The Toponome of Virus Infected Skin

摘要 病毒性皮肤感染是人类人口发病和死亡的一个重要原因，有数百万人 世界各地感染痘病毒的人，这种病毒只存在于 当免疫受损的个体出现衰弱的全身性疾病时，由宿主免疫系统进行检查 感染。相关的痘病毒痘苗病毒(VACV)被用来为数亿人接种疫苗 在根除天花计划期间，它仍然是最广泛使用的病毒疫苗的骨干 向量。在病毒感染期间，靶器官的形态会发生显著的变化，由 在病毒生命周期进程中的细胞融合，以及通过大量各种反应的涌入 免疫细胞。感染所产生的炎症也可以刺激非经典型的分化 与细胞不同的驻留或浸润性细胞群体通常处于稳定状态。 因此，这些细胞的复杂表型需要使用大的探针面板(主要是 抗体)。然而，传统的方法(显微镜、流式细胞术、蛋白质组学或基因组学)不允许 单个细胞蛋白质表达的同时分析、量化和拓扑定位 细胞和蛋白质之间的相互关系，而不破坏和混淆组织分离或分析 来自单个组织的50-100个参数。为了完成这次调查，我们将进行非侵入性的 使用Toponome成像系统分析50+参数，成像周期显微镜是一种完全 自动显微镜，允许大分子和细胞系统显示的空间分辨率 在完整的组织中有数以千计的潜在相互作用。我们将分析分子和细胞的变化 在皮肤不同部位感染后用VACV(一种痘病毒感染模型)感染小鼠皮肤。 我们假设多重拓扑学分析将揭示一个独特的分子和细胞特征 痘病毒皮肤感染。在目标1中，我们将优化染色程序并检查和验证感染， 寄居细胞和浸润性细胞在起始、控制和发育过程中的定位和表型 VACV皮肤感染的消退阶段。在目标2中，我们将研究两个单独的减数分裂细胞的作用 在确定VACV病变的形态和浸润性细胞的功能方面有重要作用。我们 预期从这些研究中获得的结果将成为未来RO1或PO1提案的基础 感染、驻留和渗透的细胞群体在病毒皮肤中相互作用的机制 感染，有可能使用临床样本来验证在人类身上的研究。

Abstract Viral skin infections are a significant cause of morbidity and mortality in the human populations, with millions of individuals infected worldwide with the poxvirus molluscum contagiosum virus (MCV), which is only held in check by the host immune system as immunocompromised individuals present with debilitating systemic infection. The related poxvirus Vaccinia virus (VACV) was used to immunize hundreds of millions of people during the smallpox eradication program, and remains a backbone of the most widely used viral vaccine vectors. During virus infection there is a significant change in the morphology of a target organ caused by cellular fusion during progression of the virus life cycle, as well as by the influx of a large variety of responding immune cells. The inflammation produced by infection can also stimulate the differentiation of non-classical populations of resident or infiltrating cells that do not resemble cells typically present in the steady state. Therefore, the complex phenotyping of these cells requires the use of large panels of probes (mostly antibodies). However, traditional methods (microscopy, flow cytometry, proteomics or genomics) do not allow simultaneous analysis of protein expression by individual cells, quantification and topological localization of cells and proteins relative to each other without damaging and confounding tissue dissociation, or analysis of 50-100 parameters from a single tissue. In order to accomplish this investigation we will perform non-invasive analysis of 50+ parameters using a Toponome Imaging System, an imaging cycler microscope that is a fully automated microscope that allows the spatial resolution of large molecular and cellular systems displaying many thousands of potential interactions in intact tissues. We will analyze the molecular and cellular changes in the skin at different points after infection of the skin of mice with using VACV (a model for poxvirus infection). We hypothesize that multiplexed toponomic analysis will reveal a unique molecular and cellular signature poxviral skin infection. In Aim 1 we will optimize staining protocols and examine and validate infection, localization and phenotype of resident and infiltrating cell populations during the initiation, control and resolution phases of VACV dermal infection. In Aim 2 we will examine the role of two individual meyloid cell populations in establishing both the morphology of the VACV lesion and the function of infiltrating cells. We anticipate that the results gained from these studies will form the basis of a future RO1 or PO1 proposal in which the mechanisms by which infected, resident and infiltrating cell populations interact in viral skin infections, with potential for the use of clinical samples to validate out studies in humans.