Defining the Complosome in Human Cells, Tissues and Disease States

摘要 在过去三年半的R01赠款支持期间，我们在以下方面取得了实质性进展： A)评估C3在新发现的细胞内补体系统(ICS或“复合体”)中的作用， 包括CD46信号的调节，b)开发一种检测方法来定量检测具有生物活性的 C3称为C3(H2O)，c)识别大多数细胞类型共同的受体介导的C3(H2O)摄取过程， 以及d)确定细胞和器官系统(呼吸道)对缺血和免疫的ICS反应 居间攻击。我们目前和正在进行的目标是发表我们的结果，描述人类受体 C3(H2O)以及研究表明，在同种异体免疫中，肺移植的C3表达减少。 居间伤害。我们还在进一步描述复合体的激活和调节机制 RNA和蛋白质水平，并将我们的分析扩展到人类外周血吞噬细胞、上皮细胞和， 尤其是内皮细胞群体。另一个目标是剖析ICS的作用，利用体外和 从肺组织供者和相应的人类细胞系中分离出原代人类细胞的模型系统， 在感染的小鼠模型中也是如此。因此，我们建议继续开发和使用模型系统 为了获得一种细胞类型的复合体如何在正常内稳态中发挥作用的详细图谱以及它的 对细胞损伤和恶性肿瘤的反应。这样的分析可能会引发对如何 这一系统的功能障碍与人类疾病有关。 这项提案的一个重要目标与肿瘤学有关。除了它作为一种抑制因子的作用之外 在宿主细胞上的补体激活和复合体的关键成分CD46正在成为一个关键 在恶性转化和癌症免疫治疗方面发挥着重要作用。一方面，CD46在细胞上过度表达 许多肿瘤，但另一方面，它是治疗性溶瘤病毒的靶标，利用它作为对接。 机制。我们为这些研究做出了贡献，现在计划剖析其过度表达的原因 及其对复合体的影响，从多发性骨髓瘤与Siteman癌协同开始 中心。所开发的模型将随后扩展到其他类型的癌症。 总而言之，我们的建议汲取了我们对以下领域长期承诺的力量 补充研究和我们雄心勃勃的承诺，确定 新发现的ICS。我们特别被这一赠款机制所吸引，因为它具有灵活性。这 提案不仅利用了私人投资公司的专业知识，而且还利用了一支才华横溢的团队。 通过创建更具创新性的模型系统并获得 全面的地图，扩展了我们相对于ICS的“运作”的知识基础。

Abstract During the past three and one-half years of R01 grant support, we have made substantial progress in: a) assessing the role of C3 in the newly discovered intracellular complement system (ICS or “complosome”), including its modulation by CD46 signaling, b) developing an assay to quantitate a biologically active form of C3 known as C3(H2O), c) identifying a receptor-mediated C3(H2O) uptake process common to most cell types, and d) determining the ICS response in cells and an organ system (the airway) to ischemic and immune- mediated attack. Our immediate and ongoing goals are to publish our results describing the human receptor for C3(H2O) as well as studies demonstrating that C3 expression by lung transplants is reduced in alloimmune- mediated injury. We are also further delineating complosome activation and regulatory mechanisms at the RNA and protein level and are extending our analyses to human peripheral blood phagocytic, epithelial and, especially, endothelial cell populations. A further goal is to dissect the role of the ICS, utilizing both an in vitro model system with primary human cells isolated from lung tissue donors and corresponding human cell lines, as well as in mouse models of infection. Thus, we propose to continue to develop and employ model systems to obtain a detailed map of how a cell type's complosome functions in normal homeostasis as well as its response to cellular damage and malignancy. Such analyses will likely trigger new insights into how dysfunctions of this system correlate with human diseases. An important goal of this proposal relates to oncology. In addition to its role as an inhibitor of complement activation on host cells and a critical component of the complosome, CD46 is emerging as a key player in both malignant transformation and cancer immunotherapy. On one hand, CD46 is overexpressed on many tumors, yet on the other hand, it is targeted by therapeutic oncolytic viruses that use it as a docking mechanism. We have contributed to these studies and now plan to dissect the rationale of its overexpression and its effect on the complosome, beginning with multiple myeloma in collaboration with the Siteman Cancer Center. The models developed will be extended subsequently to other types of cancers. In summary, our proposal draws on the strength of our long-term commitment to the field of complement research and our ambitious undertakings related to defining the mechanisms and players of the newly discovered ICS. We are especially attracted to this grant mechanism because of its flexibility. This proposal draws not only on the expertise of the PI, but also on a team of talented individuals who are well positioned to continue being leaders the field by creating more innovative model systems and obtaining a comprehensive map that expands our knowledge-base relative to the “workings” of the ICS.