Anti-neutrophil cytoplasmic autoantibodies (ANCA), targeting the neutrophil protein granules myeloperoxidase (MPO) and proteinase 3 (PR3), activate neutrophils in vitro, and are associated with a systemic autoimmune vasculitis, in which a pauci-immune crescentic glomerulonephritis is common. The in vivo study of ANCA pathogenesis is severely limited, both by the lack of a robust anti-PR3 IgG induced disease model, and by the differences found between human and mouse biology. Therefore the development of a disease model based on humanised mice, defined as mice possessing human immune cells, has the potential to overcome these limitations, while also allowing the direct study of human ANCA in vivo. The aims of this thesis were 1) to establish in vitro assays of ANCA induced neutrophil activation, and thus allow the study of ANCA:neutrophil interactions and 2) to establish a humanised mouse model of ANCA vasculitis. ANCA were purified from patient plasma, and two human neutrophil respiratory burst assays were developed. Subsequently, it was shown that Granulocyte Colony Stimulating Factor (GCSF) primes neutrophils for an anti-MPO induced respiratory burst, and the inhibition of phosphoinositol 3-kinase p/5 abrogates the ANCA induced release of superoxide. Humanised mice were produced by the engraftment of human haematopoietic stem cells into irradiated, adult NOD-scid \\-2rf1’ mice. At least 8 weeks later a population of human neutrophils were identified by flow cytometry, and this population was expanded by treatment with GCSF. It was shown that these cells could respond to inflammatory stimuli in vivo, by modulating their expression of activation markers, and in vitro, by undergoing respiratory burst and degranulation. Patient derived ANCA was passively transferred into humanised mice that had been primed with GCSF and lipopolysaccharide. Mice were culled 7 days later, but there was no histological or biochemical evidence of disease. Thus, this work has identified GCSF and phosphoinositol-3-kinase p/8 as molecules of potential importance in ANCA vasculitis. Furthermore, the passive transfer of patient ANCA into humanised mice did not prove pathogenic in this study, and possible reasons for this are discussed. The demonstration of functional responses in human neutrophils does, however, suggest that this humanised mouse model has the potential be useful for the study of human neutrophils in vivo.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628344 |
| Date | January 2013 |
| Creators | Coughlan, Alice |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://kclpure.kcl.ac.uk/portal/en/theses/the-development-of-a-humanised-mouse-model-of-anca-associated-vasculitis(0207457f-ce89-4c39-b69d-b80e9cf7eff5).html |
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