The goal of this project is to develop and refine in vivo imaging techniques to permit an analysis of the cell movements, enzymatic activity and gene expression patterns of the immune system. Microinjection of fluorescent and magnetic resonance dyes permit a prospective analysis of tissue movement and cell lineage by uniquely labeling cells that can later be recognized by the presence of the cell-autonomous dye. MRI (magnetic resonance imaging) microscopy is an emerging imaging technology, well suited to questions of the immune system. An integrated approach employing an assembled team with expertise in chemistry, experimental embryology, MRI microscopy and immunology will focus on three specific aims: 1. identify and study antigen-specific lymphocytes. This will be accomplished by the development and testing of new MRI contrast agents (T1 and T2) so that individual cells or small groups of cells can be indelibly labeled. These agents will be of three types: membrane impermeant, membrane intercalating and receptor mediated for specific delivery. These MRI """"""""dyes"""""""" will be used to follow directly cell and tissue movements within the mouse embryo by repeatedly imaging the same animal to track immune response; 2. synthesize and test new classes of biochemically-triggered in vivo MRI contrast agents designed to detect cysteine protease activity (caspases) or calcium, and will be of two types: A. agents that monitor in vivo enzymatic processes, and ultimately gene expression; B. agents that monitor in vivo activity of intracellular messengers. This will permit studies of cell signaling and regulation in intact animals and a host of diagnostic applications; and 3. refine and implement MR imaging techniques as a facile means of obtaining 3D anatomical and functional information in mice that have been labeled with the contrast agents prepared in 1 and 2. The impact of a class of MR agents that can report on gene activity in living embryos cannot be overstated. It would permit a variety of new approaches involving the expression of transgenes during development as well as offer the possibility of determining the site of cells infected with recombinant retroviruses in lineage studies. Further, specific caspase inhibition may result in the protection of functional cells. In order for this inhibition to become a model for the development of therapeutic strategies, a fundamental understanding of the in vivo mechanism is required. A MR contrast agent that is uniquely sensitive to this enzyme process would be of great utility in dissecting this mechanism.
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