Flores-Romo L, Bacon K B, Estrada-Garcia T, Shibayama M, Tsutsumi V, Martinez-Palomo A
Glaxo Institute for Molecular Biology, Plan-les-Ouates, Geneva, Switzerland.
J Immunol Methods. 1993 Dec 3;166(2):243-50. doi: 10.1016/0022-1759(93)90365-e.
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.
为了研究肠道病原体溶组织内阿米巴感染靶细胞的初始事件，我们基于人结肠癌细胞系（CaCo-2）和用荧光素标记的生物素化变形虫开发了一种定量黏附测定法。为了防止溶组织内阿米巴对结肠细胞产生强烈且快速的裂解活性（否则会妨碍对初始黏附步骤的研究），对寄生虫进行轻度固定、生物素化并用链霉亲和素-异硫氰酸荧光素标记。标记后的寄生虫与肠上皮细胞结合后，通过洗涤去除未黏附的变形虫，并使用自动荧光酶标仪对黏附的寄生虫进行定量。该生物测定法简单、无害，可在1.5小时内完成。在含有约10⁵个结肠细胞的96孔板中，我们能够检测到每微孔200至20000个荧光寄生虫的范围。荧光强度（任意单位）与每孔添加的寄生虫数量呈直接关系，且不受培养板大小（96、24或6孔）的限制。作为该测定法价值的一个例子，使用两种已知会影响内皮细胞和上皮细胞黏附特性的促炎细胞因子（白细胞介素-1（IL-1β）和干扰素-γ（IFN-γ））来评估它们对肠上皮细胞-阿米巴结合的影响。细胞因子处理肠上皮细胞后揭示的阿米巴结合增加表明，寄生虫可能利用炎症刺激来增加其与结肠上皮的结合。我们认为这种快速、灵敏且简单的方法为大规模筛选测定法提供了潜力，通过分析溶组织内阿米巴与肠上皮细胞之间初始相互作用所涉及的机制来研究这种原生动物感染的免疫生物学。
