Dendritic Cell-Based Biosensor System

描述（由申请人提供）：树突状细胞（DC）战略性地位于环境界面，作为免疫前哨细胞和组织驻留抗原呈递细胞。当暴露于感染性微生物或“危险信号”时，DC产生促炎介质，经过成熟，并迁移到淋巴结激活幼稚T细胞。因此，DC激活是连接先天免疫和适应性免疫的初始和关键事件。我们将采用基于直流的生物传感器系统来系统地研究直流激活过程，并开发新的直流刺激剂。具体目标是：1)优化基于dc的生物传感器。我们已经开发了一个DC生物传感器原型，通过转染XS106 DC细胞系，13个荧光素酶报告基因具有不同的顺式增强子，并鉴定了各种微生物，生物，药理学和物理因子，激活DC中不同的转录调控途径。我们将通过分离永久表达EGFP报告结构的DC克隆来优化该系统。2)系统验证。我们将通过测试目前已知的直流刺激剂和筛选来自NCI的化合物文库（>105复杂度）来测试直流生物传感器的实用性。一旦验证，这项技术将转移到行业，以促进他们的药物发现工作。3)合成刺激dc的类肽。类肽（n -取代甘氨酸低聚物）在抗蛋白酶性、稳定性和多样性等方面比多肽具有优势。以分裂/池方式合成的肽库（bbb107复杂度）将以头固定形式进行筛选。“Hit”化合物将转化为可溶性二聚体形式，并通过分子改组修饰以获得刺激dc的“铅”肽。4)合成直流刺激照明。使用可调照明设备，我们将通过测试bbbb107不同照明的影响来搜索宇宙的光。我们还将测试DC激活是由表面受体的光化学交联诱导的概念。5)确定不同药剂活化DC的生物学后果。新发现的DC刺激剂将检查其对DC的基因表达谱、表面表型、细胞因子和趋化因子产生、T细胞刺激能力的体外影响以及对朗格汉斯细胞的体内影响。我们将使用计算生物学工具分析实验数据，以定义控制直流激活过程的算法。我们的研究将为DC生物学提供新的见解，并可能导致新的佐剂和治疗方法的发展。

DESCRIPTION (provided by applicant): Dendritic cells (DC) are strategically located at the environmental interface, serving as immunological sentinel and tissue-resident antigen presenting cells. Upon exposure to infectious microorganisms or "danger signals", DC produce pro-inflammatory mediators, undergo maturation, and migrate to lymph nodes to activate naive T cells. Thus, DC activation is the initial and key event linking innate to adaptive immunity. We will employ the DC-based biosensor system to study DC activation processes systematically and to develop novel DC-stimulating agents. Specific aims are: 1) To optimize the DC-based biosensor. We have developed a DC biosensor prototype by transfecting XS106 DC line with 13 luciferase reporters with different cis-enhancers and identified various microbial, biological, pharmacological, and physical agents that activate distinct transcription regulatory pathways in DC. We will optimize this system by isolating DC clones permanently expressing EGFP reporter constructs. 2) System validation. We will test the utility of the DC biosensor by testing currently known DC-stimulating agents and by screening a chemical compound library (>105 complexity) from NCI. Once validated, this technology will be transferred to the industry to facilitate their drug discovery effort. 3) To synthesize DC-stimulating peptoids. Peptoids (N-substituted glycine oligomers) have advantages over peptides in protease resistance, higher stability, and higher diversity. Peptoid libraries (>107 complexity) synthesized in split/pool fashions will be screened in bead-immobilized forms. "Hit" compounds will be transformed into soluble dimeric forms and modified by molecular shuffling to obtain DC-stimulating "lead" peptoids. 4) To synthesize DC-stimulating illumination. Using a tunable illumination device, we will search the universe of light by testing the impact of >107 different illumination. We will also test the concept that DC activation is inducible by photochemical cross-linking of surface receptors. 5) To determine biological consequences of DC activation by different agents. Newly identified DC-stimulating agents will be examined for their in vitro impact on gene expression profiles, surface phenotype, cytokine and chemokine production, and T cell-stimulating capacity of DC and for in vivo impact on Langerhans cells. We will analyze experimenta data using computational biology tools to define algorithms that govern DC activation processes. Our study wil provide new insights into DC biology and may lead to the development of novel adjuvants and therapeutics.