The thyroid, the eyes and the gut: a possible connection.

INTRODUCTION: Graves' disease (GD) is an autoimmune disorder responsible for 60-90% of thyrotoxicosis, with an incidence of 1 to 2 cases per 1000 population per year in England. Graves' orbitopathy (GO) is the most frequent extrathyroidal manifestation, not provoked directly by abnormal thyroid hormone levels, but by the consequence of the underlying autoimmune process. The aetiology of autoimmune disorders is due to an interplay between susceptibility genes and environmental factors, such as infections and stress. What triggers the autoimmune reaction to a specific site of the body is not yet clearly understood. The lack of knowledge in GD and GO pathogenesis implicates therapies that only limit damage but do not prevent disease onset. MATERIAL AND METHODS: We performed on PubMed and the Cochrane Library a literature search for the articles published until July 2016 by using the search terms 'graves disease' and 'microbiome', 'orbitopathy' and 'autoimmune pathogenesis'. Reference lists of relevant studies were hand-searched for additional studies. CONCLUSION: In this scenario, a Marie Sklodowska-Curie funded project INDIGO ( http://www.indigo-iapp.eu/ ) is investigating the role of the gut bacteria in GD and GO pathogenesis. The gut is the first and the widest area of bacteria access, with the highest concentration of T cells in the human body and trained to react to microorganisms. Interestingly, all the environmental factors involved in GD and GO pathogenesis can alter the balance within the microorganisms located in the gut, and influence the immune system, in particular the proportions of regulatory Treg and inflammatory TH17 cells. It is hoped that investigating GD and GO pathogenesis from this novel aspect will identify new targets for prevention and treatment.

Moderate-intensity exercise alters markers of alternative activation in circulating monocytes in females: a putative role for PPARγ.

PURPOSE: Monocytes may be primed towards differentiation into classically activated M1 macrophages or alternatively activated M2 macrophages. M1 macrophages greatly contribute to the inflammation which promotes insulin resistance, whereas M2 macrophages resolve inflammation. We have previously shown that exercise increases M2 marker expression in mixed mononuclear cells, possibly via activation of the nuclear transcription factor PPARγ. However, these effects have not been demonstrated specifically within monocytes. Thus, we aimed to investigate whether moderate-intensity exercise elicited similar effects on monocytic M1/M2 marker expression and PPARγ activity to those reported previously in mononuclear cells, so as to further elucidate the mechanisms by which exercise may alter inflammatory status and, accordingly, prevent insulin resistance. METHODS/RESULTS: 19 sedentary females completed an 8 week moderate-intensity exercise programme (walking 45 min, thrice weekly). Monocytes were isolated from blood via immunomagnetic separation; monocyte expression of M2 markers (Dectin-1: 2.6 ± 1.9-fold; IL-10: 3.0 ± 2.8-fold) significantly increased, whilst the expression of the M1 marker MCP-1 significantly decreased (0.83 ± 0.2 cf. basal), over the duration of the programme. Serum PPARγ activity levels and PPARγ target-genes (CD36: 1.9 ± 1.5-fold; LXRα: 5.0 ± 4.7-fold) were significantly increased after the 8 week exercise programme. Associated with these effects were significant improvements in systemic insulin sensitivity (McAuley's ISI: Δ0.98 M/mU/L cf. basal). CONCLUSION: Exercise participation suppressed M1 markers and induced M2 markers in monocytes, potentially via PPARγ-triggered signalling, and these effects may contribute (perhaps via priming of monocytes for differentiation into M2 tissue-macrophages) to improved systemic insulin sensitivity in exercising participants. These findings provide an alternative mechanism by which exercise may exert its anti-inflammatory effects in order to prevent insulin resistance and type 2 diabetes.

Reversal of Pathological Features of Graves' Orbitopathy by Activation of Forkhead Transcription Factors, FOXOs.

CONTEXT: Graves' orbitopathy (GO) is a disfiguring/distressing, inflammatory autoimmune condition. This intractable problem is caused by expansion of the orbital contents around the eye by excessive fat generation (adipogenesis) and overproduction of extracellular matrix components, especially hyaluronan (HA) from preadipocytes/fibroblasts (PFs). Current immunosuppressive/antiinflammatory treatments are largely ineffective and have unpleasant side effects, and a better therapeutic strategy through understanding GO-associated pathological features is needed. OBJECTIVE: Previously we identified depot-specific HA synthase 2 regulation (HAS2; major source of HA), which facilitates orbit-specific HA accumulation during adipogenesis, and targeting phosphatidylinositol-3-kinase/mechanistic target of rapamycin-complex-1 pathways blocked both pathological features. The current study revealed low expression levels of Forkhead box O (FOXOs; critical downstream effectors of phosphatidylinositol-3-kinase) in orbital PFs through adipogenesis compared with sc levels. We aimed to dissect the role of FOXOs in GO pathogenesis to identify nonimmunosuppressive targets for GO treatment. DESIGN/SETTING/PARTICIPANTS: Human orbital and sc primary PFs were treated with small interfering RNA/chemical inhibitor (AS1842856) of FOXOs or FOXO enhancer trifluoperazine hydrochloride (TFP; Food and Drug Administration approved drug), in serum-free medium for 24 hours, or TFP treatment in adipogenic medium for 15 days. MAIN OUTCOME MEASURES: Quantitative PCR was used to measure HAS2 transcripts and the terminal adipogenesis differentiation marker lipoprotein lipase. HA accumulation in the medium was measured by an ELISA. RESULTS: Substantially increased or decreased HAS2/HA production was observed by inhibiting (small interfering RNA or chemical inhibitor) or enhancing (TFP) FOXO expression, respectively. TFP treatment is also sufficient to counteract thyrotropin receptor-activated HAS2/HA production and block adipogenesis in orbital PFs. CONCLUSIONS: FOXOs play a crucial repressor role in the regulation of HAS2/HA production and adipogenesis in orbital PFs. Our data reveal for the first time that resetting GO-associated pathological features through drug-targeted activation of FOXOs could provide a feasible nonimmunosuppressive therapeutic strategy for GO.

The sodium iodide symporter is unlikely to be a thyroid/breast shared antigen.

PURPOSE: Anti-thyroid peroxidase (TPO) autoantibodies (TPOAb) seem to be protective for patients with breast cancer (BC). Thyroid and breast tissues both express the sodium iodide symporter (NIS), similarly both have a peroxidase activity, TPO and lactoperoxidase (LPO) respectively. We hypothesize a common immune response to a thyroid/breast shared antigen suggesting three putative mechanisms: (1) TPOAb react to both TPO and LPO, (2) TPO could be expressed in BC and (3) patients with TPOAb could have autoantibodies to NIS (NISAb). Previous studies excluded NISAb that block NIS activity in sera of patients with thyroid autoimmunity (TA) and/or BC. This study investigates neutral NISAb (binding without affecting function). METHODS: Clones of CHO cells stably expressing human NIS (hNIS; CHO-NIS) were isolated following transfection of hNIS in pcDNA3 vector. Expression of hNIS mRNA and surface protein was confirmed by PCR and flow cytometry respectively using a hNIS-mouse-monoclonal-antibody. CHO-NIS and controls transfected with the empty pcDNA3 vector (CHO-Empty) were incubated with 42 heat-inactivated human sera followed by an anti-human-IgG-AlexaFluor488-conjugate: 12 with BC, 11 with TA, 10 with both BC and TA and 9 with non-autoimmune thyroid diseases. The Kolmogorov-Smirnov Test was used to compare the fluorescence intensity obtained with CHO-NIS and CHO-Empty, using sera from six young males as a negative control population. RESULTS: None of the 42 sera were positive for NISAb. CONCLUSIONS: NISAb are rare and NIS is unlikely to be a common thyroid/BC shared antigen. We have recently demonstrated TPO expression in BC tissue and are currently investigating TPOAb cross-reactivity with TPO/LPO.

Possible targets for nonimmunosuppressive therapy of Graves' orbitopathy.

CONTEXT: Graves' orbitopathy (GO) is caused by expansion of the orbital contents by excess adipogenesis and overproduction of hyaluronan (HA). Immunosuppressive and antiinflammatory treatments of GO are not always effective and can have side effects, whereas targeting GO-associated tissue remodeling might be a more logical therapeutic strategy. Previously we reported that signaling cascades through IGF1 receptor and thyrotropin receptor within orbital preadipocytes/fibroblasts drove adipogenesis and HA production. Our current study combined the stimulation of IGF1 receptor and thyrotropin receptor increase of HA accumulation, which we hypothesize is by activation of phosphatidylinositol 3-kinase (PI3K)-1A/PI3K1B, respectively. The central aim of this study was to investigate whether PI3K/mammalian target of rapamycin complex 1 (mTORC1) inhibitors affected adipogenesis and/or HA production within orbital preadipocyte/fibroblasts. METHODS: Human orbital preadipocytes were treated with/without inhibitors, LY294002 (PI3K1A/mTORC1), AS-605240 (PI3K1B), or PI103 (PI3K1A/mTORC1) in serum-free medium for 24 hours or cultured in adipogenic medium for 15 days. Quantitative PCR was used to measure hyaluronan synthases (HAS2) transcripts and the terminal adipogenesis differentiation marker lipoprotein lipase. HA accumulation in the medium was measured by an ELISA. RESULTS: Unlike AS-605240, both LY294002 (10 µM) and PI-103 (5 µM) significantly decreased HAS2 transcripts/HA accumulation and adipogenesis. Because PI-103 and LY294002 are dual PI3K/mTOR inhibitors, we investigated the inhibition of mTORC1 (rapamycin 100 nM), which significantly decreased adipogenesis but had no effect on HAS2 transcripts/HA, implicating PI3K-1A in the latter. CONCLUSIONS: The combined inhibition of PI3K1A and mTORC1 signaling in vitro decreased both HA accumulation and adipogenesis. Because PI3K and mTOR inhibitors are clinically used to treat other conditions, they have the potential to be repositioned to be used as an alternative nonimmunosuppressive therapy of GO.

Does thyroid peroxidase provide an antigenic link between thyroid autoimmunity and breast cancer?

Women with breast cancer (BC) and antithyroid peroxidase (TPO) autoantibodies (TPOAb) have a better prognosis than women lacking TPOAb. Sera from women with TPOAb displayed immunoreactivity to BC tissue by immunofluorescence that was not apparent in women without TPOAb. We hypothesize a BC/thyroid shared antigen that provides a target for humoral or cell-mediated immune activity; candidates include the sodium/iodide symporter (expressed in thyroid and BC), cross-reacting epitopes in TPO and lactoperoxidase (LPO) or TPO itself. As the association is with TPOAb, we investigated TPO expression in BC, breast peritumoral tissue (PT), other tissues (tumoral and not) and thyroid as positive control. Transcripts for known and novel TPO isoforms were detected in BC (n = 8) and PT (n = 8) but at approximately 10(4) -fold lower than in thyroid while in non-BC tumors (n = 5) they were at the limit of detection. TPO was expressed also in adipose tissue (n = 17), 10(3) -fold lower than in thyroid. Full length TPO (Mr 105-110 kDa) was detected in Western blots in the majority of examined tissues; preabsorption of the TPO antibody with recombinant TPO (but not LPO) reduced the signal, indicating specificity. The same occurred with some lower molecular weight bands, which could correspond to smaller TPO transcript isoforms, present in all samples. In conclusion, TPO is weakly expressed in BC and other tissues; this could partly explain the high frequency and protective role of TPOAb in BC patients. Further studies will investigate tissue specificity, function and immunogenicity of the novel TPO variants (some BC-specific) identified.

Dehydroepiandrosterone (DHEA) treatment in vitro inhibits adipogenesis in human omental but not subcutaneous adipose tissue.

Dehydroepiandrosterone (DHEA), a precursor sex steroid, circulates in sulphated form (DHEAS). Serum DHEAS concentrations are inversely correlated with metabolic syndrome components and in vivo/in vitro studies suggest a role in modulating adipose mass. To investigate further, we assessed the in vitro biological effect of DHEA in white (3T3-L1) and brown (PAZ6) preadipocyte cell lines and human primary preadipocytes. DHEA (from 10(-8)M) caused concentration-dependent proliferation inhibition of 3T3-L1 and PAZ6 preadipocytes. Cell cycle analysis demonstrated unaltered apoptosis but indicated blockade at G1/S or G2/M in 3T3-L1 and PAZ6, respectively. Preadipocyte cell-line adipogenesis was not affected. In human primary subcutaneous and omental preadipocytes, DHEA significantly inhibited proliferation from 10(-8)M. DHEA 10(-7)M had opposing effects on adipogenesis in the two fat depots. Subcutaneous preadipocyte differentiation was unaffected or increased whereas omental preadipocytes showed significantly reduced adipogenesis. We conclude that DHEA exerts fat depot-specific differences which modulate body composition by limiting omental fat production.

Animal-type melanoma: a clinical and histopathological study of 22 cases from a single institution.

Background Animal-type melanoma is a rare distinct melanoma subtype, characterized by proliferation of heavily pigmented epithelioid and spindled melanocytes that resembles the heavily pigmented melanomas seen in grey horses. While animal-type melanoma is generally considered to be more indolent than conventional melanoma, only a limited number of cases have been reported and, as such, the clinical characteristics of animal-type melanoma are incompletely understood. Objectives To characterize the clinical and histopathological features of animal-type melanoma, and determine any features that may predict outcome. Patients/Methods Data was extracted from a prospectively collected melanoma database (1994-2008), and a retrospective pathology database (1991-2008) for all patients with a diagnosis of both equivocal (8) and unequivocal (14) malignant animal-type melanoma. We reviewed the clinical and histopathological features, including the sentinel lymph node biopsy (SLNB) status. Results A total of 22 patients were identified, with a median age of 35 years. The median Breslow depth was 2.22 mm. A SLNB was performed in 17 patients, eight (47%) were positive. Younger age was associated with: (i) animal-type melanoma with features equivocal for malignancy (median age of 7 vs. 48 years, P = 0.01), and (ii) a negative SLNB (median age 12 vs. 53 years, P = 0.03). Four patients with unequivocal animal-type melanoma developed recurrent metastatic disease, with one patient death. No patient with an equivocal animal-type melanoma or negative SLNB developed recurrent disease; however, this did not reach statistical significance (P = 0.13 and P = 0.09, respectively). Conclusions Animal-type melanoma has a propensity for regional lymphatic metastasis and is rarely capable of disseminated metastatic disease and death. Animal-type melanoma appears to exhibit a spectrum of biological behaviour, with young patient age associated with more indolent disease.

Current insights into the pathogenesis of Graves' orbitopathy.

Graves' orbitopathy (GO) is part of an autoimmune disease constellation comprising hyperthyroidism, orbitopathy, pretibial myxedema, and acropachy. Signs and symptoms of GO occur due to inflammation of the orbital connective tissue, inflammation and fibrosis of the extraocular muscles, and adipogenesis. Stimulatory TSH receptor (TSHR) antibodies (TRAb) cause hyperthyroidism, but pathogenetic mechanisms in the orbit are less clear. The TSHR is one of the favoured candidate antigens; others such as the IGF1R might also play a role. Compared with other anatomical locations, orbital fibroblasts are extremely reactive to inflammatory stimuli, especially via CD40 activation. Orbital fibroblasts also differentiate into adipocytes, in response to the prevailing inflammatory cytokine milieu. Consequently TSHR gene expression increases together with expression of adipogenesis related genes. The same genes that confer susceptibility to Graves' disease (GD), both thyroid specific and immunoregulatory, also influence GO, although an increasing number of candidate genes with higher impact on orbitopathy are being identified. Smoking is the only environmental factor known to increase the likelihood and severity of GO developing in GD patients. A robust animal model of GO would facilitate the evaluation of new treatments. To date most models have centered on provoking autoimmune responses to the TSHR, but other antigens, alone or in combination with this receptor, hopefully will succeed in inducing the full spectrum of GD.

Tobramycin-induced aquagenic wrinkling of the palms in a patient with cystic fibrosis.

Aquagenic wrinkling of the palms (AWP) is a rare condition, defined clinically by the appearance or accentuation of an asymmetrical, translucent to white, papular eruption on the palms after immersion in water. It is associated with cystic fibrosis (CF), and approximately half of all reported cases occur in patients with documented CF. We report a case of AWP in a young woman with CF, where the AWP was related to treatment with the aminoglycoside antibiotic, tobramycin. Although the mechanism of AWP is unknown, influx of water across an osmotic gradient into eccrine ducts has been proposed. Aminoglycosides may affect AWP by blocking various cell surface channels and receptors, which may influence cell-volume regulation.

Enhancement of glycoprotein hormone alpha subunit promoter reporter gene activity in co-transfection studies--a cautionary reminder.

Reporter constructs have a wide range of application in determining eucaryotic gene regulation and expression. IN VITRO models frequently necessitate co-transfection and there have been reports of interference, predominantly inhibition, between promoters. Studies investigating the biological activity of a mutant thyrotropin receptor involved co-transfection with receptor constructs and a cAMP responsive luciferase reporter driven by the glycoprotein hormone alpha subunit promoter. We observed considerable enhancement of basal luciferase activity by co-transfecting the empty expression vector, which contained the SV40 late promoter. We have investigated the mechanism using different concentrations and several viral promoters in COS cells, following transient co-transfection. The increase was dose dependent but plateaued at 50 ng of vector DNA. This was not due to an adjuvant effect since luciferase activity was unchanged by adding increasing amounts of a promoterless plasmid. Enhancement was maintained when truncating the promoter to -346, but eliminated in the promoter truncated upstream of -244, indicating a binding site for a putative repressor of glycoprotein hormone alpha expression between -244 and -346. Enhancement was maintained with the addition of a second constitutive reporter (bos beta-gal) to correct for transfection efficiency, although this was the consequence of enhanced luciferase activity by the bos beta-gal, which in itself was inhibited by the SV40 promoter. Artefactual enhancement of reporter activity can occur and highlights the need for careful choice of controls when performing transient co-transfection experiments. In silico analysis of the promoters identified a possible shared forkhead transcription factor binding site.

A new form of familial multi-nodular goitre with progression to differentiated thyroid cancer.

We report a kindred with euthyroid multi-nodular goitre (MNG) of adolescent onset. Two of the seven subjects with MNG have progressed to papillary thyroid cancer. One affected male had nodular kidney disease, and breast cancer occurred in one affected female. Genes that were candidates on the basis of the associated kidney (PAX8) and breast diseases (sodium iodide symporter (NIS)), were sequenced. No mutations were found in the coding region, intron/exon splice sites or in the promoter sequences (from -1248 relative to the translation initiation codon) of PAX8. Similar results were obtained for NIS. Subsequently, microsatellite analyses were performed on 14 informative family members. We used 2 to 3 markers per locus for 6 loci (on chromosomes 1,2,3,14,19,X) previously reported to predispose to MNG and/or familial non-medullary thyroid cancer (FNMTC). On the basis of non-significant logarithm of the odds ratio (LOD) scores or inheritance of different alleles in affected individuals, all loci have been excluded. Thyroidectomy specimens from three members of the kindred show multiple benign lesions, with papillary cancer in two. The morphological features do not resemble those seen in familial adenomatous polyposis, Cowden syndrome, or in multiple oxyphil lesions. From these findings and from the absence of any linkage to any of the known loci associated with MNG or FNMTC, we suggest that this represents a new form of inherited MNG with a significant risk of progression to papillary carcinoma.

Quantification of cells expressing the thyrotropin receptor in extraocular muscles in thyroid associated orbitopathy.

BACKGROUND/AIM: Thyroid associated orbitopathy (TAO) and Graves' disease (GD) have an autoimmune pathogenesis, possibly related to the thyrotropin receptor (TSHR). The aim of this study was to determine whether TSHR immunoreactivity is correlated with disease severity or serum TSHR antibody (TRAB) levels. METHODS: Orbital tissues from 30 patients with TAO were compared with those of 20 patients with strabismus and four with non-thyroid orbital inflammation. TSHR was detected by immunohistochemistry and TRAB were measured by radioreceptor assay. RESULTS: No TSHR immunoreactivity was detected in the 24 control orbital tissues, whereas in all TAO biopsies elongated fibroblast-like cells, expressing TSHR, were present. These cells were located between the muscle cells, which were separated by oedema in the acute phase but fibrous tissue in the chronic phase of disease. Semi-thin sections showed numerous mast cells present in the chronic phase and in close contact with adipocytes. The number of TSHR immunostained cells was high in early disease, decreased with disease duration, and was positively correlated with TRAB levels at the onset of TAO. CONCLUSION: TSHR immunoreactivity was demonstrated specifically in TAO orbits which highlights the importance of TRAB early in the pathogenesis.

Biological activity of activating thyrotrophin receptor mutants: modulation by iodide.

Epidemiological studies have revealed a significantly higher incidence of toxic adenoma (TA) and toxic multi-nodular goitre (TMNG) in regions of iodine deficiency. Fifty to eighty percent of TA and TMNG are caused by activation of the cAMP pathway, mostly by mutations in the thyrotrophin receptor (TSHR). We aimed to investigate whether iodide could modulate the biological effects of activating TSHR mutations. We have applied an in vitro model of TA comprising FRTL-5 cells stably expressing activating TSHR. We have mimicked the in vivo situation by examining the effects of prolonged exposure to iodide on the proliferation and signal transduction etc. of these cells. We observed an iodide-induced 'inhibition of proliferation' which was significant from 10 mM in the presence of serum but from 1 mM in its absence. The inhibition of proliferation was significantly higher in the activating mutant expressing FRTL-5 compared with control Neo or wild-type TSHR, indicating that the effect was mediated via the cAMP cascade. The effect was neither due to hyper-tonicity nor was it the result of an increase in cell death either by apoptosis or necrosis. Prolonged exposure to iodide produces an increase in cells in the G2 and post-G2 phases, indicating that G2/M blockade contributes to the mechanism of inhibition. The mutant expressing FRTL-5 cells have increased proliferation when chronically exposed to TSH, and this is associated with a reduction in phosphorylated (p) CREB levels. This contrasts with the effect of iodide in which inhibition of proliferation is accompanied by an increase in pCREB. In conclusion, our studies indicate that the biological effects of activating TSHR mutations vary with the ambient iodide supply and could be masked in regions of high iodine intake.

Inducing Graves' ophthalmopathy.

The majority of patients with Graves' disease (GD) have some degree of ocular involvement and this requires surgical or medical intervention in about 5% of cases. There are autoimmune and inflammatory processes operating in Graves' ophthalmopathy (GO), which together induce glycosaminoglycan production, edema and adipogenesis resulting in an increase in the volume of the orbital contents. GO is a heterogeneous disorder, i.e.: 1) whilst usually associated with hyperthyroidism it may occur in euthyroid (and even hypothyroid) patients; 2) expansion of orbital tissues may be due to 'big-fat' or 'big muscles'. The heterogeneity is further exemplified by the spectrum of protocols which have succeeded in inducing aspects of the disease both in animal models and in humans including: 1) Production of severe hypothyroidism in guinea pigs by thyroidectomy and administration of pituitary extract (TSH); 2) Induction of T cells autoreactive to the thyrotropin receptor (TSHR) in mice; 3) Depletion of regulatory T cells in humans susceptible to autoimmunity; 4) Modulation of adipose tissue metabolism in mice and men. In addition, identical induction protocols result in different pathological features depending on the environment, e.g. TSHR primed T cells produce thyroiditis and ocular pathology in BALBc mice in Brussels but thyroid stimulating antibodies accompanied by elevated thyroxine in these animals (from the same supplier) in Cardiff. Thus, experiences in the induction of GO have confirmed the polygenic, multifactorial nature of the disorder and highlight the importance of careful disease classification to promote further progress in understanding.

Potent thyrotrophin receptor-blocking antibodies: a cause of transient congenital hypothyroidism and delayed thyroid development.

OBJECTIVE: We describe an infant with surprisingly severe neonatal hypothyroidism due to transplacental passage of thyrotrophin receptor (TSH-R)-blocking antibodies (TBAb). DESIGN AND METHODS: TBAb were detected using a cell line which stably expresses the human TSH-R and a cAMP-responsive luciferase reporter by their ability to inhibit TSH-stimulated luciferase expression. Potent TBAb were detected in maternal serum and initially in the infant's serum but, in the latter, TBAb decreased over time to within the reference range by 3-4 months of age, illustrating the transient nature of this condition. RESULTS: The thyroid function of this child did not return to normal on withdrawal of thyroxine therapy at 16 months of age when he developed transient compensated hypothyroidism. CONCLUSIONS: We propose that the presence of potent TBAb in utero and in the first weeks of life may have implications for the development of a normally sized thyroid gland. We have demonstrated the presence of TBAb in the mother's milk and, as far as we are aware, this is the first such report. However, the TBAb in the milk probably did not contribute significantly to hypothyroidism in the child, given the reducing antibody titre in his circulation.

The prevalence of anti-acetylcholinesterase antibodies in autoimmune disease.

A robust and precise enzyme linked immunosorbent assay (ELISA) with proven sensitivity and specificity has been employed to detect human antibodies (allogenic/autogenic) to human acetylcholinesterase (AChE). The sensitivity of the method has been established using mouse monoclonal antibodies (0.8 ng/ml) and uniquely, human sera positive for anti-Yt(a) allogenic antibodies, to one phenotypic form (most common) of human AChE. The latter was also used as the positive human control to ensure functionality of the assay. The ELISA method was used to establish a normal distribution curve for absorbance values employing sera from healthy blood donors Subsequently, the ELISA was employed to investigate the prevalence of anti-AChE antibodies in patients with confirmed autoimmune disease and patients with non-autoimmune thyroid disease (diseased control). The results indicate that there is not a high prevalence of anti-AChE antibodies in patients with confirmed autoimmune disease. The lack of anti-AChE autoantibodies in patients' with clinically apparent Graves' ophthalmopathy, mitigates against there being a causal role of such antibodies in Graves' associated eye disease.

Biological activity of activating thyroid-stimulating hormone receptor mutants depends on the cellular context.

Activating TSH receptor (TSHR) mutations are a major cause of toxic thyroid adenoma and familial hyperthyroidism, and more than 37 such mutations have been described. Previously their functional activity had been assessed in terms of cAMP and inositol phosphate production and predominantly in transiently transfected COS-7 (monkey embryonic kidney cells), a model that does not reflect effects on thyrocyte proliferation and function. Here we have performed a systematic comparison of wild-type and seven gain-of-function TSHR mutants, introduced into rat FRTL-5 and human thyrocytes, using retroviral vectors. Our results show that 1) biological potency of TSHR mutants in thyroid cells does not correlate with their cAMP levels in transfected COS cells, highlighting the importance of cellular context and level of expression when assessing biological effects of oncogenic mutations; 2) dissociation between stimulation of function and growth occurs with thyrocyte differentiated functions more readily stimulated than growth; 3) TSHR mutants show a similar order of potency in FRTL-5 cells and human thyrocytes; 4) mutants inducing the highest stimulation of adenylyl cyclase may paradoxically fail to induce proliferation; and 5) biological effects of cAMP activating TSHR mutants are attenuated by complex counterregulatory mechanisms at least at the level of phosphodiesterases and cAMP regulatory element modulator isoforms.

Adipose thyrotrophin receptor expression is elevated in Graves' and thyroid eye diseases ex vivo and indicates adipogenesis in progress in vivo.

The thyrotrophin receptor (TSHR) provides an autoantigenic link between the thyroid and orbit in Graves' (GD) and thyroid eye diseases (TED). We measured TSHR transcripts in different fat depots to determine whether TSHR expression levels are influenced by the autoimmune/inflammatory process and/or thyroid hormone status, using quantitative real-time PCR. Nine intact or fractionated adipose samples, from patients with GD and/or TED, were analysed ex vivo. Eight expressed the TSHR, at levels approaching the thyroid, and one was at the limit of detection. Thirteen/fifteen orbital and abdominal fat samples from patients free of GD and TED, measured ex vivo, were negative for TSHR transcripts and two were at the limit of detection. All preadipocyte samples induced to differentiate in vitro expressed the TSHR. To investigate the influence of thyroid hormone status on adipose TSHR expression, we induced hyper- and hypothyroidism in BALBc mice by administering tri-iodothyronine and propylthiouracil respectively. In euthyroid animals, whole fat samples were at the limit of detection and were not altered by thyroid hormone status. The results show that adipose TSHR expression ex vivo indicates adipogenesis in progress in vivo and is associated with the autoimmune/inflammatory process in GD and TED but is not restricted to the orbit or influenced by thyroid hormone status.

The W546X mutation of the thyrotropin receptor gene: potential major contributor to thyroid dysfunction in a Caucasian population.

Congenital hypothyroidism (CH) occurs in approximately 1 in 3000 births and can be caused by mutations in 9 known genes, including that encoding the TSH receptor (TSHR). We report on two Welsh siblings, detected by neonatal screening, who had normal sized and placed glands but negative isotope uptake. Genomic DNA was obtained from both siblings and parents, the TSHR amplified using pairs of intronic and/or overlapping exonic primers and the PCR products sequenced automatically. Both siblings were homozygous for a previously described G to A transition producing a missense mutation, W546X, in the fourth membrane spanning region of the TSHR, rendering it unresponsive to TSH. Both parents were heterozygous and unrelated; furthermore, the W546X has been described in three further families (one of which is Welsh), suggesting that it may be a relatively common mutation. We genotyped 368 euthyroid Welsh individuals using single nucleotide primer extension, and found 366 homozygous wild-type (G:G) and 2 heterozygous (G:A) for the mutation. In conclusion, CH in the siblings is due to the missense mutation, W546X, in their TSHR gene. The W546X allele was detected in approximately 1 in 180 individuals and may be a major contributor to hypothyroidism in the Welsh population.