Search

The isolation of human bronchial epithelial (HBEC) and fibroblastic cells (HBFC) from biopsies of asthmatic and non-asthmatic volunteers provided unique cellular materials to be used for the production of bioengineered bronchial equivalents (BE) in vitro. The HBEC are grown on a mesenchymal layer seeded with HBFC and the BE can be maintained for at least 15 days in culture. Under the BE culture conditions established previously, HBEC undergo differentiation into ciliated and goblet cells, within a pseudostratified organization comparable to human bronchi. We published previously the results from histologic and functional analyses of such BE produced exclusively using non-asthmatic HBEC and HBFC. We report here the comparative analyses of BE produced with non-asthmatic and asthmatic living HBEC and HBFC (naBE and aBE, respectively). Our data indicated that all asthmatic HBEC populations grown on a mesenchymal layer, containing non-asthmatic HBFC, slowly reached a confluent state but then detached from the matrix upon culture time. These BE appear to be very good models to study the mechanisms involved in asthma in vitro.

We have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serum-free medium supplemented with retinoic acid (5 x 10(-8) M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models will be useful for studying human bronchial properties in vitro.

Bronchial biopsies are currently used to study the pathophysiology of airway diseases, and comparisons are often made with biopsies from healthy volunteers. It is therefore important to evaluate the variability in each parameter analyzed in bronchial biopsies of healthy volunteers in order to be able to discriminate significant changes. We analyzed bronchial biopsies of 31 nonsmoking, nonatopic healthy subjects who volunteered as normal controls for studies on pathophysiology of asthma. Mean % epithelial desquamation was 23.7% of observed total epithelial length. No subepithelial fibrosis was observed. Inflammatory cell counts (/mm(2) connective tissue surface) were variable among subjects but not different between small (

Background: Natural exposure to pollen provokes an increase in airway responsiveness in nonasthmatic subjects with seasonal allergic rhinitis. This natural exposure may induce inflammatory cell recruitment and cytokine release, leading to lower airway inflammation., Objective: The aim of this study was to characterize lower airway inflammation in nonasthmatic pollen-sensitive subjects., Methods: We performed immunohistochemical tests on bronchial biopsy specimens from subjects with rhinitis who had no past or current history of asthma to evaluate cytokine expression and inflammatory cell numbers and activation both in and out of the pollen season., Results: The number of CD4(+), CD8(+), and CD45RO(+) lymphocyte subpopulations were significantly higher during the pollen season compared with the out-of-season period (P <.04). Furthermore, EG1(+) cells tended to increase after natural pollen exposure (P =.06). The number of IL-5(+) cells increased significantly after natural exposure to pollen compared with out-of-season numbers (P <.01). This increase in IL-5 expression was correlated with the numbers of CD3(+), CD4(+), CD45RO(+), and EG1(+) cells. The numbers of tryptase-positive, IFN-gamma(+), and IL-4(+) cells did not change after natural exposure., Conclusion: This study showed that natural pollen exposure was associated with an increase in lymphocyte numbers, eosinophil recruitment, and IL-5 expression in the bronchial mucosa of nonasthmatic subjects with allergic rhinitis.

Asthma is characterized histologically by a bronchial subepithelial fibrosis. Cytokines and other mediators released in the asthmatic chronic inflammatory microenvironment can activate the repair process that leads to fibroblast proliferation and collagen synthesis. To our knowledge, there are no data regarding the effect of a chronic inflammatory microenvironment on the phenotype of human bronchial fibroblasts. In the present study, we address this issue by comparing bronchial fibroblasts isolated from normal and asthmatic subjects in terms of: (a) proliferation over cell passage; (b) in vitro lifespan; (c) proliferative response to transforming growth factor-beta 1, platelet-derived growth factor-BB, dexamethasone, and retinoic acid; and (d) base-line synthesis of procollagens I and III. Bronchial fibroblasts from asthmatic subjects demonstrated lower DNA synthesis with cell passage than bronchial fibroblasts from normals. The in vitro lifespan of asthmatic bronchial fibroblasts was lower than in those from normal subjects and was significantly correlated with airway responsiveness. Platelet-derived growth factor-BB and dexamethasone increased 3H-thymidine incorporation in asthmatic bronchial fibroblasts without having any significant effect on normal fibroblast proliferation. Transforming growth factor-beta 1 and retinoic acid had no significant effect on bronchial fibroblast proliferation. Base-line procollagens I and III synthesis measurements showed no differences between normal and asthmatic fibroblasts. Taken together, these results indicate that the chronic inflammatory microenvironment found in asthma can modulate some aspects of bronchial fibroblast phenotype.

Asthma treatment has moved from bronchodilator therapy to an emphasis on anti-inflammatory therapy. Airway inflammation is believed to induce airway hyper-responsiveness (AHR) through the release of mediators that increase the airway response to agonists. However, the exact contribution of airway inflammation in the physiology of airway hyper-responsiveness remains undefined. Structural modifications in airways resulting from inflammation may contribute to the development and persistence of AHR and the development of asthma. This paper reviews some of the main components of airway inflammation and structural changes in asthma, and discusses how these processes may interact to modify airway function and induce respiratory symptoms.

Objectives: To evaluate the relationships between airway subepithelial collagen deposition and epithelial desquamation with airflow obstruction and hyperresponsiveness in different types of asthma and other respiratory conditions such as chronic cough and allergic rhinitis., Design and Participants: We compared the histopathologic features observed on bronchial biopsy specimens obtained from 80 subjects: 38 with different types of asthma, 19 with chronic cough, 13 with allergic rhinitis, and 10 normal control subjects. Each subject had a questionnaire on respiratory symptoms and medication needs, measurements of expiratory flows and methacholine responsiveness, allergy skin prick tests, and a bronchoscopy with bronchial biopsies. None of the subjects studied used bronchial anti-inflammatory agents., Results: Different degrees of bronchial subepithelial fibrosis were present in asthmatic subjects, the most intense being observed in occupational asthma; a subepithelial deposition of collagen was also found in subjects with allergic rhinitis, although it was less intense than in asthma and irregularly distributed under the basement membrane. On global analysis, we found a significant correlation between individual provocative concentration of methacholine inducing a 20% fall in FEV1 (PC20) and subepithelial fibrosis intensity (rs=-0.70, p<0.001). The degree of epithelial desquamation was correlated with that of subepithelial fibrosis (rs=0.36, p=0.02) in subjects with normal airway responsiveness, but it was not correlated with the PC20 (rs=0.10, p>0.05). Neither the degree of subepithelial fibrosis nor epithelial desquamation was correlated with the FEV1., Conclusion: These results suggest that structural airway changes such as subepithelial collagen deposition may be significant determinants or markers of a process that results in airway hyperresponsiveness.

Very little information is available on the acute histopathological bronchial alterations caused by reactive airways dysfunction syndrome (RADS). We had the opportunity to carry out sequential bronchial biopsies in a subject with RADS due to chlorine (60 h, 15 days, 2 and 5 months after the acute exposure), and also to assess spirometry and bronchial responsiveness to methacholine. A 36 year old worker in a water-filtration plant (nonsmoker) abruptly inhaled high concentrations of chlorine on September 12, 1994. He experienced immediate nasal and throat burning, retrosternal burning and wheezing, and these symptoms persisted during and after the workshift. Two days later, he complained of retrosternal burning, dyspnoea and wheezing. Inspiratory wheezing was documented. His forced expiratory volume in one second (FEV1) was 66% of predicted and the provocative concentration of methacholine causing a 20% fall in FEV1 (PC20) was slightly abnormal (2.5 mg.mL-1). On the following day, the patient underwent bronchial biopsies, which showed almost complete replacement of the epithelium by a fibrinohaemorhagic exsudate. The subject was prescribed inhaled steroids. Fifteen days after the accident, the PC20 was improved to 6 mg.mL-1. Bronchial biopsies showed considerable epithelial desquamation with an inflammatory exudate and swelling of the subepithelial space. Five weeks after the accident, the PC20 was normal (57 mg.mL-1). Inhaled steroids were stopped. Two months after the accident, the PC20 deteriorated to 4 mg.mL-1. Biopsies then showed regeneration of the epithelium by basal cells and there was still a pronounced inflammatory infiltrate. Inhaled steroids were restarted. Three and five months later, the PC20 was normal (24 mg.mL-1). Bronchial biopsies showed a greatly improved epithelium and reduction of the inflammatory infiltrate. This case report shows that reactive airways dysfunction syndrome can cause acute, marked, though partially reversible, histological abnormalities. Inhaled steroids may modulate changes in bronchial responsiveness in this condition.

Recent advances in biomedical sciences have led to the development of various methods for the evaluation of the physiopathology of respiratory diseases. This study reports morphologic and functional features of cells isolated by a new method from bronchial biopsies of normal and asthmatic subjects. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. The cells were cultured for several passages and stored frozen. Two selective culture media were used in order to obtain pure epithelial and fibroblastic cell populations. Immunofluorescence analysis of intermediate filaments, keratins, and vimentin confirmed the type of the isolated cells. The proportions of alpha-actin-expressing cells varied among the fibroblastic cell populations isolated from normal and asthmatic subjects. Interestingly, the population containing high numbers of alpha-actin-expressing cells and presenting the fastest collagen contraction kinetic was isolated from bronchial biopsies of an asthmatic subject. Moreover, the fibroblastic cells that showed the best contractile properties 24 h after their seeding in floating collagen gels were isolated from bronchial biopsies of asthmatic patients having PC20 values below 1 mg/ml. On the basis of these data, we propose a new approach to isolate, culture and characterize human bronchial cells in vitro.

This study was carried out to determine whether the increase in airway responsiveness induced by natural antigenic exposure in nonasthmatic subjects is associated with an increase in maximal bronchoconstrictor response (MBR), and if these changes could be due to an increase in airway wall thickness from allergen-induced increase in airway inflammation. In 11 nonasthmatic subjects with seasonal allergic rhinitis, a methacholine challenge was obtained monthly, during and out of pollen exposure. Each subject had a high-resolution chest tomography in and out of the pollen season, to determine the relative thickness of the right intermediary bronchus over its total diameter (T/D), as well as inflammatory cell counts, apparent basement membrane thickness as an indication of subepithelial fibrosis and epithelial desquamation in bronchial biopsy specimens. In season, the mean provocative concentration of methacholine producing a 20% decrease in forced expiratory volume in one second (PC20) decreased from 51.5 to 25.8 mg.mL-1, and the maximal post-methacholine fall in forced expiratory volume in one second (delta FEV1,max) or forced vital capacity (delta FVC) and the slope of the dose response curve (DRS) increased compared with out of season: delta FEV1,max 44 +/- 5 vs 25 +/- 5%; delta FVC 34 +/- 5 vs 16 +/- 4%; and slope of DRS 14.1 +/- 2.8 vs 6.9 +/- 1.3%/mg.mL-1. No significant change was observed in T/D ratio. The seasonal change in delta FVC was positively correlated with the delta FEV1,max (rs = 0.891) and the change in DRS (rs = 0.909), but not with the change in PC20, nor with changes in bronchial biopsy inflammatory features or T/D ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to determine 1) the features of airway inflammation after removal from exposure to high (HMW) and low (LMW) molecular weight agents 2) if there are any differences in the pattern of inflammation induced by these two types of agents, we studied 18 subjects with a recently confirmed diagnosis of occupational asthma (OA) due to HMW (n = 11) and LMW (n = 7) agents. The duration of asthma symptoms varied from 2 to 108 months (mean 33 months), and withdrawal from exposure to the sensitizing agent from 3 to 24 weeks (mean 10 weeks). All subjects underwent measurements of expiratory flow rates, methacholine inhalation tests, and a flexible bronchoscopy with bronchoalveolar lavage (BAL) and bronchial biopsies. Endoscopic findings were compared with a group of 10 normal subjects. At the time of the bronchoscopy, asthma symptoms were minimal in most subjects. Although 15/18 subjects had normal forced expiratory volume in one second (FEV1 > 80% pred), all subjects had increased airway responsiveness to methacholine (provocation concentration producing a 20% fall in FEV1 = 0.2-10.0 mg.ml-1). BAL analysis showed similar median percentages of the total number of cells and differentials in control subjects and those exposed to HMW and LMW agents. Bronchial biopsies showed that mean inflammatory cell count, both epithelial and sub-epithelial, was similarly raised in OA subjects exposed to either HMW or LMW agents, compared to controls, except for epithelial lymphocyte count. In contrast to the controls, bronchial biopsy of both groups with OA also showed other changes such as extensive epithelial desquamation, ciliary abnormalities of the epithelial cells, smooth muscle hyperplasia and subepithelial fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The physiopathology of chronic cough remains obscure. We evaluated the possibility that chronic cough in nonasthmatic subjects is associated with airway inflammation, and if this is so, what the relationship between this inflammation and the possible etiology of cough might be, as well as its response to inhaled steroids. Nineteen nonsmoking, nonasthmatic subjects referred for a persistent cough (mean: 3.8 yr) were evaluated and compared with 10 normal subjects. The evaluation included a respiratory questionnaire, a physical examination, allergy skin-prick tests, chest and sinus radiographs, esophageal pH monitoring, measurements of expiratory flows, methacholine and citric acid challenges, and flexible bronchoscopy for bronchoalveolar lavage (BAL) and bronchial biopsies. Fourteen subjects further accepted participation in a randomized, double-blind crossover trial of inhaled beclomethasone (500 micrograms four times daily) and a placebo for 1 mo each. Four groups of subjects were identified according to the presence of postnasal discharge (n = 4), gastroesophageal reflux (n = 6), both conditions (n = 5), or neither (n = 4). Subjects with chronic cough had an increased number of inflammatory cells in their bronchoalveolar lavage fluid (BALF), but there was no significant difference between the four subgroups of coughers. As compared with control subjects, the bronchial biopsies of subjects with chronic cough showed increased epithelial desquamation (p = 0.004) and inflammatory cells (p = 0.005), particularly mononuclear cells (p < 0.01), in addition to submucosal fibrosis, squamous-cell metaplasia, and loss of cilia. These findings were not significantly different between the different etiologic groups. In subjects with chronic cough, basement-membrane thickness was normal and not different from that of control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: This study looked at respiratory symptoms, peak expiratory flow rates (PEFRs), airway responsiveness to methacholine and inflammatory changes on bronchial biopsies, bronchial lavage (BL), and bronchoalveolar lavage (BAL) during natural antigenic exposure in nine subjects with pollen-sensitized seasonal asthma., Methods: The subjects recorded daily symptoms of asthma, cough and rhinitis, and morning and evening PEFRs between January and September, during and out of the pollen exposure. Baseline forced expiratory volume in 1 second, forced vital capacity, and methacholine responsiveness were measured every 3 to 4 weeks. BAL, BL, and bronchial biopsies were performed in the pollen season at the initial increase of asthma symptoms and out of pollen exposure., Results: At the time of bronchoscopy during the pollen season compared with out of season, asthmatic subjects had an increase in asthma symptom score (1.18 +/- 0.24/0.44 +/- 0.18, p < 0.05), a reduction of PEFR (407 +/- 23/442 +/- 20 L/min, p = 0.02), and a decrease in PC20 (1.15/1.48 mg/ml, p = 0.05). In asthmatic subjects, median BAL and BL cell counts and cell differentials during or out of antigenic exposure were similar, but BAL and BL eosinophils and metachromatic cells counts were always higher than in healthy subjects. In comparison with controls, biopsies obtained in asthmatic subjects showed airway lesions such as epithelial desquamation, squamous cell metaplasia, thickening of basal membrane, inflammatory cells (p < 0.05 for neutrophils), edema, and ciliary abnormalities. During pollen exposure, inflammatory signs increased, but this change was only significant for the extent of epithelial desquamation and neutrophil counts. No significant correlation was found between the intensity of airway inflammation and changes in airway responsiveness., Conclusions: In subjects with mild allergic asthma and pollen-induced asthma, seasonal antigenic exposure was associated with an increase in epithelial shedding and in the number of neutrophils on bronchial biopsies, suggesting a mild increase in baseline airway inflammation. However, these changes were not correlated with increases in airway responsiveness.

The pulmonary effects of chronic marijuana (M) and tobacco (T) smoke inhalation were studied in adult female dogs. The smoke was inhaled through a tracheostomy tube: four cigarettes containing either tobacco (3.2 g/dog) or marijuana (3.0 g/dog) were used per day over 900 days. At autopsy, the pulmonary volume and alveolar surface of the T group had decreased, compared to controls (C) and the M group. The tracheostomy (Tr) produced itself result in chiolitis; in order of severity the M group greater than T greater than Tr greater than C. The incidence of squamous metaplasia also followed this order but the T group showed more abnormalities affecting the mucosal membrane. Our findings reported that chronic inhalation of marijuana and tobacco produced the bronchiolitis with the macrophage infiltration in the wall of the terminal air-passages. This may thereafter induce the pulmonary emphysema.