Laboratory Core

实验室核心将提供集中的分子微生物学诊断支持， 细胞培养支持。每个项目和其他核心所需的细胞因子测量和培训。 淋球菌（GC）、沙眼衣原体（Ct）。阴道毛滴虫（电视），将培养 诊断PCR DNA阳性对照和DNA测序对照。GC为动物培养物 分泌物和组织，以测量基因缺陷型中的生物体数量（负荷） (KO)与野生型小鼠相比。GC荧光染色将用于体内感染的证据 实验和感染的巨噬细胞的形态学。核心将培养野生巨噬细胞 型和基因敲除小鼠，用GC激发以研究NLR的作用。DNA将 从小鼠阴道分泌物和组织中提取，在感染期间的不同时间点收集， 将使用特异性引物通过PCR检测GC DNA。样品中所含的微生物负荷将 使用新的终点定量PCR方法（a-PCR）进行定量，我们之前已经验证， 使用人类生殖器标本研究Ct感染和传播，并研究弗朗西斯菌 使用组织样品（脾、肝、肺和肾）在小鼠模型中检测土拉热菌（Ft）感染。这 测定与定量Ct或Ft培养具有极好的相关性。将在体内定量GC负荷 和体外实验并与培养物进行比较。生物体数量存在阈值 在将小鼠分为对细菌抗原刺激应答者和无应答者的分泌物或组织中， 追究我们假设，先天性反应是依赖于生物体的负荷， 小鼠和具有低有机体负荷的小鼠将具有较低的响应（或无响应）， 具有较高生物负荷的小鼠。我们将研究GC负载如何：1）通过先天性 感染KO小鼠和双KO小鼠免疫应答）; 2）它们如何与定量 细胞因子的测量，和3）影响感染小鼠中不育的发展。

The Laboratory Core will provide tiie centralized molecular microbiology diagnostic support, bacterial and cell culture support. Cytokine measurements, and training needed for each of the Projects and other Cores. Neisseria gononrhoeae (GC), Chlamydia trachomatis (Ct). Trichomonas vaginalis (TV), will be cultijre for diagnostic PCRs DNA positive controls and DNA sequencing controls. GC will be culture from animal secretions and tissue during in-vivo experiments to measure organism numbers (loads) in gene deficient (KO) mice compared to wild type mice. GC fluorescence staining will be use to evidence infection for in-vivo experiments, and tiie morphology of infected macrophages. The core will cultijre macrophages from wild type and knockout mice, to be challenged witii GC to study tiie role of nod- like receptors (NLRs). DNA will be extracted from mice vaginal secretions and tissue, collected at different time points during infection and GC DNA will be detected by PCR using specific primer. The organism loads contained in the sample will be quantified using a new end point quantitative PCR method (a-PCR) tiiat we have previously validated to study Ct infection and transmission using human genital specimens, and for the study of Francisella tularensis (Ft) infection in the mouse model using tissue samples (spleen, liver, lungs and kidneys). This assay has an excellent correlation with quantitative Ct or Ft culture. GC loads will be quantified in in-vivo and in-vitro experiments and compared to culture. The presence of a threshold in the number of organisms in secretions or tissue that divide mice into responder vs. non-responders to bacterial antigen stimulation will be investigated. We hypothesize that the innate response is dependent on the organism load, and that uninfected mice and mice with low organism loads will have a lower response (or no respond) compared with mice having higher organism loads. We will study how GC loads: 1) are modified by changes in innate immunity respond in infected KO mice and double KO mice); 2) how they conrelate with quantitative measurements of Cytokines, and 3) influence the development of sterility in infected mouse.