Medical Biology - Theses
Permanent URI for this collection
Search Results
1 - 3 of 3
-
ItemGM-CSF regulation in inflammatory arthritis( 2019)Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease of the joints, affecting 0.5% to 1% of global population. Current targeted therapies antagonize the debilitating effects of key inflammatory mediators and immune cells. However, few patients achieve complete remission, prompting novel therapeutic approach. Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) was first identified as a haemopoietic growth factor but is now recognised as a proinflammatory cytokine in a number of autoimmune inflammatory diseases, including RA and Multiple Sclerosis (MS). GM-CSF promotes destructive joint inflammation by priming pro-inflammatory phenotypes of myeloid cells, such as neutrophils, monocytes and macrophages. Accordingly, therapies targeting GM-CSF or its receptor are currently under clinical evaluation in RA and MS and seem promising. However, much remains to be explored how GM-CSF is dynamically regulated during arthritis, especially in autoantibody-mediated inflammation as seen in seropositive RA patients. This work aims to characterize the cellular source of GM-CSF during antibody-induced arthritis and evaluate the cell-intrinsic negative regulation of GM-CSF signalling in myeloid cells and arthritis. I utilized the autoantibody-driven, immune complex-mediated serum transfer induced arthritis (STIA) murine model, which mimics the effector phase of seropositive RA patients. Using the novel GM-CSF dual reporter mice, joint-infiltrating Natural Killer (NK) cells were found to be main GM-CSF-producing cells during STIA. By using NK-deficient (Mcl1fl/fl:Ncr1-Cre) mice, NK-depleted (anti-NK1.1 antibody-treated) mice or specifically deleting GM-CSF production by NK cells (Csf2fl/fl:Ncr1-Cre), I was able to show the importance of NK cells and their GM-CSF-producing function in maintaining arthritis (Chapter 3). GM-CSF on myeloid cells induced the Cytokine Inducible SH2-containing (CIS) protein, a member of the suppressor of cytokine signalling (SOCS) protein family. Using Cish-/- mice, I showed that CIS negatively regulated GM-CSF signalling post activation, which is evident in intracellular signalling pathways, effector cell functions and in antibody-induced arthritis (Chapter 4). Taken together, this study provides new insights into the pathogenesis of antibody-driven, GM-CSF-mediated autoimmune inflammation and provide a rationale towards designing novel anti-inflammatory agents such as NK modulator or CIS mimetics for RA.
-
ItemDistinct precursors of the dendritic cell subtypes( 2006-03)Dendritic cells (DC) are antigen-presenting cells that are critical for the initiation and regulation of the immune response. Several DC subtypes within mouse spleen have previously been characterised and these include the plasmacytoid (pDC), and conventional DC (cDC) of the CD8+ and CD8- subtypes. Each subtype appears to have a specialised role in the various arms of immunity and tolerance. Less clear is the process by which these DC develop from haematopoietic precursors, of the precursor stages and branch points from bone marrow (BM) stem cells to each of the peripheral DC subtypes. The research described herein had the aim of identifying and isolating some of the intermediate precursors of DC, downstream of stem cells, and determining whether these differed in the steady-state versus inflammation. Particular was given to DC of the spleen. Experiments that sought the identity of such precursors involved both i) transfer of cell fractions that contained DC precursors into steady-state or inflamed recipient mice to assess their in vivo development at later times, and ii) analysis of an in vitro culture system to question whether it reflected development of the steady-state DC subtypes.
-
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.