Hematopoietic reconstitution following bone marrow transplantation requires that donor-derived stem cells come under the influence of the microenvironment (ME) which consists of host-derived stromal cells as well as donor-derived macrophages and lymphocytes. The interactions that must occur between these various cellular components to establish and maintain a functioning allograft are poorly understood. Such an understanding is imperative, however, for identifying and treating graft failure. Preliminary studies designed to identify mechanisms of graft failure suggest that there may be several different processes besides host-mediated graft rejection that can result in poor marrow function. Included among these are graft-versus-host reaction which may damage the host components of the ME, compromised donor accessory cell function caused by manipulation of donor marrow prior to transplantation, and cytomegalovirus infection of the microenvironment, resulting in alterations of growth factor production. All of these mechanisms are similar in that they involve a defective ME. To better understand how the ME may be compromised, we propose to use long- term marrow cultures (LTMC) to evaluate ME function in vitro, and relate these observations to marrow function in vivo. Specifically, ongoing studies of normal LTMC function will continue to provide a more precise definition of the functional components of the microenvironment, including identification of cytokines produced and the cells responsible for their production. Data obtained from normal marrow will be compared to that obtained from patients who engraft normally and those who are experiencing graft failure. Immunocytochemistry will be used to identify the cellular components of the ME. RNase protection assays and polymerase chain reaction amplification of cDNA will identify the presence of specific cytokine mRNA. The presence of cytokine protein will be determined using a combination of bioassays, RIAs, and ELISA. Using stromal cell cultures that are initiated from isolated stromal cell precursors and therefore devoid of myeloid cells, we will investigate how the addition of specific allogeneic myeloid cells contributes to ME function. In association with project 0015, we will place special emphasis on how HLA disparity between these cell populations may affect cellular interactions. In conjunction with project 0014, stromal cell cultures will also be used to evaluate the role of anti-CD44 antibody in stromal cell-progenitor cell interactions. This requires that pure populations of progenitor cells be added to defined stromal cell cultures, and their ability to produce myeloid cells measured in the presence and absence of alpha-CD44 antibody. The studies proposed in this project should help to identify critical cellular components of the microenvironment and determine how specific transplantation procedures may interfere with their function.
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