Chronic DTH in human GA

人移植物动脉硬化症的慢性迟发性高血压移植物动脉硬化(GA)是由宿主的抗移植物免疫反应引起的，通过内膜增生、病理性血管重塑和异常的血管调节来减少移植物的灌注量。由此产生的缺血会导致终末性器官衰竭。GA是晚期心脏移植物丢失(慢性排斥)的主要原因。GA的免疫发病机制尚不清楚。我们最初的免疫细胞化学研究指出慢性迟发性超敏反应(DTH)是主要机制。根据我们的发现，我们假设动脉内膜内的同种异体反应性、非溶细胞性宿主T细胞通过分泌细胞因子(如干扰素-γ)直接响应移植物内皮细胞(EC)上的同种异体抗原，通过激活巨噬细胞细胞因子的分泌，直接或间接地作用于EC和血管平滑肌细胞(VSMC)，导致构成GA的结构和功能变化。在这个项目中，我们将询问人的EC是否能直接刺激同种异体反应性的CD4+T细胞分化为介导迟发型变态反应的效应细胞(称为TH1细胞)；他们是否能招募已经分化的TH1细胞进入内膜；他们是否能激活内膜TH1细胞分泌干扰素-γ；以及这些过程是否通过EC损伤或EC与炎性细胞因子(如肿瘤坏死因子或IL-12)的激活而促进。我们还将询问移植物EC是否能间接激活或促进TH1细胞的分化(例如，肿瘤坏死因子或IL-12)。我们还将询问移植物EC是否可以间接激活或促进TH1细胞的分化(例如，通过树突状细胞)。这些实验将同时使用细胞培养系统(目标1)和完整的人类动脉，无论是在器官培养中还是在异种移植到SCID/Beige小鼠之后(目标2)。我们将使用我们完整的动脉培养和异种移植模型来确定同种异体反应性TH1细胞是否可以在动脉壁产生结构和功能变化，这是GA的特征(目标3)。我们将使用标准的形态学方法(例如，组织病理学、免疫荧光/免疫细胞化学、原位杂交)，并用激光捕获显微镜/逆转录对这些方法进行补充，以分析这些实验组织。为了验证我们的模型，我们将使用这些样本方法以及通过使用这些相同的方法以及通过使用带有肽噬菌体展示、蛋白质组学和功能基因组学的分子表达谱来将我们的实验血管与患者来源的GA样本进行比较(目标4)。将被检验的慢性迟发性迟发性高血压假说是PPG应用的核心，与项目2中提出的干扰素-γ对血管细胞作用的研究非常吻合。项目1也是项目3中提出的GA病理生理学成像的基础。项目1的实验使用了所有四个科学核心。

Chronic DTH in Human Graft Arteriosclerosis. Graft arteriosclerosis (GA) is caused by a host anti-graft immune response that reduces allograft perfusion through a combination of intimal hyperplasia, pathological vascular remodeling and abnormal vasoregulation. Resultant ischemia produces end organ failure. GA is the major cause of late cardiac allograft loss (chronic rejection). The immunopathogenesis of GA is unknown. Our original immunocytochemical studies pointed to chronic delayed type hypersensitivity (DTH) as the primary mechanism. Based on our findings, we hypothesized that alloreactive, non-cytolytic host T cells within the arterial intima directly respond to alloantigens on graft endothelial cells (EC) by secreting cytokines (e.g. IFN-gamma) that doth directly and indirectly, through activation of macrophage cytokine secretion, act on EC and vascular smooth muscle cells (VSMC) to cause the structural and functional changes that constitute GA. In this project we will ask if human EC can directly stimulate alloreactive CD4+ T cells to differentiate into effector cells that mediate DTH (known as TH1 cells); if they can recruit already differentiate TH1 cells to enter into the intima; if they can activate intimal TH1 cells to secrete IFN-gamma, and if these processes are promoted by EC injury or by EC activation with inflammatory cytokines (e.g., TNF or IL-12). We will also ask if graft EC can indirectly activate or promote the differentiation of TH1 cells (e.g., TNF or IL-12). We will also ask if graft EC can indirectly activate or promote the differentiation of TH1 cells (e.g. via dendritic cells). These experiments will use both cell culture systems (aim 1) and intact human arteries, either in organ culture or after xenotransplantation into SCID/beige mice (aim 2). We will use our intact artery culture and xenotransplant models to determine if alloreactive TH1 cells can produce structural and functional changes in the artery wall that are characteristics of GA (aim 3). We will use standard morphological approaches (e.g., histopathology, immunofluorescence/immunocytochemistry,. In situ hybridization) and supplement these methods with laser capture microscopy/reverse transcription- to analyze these experimental tissues. To validate our models, we will compare our experimental vessels with patient-derived GA specimens using these sample approaches as well as by using these same approaches as well as by using molecular expression profiling with peptide phage display, proteomics and functional genomics (aim 4). The chronic DTH hypothesis to be tested ION Project 1 is central to this PPG application and fits thematically well with the investigations of IFN-gamma actions on vascular cells proposed in Project 2. Project 1 also serves as a basis for the proposed imaging of GA pathophysiology proposed in Project 3. The experiments of Project 1 use all four scientific cores.