Laboratory Core

实验室核心将提供集中化的分子微生物学诊断支持，细菌和 细胞培养支持。细胞因子测量，以及每个项目和其他核心所需的培训。 淋球菌(GC)、沙眼衣原体(CT)。将培养阴道毛滴虫(TV) 诊断PCRS DNA阳性对照和DNA测序对照。GC将从动物身上培养出来 体内实验期间的分泌物和组织，以测量基因缺陷的生物体数量(负荷) (KO)小鼠与野生型小鼠进行比较。GC荧光染色将用于体内感染的证据 实验，观察感染巨噬细胞的形态。核心将培养来自野生动物的巨噬细胞 分型和基因敲除小鼠，用GC研究结节样受体(NLRs)的作用。DNA将会 从小鼠阴道分泌物和组织中提取，在感染和感染期间的不同时间点收集 用特异的引物进行聚合酶链式反应检测GCDNA。样本中包含的生物体负荷将是 使用一种新的终点定量聚合酶链式反应方法(a-PCR)进行量化，这是我们之前验证过的 利用人类生殖器标本研究CT感染和传播，并用于研究弗朗西斯氏菌 利用组织标本(脾、肝、肺和肾)建立小鼠图拉氏菌(Ft)感染模型。这 测定结果与定量CT或Ft培养结果有很好的相关性。GC负荷将在体内进行量化 并进行体外实验，并与培养结果进行比较。生物体数量存在一个门槛 在将小鼠分为对细菌抗原刺激有反应者和无反应者的分泌物或组织中， 被调查。我们假设先天反应依赖于生物体负荷，而未受感染的 小鼠和低机体负荷的小鼠的反应(或无反应)比 机体负荷较高的小鼠。我们将研究GC负载是如何改变的：1)先天的变化 感染KO小鼠和双KO小鼠的免疫应答)；2)它们如何与数量 细胞因子的测定，以及3)影响感染小鼠不孕症的发展。

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.