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.
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