Chipps T J, Streeter P R, Franc D T, Neumeyer K, Planck S R, Rosenbaum J T, Smith J R
Casey Eye Institute, Oregon Health and Science University, 3375 SW Terwilliger Blvd, Portland, OR 97239, USA.
J Immunol Methods. 2006 May 30;312(1-2):209-13. doi: 10.1016/j.jim.2006.03.003. Epub 2006 Apr 27.
In the human host, infection with the protozoan parasite, Toxoplasma gondii, most commonly involves the eye and/or the brain. Previous work indicates a relative susceptibility of the human retinal vascular endothelium to infection with the T. gondii tachyzoite, which may contribute to this tissue localization. To facilitate the investigation of potential adhesive interactions between parasite and endothelium in the retina, we have modified the Woodruff-Stamper assay, originally described to study lymphocytic-endothelial binding. Vascular endothelium was identified in sections of human retina by Alexa Fluor 594-tagged anti-von Willebrand factor antibody. Binding of yellow fluorescent protein-expressing tachyzoites to endothelium under conditions of flow, simulated by rotation on an orbital shaker, was quantified in a masked fashion using imaging software. We observed multiple yellow spots in contact with Alexa Fluor 594-positive retina, indicating binding of T. gondii tachyzoites to retinal vascular endothelium. This modification of the Woodruff-Stamper assay provides an opportunity to evaluate potential host receptors for T. gondii on the retinal vascular endothelium. In addition, the assay suggests a methodology that could be used to examine adhesion of other microbes to microvasculature in different tissues.
在人类宿主中，感染原生动物寄生虫刚地弓形虫最常累及眼睛和/或大脑。先前的研究表明，人类视网膜血管内皮细胞相对易受刚地弓形虫速殖子感染，这可能是该组织定位的原因之一。为便于研究寄生虫与视网膜内皮细胞之间潜在的黏附相互作用，我们改进了最初用于研究淋巴细胞与内皮细胞结合的伍德拉夫-斯坦珀试验。通过Alexa Fluor 594标记的抗血管性血友病因子抗体在人类视网膜切片中识别血管内皮细胞。在通过轨道振荡器旋转模拟的流动条件下，使用成像软件以盲法对表达黄色荧光蛋白的速殖子与内皮细胞的结合进行定量。我们观察到多个黄色斑点与Alexa Fluor 594阳性视网膜接触，表明刚地弓形虫速殖子与视网膜血管内皮细胞发生了结合。这种对伍德拉夫-斯坦珀试验的改进为评估视网膜血管内皮细胞上刚地弓形虫的潜在宿主受体提供了机会。此外，该试验还提出了一种可用于检测其他微生物与不同组织中微血管黏附的方法。
