Marenza Leo

PREGO (presentation of Graves’ orbitopathy) study: changes in referral patterns to European Group On Graves’ Orbitopathy (EUGOGO) centres over the period from 2000 to 2012

Background/aims The epidemiology of Graves’ orbitopathy (GO) may be changing. The aim of the study was to identify trends in presentation of GO to tertiary centres and initial management over time. Methods Prospective observational study of European Group On Graves’ Orbitopathy (EUGOGO) centres. All new referrals with a diagnosis of GO over a 4-month period in 2012 were included. Clinical and demographic characteristics, referral timelines and initial decisions about management were recorded. The data were compared with a similar EUGOGO survey performed in 2000. Results The demographic characteristics of 269 patients studied in 2012 were similar to those collected in the year 2000, including smoking rates (40.0% vs 40.2%). Mild (60.5% vs 41.2%, p<0.01) and inactive GO (63.2% vs 39.9%, p<0.01) were more prevalent in 2012. The times from diagnosis of thyroid disease to being seen in EUGOGO centres (6 vs 16 months) and from first symptoms of GO (9 vs 16 months) or from diagnosis of GO (6 vs 12 months) to first consultation in EUGOGO centres were shorter in 2012 (p<0.01). The initial management plans for GO were no different except surgical treatments for patients with mild inactive disease were more frequently offered in the 2012 cohort than in 2000 (27.3% vs 17%, p<0.05), and selenium supplements were offered only in the 2012 cohort (21.2% vs 0%, p<0.01). Conclusions These findings suggest that the clinical manifestations of patients with GO may be changing over time in Europe.

Oxidative stress in graves' disease.

Increased reactive oxygen species (ROS) generation and the consequent oxidative damage are involved in the development of several diseases, including autoimmune diseases. Graves’ disease is an autoimmune disorder characterized by hyperthyroidism and, less frequently, orbitopathy. Hyperthyroidism is characterized by increased oxidative stress. Untreated hyperthyroidism is associated with an increase of several parameters of oxidative stress and in most studies (but not all) by an increase of antioxidant defense enzymes. Restoration of euthyroidism with antithyroid drug is associated with a reversal of the biochemical abnormalities associated with oxidative stress. Animal and human studies suggest that increased ROS may directly contribute to some clinical manifestation of the disease, including orbitopathy. Antioxidants administered alone improve some clinical signs and symptoms of hyperthyroidism and, when associated with antithyroid drugs, induce a more rapid control of clinical manifestations and a faster achievement of euthyroidism. A large randomized clinical trial has shown that antioxidant supplementation (selenium) may also be beneficial for mild Graves’ orbitopathy.

Total thyroid ablation in Graves’ orbitopathy

Graves’ orbitopathy (GO) is an autoimmune condition almost always associated with autoimmune thyroid disease, especially Graves’ disease (GD). According to the most widely accepted model, the autoantigens responsible for GO would include molecules expressed by thyroid epithelial cells that are present also in orbital tissues. The high likelihood that the etiologies of GO and of the underlying autoimmune thyroid diseases are somehow linked is confirmed by the very close relationship between GO, the onset and the course of Graves’ diseases, the size of the thyroid gland, and most importantly, thyroid function and thyroid treatment. Based on these considerations, it has been proposed that complete removal of thyroid antigens and of thyroid infiltrating lymphocytes, the so-called total thyroid ablation (TTA), may be followed by an attenuation of the immune reaction against orbital antigens, and ultimately by an amelioration of GO. The possibility that TTA, achieved by near total thyroidectomy followed by radioiodine, may be beneficial for GO was initially suggested by two retrospective studies and more recently by two prospective, randomized clinical trials conducted in patients with moderate GO treated with intravenous glucocorticoids. Although there seemed to be no difference in the long term, compared with near total thyroidectomy alone TTA was associated with a shorter time required for GO to improve, or anyway to reach its best possible outcome, and with a lesser requirement for additional treatments for GO to improve. Whether this is sufficient to offer ablation to patients remains a matter of discussion. At present, this procedure could be offered only to patients scheduled to thyroidectomy and glucocorticoid treatment.

Dissecting the Genetic Susceptibility to Graves’ Disease in a Cohort of Patients of Italian Origin

Graves’ disease (GD) is an autoimmune oligogenic disorder with a strong hereditary component. Several GD susceptibility genes have been identified and confirmed during the last two decades. However, there are very few studies that evaluated susceptibility genes for GD in specific geographic subsets. Previously, we mapped a new locus on chromosome 3q that was unique to GD families of Italian origin. In the present study, we used association analysis of single-nucleotide polymorphism (SNPs) at the 3q locus in a cohort of GD patients of Italian origin in order to prioritize the best candidates among the known genes in this locus to choose the one(s) best supported by the association. DNA samples were genotyped using the Illumina GoldenGate genotyping assay analyzing 690 SNP in the linked 3q locus covering all 124 linkage disequilibrium blocks in this locus. Candidate non-HLA (human-leukocyte-antigen) genes previously reported to be associated with GD and/or other autoimmune disorders were analyzed separately. Three SNPs in the 3q locus showed a nominal association (p < 0.05): rs13097181, rs763313, and rs6792646. Albeit these could not be further validated by multiple comparison correction, we were prioritizing candidate genes at a locus already known to harbor a GD-related gene, not hypothesis testing. Moreover, we found significant associations with the thyroid-stimulating hormone receptor (TSHR) gene, the cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene, and the thyroglobulin (TG) gene. In conclusion, we identified three SNPs on chromosome 3q that may map a new GD susceptibility gene in this region which is unique to the Italian population. Furthermore, we confirmed that the TSHR, the CTLA-4, and the TG genes are associated with GD in Italians. Our findings highlight the influence of ethnicity and geographic variations on the genetic susceptibility to GD.

Orbital lymphoma masquerading as thyroid-associated orbitopathy.

A 70-yr-old woman 1 was referred because of orbital pain, bilateral proptosis, eyelid swelling, inflammatory symptoms, and visual impairment. Her symptoms had begun 2 yr earlier, and a computed tomography (CT) scan of the orbits at the time had shown mild proptosis and enlargement of the left superior rectus muscle. Thyroid function tests revealed mild hypothyroidism with negative thyroid autoantibodies. A diagnosis of thyroid-associated orbitopathy (TAO) was made. The patient had a history of mild chronic lymphocytic leukemia, without lymph node or solid organ involvement. She was treated with L-thyroxine and an 18-month course of low-dose methylprednisolone and azathioprine, with a partial improvement of her eye manifestations. Her eye symptoms recurred, however, when immunosuppressive treatment was withdrawn and consequently she was referred to our department. Physical examination (Fig. 1, upper panel) showed marked, bilateral proptosis, soft tissue inflammation, and mild impairment of ocular movement (1). There was no upper lid retraction. Thyroid function tests were normal on L-thyroxine with negative thyroid autoantibodies, including TSH receptor autoantibodies. A CT scan of the orbits (Fig. 1, upper panel) showed bilateral proptosis, mild enlargement of extraocular muscles, together with a diffuse nodular infiltration of soft tissues, involving the eyelids and the orbital cavity. These features suggested ocular involvement by a neoplastic process, rather than TAO, in which the typical finding is the enlargement of extraocular muscles (rectus inferior medial superior) (2). An orbital biopsy of affected tissue (Fig. 1, lower panel) revealed a dense infiltrate of small B lymphocytes staining positive for CD20, CD5, and bcl-2, therefore supporting the diagnosis of B cell chronic lymphocytic leukemia involving the orbits (3, 4). Treatment with clorambulacil was commenced. Four weeks later, improvement of eye manifestations was evident, and this was sustained at the next follow-up visit 4 months later (Fig. 1, middle panel). Orbital manifestations went into complete remission at the latest follow-up visit (10 months after clorambucil therapy was started). Orbital lymphoma can mimic TAO. In atypical cases of TAO, like those who have no history of Graves’ disease, no TSH receptor antibodies, and absence of upper lid retraction, alternative diagnoses should be considered (5). In these cases, orbital imaging should always be performed. Indeed, the unusual eye muscle involvement

VARIABLES AFFECTING THE LONG-TERM OUTCOME OF GRAVES ORBITOPATHY FOLLOWING HIGH-DOSE INTRAVENOUS GLUCOCORTICOID PULSE THERAPY IN PATIENTS NOT TREATED WITH ORBITAL RADIOTHERAPY.

OBJECTIVE Intravenous (iv) glucocorticoids (GC) (ivGC) are used for active Graves orbitopathy (GO), but factors affecting GO outcome are poorly understood. We performed a retrospective study to investigate the variables affecting GO after ivGC. METHODS We evaluated 83 consecutive GO patients treated with ivGC but not orbital radiotherapy (ORT) and re-examined them after a median of 47 months. The endpoints were the relationships between GO outcome or additional treatments with age, sex, smoking habits, thyroid volume, thyroid treatment, time since thyroid treatment, antithyroid-stimulating hormone receptor antibodies (TRAb), GO duration, GO features, and follow-up time. RESULTS GO features improved after treatment, resulting in moderate and marked amelioration in ~75% and ~41% of patients respectively. By multivariate analysis, a moderate GO improvement correlated with diplopia at first observation, which was more severe in responders. A marked GO improvement correlated with time between first and last observation and time after thyroid treatment, which were longer in responders. This likely reflected the combination of an early effect of GC and a late, spontaneous improvement of GO, as shown by analyses of GO outcome at various time points. Additional treatments after ivGC correlated by multivariate analysis with eyelid aperture, diplopia and NOSPECS score (NOSPECS stands for no GO signs [N], only eyelid sign [O], soft tissue involvement [S], proptosis [P], extraocular motility restriction [E], corneal involvement [C], and sight loss [S]) at first observation, which were more severe in responders. CONCLUSION Our study shows that response to ivGC increases with time, likely reflecting the known tendency of GO to improve spontaneously, and is more pronounced when GO is more severe to begin with, which is associated with more additional treatments. ABBREVIATIONS ANOVA = analysis of variance CAS = clinical activity score GC = glucocorticoids GO = Graves orbitopathy 131I = radioactive iodine iv = intravenous ivGC = high-dose intravenous glucocorticoid pulse therapy MMI = methimazole OD = orbital decompression ORT = orbital radiotherapy TRAb = antithyroid-stimulating hormone receptor antibodies.

Graves’ orbitopathy, idiopathic orbital inflammatory pseudotumor and Epstein–Barr virus infection: a serological and molecular study

AbstractObjectiveOne of the hypotheses on the pathogenesis of autoimmune diseases, including Graves’ disease (GD) and Graves’ orbitopathy (GO), involves bacterial or viral infections. Recently, Epstein–Barr virus (EBV) has been proposed to play a role in the pathogenesis of idiopathic orbital inflammatory pseudotumor (IOIP) in Asians. The aim of the present study was to investigate the possible association of GO with EBV infection/exposure, as compared with IOIP, using serum and tissue samples, as well as primary cultures of orbital fibroblasts.MethodsThirty-one patients were studied, including four with IOIP, ten with GO, nine with GD without GO and eight control patients without IOIP, GD and GO. All patients with IOIP and GO underwent orbital decompression. Control patients underwent palpebral surgery. Fibroadipose orbital tissue samples were collected. Serum anti-EBV antibodies were measured in all patients. EBV-DNA was measured in blood samples, orbital tissue samples and primary cultures of orbital fibroblasts. ResultsSerum assays showed that the vast majority of patients have had a previous exposure to EBV, but no one had an acute infection. EBV-DNA was detected in ~40% of blood samples from GO, GD and control patients, but in none of the IOIP samples. EBV-DNA was not detected in any of the orbital tissue samples tested or in primary cultures of orbital fibroblasts.ConclusionsEBV infection does not seem to be associated with GD, GO and IOIP in Caucasians. Whether EBV is involved in IOIP in Asians or other populations remains to be confirmed.

Natural history of graves’ orbitopathy after treatment

Intravenous glucocorticoids are used for Graves’ orbitopathy, alone or associated with/followed by additional treatments (orbital radiotherapy, orbital decompression, palpebral or eye surgery). However, the relation between associated/additional treatments and other variables with Graves’ orbitopathy outcome following intravenous glucocorticoids is not clear. Thus, the present study was conducted to investigate retrospectively the impact of associated/additional treatments and other variables on Graves’ orbitopathy outcome after intravenous glucocorticoids. We evaluated 226 untreated Graves’ orbitopathy patients. Following first observation, patients were given intravenous glucocorticoids and re-examined after a median of 46.5 months. The end-points were the relation between Graves’ orbitopathy outcome, outcome of NOSPECS score and of the single Graves’ orbitopathy features with several variables, including associated/additional treatments. All Graves’ orbitopathy features improved significantly after treatment. Overall, Graves’ orbitopathy improved in ~60 % of patients (responders), whereas it was stable or worsened in ~40 % of patients (non-responders). Time between first and last observation and clinical activity score at first observation correlated significantly with Graves’ orbitopathy outcome. The outcomes of NOSPECS, eyelid aperture, clinical activity score and diplopia correlated with time between the first and last observation. The NOSPECS outcome correlated with gender. The outcomes of proptosis, eyelid aperture and visual acuity correlated with orbital decompression. The outcome of diplopia correlated with orbital radiotherapy. Taking into account the limitations of retrospective investigations, our findings confirm that time (i.e. the natural history of Graves’ orbitopathy) is a key factor in determining the long-term outcome of Graves’ orbitopathy, radiotherapy is effective for diplopia, and orbital decompression is followed by an amelioration of several Graves’ orbitopathy features.

Selenium rescues orbital fibroblasts from cell death induced by hydrogen peroxide: another molecular basis for the effects of selenium in graves’ orbitopathy

In vivo and in vitro studies suggest that the anti-oxidant agent selenium can be used for the treatment of Graves’ disease (GD) and Graves’ orbitopathy (GO) [1–3]. GO, the most common extrathyroidal manifestation of GD [4–6], is due to an autoimmune reaction against orbital fibroadipose tissue, following which a number of effector mechanisms, including oxidative stress, are responsible for the pathological changes of the orbit [7]. Treatment with selenium is associated with an improvement of mild GO [2], and recently we reported that selenium exerts antioxidant actions in vitro, namely in orbital fibroblasts exposed to oxidative stress by incubation with hydrogen peroxide (H2O2) [3]. H2O2 was cytotoxic if used at concentrations ≥10 μM, but at 5 μM it elicited proliferation of fibroblasts and cytokine release [3]. The effects of H2O2 were counteracted by selenium, which also caused a reduced release of hyaluronic acid [3]. When performing the experiments of our previous study, we observed casually that selenium seemed to counteract also the cytotoxic action of H2O2. Hence, we conducted an additional study aimed at investigating whether selenium is actually capable of inhibiting H2O2-dependent cytotoxicity, as well as at comprehending the mechanisms underlying H2O2 cytotoxicity and selenium actions.

Intracellular retention of thyroglobulin in the absence of the low-density lipoprotein receptor-associated protein (RAP) is likely due to premature binding to megalin in the biosynthetic pathway.

ObjectiveThe low-density lipoprotein receptor associated protein (RAP) is expressed by thyroid epithelial cells (TEC) in a TSH-dependent manner. In the thyroid RAP functions as a molecular chaperone for the thyroglobulin (Tg) endocytic receptor megalin/LRP2, which is retained intracellularly in RAP KO mice rather than being expressed on the apical membrane of TEC, its usual location. RAP binds also to Tg, which is also retained intracellularly in RAP KO mice, thereby suggesting a role of RAP in Tg secretion. Here we investigated whether Tg intracellular retention in the absence of RAP is due to premature Tg-megalin interactions during the biosynthetic pathway or to a direct action of RAP on Tg secretion.MethodsWe performed immunoprecipitation experiments in thyroid extracts from RAP KO and WT mice. In addition, we investigated Tg secretion in COS-7 cells co-transfected with human RAP (hRAP) and mouse Tg (mTg).ResultsAn anti-megalin megalin precipitated greater amounts of Tg in thyroid extracts from RAP KO than from WT mice, suggesting increased intracellular interactions between megalin and Tg in the absence of RAP. COS-7 cells transiently transfected with hRAP, mTg or both, expressed the two proteins accordingly. RAP was found almost exclusively in cell extracts, whereas Tg was found both in extracts and media, as expected from the knowledge that RAP is ER-resident and that Tg is secreted. Regardless of whether cells were transfected with mTg alone or were co-transfected with hRAP, similar proportions of the total Tg synthesized were detected in cell extracts and media.ConclusionsThe intracellular retention of Tg in the absence of RAP is likely due to its premature interaction with megalin, whereas RAP does not seem to affect Tg secretion directly.