Medical Biology - Theses
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ItemThe control of normal and leukaemic human cells by the colony stimulating factors( 1986)To examine the control of human haemopoietic cells by the haemopoietic hormones (the Colony Stimulating Factors, CSF’s) both purified factors and purified cell populations are required. Studies were initially performed to characterize the action of purified murine and purified and partially-purified human CSF’s on human marrow cells. Of the purified murine CSF’s, G-CSF was active on human bone marrow cells. The murine molecule Eosinophil Differentiation Factor (EDF) was shown to an Eosinophil-CSF with activity on murine and human cells. Recombinant human GM-CSF (rHGM-CSF) was shown to possess all the biological activities of the partially purified native molecule CSF-α. Both the glycosylated and non-glycosylated preparations of rHGM-CSF showed equivalent biological activity in vitro. Subsequent studies were performed to purify and characterize a population of normal committed-granulocyte progenitor cells and the action of CSF’s on these cells was examined. Normal human promyelocytes and myelocytes were obtained using the monoclonal antibody WEM-G11 and the fluorescence activated cell sorter. These cells demonstrated transient, CSF-stimulated proliferation in vitro and generated neutrophilic clones of less than 40 cells in size (clusters). These cells were stimulated to proliferate by human CSF-α, CSF-β and murine G-CSF. Clone transfer experiments documented the ability of clones initiated by one CSF (α or β) to proliferate when transferred to cultures stimulated by the other CSF. The cross-species activity of human CSF-β and murine G-CSF, and the ability of CSF-β to compete with radio-iodinated murine G-CSF for binding-sites on murine (and human) cells suggested that CSF-β was the human equivalent of murine G-CSF. A comparative study of leukaemic promyelocytes demonstrated that fractionated promyelocytes-myelocytes from patients with chronic myeloid leukaemia also showed transient clonal proliferation in vitro and responded to CSF-α, CSF-β and murine G-CSF. Promyelocytes from the blood of these patients generated clones of only two cells in CSF-unstimulated cultures. This behaviour was mimicked when normal promyelocytes-myelocytes were pulse-stimulated by CSF for 45 min. Leukaemic cells from patients with acute myeloid leukaemia also demonstrated CSF-stimulated proliferation in vitro and were responsive to CSF-α, CSF-β, rHGM-CSF and murine G-CSF. There was however considerable heterogeneity in the CSF-responsiveness of these cells. Differentiation-induction by CSF in leukaemic cells was examined using the human leukaemic cell line HL60. When stimulated by CSF, these cells showed increased expression of myeloid surface antigens (as monitored by three monoclonal antibodies) and decreased numbers of clonogenic cells. In some experiments the number of clonogenic cells was reduced to zero. In an attempt to establish a sensitive micro-assay for CSF, two target cell populations were examined. Normal promyelocytes-myelocytes displayed CSF-stimulated proliferation in a micro-assay system but this was not associated with a heightened sensitivity to CSF. The survival of mature human neutrophils and eosinophils in vitro was however shown to be enhanced by CSF’s in a “lineage-specific” manner and this assay was between 10^2-10^3 times more sensitive to CSF than agar cultures. These studies demonstrated that the Colony Stimulating Factors enhanced survival, stimulated proliferation and stimulated differentiation-commitment of normal and leukaemic human cells.