Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.583769
Title: In vitro and in vivo models to investigate tissue remodelling in Graves' ophthalmopathy
Author: Baker, Glynn R. C.
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2006
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Abstract:
In chapter one the aim was to establish and extend the characterization of murine models of thyroiditis and Graves' ophthalmopathy (GO), induced by transfer of TSH receptor (TSHR) primed T cells. In the first experiment, genetic immunization or TSHR fusion protein induced TSHR antibodies in all nine mice. Some of the antibodies functioned as thyroid-stimulating antibodies and/or TSH binding inhibiting Igs with 2/7 mice having elevated T4. Splenocyte transfer induced no immune response in naive BALB/cbyJico recipients. Subsequently genetic immunization or fusion protein-treated mice were maintained in either local or Brussels conditions (water, chow, and bedding). TSHR antibodies were induced in 9/9 Brussels (with decreased T4 in 1/9) but 5/9 local mice. No thyroiditis or orbital changes were induced, but misleading fixation artefacts in extraocular muscles were noted. Thyroids from many TSHR-treated and control mice contained ectopic thymus. Ectopic thymus can masquerade as thyroiditis, and care is required to avoid muscle artefacts. In chapter 2 the biological effects of TSHR activation in vitro, in adipose tissue - the site of orbital TSHR expression was examined. Activating mutant TSHR (TSHR*) or wild type (WT), were introduced into human orbital preadipocytes using retroviral vectors. Their proliferation, basal cAMP accumulation, spontaneous and PPARy induced adipogenesis were assessed and compared with non-modified cells. Expression of TSHR* significantly inhibited the proliferation of preadipocytes and produced an increase in unstimulated cAMP. PPARy induced adipogenesis in non-modified cells produced morphological changes, but not in TSHR* expressing cells. TSHR activation renders preadipocytes refractory to PPARy induced adipogenesis. The failure to develop lipid-containing vacuoles suggested the terminal stages of differentiation have been inhibited. In chapter 3 tears from patients with Graves' disease (GD), GO and smokers were examined for TSHR activation and protein changes. Reflex tears were collected from 37 normal non-smokers (NNS), 33 normal smokers (NS), 51 GD and 85 GO patients. Specific thyrotropin receptor stimulating activity (TSHRSA) was measured and also serum thyroid stimulating antibodies (TSAB). Pooled NNS, NS and GO tears were separated by SDS- PAGE and silver stained. Proteins expressed in the NS and GO pools but not NNS, were excised and analysed by MALDI-TOF mass spectrometry. Specific TSHRA was detected in 25% NS, 32% GD and 41% GO. A thyroid blocking antibody did not inhibit TSHRA. Clinical activity score correlated with serum TSAB but not tear TSHRSA. SDS-PAGE analysis revealed a higher vol/vol protein concentration in GO tears and additional proteins of 30 to 41 kD, in GO and NS compared with NNS. These were identified as zinc-alpha-2-glycoprotein (ZAG) and lactoferrin (LF). ZAG (also known as lipid mobilising factor) and LF do not have TSHRSA. Therefore, similar changes in tear composition are present in GO and NS compared with NNS. We postulate that TSHRSA is a protease enzyme. ZAG and LF expression is increased and their molecular weights are modified, suggesting degradation and/or changes in glycosylation, which may affect their bioactivities. In chapter 4, transgenic adiponectin elevation in FVB mice produced profound changes in interscapular and orbital fat depots. No previous models have demonstrated orbital fat proliferation and these changes could be followed by orbital MRI. TSHR expression increases with adipogenesis but does not in this case lead to autoimmunity directed towards the TSHR. A wide range of circulating adiponectin levels are present GO patients and no clinical correlations were evident.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.583769  DOI: Not available
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