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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Investigations into the role of endogenous Annexin-A1 in dendritic cell biology

Huggins, Anthony January 2012 (has links)
A school of literature has shown that Annexin-A1 (Anx-A1) is an endogenous anti-inflammatory protein that exerts a regulatory control over the innate immune system in order to restore homeostasis after an inflammatory reaction. Surprisingly, recent published works have highlighted that Anx-A1 has an alternate role in the adaptive immune system by positively modulating the strength of TCR signalling and biasing helper-subset differentiation. Dendritic cells are a class of innate leukocytes, poised at the environmental interface, that are the essential immune cells responsible in the initiation of T-cell driven responses. These findings provided the foundation for this PhD project, the principal aim of which is to provide a link between the disparate effects of Annexin-A1 in innate and adaptive immunity by investigating the role of endogenous Annexin-A1 in dendritic cell biology and its effector function as an antigen-presenting cell towards T cell activation and differentiation. To address this hypothesis, I cultured bone marrowderived dendritic cells from AnxA1-deficient mice or control littermates and stimulated with LPS (100ng/ml) then compared phenotypic and functional characteristics. My results demonstrate that Anx-A1-/- bone marrow derived dendritic cells show an increased number of CD11c+ cells expressing high levels of some maturation markers such as CD40, CD54 and CD80 and a decreased capacity to take up antigen compared to control Anx-A1+/+ cells. However, analysis of LPS-treated dendritic cells from Anx-A1-/- mice demonstrated a diminished up-regulation of maturation markers, a decreased migratory activity in vivo and an attenuated production of the inflammatory cytokines Interleukin (IL)- 1β, Tumour Necrosis Factor (TNF)-α and IL-12. This defect was resultant of an impaired Nuclear Factor (NF)-κB/DNAbinding activity due to lack of Anx-A1 signalling as demonstrated by the reduced activation of Extracellular-signal Regulated Kinase (ERK) 1/2 and protein kinase B (PKB)/Akt compared to cells from control littermates. As a consequence of these defects, I assessed the antigenpresenting/ T-cell activating capabilities of these DC. Anx-A1-/- DC showed an impaired capacity to stimulate T cell proliferation and differentiation in allogeneic mixed leukocyte reaction. To dissect this biologically relevant phenomenon further, I employed an antigenspecific, T-cell restricted model; a co-culture system of chicken ovalbumin peptide-pulsed, LPS-matured bone marrow-derived DC incubated with transgenic TCR T cells from OT-I/RAG-1-/- (OT-I, OTI/ CD8+) or OT-II/ RAG-1-/- (OT-II, OT-II/CD4+) mice. Peptide-pulsed, LPS-matured AnxA1-/- DC failed to initiate an appropriate T cell activation in both OT-I and OT-II T cells indicated by reduced cell proliferation when compared to T cells co-cultured with peptide6 pulsed, LPS-matured AnxA1+/+ DC. Additionally, comparison of peptide-pulsed, LPS-matured AnxA1-/- DC with AnxA1+/+ DC counterparts detected severely diminished levels of IL-2 from cocultures with OT-I T cells and ablated IFN-γ production from cocultures with both OT-I and OT-II T cells. In conclusion, AnxA1 seems to act as a positive modulator of immunogenic activation of DC, whereby the AnxA1 signal pathway has a probable synergism with the TLR4 signalling cascade. DCderived AxnA1 appears to contribute in promoting T cell activation with a larger influence on OT-I/CD8+ T cells than OT-II/CD4+ T cells. Altogether these findings suggest that inhibition of Anx-A1 expression or function in dendritic cells might represent a useful way to modulate the adaptive immune response and pathogen-induced T cell-driven immune diseases.
2

Targeting growth factors to sites of inflammation : gene therapy for multiple sclerosis

Sclanders, Michelle January 2013 (has links)
Disease progression in Multiple Sclerosis (MS), an autoimmune disease of the CNS, is widely accepted to be due to persistent myelin loss (demyelination) coinciding with lost nerve cells and nerve fibres (neuroaxonal loss). Current treatments are immunomodulatory and do not address the neuroaxonal or demyelinating pathology of the disease. It is hypothesised that a lack of growth factors within the CNS may result in the failure of remyelination. Therefore, biologics such as recombinant therapeutic proteins used for gene therapy offer a promising therapeutic intervention to the progressive stages of the disease. However, due to the short half-lives of these therapeutics and their pleiotropic effects, there is cause for concern over their safety and efficacy. Using LAP technology (the fusion of the therapeutic protein with the latent associated peptide [LAP] of TGFβ), the half-life of the therapeutic protein can be increased and can be targeted to sites of inflammation and disease. This study aimed to investigate the potential neuroprotective, remyelinating and anti-inflammatory effects of latent versions of the growth factors erythropoietin (EPO), insulin-like growth factor 1 (IGF1) and transforming growth factor beta (TGF) respectively. Firstly, using molecular cloning techniques, these growth factors were individually fused and linked to the LAP of TGF via a matrix metalloproteinase (MMP) cleavage site resulting in three latent growth factors. Secondly, these latent growth factors were shown to be expressed, and to be biologically active in vitro when released by MMP cleavage. Finally, syngeneic fibroblasts were engineered to express the latent growth factors. It was found that, in CREAE, the fibroblasts engineered to produce latent TGF significantly reduced the disease clinical score as compared to controls whilst latent EPO produced by transduced fibroblasts failed to exert a statistically significant effect on disease progression. Nonetheless, this study demonstrates the feasibility of the latency platform technology to generate latent therapeutics with the ability to act as an intervention to disease progression in MS.

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