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Biomedical subjects

E Puchelle

Publications and source records attributed to E Puchelle.

At least 91 records · Page 5Linked to original sources
PubMed1993

Quantitative analysis and cartography in scanning electron microscopy: application to the study of bacterial adhesion to respiratory epithelium.

This paper describes the coupling between a scanning electron microscope (SEM) and an image analysis workstation. The system was designed in order to drive the SEM and to analyse any sample. It allows automatic (edge detection) or semiautomatic (pointing, marking, drawing) object detection. Two types of data can be obtained: (1) topographical information, such as the location of the object within a region of interest drawn at any magnification of the microscope, or (2) quantitative data, such as morphometric characteristics of objects. In addition, high resolution maps of the section, regions of interest, and objects can be obtained with a laser printer. This software was first applied to quantitate the adhesion of the bacteria Pseudomonas aeruginosa to human respiratory epithelial cells in culture. P. aeruginosa was shown associated with ciliated cells. The second application concerned the study of the distribution of specific carbohydrate residues at the surface of the respiratory cells. The gal residues were revealed using the lectin Ricinus communis agglutinin II, adsorbed to colloidal gold particles. A relationship between the presence of adherent bacteria and labelling was shown.

Bacterial Adhesion↗
PubMed1993

Epidermal growth factor promotes wound repair of human respiratory epithelium.

Reepithelialization of the airway mucosa is an essential step toward restoring a normal functional protective barrier during the repair of airway epithelial wounds. We investigated the role of epidermal growth factor in the wound healing of human surface epithelial cells cultured from nasal polyp explants on a type I collagen gel in serum-free defined medium. By using image analysis techniques, we measured the outgrowth area, the ciliated surface, the ciliary beating frequency, and the in vitro wound repair rate in the presence of different epidermal growth factor concentrations. We observed a significant dose-dependent increase in the outgrowth area (10-fold increase with epidermal growth factor doses of 0 to 20 ng/ml), in the percentage of the outgrowth surface covered by ciliated cells (30% without epidermal growth factor and 43% with epidermal growth factor 20 ng/ml) and in the ciliary beating frequency (12.6 to 14.5 Hz). The wound repair rate was improved by 29% in the presence of epidermal growth factor 10 ng/ml. These results suggest that epidermal growth factor could be involved in the wound repair process of the airway epithelium.

Journal Article↗
PubMed1993

Carbocisteine improves the mucociliary transport rate in rats with SO2-induced bronchitis.

In order to study the effect of carbocisteine on the mucociliary function of the respiratory tract, we performed a double-blind study on rats with SO2-induced (400 ppm) hypersecretion. During the experimental bronchitis, the treated group of rats received carbocisteine through a stomach tube at a dose level of 500 mg/kg for 15 days, whereas the untreated group of rats received distilled water. After killing the rats, and following lung excision, the respiratory mucus was scraped off and collected by using a glass capillary. The mucus degree of purulence was macroscopically estimated and the mucus transport rate was measured by using the frog palate technique. The mean mucus relative transport rate, measured on the frog palate, was 0.60 +/- 0.17 in the untreated group and was significantly higher (P < 0.01) in the treated group (0.73 +/- 0.14). Carbocisteine also significantly altered (P < 0.01) the mucus macroscopical aspect, leading to a decrease in the number of rats with purulent mucus. These results suggest that carbocisteine maintains an efficient mucus transport rate, leading to a less infected respiratory tract.

Animals↗
PubMed1993

Tubule formation by human surface respiratory epithelial cells cultured in a three-dimensional collagen lattice.

Human surface respiratory epithelial (HSRE) cells from nasal polyps have been cultured within collagen lattices in a serum-free defined medium. Cell growth observed over a period of 12 days showed a population doubling time of 36 h. Under these culture conditions, we observed a contraction of the lattices. Phase-contrast light microscopy and transmission electron microscopy demonstrated that the HSRE cells formed tubular ductlike structures. Lumens formed by HSRE cells were surrounded by cuboidal-shaped polarized cells with numerous ciliated cells, secretory cells, and undifferentiated cells. Epidermal growth factor (EGF) was observed to stimulate the tubule formation and the contraction of the lattices. Videomicroscopic observations and analysis of the ciliary beating frequency (CBF) demonstrated that the cilia were homogeneously distributed on the whole apical surface of the ciliated cells and that their movement was well coordinated, with a CBF similar to that observed in outgrowth cells from cultured human nasal and tracheal epithelia. Immunofluorescent staining of basement membrane components synthesized and secreted by cells revealed the presence of type III collagen around the tubules. Type IV collagen and laminin were present in the cytoplasm and at the periphery of the cells. The biotin-streptavidin-gold immunocytochemical technique with monoclonal anti-mucin antibody showed intracellular localization of mucins in secretory granules of the secretory cells. With the use of substrate gel electrophoresis polyacrylamide gels impregnated with gelatin, collagenase activity was detected in the conditioned medium of the cultured HSRE cells. These results suggest that both three-dimensional collagen gel and soluble factors such as EGF regulate tubule formation by HSRE cells. Moreover, the capacity of the epithelial cells to contract the gel suggests they may be involved in the wound healing process.

Basement Membrane↗
PubMed1993

Studies on human porin. IX. Immunolocalization of porin and CFTR channels in human surface respiratory epithelium.

The expression of the voltage-dependent anion channel (VDAC) "Porin 31 HL" and its cellular and subcellular immunocytochemical localization in the human respiratory epithelium were studied with monoclonal and polyclonal antibodies using immunofluorescence and immunogold labelling with light (LM) and transmission electron microscopy (TEM), respectively. Porin was identified in the apical domain of the ciliated cells and in the basal cells of the respiratory epithelium. Immunogold labelling was present in the apical plasma membrane and subapical vesicles of the ciliated cells. In pre-embedded freshly dissociated surface epithelial cells, porin could also be identified with TEM at the outer part of the plasma membrane of basal cells. By LM double immunolabelling, both porin and cystic fibrosis transmembrane conductance regulator (CFTR) were identified in the apical domain of ciliated cells but not in basal cells where CFTR was never identified. On Western blots of solubilized total membrane protein preparations from the same frozen surface epithelial respiratory cells, the antibodies recognized a group of 3 proteins of 31, 60 and 130-140 kDa with a strong reactivity for a 31 kDa protein, corresponding to the porin and a protein of 170 kDa which is consistent with mature CFTR. These results suggest that porin might be part of a multi-component chloride channel complex which could interact with CFTR.

Antibodies↗
PubMed1993

Nasal epithelial cell culture as a tool in evaluating ciliary dysfunction.

Cultures of respiratory epithelial cells were obtained from nasal polyps collected in patients with and without primary ciliary defect. The ciliary beating frequency and the ciliary beating heterogeneity were determined on native and cultured tissues. We observed a significantly higher (p < 0.01) ciliary beating frequency of cultured ciliated cells, when compared with ciliated cells from the native tissue. The ciliary beating frequency of the cultured ciliated cells from the patient with primary defect (7.9 +/- 2.1 Hz) was significantly lower when compared with the beating frequency of the ciliated cells from the control subject (12.4 +/- 2.0 Hz). In addition, the percentage of ciliated cells characterized by a beating frequency lower than 8 Hz was 90.7% in the native tissue and 47.5% in the cultured tissue from the patient with ciliary primary defect. In the patient without ciliary primary defect, 90% of the cultured ciliated cells had a homogeneous ciliary beating, whereas in the patient with primary ciliary defect, only 47% of the ciliated cells had a homogeneous ciliary beating. These results suggest that the culture of respiratory cells associated with the functional activity measurement of the ciliated cells represent another way of precisely determining the extent of the primary ciliary dyskinesia defect.

Adult↗
PubMed1993

Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands.

Cystic fibrosis (CF) is caused by mutations in the gene coding for the CF transmembrane conductance regulator (CFTR). From human normal tracheal submucosal gland cells in culture, we identified endogenous CFTR as a 170 kDa protein, consistent with that of fully glycosylated, mature CFTR molecule. This observation led to the hypothesis that airway secretory glands could be an important site for the CFTR expression. Using anti-human CFTR polyclonal and monoclonal antibodies, we examined the cellular and subcellular localization of the CFTR protein in airway submucosal glands from human and bovine tracheal tissues as well as in tracheal gland cell cultures. In human tracheal tissue, CFTR immunolabelling was present along both the apical and basolateral plasma membranes of glandular mucous cells. In contrast, CFTR was associated with the secretory granules of glandular serous cells. Using immunogold electron microscopy, we demonstrated that CFTR protein was more specifically associated with the membrane of serous cell secretory granules. In bovine tracheal tissue CFTR labelling was also identified in the secretory granules of glandular serous cells. In contrast, when bovine and human tracheal gland cells were cultured, no mature secretory granules were present, but a predominantly intracytoplasmic distribution of CFTR was observed. Our data thus suggest that in airway tissues, CFTR could be involved in intracellular processes of the mucus exocytosis in submucosal secretory glands.

Animals↗
PubMed1993

Differentiation of human surface upper airway epithelial cells in primary culture on a floating collagen gel.

The differentiation of human nasal surface epithelial cells in primary three-dimensional (3D) culture was studied. The dissociated cells were seeded on type I and IV collagen gel and grown in a serum-free medium supplemented with hormones and growth factors. During the first days of culture, epithelial cells were infrequently differentiated. Detachment and retraction of collagen by the cells generally occurred after 8-10 days of culture, allowing the formation of a floating collagen gel. This induced the differentiation of epithelial cells on 3D cord-like structures consisting of a collagen core surrounded by well-differentiated cells. Under scanning and transmission electron microscopy, we observed the formation of a pseudostratified respiratory-type epithelium consisting of columnar mature ciliated cells and secretory cells, epithelial cells in the process of ciliogenesis, and small pyramidal basal cells. The videomicroscopic analysis of the ciliated cells showed that the mean ciliary beating frequency (12.2 +/- 1 Hz) was close to the values obtained on polyp explants (11.7 +/- 0.8 Hz). Immunocytochemical localization of secretion with mucin-specific antibodies showed the expression of mucous cell function. In addition, the epithelial cells within the cord-like structures maintained a differentiated morphology and active beating of ciliated cells for more than 35 days in primary culture. Conversely, when the cells were grown on a collagen gel attached to plastic, they remained more flattened and the number of differentiated cells was lower. These results suggest that human upper airway epithelial cell differentiation in culture, as assessed by mucociliary function, is enhanced by the 3D organization of the cells around the floating collagen gel substrate.

Cell Differentiation↗
PubMed1993

Distearoyl phosphatidylglycerol liposomes improve surface and transport properties of CF mucus.

We have previously shown that a decreased level of phosphatidylglycerol in cystic fibrosis (CF) respiratory mucus is partly responsible for its marked adhesiveness and stickiness, which impair mucus transport, and that distearoyl phosphatidylglycerol (DSPG) was the most efficient form of phosphatidylglycerol in the enhancement of respiratory mucus clearance. The aim of our study was to analyse the effect of distearoyl phosphatidylglycerol liposomes on the transport by cough and cilia of cystic fibrosis respiratory mucus. The surface and transport properties of mucus were measured: 1) on native cystic fibrosis mucus; 2) on cystic fibrosis mucus complemented with DSPG liposomes at a non-cytotoxic concentration; and 3) on cystic fibrosis mucus complemented with water. The work of adhesion of cystic fibrosis mucus was significantly decreased by DSPG liposomes, but not by water. For mucociliary transport, the cystic fibrosis mucus was transported at a higher rate with DSPG liposomes and water compared to native cystic fibrosis mucus. The cough clearance of cystic fibrosis respiratory mucus was significantly improved in the presence of DSPG and water, but the effect was more pronounced with DSPG liposomes than with water. We conclude that the use of DSPG liposomes as a lubricating agent proves to be an interesting therapeutic approach for improving the cough and mucociliary transport in cystic fibrosis patients.

Animals↗
PubMed1993

Cellular and molecular mechanisms of bacterial adhesion to respiratory mucosa.

Different bacterial species adhere avidly to respiratory mucus. Such adhesion, when followed by ciliary clearance, represents an important stage of the airway defense system. However, in pathological conditions, the mucociliary clearance may be severely reduced, and mucus-associated bacteria may multiply and infect the underlying epithelium. Only a few bacteria have been shown to adhere to ciliary membranes of functionally active ciliated cells. Therefore, the first way in which most of the respiratory pathogens associate with the airway epithelium is likely to be by their adhesion to mucus. Some bacteria also secrete products that may affect ciliary function and/or cause cell death and epithelial disruption. Respiratory pathogens that do not bind to normal ciliated cells may readily adhere to injured epithelial cells, or to the unmasked extracellular matrix. Furthermore, following injury, epithelial respiratory cells in the process of migration, in order to repair the wounds, may present receptors to which bacteria adhere. The adhesion to all of these epithelial receptors may contribute to the chronicity of many bacterial respiratory infections.

Animals↗
PubMed1993

Vectorial delivery of newly-synthesized secretory proteins by human tracheal gland cells in culture.

The polarized secretion of newly-synthesized proteins of human tracheal submucosal gland cells was studied. Human tracheal gland cells were cultured on permeable filter supports allowing a separate biochemical analysis of apical and basal secretion. By transmission electron microscopy, confluent filter-grown cells were seen to form a continuous sheet of both multilayer and monolayer epithelial cells. Junctional complexes between adjacent cells were observed. On immunofluorescence microscopy, human tracheal gland cells in cultured exhibited characteristics of epithelial and secretory cells, including cytoplasmic staining for cytokeratin and for two secretory protein markers specific to the glandular serous type cell: lysozyme and antileucoprotease. [35S]methionine metabolic labelling experiments demonstrated that at least 90% of newly-synthesized secretory proteins were recovered in the apical medium. Moreover, lysozyme secretion was strongly polarized since 85% was released into the apical medium. Conversely, antileucoprotease secretion was more bidirectional since nearly 40% of released antileucoprotease was present in the basal medium. The fact that these two secretory proteins are released with differing relative polarity emphasizes that human tracheal gland cells exhibit at least two different exocytotic routing operations.

Cell Polarity↗
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↗