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E Puchelle

Publications and source records attributed to E Puchelle.

At least 19 recordsLinked to original sources
PubMed1992

Phospholipid composition and surface-active properties of tracheobronchial secretions from patients with cystic fibrosis and chronic obstructive pulmonary diseases.

Among the various components of tracheobronchial secretions, lipids and particularly phospholipids have been shown to influence rheological properties of airway secretions in patients with cystic fibrosis. We studied the phospholipid composition of tracheobronchial secretions, collected from patients suffering from cystic fibrosis (CF) and other chronic obstructive pulmonary diseases (COPD), and we analyzed the possible relationship between the phospholipid profile and the wettability of tracheobronchial secretions evaluated by the measurement of contact angle. Although total phospholipid content and contact angle of tracheobronchial secretions were significantly increased (P less than 0.01) in CF compared to COPD, no significant relationship existed between these two parameters. The concentrations of the different phospholipid subclasses were not homogeneously modified according to the origin of the secretions. Compared to COPD secretions, the CF secretions were characterized by a significant (P less than 0.001) increase in rigidifying fractions such as sphingomyelin and phosphatidylserine/phosphatidylinositol and a significant (P less than 0.001) decrease in surface-active fractions, such as phosphatidylcholine and phosphatidylglycerol (PG) (P less than 0.001). In the two groups, the surface-active phospholipid fraction, PG, was negatively correlated to the contact angle of tracheobronchial secretions. These results suggest that a decrease in PG content in CF secretions may be one factor responsible for an increase in their adhesivity to the respiratory mucosa, and, consequently, for mucus stasis and severity of bronchial obstruction in cystic fibrosis.

Adolescent↗
PubMed1992

Immunocytochemical analysis reveals differences between the subcellular localization of normal and delta Phe508 recombinant cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation responsible for CF is the deletion of amino acid residue Phe508, with an average allelic frequency of 70%. We have isolated an anti-CFTR monoclonal antibody which specifically recognizes recombinant normal and delta Phe508-CFTR produced by a vaccinia virus expression system. Immunocytochemical analysis of L cells expressing either normal or delta Phe508-CFTR showed a marked difference in subcellular distribution. Normal CFTR had a distinct localization in the perinuclear area and was also associated with the plasma membrane. delta Phe508-CFTR essentially lacked the membrane-associated distribution and was present throughout the cytoplasm. This heterologous expression system thus provides a model system for studying the subcellular localization of different mutant forms of CFTR.

Animals↗
PubMed1992

Epithelial respiratory cells from cystic fibrosis patients do not possess specific Pseudomonas aeruginosa-adhesive properties.

Nasal polyp cells in primary culture from cystic fibrosis (CF) and non-CF patients were compared for the ability to bind Pseudomonas aeruginosa cells and for the presence of sulphated glycoconjugates at the epithelial cell surface. Quantitation of bacterial adhesion, by scanning electronmicroscopy, showed no significant difference between the cells cultured from CF and non-CF patients. Micro-organisms associated with ciliated cells were mainly aggregated, in contrast with those from non-ciliated cells. Sulphated glycoconjugates were identified on cells cultured from both CF and non-CF patients, regardless of whether or not these cells had attached bacteria. A matrix-like material that surrounded the aggregated bacteria was more prominent on cells cultured from CF patients than on those from non-CF patients. The interaction of aggregated P aeruginosa cells with polyp cells cultured from both CF and non-CF patients appeared to occur by means of this matrix material. Our findings suggest that chronic colonisation of the airways of CF patients cannot be explained by an increased affinity between the P. aeruginosa cells and the respiratory cell surface receptors in the CF patient. Nevertheless, the in-vitro observation that the matrix surrounding the bacteria reacted with a monoclonal antibody against respiratory mucins allows us to speculate that increased mucin secretion by cells from CF patients might, in vivo, play a decisive role in the interaction between P. aeruginosa and the respiratory epithelium.

Bacterial Adhesion↗
PubMed1992

Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium.

Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.

Cystic Fibrosis↗
PubMed1992

Pseudomonas aeruginosa adhesion to normal and injured respiratory mucosa.

Human nasal polyps in outgrowth culture were used to study the adhesion of Pseudomonas aeruginosa to respiratory cells. By transmission electron microscopy, bacteria associated with ciliated cells were identified trapped at the extremities of cilia, usually as aggregates of several bacterial cells. They were never seen at the interciliary spaces or attached along cilia. Bacteria were also seen to adhere avidly to migrating cells of the periphery of the outgrowth culture. Using a model of repair of wounded respiratory epithelial cells in culture, we observed that the adhesion of P. aeruginosa to migrating cells of the edges of the repairing wounds was significantly higher than the adhesion to non-migrating cells and that adherent bacteria were surrounded by a fibronectin-containing fibrillar material. The secretion of extracellular matrix components is involved in the process of epithelium repair following injury. To investigate the molecular basis of P. aeruginosa adhesion to migrating cells, bacteria were treated with a fibronectin solution before their incubation with the respiratory cells. P. aeruginosa treatment by fibronectin significantly increased their adhesion to migrating cells. Accordingly, we hypothesize that during cell migration, fibronectin secreted by epithelial cells may favour P. aeruginosa adhesion by establishing a bridge between the bacteria and the epithelial cell receptors. Such a mechanism may represent a critical step for P. aeruginosa infection of healing injured epithelium.

Bacterial Adhesion↗
PubMed1992

Role of the physiochemical properties of mucus in the protection of the respiratory epithelium.

The respiratory mucus is a very complex biological material, which possesses both flow and deformation rheological properties, characterized by non-linear and time-dependent viscoelasticity and physical properties of adhesiveness and wettability. Viscosity and elasticity are directly involved in the transport capacity of mucus, whereas wettability and adhesiveness contribute to the optimal interface properties between the mucus and the epithelial surface. Optimal conditions for the protective and lubricant properties of respiratory mucus are represented by high wettability, and adhesiveness high enough not to induce flow of mucus in the respiratory bronchioles under gravity but low enough to mobilize mucus by airflow during coughing. An intermediate viscoelasticity is also required for an optimal mucociliary transport. Different biochemical constituents such as glycoproteins, proteins, proteoglycans and lipids are involved in the gel properties of respiratory mucus. During bronchial infection and particularly in cystic fibrosis, the loss of water and the increase in macromolecules result in a marked increase in viscosity and adhesiveness responsible for the mucus transport impairment. The various lipids present in mucus contribute differently to the physicochemical properties. Surface-active phospholipids, such as phosphatidylcholine and phosphatidylglycerol improve the wettability of mucus, whereas neutral lipids and glycosphyngolipids contribute to the hyperviscosity of mucus during infection. Phospholipids and associated mucins are also implicated in the interaction between bacteria and epithelial cells. Therefore, the respiratory mucus needs appropriate physicochemical properties for the protection, hydration and lubrication of the underlying airway epithelium.

Adhesiveness↗
PubMed1991

Semi-automatic quantitation of dense markers in cytochemistry.

The quantitation of electron dense labelling is very tedious when it is done "by hand". Accordingly we developed software allowing, at electron microscopic level, a semi-automatic counting of dense markers in biological specimens. It includes the digitization of images and extraction of dense particles from the grey level of the background. The definition of the areas of interest was carried out by the observer but all quantitative calculations were done automatically. This method was applied to different biological materials (phospholipid and lysozyme labelling in secretory granules of human submucosal bronchial gland cells). The results obtained by this semi-automatic procedure were in good agreement with those obtained by manual counting of colloidal gold labelling (r = 0.97).

Automation↗
PubMed1991

Proliferation, differentiation and ciliary beating of human respiratory ciliated cells in primary culture.

The growth, differentiation, ciliary beating pattern and frequency of human respiratory ciliated cells in primary culture were studied by scanning and transmission electron microscopy and by videomicroscopy. The epithelial cells were obtained as outgrowth from explants of adult nasal polyps. When the explants were grown on type-I and type-IV collagen substrates in a standard serum-free, hormone-supplemented medium, a high percentage of ciliated cells (range 29 +/- 5% to 37 +/- 6%) was present within 2 days of culture. After 5 days of culture, the percentage of ciliated cells near the explant was 51 +/- 5%. Most of the cultured ciliated cells (85%) were characterized by individual cilia showing a coordinated movement during the beat cycle and a beating frequency (13.3 +/- 1.3 Hz) similar to that reported in vivo. In the other 15% of the ciliated cells, the dyskinetic cilia were aggregated into clumps and characterized by a rigid and planar bending movement and a lower (P less than 0.01) beating frequency (10.7 +/- 1.4 Hz). It is suggested that the latter type of cell, already described during fetal development, might be an intermediate type of ciliated cell which appears temporarily during the surface respiratory epithelial differentiation.

Cell Differentiation↗
PubMed1991

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1991

Three-dimensional imaging of the mucus secretory process in the cryofixed frog respiratory epithelium.

Using the frog palate as a representative model of human mucociliary epithelium, we analyzed, after quick freezing fixation, the three-dimensional (3-D) respiratory mucus secretory release with high voltage (200-300 kV) transmission electron microscopy (TEM). The 3-D vision of the mucus release from the secretory cells was obtained as stereo-pairs and "bas-relief" images after analysis of stereo-pairs using an image analyzer. After standard glutaraldehyde fixation, the secretory cells showed a typical goblet shape with secretory granules heterogeneous in size and electron-density which often fuse together. On the other hand, quick-frozen secretory cells exhibited a columnar shape and their membrane-bound secretory granules contained a homogeneously dark matrix. The expanded gel mucus layer was preserved and its depth never exceeded 2 microns. When the epithelium was immersed in culture medium in presence of cholinergic agonist, a marked discharge of mucus was observed and the granules swelled at the apex of the secretory cell before being discharged in the lumen. In native cryofixed epithelium, the secretory granules exhibited a marked deformability during the process of their extrusion from the secretory cell. Clusters of secretory granules surrounded by cytoplasmic material were observed in the extracellular lumen, suggesting an apocrine-type secretion. These observations indicate that rapid cryofixation and 3-D stereoscopic imaging enable a unique opportunity to analyze, without artifact, the mucous secretory process. We speculate that, apart from the classical merocrine-type secretion mechanism, the respiratory mucus may be released, at least partly by an apocrine-type secretion.

Animals↗
PubMed1991

Role of phospholipid lining on respiratory mucus clearance by cough.

Phospholipid lining, present at the respiratory mucus-mucosa interface, may have an important role in the protective function of the airways by its abhesive properties and may also facilitate mucus transport. To mimic respiratory mucus-mucosa interface, monolayers of three different forms of phosphatidylglycerol (PG) have been deposited on glass slides by the Langmuir-Blodgett technique. Mucus adhesion and clearance by cough of mucus on these PG-coated or noncoated surfaces have been analyzed and compared, using frog respiratory mucus as "normal" mucus. Among the three PG types studied, the phosphatidylglycerol distearoyl, which is the phospholipid with the longest saturated fatty acid chain, was found to significantly improve the mucus cough clearance by decreasing the mucus work of adhesion compared with the noncoated surfaces. On the other hand, phosphatidylglycerol dipalmitoyl did not improve mucus cough clearance although it decreased mucus adhesion, and phosphatidylglycerol dioleyl did not improve either mucus cough clearance or mucus adhesion.

Animals↗
PubMed1991

Wound repair of human surface respiratory epithelium.

Surface airway epithelium is frequently injured by noxious inhaled agents, epithelial wound repair may be an important process by which the epithelial barrier integrity is maintained. To evaluate the role of surface airway cells in the wound repair process, we developed an in vitro wounding model of human nasal epithelial respiratory cells in primary culture. Circular wounds were made in the epithelial cell culture by detaching, with a glass capillary, approximately 50 cells from the collagen matrix. Video microscopy and electron microscopy observations demonstrated the contribution of two main events during the repair process: the spreading of the cells at the edge of the wounded surface, and the migration of epithelial cell sheets. Complete wound closure occurred within 5 to 8 h. The inhibition of wound repair by cytoskeleton inhibitors or cellular protein synthesis inhibitors suggested that these factors are involved in the wound repair process of surface airway epithelium.

Actin Cytoskeleton↗
PubMed1991

Differential adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells in primary culture.

Human nasal polyps in outgrowth culture were used to study the Pseudomonas aeruginosa adhesion to respiratory cells. By scanning electron microscopy, P. aeruginosa were seen associated with ciliated cells, but by transmission electron microscopy, bacteria were never seen at the interciliary spaces or attached along cilia, but were identified trapped at the extremities of cilia, usually as bacterial aggregates. A fibronectin-containing fibrillar material was seen associated with aggregated bacteria. By time-lapse video microscopy, bacteria were seen to aggregate in the culture medium following their addition to the culture wells. Progressively, these aggregates were trapped by cilia or attached to migrating cells of a lower cell layer that protruded beneath the upper layer cells, at the outgrowth periphery. P. aeruginosa adhesion to these lower cell layer migrating cells was significantly higher than to ciliated or nonciliated cells of the upper cell layer. Migrating cells were intensely labeled by the complexes Con A and arachis hypogea agglutinin (PNA)-FITC, in contrast to the other cells. The percentage of PNA-labeled cells with attached bacteria was significantly higher than that without bacteria. These results suggest that changes of cell surface glycoconjugates related with cell migration may favor P. aeruginosa adhesion to respiratory cells.

Bacterial Adhesion↗
PubMed1991

Identification of phospholipids in secretory granules of human submucosal gland respiratory cells.

Although it has been shown that tracheal epithelial cells in culture synthesize and secrete phospholipids, no direct evidence for in situ phospholipid storage in human respiratory secretory epithelial cells has been demonstrated. We used a high-resolution cytochemical enzyme-gold technique to identify and precisely localize phospholipids in human submucosal gland secretory cells. In addition, lysozyme, a specific serous cell marker, was identified using the biotinstreptavidin gold technique with lysozyme antiserum. This double labeling of phospholipids and lysozyme was performed using gold particles of diameters 15 nm and 5 nm, respectively. Quantitation of phospholipid labeling was performed on an image analyzer. Phospholipids were identified in serous granules (8.87 +/- 2.21 gold particles/microns 2) in a significantly (p less than 0.05) higher density than in mucous granules (5.57 +/- 3.07 gold particles/microns 2). These results support the hypothesis that submucosal human airway serous and mucous secretory cells produce phospholipids which may be secreted in the airway lumen.

Bronchoalveolar Lavage Fluid↗
PubMed1991

Role of simulated repetitive coughing in mucus clearance.

The role of repetitive simulated coughing on the clearance of gel mucus simulant was investigated in vitro, by using a simulated cough machine. The repetition of cough induced a significant increase (p less than 0.01) in mucus simulant clearance (139.3 +/- 78.7 mm) in comparison to a single cough (24.9 +/- 27.5 mm). Moreover, the increase in frequency of the repetitive coughing induced a marked and significant increase in mucus simulant clearance (75.4 +/- 51.1 mm and 139.3 +/- 78.7 mm at 0.1 Hz and 1.6 Hz frequency, respectively). A significant (p less than 0.05) correlation was observed between the percentage increase of clearance and both shear-thinning index (r = 0.62) and the thixotropic index (r = 0.63). These results suggest that the shear-dependent properties of mucus, associated with a repetitive coughing, may increase the efficiency of mucus clearance by air flow mechanisms.

Cough↗
PubMed1990

Production of lipocortin-like proteins by cultured human tracheal submucosal gland cells.

Evidence is obtained for the presence of lipocortin-like proteins in human tracheal gland cells in culture. Using polyclonal antibodies to lipocortin I, indirect immunofluorescence studies demonstrate that lipocortin I is mainly confined to the tracheal gland cell surface. From cell membranes, four Ca2(+)-dependent proteins (35, 40, 45 and 67 kDa) were identified as lipocortin related proteins by using immunoblotting and fluorography following [35S]methionine metabolic labeling experiments. A strong immunoreactivity for the 35 kDa protein was observed. In addition, lipocortin-like proteins with apparent Mr33, 35, 37 and 67 kDa, respectively, were released in the apical culture medium by tracheal gland cells cultured on microporous membrane of a double chamber culture system.

Annexins↗