http://repub.eur.nl/res/aut/1926/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/8938/ 1998-01-01T00:00:00Z Radiation-induced pancytopenia proved to be a suitable model system in mice and rhesus monkeys for studying thrombopoietin (TPO) target cell range and efficacy. TPO was highly effective in rhesus monkeys exposed to the mid-lethal dose of 5 Gy (300 kV x-rays) TBI, a model in which it alleviated thrombocytopenia, promoted red cell reconstitution, accelerated reconstitution of immature CD34+ bone marrow cells, and potentiated the response to growth factors such as GM-CSF and G-CSF. In contrast to the results in the 5 Gy TBI model, TPO was ineffective following transplantation of limited numbers of autologous bone marrow or highly purified stem cells in monkeys conditioned with 8 Gy TBI. In the 5 Gy model, a single dose of TPO augmented by GM-CSF 24 h after TBI was effective in preventing thrombocytopenia. The strong erythropoietic stimulation may result in iron depletion, and TPO treatment should be accompanied by monitoring of iron status. This preclinical evaluation thus identified TPO as a potential major therapeutic agent for counteracting radiation-induced pancytopenia and demonstrated pronounced stimulatory effects on the reconstitution of immature CD34+ hemopoietic cells with multilineage potential. The latter observation explains the potentiation of the hematopoietic responses to G-CSF and GM-CSF when administered concomitantly. It also predicts the effective use of TPO to accelerate reconstitution of immature hematopoietic cells as well as possible synergistic effects in vivo with various other growth factors acting on immature stem cells and their direct lineage-committed progeny. The finding that a single dose of TPO might be sufficient for a clinically significant response emphasizes its potency and is of practical relevance. The heterogeneity of the TPO response encountered in the various models used for evaluation points to multiple mechanisms operating on the TPO response and heterogeneity of its target cells. Mechanistic mouse studies made apparent that the response of multilineage cells shortly after TBI to a single administration of TPO is quantitatively more important for optimal efficacy than the lineage-restricted response obtained at later intervals after TBI and emphasized the importance of a relatively high dose of TPO to overcome initial c-mpl-mediated clearance. Further elucidation of mechanisms determining efficacy might very well result in a further improvement, e.g., following transplantation of limited numbers of stem cells. Adverse effects of TPO administration to myelosuppressed or stem cell transplanted experimental animals were not observed. http://repub.eur.nl/res/pub/8706/ 1997-01-01T00:00:00Z The detection of functional growth factor (GF) receptors on subpopulations of hemopoietic cells may provide a further dissection of immature cell subsets. Since little information is available about coexpression of different GF receptors at the level of single hemopoietic cells, we studied the feasibility of simultaneous cell staining with a combination of biotin- and digoxigenin-labeled GFs for flow cytometric detection of functional receptors. Using this methodology, coexpression of Kit and receptors for erythropoietin (EPO), interleukin 6 (IL-6), and GM-CSF on hemopoietic cells was studied by triple-staining of rhesus monkey bone marrow (BM) cells with labeled GFs and antibodies against other cell surface markers. Most of the immature, CD34+2 cells were Kit+ but did not display detectable levels of EPO-receptors (EPO-Rs) or GM-CSF-R. Approximately 60% of these CD34+2/Kit+ cells coexpressed the IL-6-R, demonstrating that immature cells are heterogeneous with respect to IL-6-R expression. Maturation of monomyeloid progenitors, as demonstrated by decreasing CD34 and increasing CD11b expression, is accompanied by a decline of Kit and an increase in GM-CSF-R expression in such a way that Kit+/GM-CSF-R+ cells are hardly detectable. IL-6-R expression is maintained or even increased during monomyeloid differentiation. IL-6-R and GM-CSF-R were not identified on most CD71+2 cells, which indicated that these receptors are probably not expressed during erythroid differentiation. Together with previous results, our data show that both Kit and CD71 are upregulated with erythroid commitment of immature progenitors. Upon further differentiation, Kit+/EPO-R-cells lose CD34 and acquire EPO-R. Maturing erythroid cells eventually lose CD71 and Kit expression but retain the EPO-R. In conclusion, this approach enables further characterization of the specificity of GFs for different bone marrow subpopulations. Apart from insight into the differentiation stages on which individual GFs may act, information about receptor coexpression may be used to identify individual cells that can respond to multiple GFs, and allows for further characterization of the regulation of lineage-specific differentiation.