Molecular Identification of an Endogenous Adjuvant

描述（由申请人提供）：在多种病理情况下，细胞损伤导致免疫反应的诱导。有人提出，细胞死亡提供危险信号，帮助模拟对细胞抗原的免疫。随着最近尿酸作为参与因子之一的分子鉴定，细胞质中存在的另一种免疫调节因子的身份仍然难以捉摸。该项目的具体假设是，在细胞损伤过程中，细胞解体释放出在生理条件下通常被隔离的分子，一旦释放，这些物质通过激活抗原提呈细胞发挥辅助活性。我们的假设基于以下观察结果。损伤细胞的胞浆在CTL诱导中显示出佐剂活性；2)。从施胶柱中纯化的高分子量物质（40 ~ 100kd）激活抗原提呈细胞。本研究的目的是对这种高分子量物质进行生物化学鉴定和表征。我们有两个具体目标。1. 高分子量物质的分子鉴定。我们将采用色谱/质谱联用方法进行鉴定。我们将通过CTL诱导/树突状细胞活化来监测高效液相色谱馏分中的佐剂活性。实质上纯化的活性组分将用质谱仪分析。2. 高分子量佐剂活性的表征。项目的这一部分将侧重于在目标1中确定的物质的表征。它可以直接从细胞中纯化到均匀或在适当的细胞中表达/合成。该物质将被测试其促进CTL反应、树突状细胞激活和其他免疫增强能力的能力。我们还将尝试制造针对该物质的抗体或使用siRNA干扰来阻断佐剂的活性。在所有的分析中，该物质将与尿酸晶体一起进行测试，以直接比较它们的效力。

DESCRIPTION (provided by applicant): Cell injury leads to the induction of immune responses under a variety of pathological circumstances. It has been proposed that cell death provides danger signals that help simulate immunity against cellular antigens. With the recent molecular identification of uric acid as one of the participating factors, the identity of another immune regulator present in the cytosol remains elusive. The specific hypothesis of the project is that cellular disintegration during cell injury releases molecules that are normally sequestered under physiological conditions, and that once released, these substances exert adjuvant activities by activating antigen presenting cells. We base our hypothesis on the following observations 1.) The cytosol from injured cells has been shown to have adjuvant activity in CTL induction; 2.) A high molecular weight substance(s) (40 to 100 KD) purified from sizing columns activates antigen presenting cells. The goal of this study is to biochemically identify and characterize this high molecular weight substance. We have two specific aims. 1. Molecular identification of the high molecular weight substance(s). We will use the combined approach of chromatography/mass spectrometry for identification. We will monitor adjuvant activities in HPLC fractions by CTL induction/dendritic cell activation. Substantially purified active fractions will be analyzed by mass spectrometers. 2. Characterization of high molecular weight adjuvant activities. This part of the project will focus on the characterization of the substance identified in Aim 1. It will be either purified directly from cells to homogeneity or expressed/synthesized in appropriate cells. The substance will be tested for its ability to promote CTL responses, dendritic cell activation and other immuno-enhancing capacities. We will also attempt to make antibodies against the substance or use siRNA interference to block the adjuvant activities. In all assays, the substance will be tested side by side with uric acid crystals for a direct comparison of their potencies.