ADHERENCE OF CANDIDA-ALBICANS TO EPITHELIAL-CELLS - STUDIES USING FLUORESCENTLY LABELED YEASTS AND FLOW-CYTOMETRY

Candida albicans adherence to epithelial cells is the first step in th e infectious process, but in spite of its importance, current methods for the quantitative measurement of adherence of C. albicans to epithe lial cells in vitro have some serious limitations. They are based on f iltration assays and either microscopic or radiometric analysis. The a dherence reaction is usually carried out with a large excess of yeasts (100-fold) over epithelial cells in order to perform the microscopic analysis, which is slow, subjective and limited to 100-200 cells and t hus lacks statistical power. The radiometric analysis fails to measure individual cells. A method for measuring yeast adherence that overcom es these problems has been developed. It is based on labelling the yea sts with the fluorogenic marker 2',7'-bis-(2-carboxyethyl)-5(6)-carbox yfluorescein acetoxymethyl ester (BCECF) prior to the adherence reacti on, and analysing 10(4) epithelial cells by flow cytometry, while nonb ound yeasts are excluded by gating. two subpopulations of buccal epith elial cells (BECs) which differ in their mean fluorescence intensities per cell (MFIs) were observed: one with MFI which did not exceed nons pecific fluorescence, and the other with MFI as high or higher than th e MFI of labelled yeasts. The two subpopulations represent yeast-free and yeast-binding epithelial cells, respectively, and the MFI incremen t of the BECs is a quantitative measure of the extent of yeast adheren ce. Control experiments confirming previously described basic features of adherence, such as enhanced adherence at increasing yeast excess, diminished adherence of trypsin-treated or heat-inactivated yeasts, an d the differential adherence of various Candida species, supported the validity of the assay. The possibility of studying adherence reliably at low yeast:epithelial cell ratios, which better mimic adhesion as i t occurs in vivo, is an important advantage of the assay. New findings , using this method, included the observation that exfoliated BECs fro m diabetic patients exhibited the same capacity for C. albicans adhere nce as cells from healthy controls, and that epithelial cells from ear ly human ontogenic stages had a significantly lower adherence level th an those from later stages.