Creaney J, Wilson L, Dosen M, Sandeman R M, Spithill T W, Parsons J C
Victorian Institute of Animal Science, Attwood, Australia.
Exp Parasitol. 1996 Jul;83(2):202-15. doi: 10.1006/expr.1996.0067.
Irradiation has been successful in the attenuation of infective stage parasites for use as vaccines against a number of parasites including Fasciola spp. The mechanisms of action of irradiation-attenuated vaccines, however, are not clearly understood. In this study, we examined the effect of 3, 10, and 40 krad of gamma-irradiation on the expression of carbohydrates and cathepsin-B by newly excysted juvenile Fasciola hepatica (NEJ). Following irradiation of metacercariae, the expression of concanavalin A (ConA)-specific sugars was decreased on the surface of NEJ and the expression of wheat germ agglutinin (WGA)-specific sugars was increased in the gut and reduced on the surface of NEJ. Cathepsin proteases are a major component of liver fluke excretory/secretory material (ES) and can cleave host immunoglobulin (Ig). Cathepsin-B protease was localized in nonirradiated NEJ to the gut lumen and to secretory granules within the gut epithelia. Irradiation of fluke with 3, 10, and 40 krad of gamma-rays significantly reduced the tissue expression of cathepsin-B at 8 hr postirradiation in an apparently dose-dependent manner. After a further 24 hr culture tissue expression of cathepsin-B was significantly reduced in 10- and 40-krad-irradiated NEJ. Protease activity of ES samples collected over a 24-hr period from irradiated and nonirradiated NEJ cultured in vitro were tested using a rabbit Ig cleavage assay. The proteolytic activity of ES from 10- and 40-krad-irradiated NEJ was reduced during the initial 6 hr in culture and between 12 and 24 hr when compared to ES from nonirradiated controls. Biosynthetic labeling experiments using [35S]methionine and [35S]cysteine indicated that ES material was actively synthesised during 48 hr in vitro culture. Therefore, from this study, we conclude that gamma-irradiation of NEJ alters expression of cathepsin-B protease and WGA- and ConA-specific sugars which may be detrimental to parasite invasion and contribute to the protective immune responses generated in the host by irradiation-attenuated metacercariae of Fasciola spp.
辐射已成功用于减毒感染阶段的寄生虫，以制备针对包括肝片吸虫属在内的多种寄生虫的疫苗。然而，辐射减毒疫苗的作用机制尚不清楚。在本研究中，我们检测了3、10和40千拉德的γ射线辐射对新脱囊的肝片吸虫幼虫（NEJ）碳水化合物表达和组织蛋白酶B表达的影响。对囊蚴进行辐射后，NEJ表面伴刀豆球蛋白A（ConA）特异性糖的表达降低，而麦胚凝集素（WGA）特异性糖在肠道中的表达增加，在NEJ表面的表达减少。组织蛋白酶是肝吸虫排泄/分泌物质（ES）的主要成分，可裂解宿主免疫球蛋白（Ig）。组织蛋白酶B蛋白酶在未辐射的NEJ中定位于肠腔和肠上皮内的分泌颗粒中。用3、10和40千拉德的γ射线辐射吸虫后，在辐射后8小时，组织蛋白酶B的组织表达以明显的剂量依赖性方式显著降低。在进一步培养24小时后，10和40千拉德辐射的NEJ中组织蛋白酶B的表达显著降低。使用兔Ig裂解试验检测了在体外培养24小时期间从辐射和未辐射的NEJ收集的ES样品的蛋白酶活性。与未辐射对照的ES相比，10和40千拉德辐射的NEJ的ES在培养的最初6小时以及12至24小时期间的蛋白水解活性降低。使用[35S]甲硫氨酸和[35S]半胱氨酸的生物合成标记实验表明，ES物质在体外培养48小时期间被积极合成。因此，从本研究中我们得出结论，NEJ的γ射线辐射改变了组织蛋白酶B蛋白酶以及WGA和ConA特异性糖的表达，这可能对寄生虫入侵不利，并有助于宿主对肝片吸虫属辐射减毒囊蚴产生保护性免疫反应。
