Biomedical subjects
Publications and source records attributed to E Puchelle.
Rat trachea and frog palate explants were continuously perfused for 2 hr with 199 tissue culture medium. The ciliary beating frequency of the frog palate exhibited fluctuations twice as large as compared to the rat trachea. The rat trachea model should be preferred to the frog palate for long-term studies on ciliary beat frequency.
Bacterial adherence to virus-infected respiratory tract cells may be one of the several mechanisms whereby virus predisposes to bacterial pneumonia. To evaluate the effect of influenza virus infection on pneumococcus adhesion, 39 mice were infected with PR8/A influenza virus. The adherence of radiolabeled pneumococcus to mice tracheal cells was determined 2, 4, and 6 days after viral inoculation. The pneumococcal adhesion to infected tracheas was significantly enhanced on Day 6 (p less than 0.001). Scanning and transmission electron microscopy revealed that by the fourth and sixth days after virus inoculation, the ciliated and the secretory cells of the tracheal epithelium had desquamated and the mucosa were coated with a continuous layer of basal cells. In a few cases, a desquamation of the basal layer was observed and the exposed basement membrane appeared as a pole of attraction for bacteria. Pneumococci were never seen attached to control tracheas. In contrast, they were observed adhered to the microvilli of the basal cells and, to a greater extent, to the exposed basement membrane.
It has recently been suggested that in cystic fibrosis (CF), there is no rheological abnormality of airway secretions other than that associated with purulence, and that the apparent inhibition in the mucociliary transport rate might be partly due to a ciliary inhibitor present in these secretions. In order to ascertain this assumption, expectorated airway secretions were collected without salivary contamination in twenty-four CF patients and the rheological properties were measured. Using a photometric method, the effects of CF sputum samples were analysed on the ciliary beat frequency (Fm) of the frog palate, and we measured their mucociliary transport rate (TR). In all but one CF sputum, TR and Fm were lower than that of the control frog mucus (median TR: 18.7 and 11.6 mm min-1; median Fm: 12.3 and 11.3 Hz, respectively). In the eighteen patients in whom the rheological properties were outside the range for optimal mucociliary transport, the clinical Shwachman score was significantly (P less than 0.05) lower (median score: 66.2 points) than in the six patients with optimal rheologic properties (median score: 73 points). In the eleven CF patients with superinfection, the apparent viscosity (eta o) was significantly higher (P less than 0.01; median eta o: 24.4 Pa. s) and TR, expressed as a percentage of the reference value, was significantly lower (P less than 0.05; median Tr: 54.5%) in comparison with the values obtained for the thirteen non-superinfected CF patients (median eta o: 15 Pa. s and median TR: 66% respectively). The CF patients with markedly hyperviscous sputum (eta o higher than 30 Pa. s) exhibited a low Shwachman score.
Gold immunolabelling of human airways using a polyclonal antibody specific for the bronchial inhibitor (brI) demonstrates that this protease inhibitor is present in the serous cells of the bronchial glands. Most of the largest secretory granules (89%) are uniformly stained for brI whereas the smallest granules (diameter less than 80 nm) appear frequently (68%) devoid of brI.
The in vitro effects of Pseudomonas aeruginosa elastase (P. aeruginosa E) and of human leukocyte elastase on human airway lysozyme (HAL) were investigated. P. aeruginosa E inactivated and cleaved HAL, whereas human leukocyte elastase had no effect. Total inactivation of HAL by P. aeruginosa E was observed after 120 min of incubation at 37 degrees C, for an elastase-to-lysozyme molar ratio of 1:5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reaction mixtures containing HAL and P. aeruginosa E in an elastase-to-lysozyme molar ratio of 1:10 showed a progressive disappearance of the HAL band upon increasing the incubation time with P. aeruginosa E. Gel filtration chromatography indicated that HAL was cleaved into at least three peptide fragments. The cleavage of HAL by P. aeruginosa E was accompanied by parallel losses of its bacteriolytic activity and its immunoreactive property.
Pseudomonas aeruginosa elastase has recently been shown to inactivate bronchial inhibitor, the major leukoproteinase inhibitor of the bronchial tree. To test if this in vitro finding is relevant to pathology, we have measured the leukocyte elastase inhibitory capacity and the immunoreactive levels of bronchial inhibitor in the acidified and neutralized sputum specimens from 15 patients with cystic fibrosis, 11 of whom were infected by P. aeruginosa and 10 of whom exhibited free bacterial elastase activity. The percentage of functionally active bronchial inhibitor in these acid-treated sputum specimens (i.e., concentration of active inhibitor/concentration of immunoreactive inhibitor X 100) was found to be 125 +/- 17%. It was positively correlated with the free leukocyte elastase concentration (r = 0.77, p less than 0.001) but not with the free bacterial elastase concentration (r = 0.46, p greater than 0.05). Therefore, in an in vivo situation, P. aeruginosa elastase does not necessarily inactivate the bronchial inhibitor. Experiments using pure proteins show that the inactivation process does not take place when leukocyte and P. aeruginosa elastase are added simultaneously to the inhibitor. This suggests that the bronchial inhibitor reacts preferentially with leukocyte elastase and that in its complexed form it is shielded from the inactivating action of P. aeruginosa elastase. In addition, the inhibitor recovered from the acid-induced dissociation of the leukocyte elastase-inhibitor complexes, exhibits a substantially higher specific activity than does the native molecule. This explains why the average functional activity of the bronchial inhibitor is significantly higher than 100% in sputum samples from patients with cystic fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)
The non-newtonian viscous and elasto-thixotropic properties of native and lyophilized pathological bronchial mucus and of polymer solutions (3% and 6% PIB in decalin) used as mucus analogs were analyzed using a cone-plate Carri-Med rheometer and a Couette viscoelastometer that we have specifically developed for measuring the rheological properties of bronchial mucus in clinical practice. The master curves obtained for apparent viscosity under steady conditions as a function of shear rates (gamma: 2.6 X 10(-3) to 6.9 X 10(1) sec-1) were fairly similar, whatever the apparatus used. Under transient conditions, at low shear rate (gamma less than 1.4 sec-1), PIB and mucus exhibited a typical viscoelastic behavior: the shear stress increased slightly up to a steady-state value. At higher gamma, a transitory overshoot of sigma characteristic of the elastothixotropic systems appeared. Such a behavior can be interpreted as resulting from structural changes such as formation and rupture of the three-dimensional network present in bronchial mucus as in polymer solutions.
Prescription of bronchial mucus modifiers has benefited from recent advances in the knowledge of biochemical constituents of bronchial fluids, their regulation and disturbances on the one hand, and the consequences of biochemical changes in these fluids on rheological characteristics of bronchial secretions on the other. In addition, the significance of these characteristics has been evidenced by the study of mucociliary transportation. Drugs which modify bronchial fluids are studied according to their mode of action upon the organized gel phase, mobilized by ciliary activity, and the aqueous sol phase, which enables this ciliary activity. Drugs which modify the gel phase are either true mucolytic agents or muco-regulators. True mucolytic drugs include reducing agents with a free thiol group, chiefly N-acetyl-cysteine which breaks disulfide bonds linking different protein chains, and proteolytic enzymes which break protein chains. The latter also have an appreciable anti-inflammatory effect but are rarely used at present owing to allergic reactions. Mucoregulating drugs are cysteine derivatives whose thiol group is not free. They are headed by carbocysteine which activates the synthesis of sialidized acid mucins. They enable reorganization of bronchial mucus, reversing former drops in viscosity and elasticity, thereby improving mucociliary transportation. They have a complementary anti-inflammatory action.
The histochemical nature of the mucosubstances of the respiratory epithelium of the frog palate was studied. 3 main types of epithelial cells were identified: Goblet cells which contained some glycogen, some neutral mucins and a mixture of sialidase-resistant and sialidase-labile sialomucins, non ciliated columnar cells with a secretory activity restricted to their apical pole. These cells contained abundant glycogen, some neutral mucins and sialidase-resistant sialomucins, ciliated cells with no secretory activity. The surface mucous blanket was composed of a mixture of predominant sialomucins and some neutral mucins. The histochemistry of mucosubstances in the respiratory epithelium of the frog palate differed from that of human and most respiratory systems by the presence of glycogen and the absence of sulphated mucins.
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We studied the relations between the mucociliary beat frequency (MF) measured photometrically on the depleted frog palate and the rheological properties of sputum collected in patients with chronic obstructive pulmonary disease. MF was lower (p less than 0.001) with sputa (11.3 +/- 3.3 Hz) than with frog mucus (16.9 +/- 3.3 Hz) used as controls. The relative transport rate (Tr) of sputa was closely correlated (r = 0.81, p less than 0.001) to the relative MF. Significant correlations were observed between MF on the one hand and viscosity (r = -0.68, p less than 0.01), elastic modulus (r = -0.70, p less than 0.01) and spinability (r = +0.49, p less than 0.05) on the other. These results suggest that abnormalities in the rheological properties of bronchial secretions may impair the mucociliary transport rate by first decreasing the ciliary beat frequency.
In clinical practice, a wide variety of drugs are given to modify the rheological properties of bronchial mucus but they are frequently not correctly chosen because there is not a simple apparatus available for the routine measurement of the mucus viscoelasticity. We designed a new viscoelastometer which is easy to operate and permit accurate measurement of the viscoelastic properties of mucus. The system has a Couette geometry and requires a sample of 1 ml. The outer cylinder is thermostated and rotates at a constant rate. The resulting torque is measured on the inner cylinder using a microdisplacement transducer. The inner cylinder is suspended by a special wire system which maintains a constant 0.75 mm gap between the two cylinders. The apparent viscosity is calculated up to 100 Pa.s. at a low shear rate (0.4 s-1) and the elasticity is analyzed by a stress relaxation method. The characteristics of the apparatus and the typical viscoelasticity behaviour of several sputum samples are reported.
Spinability is a rheological parameter which characterizes the property of fluids to be drawn into threads when stretched. It has been shown that this property is of particular importance in the mucus transport process. This paper describes an automatic apparatus which measures the spinability of a calibrated volume (20 microliters) of sample at a stretching rate of 10 mm.sec.-1. The reproducibility of the measurements is analyzed on mucus simulants and on different types of bronchial mucus. Using this technique, we observed that the spinability of bronchial secretions is significantly correlated (r = 0.69, p less than 0.001) with their transport rate measured on the depleted frog palate. This device is suitable for measuring the spinability of all conductive types of mucus and other biological fluids.
The mucociliary epithelium (MCE) of the frog palate is a pseudostratified columnar respiratory epithelium supported by connective tissue with no submucosal glands. Morphologically different types of cells have been identified using scanning and transmission electron microscopy: ciliated cells, mucous and serous cells, intermediate cells, basal cells, and patches of vomero-nasal epithelium with sensory receptors and supporting cells. The cilia of the MCE of the frog palate are 5 to 7 microns long and a cluster of 3 to 7 projections forms a ciliary crown at their tips. Using cationized ferritin, anionic charges are identified on their whole outer surface. The mucus discharged by the secretory cells forms a continuous blanket which lies at the tips of the cilia.
Rheological studies of sputum are often considered unreliable because expectorated secretions can be contaminated by saliva. The in vitro dilution of gel mucus by 50% of saliva results in a marked decrease in apparent viscosity and elasticity. A simple technique for collecting sputum "protected" from salivary contamination is described. It consists of using dental cotton-wool swabs placed immediately before expectoration between the cheek and the gum and under the tongue to absorb saliva. The rheological and transport properties of sputum, collected by the protected and unprotected technique in 20 patients with either recurrent or chronic bronchitis, are compared. The protected sputum samples are characterized by a significantly lower pourability (p = 0.02) and sol phase percentage (p = 0.01) and a higher apparent viscosity (p = 0.05). A marked, although not significant, increase in viscoelastic modulus is also noticed with the protected sputa. No difference is observed in the mucociliary transport velocity according to whether the sputum is protected or not. The reproducibility and reliability of the measurements are considerably improved when sputum is collected by the protected technique. The ratio of inter- to intrasample variability is twice to three times as high for the unprotected samples.
Spinability of normal mucus and of sputum collected in chronic bronchitic patients was measured using an automatic device derived from that developed by CHRETIEN et al (1977) for cervical mucus. Spinability of sputum decreased as the purulence increased. Although significant correlations were found between spinability and apparent viscosity (r = - 0.50, p less than 0.05) or elasticity (r = 0.54, p less than 0.02), large variations in spinability were observed for sputum samples ranging in a zone of low viscosity and elasticity. Sputum with high spinability exhibited normal transport rate on the depleted frog palate although their viscosity and elasticity were abnormally low. The significant correlation obtained between spinability and sputum transport rate (r = 0.61, p less than 0.01) suggests that this rheological factor, along with viscosity and elasticity, plays an important role in the mucociliary transport mechanism.
The palate of the frog possess a pseudostratified epithelium with mucus secreting cells and numerous ciliated cells covered with a continuous mucus blanket of 4 to 8 micron thickness. This palate can be used as a simple model for studying mucus transport and giving rapid information on the transportability of pathological bronchial secretion by the ciliary mechanism. The analysis of the relationship between the rheological properties of sputum an their transport rates on the frog palate showed that the highest transport rates were obtained with sputum samples characterized by a high spinability (Sp greater than 70 mm) and an intermediate range of viscosity (25-180 P) and elasticity (SR = 4-12 units). The mucociliary frequency (Fm) of the frog palate was measured by a photoelectric method and analyzed as a distribution function of the ciliary frequencies by the Fast Fourier Transform method. Fm decreased significantly after mucus depletion and was restored when adding a drop of frog mucus collected on a freshly excised frog palate. On the other hand, it remained about 20% lower than the original predepleted value, when adding purulent sputum collected in patients with chronic bronchitis and cystic fibrosis. The frog palate appears as a valuable model for analyzing the mucociliary transport rate and also for studying the effect of pathological secretions on the ciliary beating frequency.
The concentrations of immunoglobulins A (IgA) and serum albumin (SA) determined in sputum by electro-immunoassay have been compared after the extraction of proteins by two methods: 1) ion exchange chromatography and 2) mechanical agitation at high ionic strength. No significant difference of IgA is observed between the two methods. On the contrary, a very significant decrease in the concentration of SA is observed by the chromatographic extraction which is incomplete when pH - value of extract is lower than that of the isoelectric point of SA.