Michel Devleeschouwer

Modulation by LL-37 of the responses of salivary glands to purinergic agonists.

The interaction of mice submandibular gland cells with LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), a cationic peptide with immunomodulatory properties, was investigated. LL-37 at a concentration that did not affect the integrity of the cells increased the uptake of calcium and activated a calcium-insensitive phospholipase A2 (PLA2). The small release of ATP induced by LL-37 could not account for this stimulation because apyrase did not significantly block the response to LL-37. The divalent cation magnesium inhibited the response to LL-37, but this inhibition was probably nonspecific because it also inhibited the in vitro bacteriostatic effect of the peptide. The increase of calcium uptake by LL-37 was not affected by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62), a rather specific inhibitor of P2X7 receptors in mice. LL-37 also increased [Ca2+]i in cells from mice invalidated for these receptors. LL-37 had no effect on the response to carbachol. It inhibited the increase of [Ca2+]i and the activation of phospholipase D by ATP. It potentiated the activation of the PLA2 by the nucleotide. Finally, LL-37 increased the fluidity of the plasma membrane of submandibular gland cells. In conclusion, our results suggest that LL-37 is an autocrine regulator of submandibular gland cells. It does not stimulate mouse P2X7 receptors but modulates their responses.

Interaction between tobramycin and CSA-13 on clinical isolates of Pseudomonas aeruginosa in a model of young and mature biofilms

The bactericidal activity of a cholic acid antimicrobial derivative, CSA-13, was tested against eight strains of Pseudomonas aeruginosa (both reference and clinical strains) and compared with the response to tobramycin. In planktonic cultures, the minimal inhibitory and minimal bactericidal concentrations of CSA-13 and tobramycin were in the 1–25xa0mg/L range except for one mucoid clinical strain which was much less sensitive to tobramycin (minimal bactericidal concentration, 65–125xa0mg/L). In young (24xa0h) biofilms, the sensitivity to CSA-13 was reduced (half-maximal concentration CSA-13 averaged 88xa0mg/L) and varied among the eight strains. The sensitivity to tobramycin was also very variable among the strains and some were fully resistant to the aminoglycoside. The combination of tobramycin with CSA-13 was synergistic in five strains. Only one strain showed antagonism between the two drugs at low concentrations of CSA-13. One reference and five clinical strains were tested in mature (12xa0days) biofilms. The effect of CSA-13 was delayed, some strains requiring 9xa0days exposure to the drug to observe a bactericidal effect. All the strains were tolerant to tobramycin but the addition of CSA-13 with tobramycin was synergistic in three strains. CSA-13 permeabilized the outer membrane of the bacteria (half-maximal concentration, 4.4xa0mg/L). At concentrations higher than 20xa0mg/L, it also permeabilized the plasma membrane of human umbilical vein endothelial cells. In conclusion, CSA-13 has bactericidal activity against P. aeruginosa even in mature biofilms and cationic steroid antibiotics can thus be considered as potential candidates for the treatment of chronic pulmonary infections of patients with cystic fibrosis. Considering its interaction with the plasma membrane of eukaryotic cells, less toxic derivatives of CSA-13 should be developed.

Determination de l'Acide l-Ascorbique a l'Aide d'Electrodes Bacteriennes, Tissulaires et Enzymatiques

Abstract L-ascorbic acid membrane electrodes based upon the use of four classes of biocatalysts immobilized at an oxygen electrode are evaluated and compared in terms of electrode properties and operating requirements. Isolated ascorbate oxydase enzyme in soluble form and in covalent binding matrices, peel of cucumber and living bacterial cells of Enterobacter agglomerans strain, respectively, are employed as biocatalytic layers. The physico-chemical factors, life times and interferences are discussed in details. The low stability of the soluble enzyme sensor does not allow its analytical utilization, but the immobilized enzyme, bacterial and tissue electrodes can be used, even in multivitamin pharmaceutical formulations. The common linear range of those three biosensors are of 4.10−6 M to 7.10−4 M with a precision and a reproducibility of ± 3%.

Electrodes Potentiometriques Et Amperometriques a Levures Permeabilisees: Determination Du L-Lactate

Abstract A biosensor using permeabilized yeasts (Hansenula anomala) is described for the determination of L-lactate. The same electrode can be used either potentiometrically or amperometrically. The linear ranges are respectively of 4.10−5M to 2.10−3M for potentiometry and 8.10−7M to 3.10−3M for amperometry with a reproducibility and a precision of ± 2 to 3%. The parameters involved in the optimization of electrode response, such as pH, temperature, cofactor concentration and ionic strength of the buffers, are discussed in details for both types of measurements. The amperometric technique is suitable for lactate determinations in biological media. In this case, differential measurements are used in order to eliminate interferences of biological redox reactions.

Contribution au Developpement d'Un Nouveau Modele d'Electrode: L'Electrode Bacterienne

Abstract A new type of selective electrode is proposed, based on the immobilization of bacteria on a cellulose acetate membrane. Proteus mirabilis (Urease +) is concentrated on the membrane. This electrode gives good results for urea determination, is very selective, stable during three weeks, may be regenerated after being used and is of low cost. The parameters involved in the typeof electrode manufacturing are discussed in details. Precision may be compared to the usual spectrophotometric techniques in biological media. Other types of applications will be published in a nearly future.

Study on the activation of phospholipases A2 by purinergic agonists in rat submandibular ductal cells

Extracellular ATP and benzoyl-ATP (Bz-ATP) increased the release of [3H]arachidonic acid ([3H]AA) from prelabeled rat submandibular gland (RSMG) ductal cells respectively two- and threefold. Both agonists also increased the release of [3H]AA from acini but at a lower level (+50% and +100% respectively). Carbachol had no significant effect on either cellular population. In ductal cells phorbol myristate acetate, an activator of protein kinase C, slightly increased the basal release of [3H]AA but did not affect the release of [3H]AA in response to ATP. Staurosporine, an inhibitor of protein kinases, inhibited the response to the purines. The removal of calcium from the extracellular medium decreased the response to ATP and Bz-ATP. Only barium could partly substitute for calcium to restore the purinergic response. Zinc inhibited the release of [3H]AA. Permeabilization of the cells with streptolysin O (SLO) activated the calcium-independent phospholipase A2 activity (iPLA2). The iPLA2, not the calcium-dependent PLA2 (cPLA2), released [3H]oleic acid ([3H]OA) from RSMG ductal cells. It is concluded that RSMG ducts have a higher PLA2 activity when compared to acini. This activity is accounted for by iPLA2 and cPLA2. Both enzymes are activated by P2X agonists by a staurosporine-sensitive mechanism. Cells permeabilized with SLO or membranes from Escherichia coli as a substrate are not good models to study the regulation of these enzymes. In intact RSMG ductal cells the two activities can be distinguished by rather specific inhibitors, by different ionic conditions and also by the fatty acid used to label the cells.

Adhesion of Klebsiellapneumoniae to human epithelial cells and influence of protamine

Bacterial adhesion is an obligatory step leading to colonisation of the host. In vitro adhesion of Klebsiella pneumoniae, at 10 8 and 10 9 bacteria ml -1 , to buccal cells from three non-smoking donors showed that at 10 9 bacteria ml -1 the number of adherent microorganisms is more statistically significant (p<0.01, ANOVA) THAN AT 10 8 bacteria ml -1 . With protamine at a concentration of 100 μg ml -1 , under our experimental conditions this basic protein abolished 60% of the adhesion on buccal and urinary cells. For urinary cells from one donor, the number of adherent bacteria was generally greater when cells were collected in the second part of the menstrual cycle

Adhesion of Pseudomonasaeruginosa to human buccal epithelial cells: adjustment of a radioisotopic method of measurement

Abstract A detailed study was performed of the different steps of an adhesion assay based on 14 C-radiolabelling of Pseudomonas aeruginosa and measurement of the radioactivity retained by buccal epithelial cells (BECs) after attachment of the bacteria. A minimum medium containing 0.2% (w/v) glucose appeared to allow an efficient 14 C-lysine incorporation into the bacteria, with a maximum after 7 h incubation. Quantitative separation of the unattached bacteria from BECs with adhering P. aeruginosa was obtained by filtration on a polycarbonate membrane filter of 12 μm pore size, presenting minimal bacterial retention. This procedure may be preceded by centrifugation (14000 g , 10 min) on a 30–40% Percoll density gradient preformed at 12000 g for 20 min, to achieve a better separation of free P. aeruginosa from BECs, in the presence of large amounts of bacteria. This radioisotopic adhesion assay was compared with the classic microscopic adhesion assay. Both methods gave similar results in terms of the mean number of adherent bacteria estimated per epithelial cell. The dispersion of the experimental values, however, was higher with the microscopic assay. These observations and the undeniable advantages of the experimental procedure underlying the radioisotopic method prompt the authors to propose the use of this radioisotopic adhesion assay as an interesting alternative to the classic microscopic assay.