Fibronectin-binding proteins of Staphylococcus aureus are involved in adherence to human airway epithelium.

This study was designed to investigate the molecular mechanisms of Staphylococcus aureus adherence to human airway epithelium. Using a humanized bronchial xenograft model in the nude mouse and primary cultures of human airway epithelial cells (HAEC), we showed that S. aureus adhered mainly to undifferentiated HAEC whereas weak adherence (11- to 20-fold lower) to differentiated HAEC was observed (P < 0.

Differentiated and functional human airway epithelium regeneration in tracheal xenografts.

To investigate the regeneration process of a well-differentiated and functional human airway epithelium, we adapted an in vivo xenograft model in which adult human nasal epithelial cells adhere and progressively repopulate denuded rat tracheae grafted in nude mice. The proliferating activity, the degree of differentiation, and the barrier integrity of the repopulated epithelium were studied during the regeneration process at optical and ultrastructural levels with immunocytochemistry and a permeability tracer. Three days after implantation in nude mice, tracheal xenografts were partially repopulated with a flattened nonciliated and poorly differentiated leaky epithelium.

Induction of a cAMP-stimulated chloride secretion in regenerating poorly differentiated airway epithelial cells by adenovirus-mediated CFTR gene transfer.

In cystic fibrosis (CF), the airway epithelium is in the process of injury and regeneration. In the context of the CF gene therapy, we previously reported that regenerating poorly differentiated (PD) cells of human airway epithelium represent preferential cell targets for recombinant adenoviral gene vectors. To define whether PD non-CF and CF epithelial cells possess a functional cystic fibrosis transmembrane conductance regulator protein (CFTR) chloride channel, we analyzed the CFTR expression and the regulation of chloride secretion under cyclic (c)AMP stimulation in these regenerating PD epithelial cells of non-CF and CF airway tissue.

Early alterations in airway mucociliary clearance and inflammation of the lamina propria in CF mice.

In cystic fibrosis (CF), whether cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction leads to decreased mucociliary clearance and mucus hypersecretion, before bacterial infection, remains an open question. To answer this question, we quantified in a blind trial the mucociliary transport velocity, the histological state, and the degree of inflammation of the tracheal mucosa in 23 cftr(m1HGU/cftr(m1HGU) transgenic mice (Dorin, J. R.

Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells.

We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients. Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model. At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury.

Targeting cell-specific gene expression with an adenovirus vector containing the lacZ gene under the control of the CFTR promoter.

In vivo gene therapy requires the development of vectors able to deliver and express therapeutic genes preferentially into specific cell populations. This can be achieved by the manipulation of viral proteins mediating target-cell recognition, as well as by the introduction of tissue-specific promoters into viral vectors. As a first approach towards this goal, we describe here the construction and testing of a recombinant adenovirus expressing the lacZ gene encoding beta-galactosidase under the control of 2 kilobase pairs (kbp) of 5' untranslated DNA sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Regenerating cells in human airway surface epithelium represent preferential targets for recombinant adenovirus.

To investigate the efficiency of adenovirus-mediated gene delivery in regenerating human respiratory epithelium, we have performed infections with an E1- and E3-deleted type 5 recombinant adenovirus containing the Escherichia coli LacZ reporter gene on different culture models of regenerating human nasal polyp surface epithelium. These models included: (i) an ex vivo organ culture of nasal polyp tissue, (ii) an explant outgrowth cell culture, and (iii) an in vitro wound repair model, on dissociated cells. In ex vivo nasal polyp tissue, transduced cells were not detected in normal pseudostratified areas, but were found in areas of the surface epithelium with a morphology reminiscent of regenerating airway tissue.

CFTR and differentiation markers expression in non-CF and delta F 508 homozygous CF nasal epithelium.

Human nasal polyps from non-CF and delta F 508 homozygous CF patients were used to compare the expression of CFTR and markers epithelial differentiation, such as cytokeratins (CK) and desmoplakins (DP), at the transcriptional and translational levels. mRNA expression was assessed by semiquantitative RT/PCR kinetic assays while the expression and distribution of proteins were evaluated by immunofluorescence analysis. In parallel, for each nasal tissue specimen, the importance of surface epithelium remodeling and inflammation was estimated after histological observations.

Expression and localization of CFTR in the rhesus monkey surface airway epithelium.

The Rhesus monkey has been used as a model for evaluating the possibility of introducing the CFTR gene into the airway epithelium in vivo. We addressed the question of whether the simian airway surface epithelium exhibits a CFTR distribution and functional activity (ciliary beating frequency) similar to that of human airway surface epithelium. Expression of CFTR mRNA was demonstrated on Rhesus monkey tracheobronchial tissue by reverse transcription polymerase chain reaction (RT-PCR) analysis.

Decreased expression of the CFTR protein in remodeled human nasal epithelium from non-cystic fibrosis patients.

Background: In normal adult pseudostratified human nasal surface epithelium, the cystic fibrosis transmembrane conductance regulator (CFTR) is localized to the apical domain of the ciliated cells, whereas in cystic fibrosis (CF), the mutated delta F 508 CFTR exhibits an abnormal cytoplasmic localization. Frequent airway injuries either in CF or non-CF patients may induce a remodeling of the surface epithelium characterized by a change in the morphological structure from normal columnar pseudostratified epithelium to either basal cell hyperplasia, mucous cell hyperplasia, or squamous metaplasia.

Experimental Design: The localization of CFTR parallel to markers of cell differentiation, such as cytokeratin 14 (CK14, a marker of basal cells), cytokeratin 18 (CK 18, a marker of ciliated and mucous cells), cytokeratin 13 (CK13, a marker of squamous metaplasia cells), and desmoplakins (DP) 1 and 2 (markers of desmosomes) was analyzed by indirect immunofluorescence.

Protection of human respiratory epithelium from Pseudomonas aeruginosa adherence by phosphatidylglycerol liposomes.

The ability of phosphatidylglycerol (DSPG) liposomes to prevent adherence of Pseudomonas aeruginosa to primary cultures of non-cystic fibrosis (CF) and delta F508 homozygous CF human respiratory epithelium was studied. The culture model was characterized by the simultaneous presence of various cellular phenotypes: well-differentiated respiratory epithelial cells, ciliated and nonciliated cells, and migrating cells which can be assimilated into a regenerating epithelium after injury. DSPG liposomes significantly decreased the binding of P.

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.

[Structure and secretory functions of the respiratory epithelium].

Airway secretions actively participate in respiratory epithelium protection. Apart from its main participation in transport of inhaled microorganisms and particles by mucociliary clearance, respiratory mucus also contributes to other protective purposes such as the control of airway humidification. Biochemical components found in secretions, such as mucins, lipids, antibacterial agents (secretory IgA, lysozyme, lactoferrin), antioxidant and antiprotease components, contribute significantly to the airway epithelium defense.

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.

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.

Modulation of albumin-like protein and lysozyme production by bovine tracheal gland serous cells. Dependence on culture conditions.

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol-stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with Mr values ranging from 15,000 to 95,000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G.

[Annulo-aortic ectasia].

On the basis of a literature review and the study of 18 cases, the authors examine annuloaortic ectasia, which involves dilatation of the fibrous ring of the heart at the aortic orifice, marked by aortic regurgitation and dystrophic aneurysm of the ascending aorta. The authors noted a predominantly muscular disorder (14 males, 4 females) often detected from an assessment of aortic regurgitation without clinical, radiologic or specific electrical features. The value of echocardiography and Doppler echocardiography is clearly apparent from the diagnosis of the disease, monitoring of progress, selection of patients for invasive pre-operative explorations and post-operative surveillance.

Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix.

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes.