This application represents a response to an RFA on hematopoietic stem cells. The most promising results during this period of funding related to their studies on homing and traffic of stem cells and progenitors. In carefully done preliminary studies the applicant has determined that treatment of transplanted cells with an antibody to VLA4 integrin or an antibody to VCAM-1 inhibited the seeding of progenitor cells in the bone marrow and also discovered that seeding of the spleen increased dramatically as did the number of progenitors circulating in the blood. In addition, the applicant has discovered that administration of anti VLA4 in vivo leads up to a 100-fold increase in circulating progenitor cells in baboons. There are three Specific Aims. The first, to delineate molecular pathways that control homing will investigate the role of the CS-1 region of the fibronectin molecule using blocking peptides and antibodies, will investigate the role of carbohydrate ligands in the lodgment assay, will test radiated vs. unirradiated endothelial cells and stroma (for this purpose innovative studies using the ROSA beta- geo26 mouse as donor will permit studies on seeding in vivo), and finally will assess the homing properties of cytokine mobilized peripheral blood stem cells and of in vitro cytokine treated BM cells.
The Second Aim i s designed to study mechanism of mobilization of hematopoietic progenitor cells with G-CSF and kit- ligand (steel factor). Specifically, the Applicant will test whether simultaneous treatment of animals with G-CSF and anti VLA4 leads to additive mobilization. In addition, she will investigate the possible role of soluble VCAM-1 in blocking alpha 4 function, determine whether Steel factor mobilization will be different than G- CSF mobilization and will determine whether long term repopulating cells are mobilized with VLA4. In addition, the activation status of VLA4 will be examined in studies that utilize a soluble human VCAM-1IgG1 fusion protein that binds to human and murine VLA4 expressing cells only after receptor activation. In addition, the applicant will compare early mobilization kinetics elicited by anti- VLA4 or anti beta 1 integrin antibodies and by chemokines IL-1 and IL-8. The Third Specific Aim is to develop an animal model for the study of the role of cytoadhesion molecules in hematopoiesis. Because VCAM-1 and VLA4 knockout mice are largely lethal, the applicant seeks to develop an in vivo model for tissue specific knockouts known as the Cre-lox mouse. The Applicant proposes to use this method to disrupt the alpha 4 chain gee of VLA4, but knock it out only in hematopoietic tissues. The approach uses the bacteriophage P1 Cre recombinase to catalyze recombination between two loxP sequences, a reaction that splices out intervening DNA between the two lox P sites. Only one intact loxP site is left after recombination Tissue specific deletion of any gene can be theoretically achieved if Cre expression is under the control of cis elements that function in a tissue specific manner. The approach of the Applicant is outlined on page 44. The applicant will generate LCR globin promoter Cre transgenic mice or CD34 promoter driven Cre transgenic mice to breed with mice containing the gene for alpha 4 between loxP sites.
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