title: A fluorescence-based quantitative adhesion assay to study interactions between Entamoeba histolytica and human enterocytes. Effect of proinflammatory cytokines.
abstract: In order to study the initial events during infection of target cells by the enteric pathogen Entamoeba histolytica, we developed a quantitative adhesion assay based on the use of a human colonic cell line (CaCo-2) and biotinylated amoebae tagged with fluorescein. To prevent the strong and rapid lytic activity of Entamoeba histolytica on colonic cells, which would otherwise impede the study of the primary adhesion steps, parasites were mildly fixed, biotinylated and labelled with streptavidin-FITC. After labelled parasites have bound to enterocytes, nonadhered amoebae are removed by washing and attached parasites quantified by means of an automated fluorescence plate reader. The bioassay is simple, nonhazardous and can be completed in 1.5 h. We were able to detect ranges from 200 to 20,000 fluorescent parasites per microwell in a 96-well plate, containing approximately 10(5) colonic cells. Fluorescence intensity (arbitrary units) increased in direct relationship to the number of parasites added per well, and was not limited by the size of the culture plate (96, 24 or six wells). As an example of the value of this assay, two proinflammatory cytokines (interleukin-1, (IL-1 beta) and interferon-gamma (IFN-gamma) known to influence the adhesion properties of endothelial and epithelial cells, were used to assess their effects upon enterocyte-entamoeba binding. The increase in amoebae binding revealed by cytokine treatment to enterocytes suggests that the parasite may take advantage of inflammatory stimuli in order to increase its binding to colonic epithelium. We believe this rapid, sensitive and simple method offers the potential for large scale screening assays to study the immunobiology of this protozoal infection by analysing the mechanisms involved in the primary interactions between Entamoeba histolytica and enterocytes.
authors.affiliation: Glaxo Institute for Molecular Biology, Plan-les-Ouates, Geneva, Switzerland.
authors.forename: L
authors.initials: L
authors.lastname: Flores-Romo
authors.forename: K B
authors.initials: KB
authors.lastname: Bacon
authors.forename: T
authors.initials: T
authors.lastname: Estrada-Garcia
authors.forename: M
authors.initials: M
authors.lastname: Shibayama
authors.forename: V
authors.initials: V
authors.lastname: Tsutsumi
authors.forename: A
authors.initials: A
authors.lastname: Martinez-Palomo
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology / Q000378:metabolism / Q000469:parasitology*; D016207:Cytokines / Q000494:pharmacology*; D004748:Entamoeba histolytica / Q000378:metabolism / Q000472:pathogenicity / Q000502:physiology*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D006790:Host-Parasite Interactions; D006801:Humans; D010641:Phenotype; D012680:Sensitivity and Specificity; D014774:Virulence
chemical_list: D016207:Cytokines; D016650:Fluorescein-5-isothiocyanate