RESUMEN
Pseudomonas aeruginosa (PA) can cause infections in compromised hosts by interacting with the glycocalyx of host epithelial cells. It binds to glycostructures on mucosal surfaces via two lectins, which are carbohydrate-binding proteins, named PA-IL and PA-IIL, and blocking this interaction is, thus, an attractive anti-adhesive strategy. The aim of this study was to determine by ciliary beat frequency (CBF) analysis whether monosaccharides or peptides mimicking glycostructures represent blockers of PA lectin binding to human airway cilia. The treatment with monosaccharides and peptides alone did not change the CBF compared to controls and the tested compounds did not influence the cell morphology or survival, with the exception of peptide pOM3. PA-IL caused a decrease of the CBF within 24 h. D-galactose as well as the peptides mimicking HNK-1, polysialic acid and fucose compensated the CBF-modulating effect of PA-IL with different affinities. PA-IIL also bound to the human airway cilia in cell culture and resulted in a decrease of the CBF within 24 h. L(-)-fucose and pHNK-1 blocked the CBF-decreasing effect of PA-IIL. The HNK-1-specific glycomimetic peptide had a high affinity for binding to both PA-IL and PA-IIL, and inhibited the ciliotoxic effect of both lectins, thus, making it a strong candidate for a therapeutic anti-adhesive drug.
Asunto(s)
Cilios/efectos de los fármacos , Lectinas/antagonistas & inhibidores , Monosacáridos/farmacología , Péptidos/farmacología , Pseudomonas aeruginosa/metabolismo , Adhesinas Bacterianas/metabolismo , Secuencia de Aminoácidos , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Bronquios/metabolismo , Bronquios/microbiología , Antígenos CD57/química , Antígenos CD57/metabolismo , Cilios/metabolismo , Fucosa/química , Fucosa/metabolismo , Galactosa/química , Galactosa/metabolismo , Humanos , Lectinas/metabolismo , Imitación Molecular , Datos de Secuencia Molecular , Péptidos/química , Pseudomonas aeruginosa/patogenicidadRESUMEN
The formation of metastases is often investigated in xenografted human tumors in mice and the need arises to detect disseminated human tumor cells in small volumes of mouse blood. Two techniques, namely quantitative real-time polymerase chain reaction (qRT-PCR) and laser scanning cytometry (LSC), were compared for screening of 100 microl blood samples from immunodeficient mice spiked with a defined number of human HT29 colon carcinoma cells. With both techniques (qRT-PCR for amplifying of human Alu-sequences and LSC techniques for screening of fluorescence labelled cells), it was possible to detect single disseminated human tumor cells. Using the qRT-PCR technique, a recovery rate of nearly 100% was found when 10-10,000 cells were added to 100 microl blood. In contrast, the median recovery rate of the LSC technique varied between 20% (10 cells/100 microl blood) and 7.5% (10,000 cells/100 microl blood). Thus, it is advisable to quantify the number of human tumor cells in mouse blood by qRT-PCR and to use LSC for phenotyping of disseminated tumor cells only.