Analysis of the human endothelial cell response to pathogenic Leptospira interrogans vs. non-pathogenic Leptospira biflexa (Eahy1 data). Homo sapiens

The overall goal of these experiments was to determine how human endothelial cells respond to pathogenic Leptospira interrogans. Leptospira interrogans causes leptospirosis, the most widespread zoonotic infection in the world. A hallmark of leptospirosis is widespread endothelial damage, which in severe cases leads to hemorrhage. In these experiments, we infected two endothelial cell lines with pathogenic Leptospira interrogans serovar Canicola strain Ca12-005, and as controls, with the non-pathogenic Leptospira biflexa serovar Patoc strain Pfra. As additional controls, uninfected cells were also included in the analyses. Overall design: The cell line used was Ea.hy926, a macrovascular line (Edgell, C. J.,et al. 1990. In vitro Cell. & Dev. Biol. 26:1167-1172, and Edgell, C. J., et al. 1983. Proc. Natl. Acad. Sci. 80:3734-3737). Infection times were performed at a multiplicity of infection (# bacteria/endothelial cell) of 10 for either 1 hour or 3 hours, after which RNA was harvested and reverse transcribed. Labeled cDNAs were used to probe HEEBO arrays purchased from Microarrays Inc. (Nashville, TN). In each of three biological replicate experiments, for each time point, three comparisons were made. First, the L. interrogans-infected cells were compared to the L. biflexa-infected cells. Second, the L. Interrogans-infected cells were compared to the uninfected cells. Third, the L. biflexa-infected cells were compared to the uninfected cells. A second endothelial cell line, HMEC (Ades et al, 1992. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 99:683-690), which is of microvascular origin, was also used; raw data files are provided separately.

本实验的整体目标是解析人类内皮细胞对致病问号钩端螺旋体（Leptospira interrogans）的应答模式。问号钩端螺旋体可引发钩端螺旋体病（leptospirosis），这是全球分布最为广泛的人畜共患传染病。钩端螺旋体病的典型病理特征为广泛的内皮细胞损伤，重症病例可引发出血症状。

本实验中，我们使用致病问号钩端螺旋体犬型血清型菌株Ca12-005感染两种内皮细胞系，并以非致病的双曲钩端螺旋体Patoc血清型菌株Pfra作为对照；同时设置未感染细胞作为额外对照纳入分析。

实验总体设计如下：所用细胞系为Ea.hy926，属于大血管内皮细胞系（Edgell CJ等，1990，《In vitro Cellular & Developmental Biology》26:1167-1172；Edgell CJ等，1983，《美国国家科学院院刊》80:3734-3737）。感染复数（multiplicity of infection，即细菌/内皮细胞数量比）设置为10，分别感染1小时与3小时，随后收集RNA并进行反转录。将标记后的cDNA用于探针杂交，所用的HEEBO微阵列购自Microarrays Inc.（美国田纳西州纳什维尔市）。

在3次生物学重复（biological replicate）实验中，针对每个时间点共设置3组比较：其一，将问号钩端螺旋体感染组与双曲钩端螺旋体感染组进行对比；其二，将问号钩端螺旋体感染组与未感染对照组进行对比；其三，将双曲钩端螺旋体感染组与未感染对照组进行对比。

本研究同时使用了另一种微血管来源的内皮细胞系HMEC（Ades等，1992，《HMEC-1：永生化人微血管内皮细胞系的构建》，《Journal of Investigative Dermatology》99:683-690），相关原始数据文件将单独提供。