Thyroid stimulating antibodies (TSAB) cause Graves’ disease and contribute to Graves’ Orbitopathy (GO) pathogenesis. We hypothesise that the presence of TSH binding proteins (truncated TSHR variants (TSHRv)) and/or nonclassical ligands such as thyrostimulin (α2β5) might provide a mechanism to protect against or exacerbate GO. We analysed primary human orbital preadipocyte-fibroblasts (OF) from GO patients and people free of GO (non-GO). Transcript (QPCR) and protein (western blot) expression levels of TSHRv were measured through an adipogenesis differentiation process. Cyclic-AMP production by TSHR activation was studied using luciferase-reporter and RIA assays. After differentiation, TSHRv levels in OF from GO were significantly higher than non-GO (p = 0.039), and confirmed in ex vivo analysis of orbital adipose samples. TSHRv western blot revealed a positive signal at 46 kDa in cell lysates and culture media (CM) from non-GO and GO-OF. Cyclic-AMP decreased from basal levels when OF were stimulated with TSH or Monoclonal TSAB (M22) before differentiation protocol, but increased in differentiated cells, and was inversely correlated with the TSHRv:TSHR ratio (Spearman correlation: TSH r = −0.55, p = 0.23, M22 r = 0.87, p = 0.03). In the bioassay, TSH/M22 induced luciferase-light was lower in CM from differentiated GO-OF than non-GO, suggesting that secreted TSHRv had neutralised their effects. α2 transcripts were present but reduced during adipogenesis (p < 0.005) with no difference observed between non-GO and GO. β5 transcripts were at the limit of detection. Our work demonstrated that TSHRv transcripts are expressed as protein, are more abundant in GO than non-GO OF and have the capacity to regulate signalling via the TSHR.

Background: Previous in vitro experiments have demonstrated that PGF2α reduced proliferation and adipogenesis in a murine cell line and human orbital fibroblasts derived from subjects with inactive Graves' orbitopathy (GO). The objective of this study was to determine if the PGF2α analogue Bimatoprost is effective at reducing proptosis in this population.Methods: A randomized controlled double-masked crossover trial was conducted in a single tertiary care academic medical center. Patients with longstanding, inactive GO but persistent proptosis (> 20 mm in at least one eye) were recruited. Allowing for a 15% dropout rate, 31 patients (26 females) were randomized in order to identify a treatment effect of 2.0 mm (p=0.05, two-sided paired t-test, power 0.88). Following informed consent, participants were randomized to receive Bimatoprost or placebo for three months after which they underwent a two-month washout, before switching to the opposite treatment. The primary outcome was the change in exophthalmometry readings over the two 3-month treatment periods.Results: The mean exophthalmometer at baseline was 23.6 (range 20.0-30.5) mm and the mean age was 55 (range 28-74) years. The median duration of GO was 7.6 (IQR 3.6-12.3) years. The majority were still suffering from diplopia (61.3%) with bilateral involvement (61.3%). Using multilevel modeling adjusted for baseline, period and carryover, Bimatoprost resulted in a -0.17 mm (reduction) exophthalmometry change (95% CI -0.67 to +0.32) p=0.490. Intraocular pressure was reduced -2.7 mmHg (95% CI -4.0 to -1.4) p=0.0070. One patient showed periorbital fat atrophy (PAP) on treatment which resolved on stopping treatment. Independent analysis of proptosis by

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