normal nonsmokers (n=15); the plasma elastase-alpha1-PI complex value (mean+/-SE) of patients with IPF (668.5+/-112.4 ng/mL) was significantly high as compared with that of normal nonsmokers (130.3+/-21.3, p<0.001). In addition, the BALF elastase-alpha1-PI complex value (mean+/-SE) of patients with IPF was also significantly high (333.1+/-87.0 ng/mg albumin) as compared with that of normal nonsmokers (83.1+/-29.3 ng/mg albumin, p<0.05). Immunohistochemistry demonstrated considerable numbers of neutrophils infiltrating the lung parenchyma in biopsy specimens obtained by open lung biopsy. CONCLUSIONS: These results suggested that although the neutrophil count in BALF could not represent the distribution of neutrophil in the lung, high levels of neutrophil elastase were demonstrated in lung parenchyma and also in both BALF and sera. Therefore, neutrophils might indeed play an important role in the pathogenesis of IPF.     

Decreased surfactant protein A in patients with bacterial pneumonia

Abnormalities have been previously noted in the lipid content of the lavage fluid of patients with bacterial pneumonia. In order to determine if these changes were also seen in surfactant apoproteins, we studied levels of surfactant protein A (SP-A) in patients with bacterial pneumonia. Patients without human immunodeficiency virus who were being evaluated for pulmonary infiltrates underwent bronchoscopy with bronchoalveolar lavage (BAL). Twenty-two patients with pneumonia, 12 caused by gram-positive organisms (Gm+ PNEU) and 10 caused by gram-negative organisms (Gm- PNEU), were compared with 10 patients with idiopathic pulmonary fibrosis (IPF) and 11 control subjects (CON). The percentage of neutrophils in the BAL was significantly higher in the patients with IPF and the pneumonia groups than in the control group (CON: mean, 1; range, 0 to 3. IPF: mean, 26; range, 13 to 42). Gm+ PNEU: mean, 33; range, 8 to 99. Gm- PNEU: mean, 64; range, 10 to 92; p < 0.0001). The amount of SP-A in the BAL fluid was similar for the CON and the IPF groups (CON: mean, 15; range, 5.75 to 26.5 micrograms/ml BAL. IPF: mean, 18.4; range, 6.49 to 45.64 micrograms/ml), whereas both pneumonia groups had significantly less SP-A (Gm- PNEU: mean, 5.54; range, 0.58 to 12.7. G+ PNEU: mean, 1.93; range, 0.47 to 6.74; p < 0.001). There was significantly less SP-A in the Gm+ PNEU group than in the Gm- PNEU group (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of nitric oxide synthesis increases adenosine production via an extracellular pathway through activation of protein kinase C

At the time of diagnosis, many sarcoidosis patients have no clinical indication for corticosteroid therapy, and prognostic parameters predicting deterioration are missing. In the present study, we investigated parameters derived from bronchoalveolar lavage (BAL) and serum in 77 patients with recently diagnosed sarcoidosis to test their predictive value. Patients were divided into a group with (Group A, n = 37) and a group without (Group B, n = 40) indications for therapy, and the course of the disease was evaluated after 5.7 +/- 0.4 mo. The CD4+/CD8+ lymphocyte ratio and percentage of BAL lymphocytes were of no predictive value. Release of tumor necrosis factor-alpha (TNF-alpha) from cultured alveolar macrophages (AM) was significantly increased in both groups (Group A = 1,872 +/- 428 pg/ml; Group B = 1,561 +/- 449 pg/ml) as compared with controls (220 +/- 37 pg/ml). In Group B, however, patients with a high level of TNF-alpha release had a significantly greater risk of disease progression than did those with normal TNF-alpha release (43.8% versus 8.3%, respectively). From the serologic parameters investigated, consisting of neopterin, angiotensin converting enzyme (ACE), and soluble interleukin-2 receptor (sIL-2R), only the last was of significant predictive value; 42.1% of sarcoidosis patients in Group B with a high level of sIL-2R experienced disease progression, whereas none of those with a normal level did. We conclude that TNF-alpha release and sIL-2R are suitable parameters for predicting disease progression in sarcoid patients who have no indication for therapy at the time of disease diagnosis.     

Interactions with the pharmaceutical industry: experiences and attitudes of psychiatry residents, interns and clerks

Alteration in leukocyte activation has been implicated as an etiological factor in the development of chronic venous stasis ulcers (CVSU). The purpose of this study was to determine differences in expression of cell surface activation markers on circulating leukocytes and systemic, soluble, serum cytokine levels between healthy controls and patients with CVSU. Twenty-three patients were separated into two groups. Group I consisted of 12 healthy, adult, age-matched male patients with no venous disease. Group II consisted of 11 adult male patients with CVSU who underwent air plethysmography (APG) and duplex scanning to determine the severity of venous insufficiency. All patients had measurements of systemic, serum-based, soluble IL-1 beta, IL-2, IL-6, TNF-alpha, and beta 2 microglobulin levels. Using fluorescence flow cytometry, we measured the percentage of lymphocytes (CD3), monocytes (CD14), and granulocytes (CD15) expressing various cell surface activation markers. By APG and duplex scan, all group II patients exhibited venous insufficiency, with a mean venous filling index of 6.9 +/- 3.9 sec. Relative to group I, group II patients demonstrated a decreased expression of the CD3+/DR+ (13.3 +/- 1.5, P < or = 0.01) and CD3+/CD38+ (31.1 +/- 2.1, P < or = 0.04) markers on T-lymphocytes and an increased expression of CD14+/CD38+ (99.6 +/- 0.2, P < or = 0.008) markers on monocytes. Circulating neutrophils showed no evidence of activation. In addition, a significant elevation in the T-helper to T-suppressor ratio (2.9 +/- 0.6, P < or = 0.0001) between groups I and II was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between lung inflammation, changes in lung function and severity of exposure in victims of the Bhopal tragedy

The world's worst chemical industrial disaster, which occurred at Bhopal on 2-3 December, 1984, resulted in considerable respiratory morbidity in the exposed population. Therefore, a study was planned to evaluate the relationship between lower respiratory tract inflammation, lung function and severity of exposure. Sixty patients exposed to methyl isocyanate and presenting with respiratory symptoms were studied using bronchoalveolar lavage (BAL) 1-7 yrs after the accident. Pulmonary function tests included forced vital capacity (FVC) and forced expiratory volume in one second (FEV1). An index of severity of exposure was derived retrospectively on the basis of the acute symptoms in the victims themselves or the occurrence of death among their family members. Total lung inflammatory cells (p < 0.01) and absolute numbers of macrophages (p = 0.01) and lymphocytes (p < 0.05) increased as severity of exposure increased. FEV1/FVC % (p = 0.05) was also significantly lower as severity of exposure increased. Moderately exposed subjects had significantly lower FEV1/FVC % (p < 0.05) compared to those mildly exposed. In nonsmokers, BAL neutrophils, both percentage and absolute numbers, showed significant negative correlations with FEV1 % predicted (rs = -0.350, p < 0.05; and rs = -0.374, p < 0.01, respectively). Neutrophil percentage was negatively correlated with FEV1/FVC % (rs = -0.378; p < 0.01). Absolute lymphocytes had significant negative correlations with FVC % pred (rs = -0.318; p < 0.05). Macrophages had significant positive correlations with FVC % pred (rs = 0.322; p < 0.05) and FEV1 % pred (rs = 0.433; p < 0.01). Radiographic abnormalities (International Labour Organization (ILO) classification) were associated with decline in FEV1 % pred (p < 0.05). This study suggests that pulmonary function abnormalities occur in gas-exposed subjects as a consequence of an abnormal accumulation of lung inflammatory cells (lymphocytes and neutrophils), and that the intensity of lung inflammation and reduction in pulmonary function are greater in severely exposed subjects. As it has been observed that decline in pulmonary function is associated with radiographic abnormalities, there is a suggestion that injury following toxic gas exposure can lead to irreversible lung damage.     

BAL neutrophilia in asthmatic patients. A by-product of eosinophil recruitment?

Although neutrophil number may be increased in the airways of patients with asthma, its pathogenetic role in this disorder remains unclear. We evaluated BAL of 8 normal control subjects, 30 +/- 2 years of age, and 24 patients with mild asthma: 17 patients with allergic asthma, 24 +/- 1 years of age, and 7 patients with nonallergic asthma, 30 +/- 1 years of age. The BAL of asthmatic patients showed increased numbers of neutrophils (p < 0.01), eosinophils (p < 0.01), and ciliated epithelial cells (p < 0.05) and increased concentrations of myeloperoxidase (MPO) (p < 0.01) compared with control subjects. Positive correlations were observed between the number of BAL neutrophils and eosinophils (Rs = 0.780, p < 0.0001) and between BAL neutrophil numbers and BAL MPO levels (Rs = 0.40, p < 0.05). No correlations were found between the following: (1) BAL eosinophils or neutrophils and BAL epithelial cells (p > 0.05, each comparison); (2) BAL neutrophils or eosinophils and log Pd15 methacholine (MCh) (p > 0.05, each comparison); or (3) BAL epithelial cells or log Pd15 MCh and BAL MPO (p > 0.05, each comparison). Dividing the patient population into two groups, allergic asthmatics and nonallergic asthmatics, similar BAL neutrophil, eosinophil, and epithelial cell numbers and similar MPO levels were found (p > 0.05, each comparison). In addition, the correlations between BAL neutrophils and eosinophils showed similar significance in the two patient subgroups (p > 0.05, each comparison). These results suggest that, both in allergic and nonallergic asthma, airway recruitment and activation of neutrophils occur as does parallel eosinophil migration. However, airway neutrophils do not seem to contribute significantly to epithelial cell injury or to airway hyperresponsiveness in the steady state.     

Bronchoalveolar lavage in horses: effect of exercise and repeated sampling on cytology

Bronchoalveolar lavage (BAL) was performed at weekly intervals in 10 Thoroughbred horses in race training (group 1) and in 4 rested horses (group 2) for 10 weeks. Lavages were continued on a weekly basis in 4 group 1 horses for an additional 9 weeks (group 3). Cytological analysis of samples included leukocyte counts, erythrocyte counts, differential leukocyte counts, and haemosiderophage score. The mean leukocyte concentration was significantly lower in group 1 (92.1 +/- 4.6 cells/microL) when compared with group 2 (133.5 +/- 8.2 cells/microL), P = 0.037. The differential leukocyte data were not significantly different between groups. There was a large amount of variability in the percentage of macrophages and lymphocytes in the differential counts over time with no obvious trends. The proportion of neutrophils demonstrated a tendency to decrease over time for both groups 1 and 2. Erythrocyte counts and haemosiderin scores were significantly higher in the exercising group than the rested horses. Neither exercise nor the technique itself evoked an inflammatory response in the BAL fluid.     

Concentration-dependent block of sodium current in guinea pig ventricular myocytes by a class III antiarrhythmic agent, MS-551

The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 microM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7 +/- 2.9 adducts/10(8) nucleotides, n = 8), followed by lymphocytes (5.9 +/- 1.7, n = 8), and granulocytes (0.5 +/- 0.2, n = 8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7 +/- 1.0, n = 5) > monocytes (2.0 +/- 0.5, n = 8) > or = lymphocytes (1.6 +/- 0.4, n = 8) > granulocytes (0.8 +/- 0.2, n = 8) by NP1-enrichment and monocytes (9.0 +/- 3.2, n = 5) > or = lymphocytes (8.0 +/- 2.1, n = 6) > granulocytes (2.1 +/- 0.3, n = 7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non-smokers, except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n = 78). Adduct levels in MNC were related to daily exposure to cigarette-tar (r = 0.31, P < 0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6-fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r = 0.86, P < 0.001, n = 12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts.     

Grain dust-induced airflow obstruction and inflammation of the lower respiratory tract

To investigate the relationship between the physiologic and biologic effects of grain dust inhalation, we exposed 15 nonsmoking, nonasthmatic, nonatopic male grain handlers to buffered saline and aqueous corn dust extract by inhalation challenge in a crossover study. The inhalation challenges to buffered saline and corn dust extract were separated by at least 14 d. Compared with buffered saline, inhalation of corn dust extract resulted in significant airflow obstruction, which was observed within 30 min of exposure and persisted for 5 h. Inhalation of corn dust extract resulted in an acute inflammatory response characterized by higher concentrations of neutrophils (p = 0.001), IL-1 beta (p = 0.001), IL-1RA (p = 0.001), IL-6 (p = 0.001), IL-8 (p = 0.001), and TNF-alpha (p = 0.04) in bronchoalveolar lavage (BAL) fluid. mRNA levels specific for IL-1 beta, IL-1RA, IL-6, and IL-8 from cells present in the BAL fluid were significantly greater after challenge with corn dust extract than after challenge with buffered saline. Importantly, no significant differences were observed in the concentration of lymphocytes or eosinophils in the BAL fluid following inhalation of corn dust extract, and the concentrations of histamine and 15-HETE were similar in BAL fluid after the two challenges. The maximal percentage decrease in FEV1 was significantly associated with the absolute neutrophil concentration in the BAL fluid (p = 0.001), as well as the concentration of TNF-alpha (p = 0.03), IL-1 beta (p = 0.005), IL-1RA (p = 0.001), IL-6 (p = 0.001), and IL-8 (p = 0.001) in the BAL fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutrophil chemokines in bronchoalveolar lavage fluid and leukocyte-conditioned medium from nonsmokers and smokers

Polymorphonuclear neutrophils (PMN) have been implicated in the pathogenesis of emphysema. The chemokines interleukin-8(IL-8), growth-related oncogene (GRO-alpha) and extractable nuclear antigen (ENA)-78 may be involved in the increased numbers of PMN in smokers' airspaces. The levels of these cytokines in bronchoalveolar lavage fluid (BALF) and bronchoalveolar lavage leukocyte conditioned medium (LCM), along with BALF PMN numbers in 12 smokers who abstained for 12 h (chronic smoking) or continued to smoke until I h before study (acute smoking) and seven nonsmokers were compared. Neutrophils in BALF increased in acute (1.96+/-0.53%, 0.99+/-0.32x10(6) cells) compared with chronic smokers (0.59+/-0.25%, 0.61+/-0.24x10(6) cells, p<0.05 nonsmokers) and nonsmokers (0.79+/-0.29%, 0.05+/-0.01x 10(6) cells, p<0.05). There were no differences in IL-8 or GRO-alpha in BALF between smokers and nonsmokers. ENA-78 levels were lower in smokers (p=0.006). There was no difference in IL-8, GRO-alpha or ENA-78 in LCM from unstimulated cells in smokers versus nonsmokers. After stimulation with lipopolysaccharide (LPS) 10 ng mL(-1), IL-8 release in acute smokers (p=0.04) and GRO-alpha release in smokers (p=0.009) were significantly higher than in nonsmokers. Following stimulation with LPS 100 ng.mL(-1), GRO-alpha release was higher in smokers (p=0.03) and increased further in acute smokers (p=0.02 versus nonsmokers, p=0.04 versus chronic smokers) and ENA-78 release increased in smokers (p=0.02 versus non-smokers). In conclusion, influx of polymorphonuclear neutrophils into smokers' airspaces is an acute phenomenon and neutrophil chemokine release from mixed bronchoalveolar lavage leukocytes is influenced by cigarette smoking and endotoxins.     

Persistent airway hyperresponsiveness and histologic alterations after chronic antigen challenge in cats

We studied the effect of chronic immune sensitization on the airway reactivity and associated cytologic and histologic alterations in initially nonatopic cats, a species that spontaneously develops idiopathic asthma. Seven cats were sensitized by intramuscular injection of Ascaris suum antigen (AA) for 4 wk, and four other cats served as sham controls. Airway sensitization was demonstrated by an increased response to nebulized AA in sensitized animals (RL = 45.9 +/- 6.1 cm H2O/L/s, versus a baseline response of 24.7 +/- 1.5 cm H2O/L/s, p < 0.01), and hyperresponsiveness was demonstrated by an increased response to acetylcholine (ACh)-challenge 24 h after AA (approximately 1.0 log decrease in PD200, p < 0.01). The number of eosinophils in the sensitized animals' bronchoalveolar lavage (BAL) fluid increased 12-fold (p < 0.01 versus control) in response to AA challenge; 32 +/- 5% of the BAL eosinophils had a specific density < 1.050, versus 8 +/- 2% prior to AA challenge (p < 0.05). There was no change in airway reactivity, eosinophil recovery, or density in the control group 24 h after sham challenge with saline. The same seven sensitized cats further received nebulized AA three times weekly for 4 to 6 wk, after which BAL samples were again obtained and ACh dose-response curves generated 72 h after the final administration of nebulized AA. Airway hyperresponsiveness increased (approximately 1.5 log decrease in PD200, p < 0.001) and the number of eosinophils recovered in BAL fluid was increased 11-fold (p < 0.05). Necropsy specimens demonstrated bronchoconstriction in AA-challenged animals but not controls; luminal narrowing was accompanied by: (1) a 29.0 +/- 0.34% increase in smooth-muscle thickness (p < 0.05); (2) goblet-cell and submucosal-gland hypertrophy and hyperplasia; and (3) epithelial erosion and eosinophilic infiltration. We demonstrate in nonhuman species persistent airway hyperreactivity associated with a complete constellation of histologic changes in epithelium, smooth muscle, and mucus glands, and cytologic changes in BAL fluid, all induced by immune sensitization. Our data suggest that chronic immune sensitization per se could be a salient factor in causing many of the changes associated with chronic bronchial asthma.     

Setting Priorities in Occupational Health Research in Vietnam

It is well known that silica exposure leads in an experimental model to the development of an acute fibrotic process. In human beings two main observations have already been done: (1) silica exposure is frequently associated with the development of connective tissue disease (CTD), especially progressive systemic sclerosis; (2) 10 to 20% patients with CTD developed pulmonary fibrosis. In this context we report 26 cases of coal miners who presented with clinical, radiological, biological and functional characteristics mimicking idiopathic pulmonary fibrosis (IPF), with or without associated coal worker's pneumoconiosis (CWP). All were men; mean age was 68 +/- 9.2 years. Twenty-three were smokers. Duration of exposure was 28.8 +/- 9.1 years. All the patients had dyspnea (stage III, IV in the NHYA classification) and diffuse crackles. Eleven out of 26 had finger clubbing. Computed tomography showed honeycombing (23 cases), and/or ground glass opacities (6 cases) with bronchiectasis (3 cases) predominant in the lower lobes; 19 had radiological signs of CWP, micronodules (n = 16) and nodules (n = 3) predominant in the upper lobes. BAL exhibited an increased % of neutrophils (11.9 +/- 16.1%). Lung function demonstrated a restrictive pattern (TLC = 73 +/- 15.6% and VC = 80 +/- 18% of predicted values) associated with a decreased DLCO (51.8 +/- 23.6% of predicted values) and hypoxemia (at rest = 66.5 +/- 11.2 mmHg, upon effort = 56 +/- 12 mmHg). Lung biopsies were performed in four cases and demonstrated interstitial fibrosis of intraalveolar septum with an accumulation of immune and inflammatory cells similar to the one described in IPF. The association between IPF and silica exposure with or without associated CWP points out the problem of legal recognition of idiopathic-like pulmonary fibrosis as a complication of the occupational exposure of coal workers.     

Use of a novel electronic data collection system in multicenter studies of irritable bowel syndrome

To evaluate whether atherosclerosis may be associated with altered leucocyte rheology, we assessed leucocyte count (by Coulter counter), aggregation (by means of the leukergy test) and expression of adhesion molecules integrin LFA-1 and CD 44 (by means of immunofluorescence staining and flow cytometry) in 9 patients with carotid plus lower limb artery atherosclerosis (group A), 14 patients with carotid atherosclerosis only (group B) and 23 controls without atherosclerosis (group C). The level of LFA-1 (calculated as mean fluorescence channels-MFCs) on neutrophils, lymphocytes and monocytes was significantly higher (p < 0.05) in group A and B patients than in controls (group A-mean +/- SE: 383.77 +/- 9.42 vs 295.45 +/- 5.76; 474.22 +/- 8.86 vs 388.35 +/- 7.84; 457.66 +/- 12.03 vs 396.25 +/- 4.37. Group B: 322.42 +/- 6.36 vs 295.45 +/- 5.76; 421.42 +/- 7.21 vs 388.35 +/- 7.84; 415.71 +/- 7.73 vs 396.25 +/- 4.37, respectively); furthermore, the MFC of LFA-1 on neutrophils was significantly different (p < 0.05) between group A and B patients. The percentage of aggregated leucocytes was significantly higher (p < 0.05) in group A patients (4.46 +/- 1.07) than those in groups B (1.75 +/- 0.38) and C (1.43 +/- 0.25), whereas no significant difference was detected between groups B and C. Leucocyte number and expression of CD44 were not significantly different among the 3 groups. In conclusion, changes in leucocyte rheology are present in patients with atherosclerosis and may contribute to chronic ischaemia.     

Alloantigen presenting capacity, T cell alloreactivity and NK function of G-CSF-mobilized peripheral blood cells

In this study we addressed whether the proportion and the function of antigen presenting cells (APC), T and NK lymphocytes are modified in the apheresis product of six healthy donors who received a stem cell mobilizing treatment with glycosylated G-CSF at 10 microg/kg/day x 5 days s.c. Flow cytometry analysis showed comparable percentages of HLA-DR+, CD19+, CD86+, CD80+ and CD1a+ cells in preG-CSF-peripheral blood mononuclear cells (preG-PBMC) and after mobilization in G-PBMC, whereas the proportion of CD14+ monocytes significantly increased in G-PBMC (3+/-1% vs 17+/-8%, P = 0.003). Analysis of lymphocyte subsets in preG-PBMC and G-PBMC showed similar proportions of CD3+, CD4+, CD8+ and CD28+ T cells, but a significantly lower percentage of CD16+ (11+/-7% vs 4+/-1%, P=0.01), CD56+ (15+/-6% vs 5+/-2%, P= 0.008), CD57+ (16+/-9% vs 5+/-2%, P=0.04), CD25+ (19+/-2% vs 9+/-6%, p=0.009) and CD122+ (5+/-2% vs 2+/-1%, P = 0.05) cells in G-PBMC. Unfractionated preG-PBMC and G-PBMC were irradiated and tested in primary mixed leukocyte culture (MLC) with two HLA-incompatible responders and induced efficient alloresponses in four of six cases, whereas G-PBMC stimulated poorly in the remaining two cases. Also, in allo-MLC with irradiated G-PBMC we detected lower amounts of IFN-gamma (P = 0.04) and of IL-2 (P = 0.06) than in allo-MLC with preG-PBMC. Furthermore, freshly isolated preG-PBMC and G-PBMC from each donor exerted comparable allogeneic responses to HLA-incompatible irradiated mononuclear cells in all cases. However, G-PBMC showed no NK activity against K562 target cells at any effector:target ratio tested. These data suggest that normal G-PBMC may prevent Thl alloresponses, maintain efficient alloreactivity to HLA mismatched antigens and have impaired NK activity.