Human anti-Ro autoantibodies bind multiple conformational epitopes of 60-kD Ro autoantigen

Bronchial asthma is characterized by eosinophil infiltration and tissue remodeling. Matrix metalloproteinases (MMPs) are thought to play critical roles by degradating interstitial matrices in a wide range of lung diseases associated with reorganization of the airway architecture. To investigate whether MMPs are involved in the pathologic processes of bronchial asthma, we examined MMP expression in asthmatic subjects. In situ hybridization revealed abundant expression of MMP-9 (gelatinase B) mRNA in biopsy specimens from asthmatic subjects (n = 5), with an average positive cell distribution of 117.8 +/- 41.1 (mean +/- SEM)/mm2. In contrast, sparse expression of the mRNA (10.8 +/- 4.8 /mm2) was observed in specimens from normal subjects (n = 4). The vast majority of cells expressing the mRNA were eosinophils in asthmatic tissues (92.2 +/- 1.2%). MMP-9 protein, which was confined to the submucosal cells in the normal subjects, was not abundantly expressed in inflammatory cells, but there was positive reactivity for MMP-9 protein in the extracellular matrix. Immunoelectron microscopic analysis showed sparse immunolocalization of MMP-9 in the perinuclear spaces of eosinophils, but not in the granules. These findings suggest the overexpression of MMP-9 by eosinophils in bronchial tissues of asthmatic individuals, and the participation of MMPs in the pathologic changes in asthmatic airways.     

15-lipoxygenase immunoreactivity in normal and in asthmatic airways

Products of the 15-lipoxygenase (15-LO) pathway of arachidonic acid metabolism such as the mono- and di-hydroxyeicosatetraenoic acids (HETEs) may contribute to the pathophysiology of allergic airway inflammation through the recruitment and activation of inflammatory cells and stimulation of glandular secretion. In this study we have examined the expression of 15-LO and its cellular localization in the asthmatic and normal bronchial mucosa. Bronchial mucosal biopsies were obtained by fiberoptic bronchoscopy from 10 patients with symptomatic allergic asthma and six normal control subjects and processed into glycolmethacrylate resin. Sections 2 microns thick were immunostained using a specific rabbit polyclonal antihuman 15-LO antibody. Strong immunoreactivity for 15-LO was present throughout the epithelium in both the asthmatic and the normal subjects, with no difference between the two groups. Cells expressing 15-LO immunoreactivity were also present in the submucosa of both groups, with a significantly greater number present in the asthmatic group (median, 15.3 cells/mm2) than in the normal group (median, 6.9 cells/mm2) (p = 0.01). The majority (85%) of the submucosal 15-LO+ cells were eosinophils. Patchy 15-LO immunoreactivity was also seen in the vascular endothelium in both groups. These findings demonstrated increased 15-LO expression in the bronchial submucosa of asthmatic subjects, and they suggest that 15-LO products in asthma originate from both bronchial epithelium and infiltrating eosinophils.     

Airways remodeling is a distinctive feature of asthma and is related to severity of disease

PURPOSE: Airways remodeling, evaluated as the subepithelial layer thickness, was compared in asthmatic patients with that of healthy subjects, and was related to clinical grading of disease, presence of atopy, and length of asthmatic history. SUBJECTS AND METHODS: Thirty-four patients with stable asthma (mean age+/-SD: 26.5+/-9.2 years; 10 female) treated with only inhaled beta2-agonists and eight healthy volunteers (mean age+/-SD: 24.6+/-2.5 years; four female) were recruited for the study. Twenty-seven of 34 asthmatics had atopy. Eleven patients had newly diagnosed conditions (duration of disease < or = 1 year), nine patients had long asthmatic history (> 1 year and < or = 10 years), and 14 had prolonged asthmatic history (> 10 years). Bronchial responsiveness to methacholine (M) was expressed as provocative concentration of M causing a 20% fall in FEV1 (PC20) (mg/mL). Degree of asthma severity was assessed using a 0- to 12-point score based on symptoms, bronchodilator use, and daily peak expiratory flow variability over a 3-week period. Bronchoscopy and bronchial biopsy were performed successfully for all subjects; the subepithelial layer thickness, in biopsy samples, was measured from the base of bronchial epithelium to the outer limit of reticular lamina. RESULTS: In asthmatics, baseline FEV1 values (percent of predicted) ranged from 75.7 to 137.0%, and PC20 M ranged from 0.15 to 14.4 mg/mL. According to the asthma severity score, 14 asthmatics were classified as having mild disease, 14 as having moderate disease, and six as having severe disease. The mean values of subepithelial layer thickness were 12.4+/-3.3 microm (range, 6.8 to 22.1 microm) in asthmatics, and 4.4+/-0.5 microm (range, 3.8 to 5.2 microm) in healthy subjects (p<0.001). Subepithelial layer thickness of those with severe asthma differed significantly from that of patients with moderate and mild asthma (16.7+/-3.1 microm vs 12.1+/-2.7 microm and 10.8+/-2.4 microm, p<0.01 and p<0.003, respectively). Moreover, in asthmatics, degree of thickening was positively correlated to asthma severity score (Spearman rank correlation coefficient [rs]=0.581; p<0.001), and negatively correlated with baseline FEV1 (rs=-0.553; p<0.001) and PC20 M (rs=-0.510; p<0.01). No difference was found between degree of thickening observed in atopic asthmatics, compared with that of nonatopic asthmatics, or between degree of thickening in patients with different lengths of asthmatic history. Lastly, multiple regression analysis revealed that asthma severity score was the significant predictive factor for thickness of subepithelial layer. CONCLUSIONS: We confirmed that airways remodeling is a very distinctive and characteristic pathologic finding of asthma. We also demonstrated that it is related to the clinical and functional severity of asthma, but not to atopy or length of asthmatic history.     

Expression of interleukin (IL)-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI)

BACKGROUND: TDI-induced asthma exhibits clinical, functional and morphological similarities with allergen-induced asthma, suggesting that an immunological mechanism is involved in the sensitization to TDI. In vitro studies using the technique of cloning lymphocytes demonstrated that a great proportion of T-cell clones derived from bronchial mucosa of subjects with TDI-induced asthma produced IL-5 and interferon-gamma, but not IL-4, upon in vitro stimulation. OBJECTIVES: To investigate in vivo the role of IL-4 and IL-5 on the inflammatory response of the bronchial mucosa to TDI in sensitized subjects, we performed a quantitative analysis of bronchial biopsies. METHODS: We obtained bronchial biopsies from six subjects with TDI asthma 48 h after an asthmatic reaction induced by TDI challenge (challenged group), in six subjects with TDI asthma 1-4 weeks after the last exposure to TDI (chronic group), and in six non-asthmatic controls. The number of eosinophils, mast cells, T-lymphocytes, and IL-4 and IL-5 protein positive cells was determined by immunohistochemistry in the area 100 microm beneath the epithelial basement membrane. RESULTS: The characteristic increase of submucosal eosinophils, but not of mast cells and T-lymphocytes, was observed in the subjects with TDI-induced asthma when compared with controls. No differences were detected between the two groups of asthmatics. In the subjects with TDI-induced asthma, cell immunoreactivity for IL-5 was increased when compared with normal controls. There was no difference in the expression of IL-5 protein between challenged and chronic asthmatics. In contrast, the expression of IL-4 protein was increased only in the asthmatic subjects tested after recent exposure to TDI. CONCLUSIONS: We demonstrated that TDI asthma 48 h after specific bronchial challenge was associated with increased numbers of cells expressing IL-4 and IL-5, whereas chronic TDI asthma was associated with increased expression of IL-5, but not of IL-4. The results suggest that subjects who developed TDI asthma exhibit increased production of IL-5 even in the absence of a recent trigger by the exogenous sensitizer and that production of TH2-like cytokines in TDI-induced asthma may not always be co-ordinately regulated in vivo.     

Lung epithelial permeability and bronchial responsiveness in subjects with stable asthma

Lung epithelial permeability of asthmatic patients has been reported to be similar or lower than that of healthy subjects and to be correlated or not to bronchial hyperresponsiveness. To clarify these discrepancies, we evaluated 99mTc-DTPA pulmonary clearance in a group of carefully selected asthmatic patients with mild, stable asthma (n = 13; seven women; mean age +/- SD = 27.69 +/- 6.63 years), and compared them with a group of healthy, nonsmoking subjects (n = 8; six women; mean age +/- SD = 24.38 +/- 5.15 years). Selection criteria for asthmatics were as follows: baseline FEV1 > or = 80% of predicted values, no bronchial infections, and/or no asthma attacks during 4 weeks prior to study and peak expiratory flow rate variability lower than 20%, over a period of 3 weeks. Patients controlled symptoms with beta 2-adrenergic drugs only, regularly or on demand. Mean baseline FEV1 (+/-SD) as percent of predicted was 102.38 +/- 13.97 and 112.88 +/- 18.36, respectively (p < 0.05). In the asthmatic group, bronchial responsiveness to methacholine (PC20 M FEV1) ranged between 0.55 and 28.5 mg/mL. Mean value (+/-SD) of DTPA clearance from lungs to blood (evaluated on the first 10 min out of 30 min of the curves) in the asthmatic group was not different from that of control group (68.31 +/- 21.46 and 69.5 +/- 15.73). In the asthmatic patients, there was no correlation between PC20 M values and DTPA T1/2 min of the whole lung, nor between PC20 M and inner and outer lung clearance zones. Moreover, both in asthmatics and healthy subjects, DTPA clearance of outer (alveolar) zones was significantly faster than that of inner (bronchial) zones (57.69 +/- 19.94 vs 102.08 +/- 38.19, p < 0.001, and 59.75 +/- 12.49 vs 103.5 +/- 31.86, p < 0.003, respectively). Our data show that DTPA clearance in patients with stable asthma is similar to that found in healthy subjects; it is not correlated to degree of bronchial responsiveness and occurs more rapidly in the outer zones than in the inner zones, both in asthmatic patients and in healthy subjects. Thus, to date, DTPA clearance index is not a valid tool for identifying and/or monitoring asthmatic patients.     

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.     

Functional characteristics of bronchial epithelium obtained by brushing from asthmatic and normal subjects

Airways epithelial cells may be involved in the pathogenesis of asthma, but their role remains to be determined. Epithelial cells can release large amounts of 15-hydroxy-eicosatetranoic acid (15-HETE) and smaller amounts of prostaglandin E2 (PGE2) as well as fibronectin, a mediator involved in epithelial repair after injury. Epithelial cells obtained after bronchial brushing of 16 asthmatic (age 38 +/- 5 yr) and 11 normal subjects (age 36 +/- 5 yr) were studied. The percentage of epithelial cells was assessed by immunocytochemistry using an anti-cytokeratin antibody. The viability of the cells was assessed by trypan blue exclusion. The release of 15-HETE PGE2 and fibronectin was studied in resting cells and after A23187 calcium ionophore stimulation. Epithelial cells always comprised more than 86% of cells recovered, and the viability of epithelial cells was significantly (p < 0.001, Mann-Whitney U test) greater in normal subjects (54 +/- 5%) compared with asthmatic subjects (13 +/- 1%). The release of 15-HETE and fibronectin by resting epithelial cells was significantly greater in asthmatics (p < 0.05, Mann-Whitney U test) than in normal subjects. A23187 significantly (p < 0.05, Wilcoxon W test) increased the release of 15-HETE and fibronectin. There was no significant difference in the release of PGE2 by resting cells from either asthmatics or normal subjects, but challenge with A23187 induced a significant (p < 0.03, Wilcoxon W test) increase in PGE2 from cells of asthmatics but not from cells of normal subjects. This study shows that epithelial cells are activated and less viable in asthma and suggests a role for these cells in asthma.     

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.     

Central administration of the neurotensin receptor antagonist, SR48692, modulates diurnal and stress-related hypothalamic-pituitary-adrenal activity

BACKGROUND: The mechanism of bronchoobstruction in asthmatics following inhalation of lipopolysaccharide (LPS) is unclear. OBJECTIVE: In this study we have investigated the effects of the LPS on eosinophil survival rate in stimulating peripheral blood mononuclear cells (PBMCs) from patients with asthma. METHODS: PBMC supernatants from 10 asthmatic patients and 4 healthy subjects were compared for eosinophil survival rate using the same purified eosinophils. Two x 10(6) cells/ml of PBMCs were cultured for 24 hours in RPMI 1640 containing 10% fetal bovine serum along with 10 micrograms/ml of LPS. 10(6) cells/ml of eosinophils were incubated for 96 hours in the presence of PBMC-derived culture supernatants at 75% in volume. RESULTS: The viability of eosinophils incubated with PBMC supernatants from asthmatic patients stimulated with LPS was higher, 62.5% +/- 10.6% (mean +/- SD), than that without LPS, 26.9% +/- 10.8%, and also higher than that of PBMCs from healthy subjects with LPS, 40.0% +/- 15.4%. This increasing activity of asthmatic PBMC stimulated with LPS was markedly inhibited from 72.2% +/- 9.7% to 38.5% +/- 6.8% by addition of mouse anti-human GM-CSF antibody to the PBMC supernatant but not mouse anti-human IL-5 antibody. CONCLUSION: These results suggest that LPS enhances eosinophil survival in asthmatics by increasing the production of GM-CSF from PBMCs.     

Increased LTB4 metabolites and PGD2 in BAL fluid after methacholine challenge in asthmatic subjects

The bronchoconstrictor potency of inhaled methacholine is widely used to assess airway responsiveness. However, evidence has accumulated that methacholine inhalation challenge may lead to an inflammatory response in the lower respiratory tract. We therefore compared cellular, leukotriene and prostanoid profiles in bronchoalveolar lavages (BAL) obtained five hours after methacholine challenge to control lavages without prior challenge. Eight subjects with asymptomatic to mild bronchial asthma and nine nonatopic healthy controls were enrolled in the study. Without prior challenge, the percentage of BAL eosinophils was higher in the asthmatic subjects ((mean +/- SD), 1.1 +/- 0.9%) than in the control subjects (0.1 +/- 0.1%. Leukotriene B4 (LTB4), and its omega-oxidation products (20-OH-LTB4 and 20-COOH-LTB4) were the only leukotrienes detectable in the baseline BAL fluids in five of the eight asthmatic patients. After methacholine challenge, no change in BAL cell profile occurred, but in the asthmatic patients, the total amounts of LTB4 and its omega-oxidation products rose from 0.52 +/- 0.50 ng.ml-1 (pre-challenge) to 1.55 +/- 1.32 ng.ml-1 (post-challenge), and prostaglandin D2 (PGD2) rose from 49.1 +/- 15.7 (pre-challenge) to 94.4 +/- 25.4 pg.ml-1 (post-challenge), with no change in 6-keto-PGF1 alpha, thromboxane B2 (TXB2), and prostaglandins F2 alpha and E2 (PGF2 alpha and PGE2). In the healthy controls, no consistent change in BAL cell profile and mediators occurred after methacholine provocation. We conclude that inhaled methacholine stimulates LTB4 and PGD2 release in asthmatics, but not in healthy controls, without affecting the number of inflammatory cells in BAL fluid.     

Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma

Oxidative stress contributes to airway inflammation and exhaled hydrogen peroxide (H2O2) and nitric oxide (NO) are elevated in asthmatic patients. We determined the concentrations of expired H2O2 and NO in 116 asthmatic (72 stable steroid-naive, 30 stable steroid-treated, and 14 severe steroid-treated unstable patients) and in 35 healthy subjects, and studied the relation between exhaled H2O2, NO, FEV1, airway responsiveness, and eosinophils in induced sputum. Both exhaled H2O2 and NO levels were elevated in steroid-naive asthmatic patients compared with normal subjects (0.72 +/- 0.06 versus 0.27 +/- 0.04 microM and 29 +/- 1.9 versus 6.5 +/- 0. 32 ppb, respectively; p < 0.001) and were reduced in stable steroid-treated patients (0.43 +/- 0.08 microM, p < 0.05, and 9.9 +/- 0.97 ppb, p < 0.001). In unstable steroid-treated asthmatics, however, H2O2 levels were increased, but exhaled NO levels were low (0.78 +/- 0.16 microM and 6.7 +/- 1.0 ppb, respectively). There was a correlation between expired H2O2, sputum eosinophils and airway hyperresponsiveness (methacholine PC20). Exhaled NO also correlated with sputum eosinophils, but not with airway hyperresponsiveness. Our findings indicate that measurement of expired H2O2 and NO in asthmatic patients provides complementary data for monitoring of disease activity.     

Anti-inflammatory effects of inhaled beclomethasone dipropionate in nonatopic asthmatics

The effects of inhaled beclomethasone dipropionate (BDP) on asthma symptoms and infiltration of the bronchial mucosa by inflammatory cells were investigated in an open study of 10 patients with mild-to-moderate nonatopic bronchial asthma. Asthma scores were recorded in an asthma diary. Peak expiratory flow (PEF), PEF diurnal variation (PEF%), forced expiratory volume in one second (FEV1%), methacholine airway hypersensitivity (minimum dose of methacholine) (Dmin) were measured. Biopsy of the bronchial mucosa was performed before and after 8 weeks of treatment with BDP (400 micrograms.day-1). The following inflammatory cells were immunostained: eosinophils with anti-EG2; mast cells with AA1; neutrophils with NP57; T-lymphocytes with anti-CD3, CD4, and CD8; and activated T-lymphocytes with anti-CD25. There was a significant improvement in the asthma symptom score, PEF%, FEV1%, and Dmin after BDP therapy and the number of EG2-, AA1-, CD3-, CD4-, and CD25-positive cells decreased significantly. We conclude that inhaled beclomethasone dipropionate inhibited inflammatory cell infiltration of airway tissue and that associated with this there was an improvement of symptoms in this open study of inhaled beclomethasone dipropionate in a group of nonatopic asthmatic subjects.     

Comparison between nasal and bronchial inflammation in asthmatic and control subjects

Although asthma and rhinitis often coexist, it is still unknown whether they are characterized by a similar inflammatory profile. We studied eosinophilic infiltration, epithelial shedding and reticular basement membrane thickness in nasal and bronchial biopsies of six control subjects, 15 untreated allergic asthmatics with perennial rhinitis, and six corticosteroid-dependent (CSD) asthmatics. In nasal and bronchial biopsies, eosinophils were greater in untreated asthmatics than in control subjects and CSD asthmatics (p = 0.001). In untreated asthmatics, eosinophils were higher in bronchial than in nasal biopsies (p = 0.002). In nasal and bronchial biopsies, reticular basement membrane thickness was greater in untreated and CSD asthmatics than in control subjects (nasal: p < 0.008 and p < 0. 004; bronchial: p < 0.001 and p < 0.008). In untreated and CSD asthmatics, reticular basement membrane thickness was greater in bronchial than in nasal biopsies (p = 0.001; Wilcoxon's W test). Nasal epithelium was not shed in all the study groups. In untreated asthmatics, bronchial epithelium shedding was greater than in control subjects or CSD asthmatics (p < 0.005), and it was greater than nasal epithelium shedding (p < 0.006). This study has shown that, although concomitant, the extent of eosinophilic inflammation of reticular basement membrane thickness and of the epithelium shedding is greater in bronchial than in nasal mucosa of asthmatic patients with perennial rhinitis.     

Granulocyte macrophage colony-stimulating factor is the main cytokine enhancing survival of eosinophils in asthmatic airways

Interleukin (IL)-3, IL-5 and granulocyte macrophage colony-stimulating factor (GM-CSF) prolong the survival of eosinophils, which are conspicuous in asthmatic airways, but it is still controversial which one plays a major role in enhancing the survival of eosinophils in asthmatic airways. The role of these cytokines in airway eosinophilia was investigated using bronchoalveolar lavage (BAL) fluids from 11 symptomatic and nine asymptomatic patients with asthma and eight normal subjects. Eosinophil survival-enhancing activity (ESEA) was measured by a numerical change in viable eosinophils isolated from the peripheral blood of atopic patients and cultured with BAL fluids. ESEA was characterized by neutralization with antibodies to IL-3, IL-5 and/or GM-CSF. The differential count of BAL cells was achieved using Diff-Quik stain. T-cell subsets and activated T-cells were analysed by flow cytometry with dual stain using monoclonal antibodies to CD3, CD4, CD8 and CD25. ESEA was detected in eight of 11 BAL fluids of symptomatic asthma, but not in those of normal controls or asymptomatic asthmatics. In six symptomatic asthmatics, the mean percentage of inhibition in ESEA by anti-GM-CSF was higher than that of anti-IL-5 as well as anti-IL-3 (p<0.05). A mixture of antibodies to IL-3, IL-5 and GM-CSF totally inhibited the ESEA in four cases. The ESEA correlated with the percentage of eosinophils (p<0.05) and that of CD25(+)CD4 lymphocytes (p<0.05) of BAL cells. In conclusion, granulocyte macrophage colony-stimulating factor, rather than interleukin-3 or -5, is associated with eosinophil survival-enhancing activity inside the airways of symptomatic asthmatics. The activation of CD4 lymphocytes is related to the elevation of such activity.     

An accelerated increase of plasma adrenomedullin in acute asthma

A novel vasorelaxant peptide, adrenomedullin (AM), has been isolated from the acid extract of human pheochromocytoma. We have recently shown that AM inhibits histamine- and acetylcholine-induced bronchoconstriction in anesthetized guinea pigs in vivo, and this bronchodilatory effect is long-lasting. Here, we measured plasma AM concentrations in nine patients with an acute attack of bronchial asthma. The results were compared with values in 30 age-matched normal control subjects and seven age-matched stable asthmatic patients. The mean AM concentrations of patients with an acute asthma attack (98 +/- 22 pg/mL) were clearly higher than those of normal control subjects (18 +/- 2 pg/mL) and stable asthmatic patients (21 +/- 3 pg/mL). Reverse-phase high-performance liquid chromatography (HPLC) showed that the major component of plasma immunoreactive AM in patients with an asthma attack and in normal subjects equally corresponded to authentic human AM(1-52). Our results suggest that plasma AM is markedly increased in many of the patients during an acute attack of bronchial asthma, but it is not observed in stable asthmatic patients. Although this report is preliminary, the observed increase of circulating AM during an acute asthma attack may represent a compensatory mechanism against the bronchoconstriction, probably through its bronchodilatory action.     

Citric acid-induced cough thresholds in normal subjects, patients with bronchial asthma, and smokers

Several challenge procedures have been developed to characterize the cough reflex in patients with airway diseases. This study was performed to compare the interindividual range of cough sensitivity in asthmatic and normal subjects as well as smokers using an identical method. Sixteen normal subjects, 20 patients with mild bronchial asthma, 6 patients with moderate to severe bronchial asthma, 9 current smokers, and 7 occasional smokers were included. In all subjects, methacholine challenges and standardized citric acid challenges were performed. Sensitivity of the cough reflex was expressed as cough threshold, i.e., as concentration at which coughing occurred. Reproducibility was assessed in 23 subjects. Within a concentration range of 0.625-320.0 mg/ml, inhaled citric acid caused cough in all subjects. Geometric mean (range) cough threshold was 13 (2.5-160) in normal subjects, 14 (5-40) in patients with mild, and 32 (20-40) mg/ml in patients with moderate to severe asthma, 40 (20-80) in current smokers, and 119 (80-160) in occasional smokers. Cough thresholds were reproducible within one doubling concentration. In normal subjects and patients with mild bronchial asthma, thresholds were not significantly different from each other but lower than those of the other groups (p<0.05 each). Cough thresholds in smokers and patients with moderate to severe asthma did also not differ significantly and were lower than in occasional smokers (p<0.05). There was no significant correlation between cough threshold, baseline FEV subset1 , and methacholine responsiveness. Our data indicate that (1) subjects with mild asthma showed on average similar cough thresholds as normal subjects, (2) there was a large variation in cough thresholds within groups, (3) the reproducibility of cough thresholds was within one doubling concentration, (4) cough thresholds did not correlate with methacholine responsiveness or baseline airway tone. In view of the prevalence of cough as a symptom of bronchial asthma, it appears that the determination of citric acid-induced cough thresholds does not yield additional diagnostic information in these subjects.     

Pathological changes according to the severity of asthma

BACKGROUND: There have been many studies concerning pathological changes in bronchial mucosa from asthmatics; however, few studies has been carried out to evaluate pathological changes according to the severity of asthma. OBJECTIVE: This study was designed to evaluate the cellular components in bronchoalveolar lavage fluid (BALF) and histological abnormalities in asthmatics according to the severity of asthma. METHODS: Bronchoalveolar lavages, bronchoscopic biopsies and ultrastructural examinations were performed in 13 asthmatics and 11 (BAL) or four (biopsies) non-asthmatic controls. The proportions of epithelial cells and correlations with PC20Meth which reflects bronchial hyperresponsiveness. Light microscopic examination revealed loss of epithelium, inflammatory cell infiltrations and thickening of the basement membrane which also showed significant correlation with PC20Meth. Hypertrophy of airway smooth muscles and hyperplasia of mucous glands were prominent in asthmatics but there was no difference according to the severity of asthma. Ultrastructural examination revealed that basement membrane thickening on light microscopic examination is due to the increased subepithelial collagen deposition with normal thickness of basal lamina. CONCLUSION: These data suggest that loss of epithelial cells, infiltration of inflammatory cells, especially eosinophils, and increased deposition of subepithelial collagen play major roles in determining the severity of asthma and non-specific bronchial hyperresponsiveness.     

Upregulation of alpha GM-CSF-receptor in nonatopic asthma but not in atopic asthma

BACKGROUND: Intrinsic asthma is characterized by an increased number of activated eosinophils and macrophages and an increased expression of the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) in the bronchial mucosa. OBJECTIVE: This study was carried out to investigate the expression of alpha GM-CSF receptor (alpha GM-CSFr) messenger RNA and protein in the bronchial mucosa of patients with intrinsic or atopic asthma and of control subjects and to correlate the expression of alpha GM-CSFr to the number of EG2+ cells (eosinophils) and CD68+ cells (macrophages) and pulmonary function. METHODS: Nineteen patients with stable asthma (9 with atopic and 10 with intrinsic asthma) and 22 normal control subjects (12 atopic and 10 nonatopic subjects) were recruited, and FEV1 (percent predicted) and PC20 were measured before bronchoscopy. Endobronchial biopsy specimens were obtained and examined for membrane-bound alpha GM-CSFr by using in situ hybridization and immunocytochemistry. RESULTS: alpha GM-CSFr mRNA- and protein-positive cells were identified in biopsy specimens from all four groups studied. There was no significant difference in the number of cells expressing alpha GM-CSFr mRNA and protein in patients with atopic asthma compared with atopic and nonatopic control subjects. However, the numbers of alpha GM-CSFr mRNA- and protein-positive cells were significantly higher in nonatopic patients with asthma compared with atopic patients with asthma and atopic and nonatopic control subjects (p < 0.001). In the patients with intrinsic asthma, the number of alpha GM-CSFr mRNA-positive cells per millimeter of basement membrane correlated with numbers of CD68+ cells (r2 = 0.87, p < 0.001) but not with EG2+ cells, and colocalization studies demonstrated that 80% of the cells expressing alpha GMCSFr mRNA were CD68+. The expression of GM-CSF was also significantly increased in patients with intrinsic asthma compared with those with atopic asthma and control subjects (p < 0.05). In addition, in intrinsic asthma, there was a correlation between alpha GM-CSFr mRNA and FEV1 (r2 = 0.61, p < 0.05). CONCLUSION: These results demonstrate that elevated numbers of cells expressing alpha GM-CSFr can be detected in nonatopic asthma but not in atopic asthma and suggest that this increased expression is predominantly macrophage-associated and may play an important pathophysiologic role in intrinsic asthma.