The long-term objectives of this application are to study the functional recovery of myeloid cells following autologous bone marrow transplantation (ABMT) and to investigate means of enhancing their cytotoxic and phagocytic abilities. The hypothesis is that immature myeloid cells can be induced with the cytokines GM-CSF, G-CSF, IL-3, and IFN-gamma to become functional earlier in myeloid maturation through expression of the receptors for the constant portion of IgG (FcgammaRs), and through enhanced maturation of the cellular cytotoxic components of the cells.
Specific aim 1 is: To determine if the maturational stage when hematopoietic progenitor cells acquire cytotoxic functions requires maturation of cytoplasmic components and/or specific levels of expression of FcgammaRs and adhesion molecules. In particular, we will (i) determine the sequence of functional maturation of myeloid cells in vitro as indicated by their ability to perform ADCC, perform phagocytosis, generate superoxide, and release cytokines, (ii) determine the time sequence when the surface markers FcgammaRs and the adhesion molecules are expressed, and (iii) determine if cytokines act in functional maturation of myeloid cells by inducing and enhancing expression of surface markers critical for triggering functional events.
Specific aim 2 is: To determine whether the functional maturation of myeloid cells in vitro reflects myeloid cell functional recovery in vivo after ABMT. The experimental design is to initially induce maturation of purified hematopoietic CD34+FcgammaR-Lin- cells in vitro as a model of myeloid functional maturation. Morphologic changes of the maturing cells will be assessed by cytochemical methods; flow cytometric analysis will be used to determine the time sequence at which the FcgammaRs and adhesion molecules are expressed. The in vitro cytotoxic potential of the cells will be determined serially by assays evaluating the functions of antibody-dependent cellular cytotoxicity (ADCC), superoxide generation, NBT reduction, and phagocytosis, allowing insight into the mechanism(s) of lysing target cells at the different stages of myeloid maturation. The second phase of this study will evaluate, by determination of mRNA synthesis and phenotypic characteristics of cells, if regulation of expression of the functionally important surface receptors FcgammaRI, FcgammaRII and of CD11b can be modified by the use of the cytokines GM- CSF, IL-3 and IFN-gamma. The third phase of the project will study the cytotoxic functional abilities of peripheral blood myeloid cells obtained serially from patients after ABMT, using a comparable set of studies as done in vitro. The findings from this study will be used to predict and direct cytokine treatment in vivo thereby optimizing myeloid cell functional recovery following ABMT.
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