mTOR is involved in the pathogenesis of Graves’ orbitopathy (GO). Because mTOR can be detected in the bloodstream, we postulated that serum mTOR could be associated with GO. Thus, we evaluated serum mTOR in GO. mTOR was measured by ELISA in serum samples from 77 consecutive patients with Graves’ hyperthyroidism (GH), 57 of whom with GO and in 19 healthy subjects. The primary endpoint was serum mTOR in GH patients with GO vs GH patients without GO. Median serum mTOR was 2440 pg/ml (1236–3053) in GO patients, 1236 pg/ml (388.5–1770) in GH without GO, and 247 pg/ml (0.9–705) in healthy subjects. mTOR was greater in GH than in healthy subjects (P=0.00012), and, within GH, in patients with GO (P = 0.0023). In GO patients, there was a direct correlation between mTOR and the clinical activity score (R = 0.377; P = 0.0038) and mTOR was higher in moderate-to-severe than in mild GO (P = 0.016). We established a cut-off value of 1445.8 pg/ml of serum mTOR (97th percentile in healthy subjects) that distinguished GO patients within GH [positive predictive value (PPV): 79.2

The natural history of Graves’ orbitopathy (GO) is believed to be characterized by a late phase of inactivation, possibly related to fibrosis involving orbital tissues. However, to our knowledge, this notion is anecdotal. Thus, the present study aimed to investigate fibrosis in orbital tissues and relate it with the features of GO. This retrospective cohort study included 56 consecutive patients with GO who underwent orbital decompression surgery. Fibronectin, CD3- and CD20-positive cells were detected by immunohistochemistry in orbital tissue samples. Collagen was detected by Masson’s trichrome staining. The primary endpoint was the relationship between staining for fibronectin and duration of GO. Staining for fibronectin was observed in 47 patients (83.9

Background: Treatment for Graves' hyperthyroidism (GH) in patients with Graves' orbitopathy (GO) remains a topic of debate. This study aimed to investigate the outcome of GO following glucocorticoids, depending on the chosen thyroid treatment. Methods: This retrospective cohort study included 49 consecutive patients with GH and moderate-to-severe, active GO, as defined by the European Group on Graves' Orbitopathy guidelines. Twenty-four patients were treated with radioactive iodine (RAI) and 25 with methimazole (MMI). All patients were administered intravenous methylprednisolone. Follow-up visits occurred at weeks 24, 48, and 72. The primary endpoint was the overall outcome of GO at week 24. Response was defined as a change in at least two of the following eye features: reduction ≥1 point in clinical activity score; proptosis reduction ≥2 mm; eyelid aperture reduction ≥2 mm; increase in eye ductions ≥8 degrees. Results: Follow-up duration was 72 weeks for both groups (interquartile range 66-72 for RAI and 48-72 for MMI). The proportion of responders for week 24 overall GO outcome was greater in RAI (54.1% vs. 16%; odds ratio [OR] 6.2 [confidence interval (CI): 1.6-23.6], p = 0.0075), but it increased in MMI at weeks 48 and 72, with no differences between groups. There was a trend indicating a better response in RAI regarding individual eye features. Improvement in GO-specific quality of life questionnaire at week 24 was trendily more pronounced in RAI (responders 50% vs. 28% in MMI; OR = 2.5 [CI: 0.7-8.4], p = 0.11), although results were similar in both groups at later time points. At week 24, only one patient (4%) in RAI and three (12%) in MMI experienced worsening of GO. Fifty-nine adverse events were recorded among 36 patients, with no differences between groups, except for infections, which were more frequent in RAI (53.8% vs. 15.3% in MMI; OR = 6.41 [CI: 1.7-23.9], p = 0.0056). Conclusions: RAI appears to be associated with an earlier response of GO to intravenous glucocorticoids. In the long term, a conservative approach also seems to be effective. RAI appears to be relatively safe when patients are concurrently treated with glucocorticoids. However, randomized clinical trials are necessary to confirm these findings.

Compared with methylprednisolone, a greater response of Graves’ orbitopathy (GO) at 24 weeks was reported in patients treated with sirolimus (rapamycin) for 12 weeks. We investigated whether serum levels of sirolimus at week-12 predict the outcome of GO at week-24. Retrospective investigation in 30 patients [males: 4; females: 26; age 61.3 (9.62) yr] with moderate-to-severe, active GO, treated with sirolimus (2 mg on day-one, followed by 0.5 mg QD for 12 weeks). Primary outcome: serum sirolimus at week-12 in week-24 GO responders vs nonresponders, assessed by a composite evaluation. Secondary outcome: establishment of a cut-off value of week-12 sirolimus concentrations predictive of response to treatment. At week-24, overall GO responders and nonresponders were 18 (60

The insulin-like growth factor-1 receptor (IGF-1R) plays a role in the pathogenesis of Graves’ orbitopathy (GO). A possible protective role of autoantibodies against IGF-1R (IGF-1R-Abs) on GO has been suggested. We conducted a cross-sectional study to investigate IGF-1R-Abs in 147 consecutive Graves’ disease (GD) patients, with (n = 92) or without (n = 55) GO (primary outcome), their relationship with GO features and their effect on cell proliferation in primary cultures of orbital fibroblasts. Serum IGF-1R-Abs levels were higher (29.3 ng/mL, IQR 17.4–36.6) in patients without GO than in those with GO (19.8 ng/mL, IQR 11.2–29.8; Mann Whitney U 1819, P = 0.00509). The prevalence of IGF-1R-Abs levels above the previously established cut-off value of 55 ng/mL did not differ statistically between the two groups, despite a trend towards a greater prevalence in patients without GO (9 vs 3.2

L’orbitopatia basedowiana (OB) tende a presentarsi con maggior frequenza in pazienti di sesso femminile ed etnia caucasica e l’esposizione al fumo di sigaretta rimane ad oggi il fattore di rischio modificabile più importante. Oltre ai fattori di rischio “storici” (controllo della funzione tiroidea, livello di TRAb, terapia radiometabolica con 131-I), nel corso degli ultimi anni sono stati individuati “nuovi” fattori di rischio che potrebbero essere coinvolti nell’insorgenza o nella recidiva della OB, come l’ipercolesterolemia, anticorpi monoclonali ad azione immunosoppressiva o alterazioni del microbiota intestinale. Questa rassegna descrive i fattori di rischio storici dell’OB e i nuovi fattori di rischio emersi più recentemente.

Objectives: The pathogenesis of Graves’ orbitopathy (GO) remains to be fully elucidated. Here, we reviewed the role of genetics and epigenetics. Design: We conducted a PubMed search with the following keywords: GO, thyroid eye disease; or Graves’ ophthalmopathy; or thyroid-associated ophthalmopathy; and: genetic, or epigenetic, or gene expression, or gene mutation, or gene variant, or gene polymorphism, or DNA methylation, or DNA acetylation. Articles in which whole DNA and/or RNA sequencing, proteome, and methylome analyses were performed were chosen. Results: The different prevalence of GO in the two sexes, as well as racial differences, suggest that genetics play a role in GO pathogenesis. In addition, the long-lasting phenotype of GO and patient-derived orbital fibroblasts suggests a genetic or epigenetic mechanism. Although no genes have been found to confer a specific risk for GO, differential gene expression has been reported in orbital fibroblasts from GO patients vs control fibroblasts, suggesting that an epigenetic mechanism may be involved. In this regard, a different degree of DNA methylation, which affects gene expression, has been found between GO and control fibroblasts, which was confirmed by whole methylome analysis. Histone acetylation and deacetylation, which also affect gene expression, remain to be investigated. Conclusions: Although no pathogenic gene variants have been reported, epigenetic mechanisms elicited by an initial autoimmune insult seem to be needed for differential gene expression to occur and, thus, for GO to develop and persist over time.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Sirolimus was found to be associated with a better outcome of Graves’ orbitopathy (GO) at 24 weeks compared to methylprednisolone. We conducted a retrospective study to investigate its efficacy and safety over a longer period. Data from 40 consecutive patients with moderate-to-severe, active GO, 20 treated with sirolimus and 20 with methylprednisolone, were collected. Primary outcome: overall outcome (composite evaluation) of GO at 48 weeks. Secondary outcomes: (1) GO outcome at 24 weeks, and, at 24 and 48 weeks: (2) outcome of single eye features; (3) quality of life (GO-QoL); (4) TSH-receptor antibodies; (5) GO relapse at 48 weeks; (6) adverse events. The overall GO outcome at 48 weeks did not differ between the two groups (responders: 55

Graves disease is the most common cause of hyperthyroidism in iodine-sufficient areas. The main responsible mechanism is related to autoantibodies that bind and activate the thyrotropin receptor (TSHR). Although Graves hyperthyroidism is relatively common, no causal treatment options are available. Established treatment modalities are antithyroid drugs, which reduce thyroid hormone synthesis, radioactive iodine and surgery. However, emerging drugs that target the main autoantigen (monoclonal antibodies, small molecules, peptides) or block the immune pathway have been recently tested in clinical trials. Graves disease can involve the thyroid exclusively or it can be associated with extrathyroidal manifestations, among which Graves orbitopathy is the most common. The presence of Graves orbitopathy can change the management of the disease. An established treatment for moderate-to-severe Graves orbitopathy is intravenous glucocorticoids. However, recent advances in understanding the pathogenesis of Graves orbitopathy have allowed the development of new target-based therapies by blocking pro-inflammatory cytokine receptors, lymphocytic infiltration or the insulin-like growth factor 1 receptor (IGF1R), with several clinical trials providing promising results. This article reviews the new discoveries in the pathogenesis of Graves hyperthyroidism and Graves orbitopathy that offer several important tools in disease management. Graves disease, an autoimmune disease characterized by an enlarged and overactive thyroid gland, is the most common cause of hyperthyroidism in iodine-sufficient areas. In this Review, the authors describe the epidemiology, pathogenesis, and conventional treatment of Graves hyperthyroidism and Graves orbitopathy and discuss advances that have enabled development of novel treatment modalities. Graves disease is an autoimmune condition that can involve the thyroid exclusively or can be associated with extrathyroidal manifestations, of which Graves orbitopathy is the most common.The main mechanism responsible for Graves hyperthyroidism is the activation of the thyroid-stimulating hormone receptor (TSHR) by autoantibodies that act as agonists, causing thyrocyte proliferation and hyperfunction.None of the conventional treatments for Graves hyperthyroidism act on its pathogenesis; new molecules that target early recognition of TSHR peptides, T cell activation, B cell stimulation and survival, production of TSHR autoantibodies, and TSHR activation have been tested with encouraging results.Graves orbitopathy is characterized by immune-mediated inflammatory reactions against autoantigens shared by thyroid epithelial cells and orbital fibroblasts; intravenous glucocorticoids are, to date, the first-line treatments for Graves orbitopathy.New target-based therapies that block pro-inflammatory cytokine receptors, lymphocytic infiltration or the insulin-like growth factor 1 receptor (IGF1R) have been tested in several clinical trials and show promising results in Graves orbitopathy.

Background: Autoimmune thyroid diseases (AITD) represent the most common autoimmune diseases. However, current therapies focus on relieving the symptoms instead of curing AITD, and new therapies to reverse the autoimmune attack on the thyroid are needed. HLA-DRβ1-Arg74 is the key HLA class II allele that triggers AITD by presenting pathogenic thyroglobulin (Tg) peptides that activate thyroid self-reactive T cells. We hypothesized that blocking the presentation of Tg peptides to T cells within the HLA-DRβ1-Arg74 peptide binding cleft could reverse the autoimmune response to the thyroid in AITD. Methods: The approach we used to block Tg peptide presentation within HLA-DRβ1-Arg74 is to design retro-inverso D-amino acid (RID) peptides that have high affinity to the HLA-DRβ1-Arg74 peptide binding pocket. Results: By using computational approaches and molecular dynamics simulations, we designed two RID peptides, RT-15 and VT-15, that blocked peptide binding to recombinant HLA-DRβ1-Arg74 molecule, as well as T cell activation in vitro. Furthermore, RT-15 and VT-15 blocked in vivo T cell activation by thyroglobulin in humanized NOD-DR3 mice induced with experimental autoimmune thyroiditis. Conclusions: In summary, we discovered two RID peptides that block thyroglobulin peptide binding to HLA-DRβ1-Arg74 and their presentation to T cells in AITD. These findings set the stage for a personalized medicine therapeutic approach for AITD patients who carry the DRβ1-Arg74 allele. This antigen-specific therapeutic strategy can potentially be extended to other autoimmune diseases.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

CONTEXT:A role of DNA methylation in Graves orbitopathy (GO) has been proposed.OBJECTIVE:This work aimed to investigate DNA methylation and gene expression in orbital fibroblasts from control and GO patients, under basal conditions or following challenge with an anti- thyrotropin (TSH) receptor antibody (M22) or cytokines involved in GO; to investigate the relationship between DNA methylation and cell function (proliferation); and to perform a methylome analysis.METHODS:Orbital fibroblasts from 6 GO and 6 control patients from a referral center underwent methylome analysis of the whole genome.RESULTS:Global DNA methylation increased significantly both in control and GO fibroblasts on incubation with M22. Expression of 2 selected genes (CYP19A1 and AIFM2) was variably affected by M22 and interleukin-6. M22 increased cell proliferation in control and GO fibroblasts, which correlated with global DNA methylation. Methylome analysis revealed 19 869 DNA regions differently methylated in GO fibroblasts, encompassing 3957 genes and involving CpG islands, shores, and shelves. A total of 119 gene families and subfamilies, 89 protein groups, 402 biological processes, and 7 pathways were involved. Three genes found to be differentially expressed were concordantly hypermethylated or hypomethylated. Among the differently methylated genes, insulin-like growth factor-1 receptor and several fibroblast growth factors and receptors were included.CONCLUSION:We propose that, when exposed to an autoimmune environment, orbital fibroblasts undergo hypermethylation or hypomethylation of certain genes, involving CpG promoters, which results in differential gene expression, which may be responsible for functional alterations, in particular higher proliferation, and ultimately for the GO phenotype in vivo.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract Significant progress has been made in the identification and characterization of susceptibility genes for the autoimmune thyroid diseases (AITD) which includes Graves’ disease (GD) and Hashimoto’s thyroiditis (HT), and are among the commonest autoimmune endocrine diseases, affecting up to 5% of the general population. There is a strong familial aggregation with these diseases, a high sibling risk ratio, and a high concordance rate in monozygotic twins. Several major AITD susceptibility genes have been identified which can be categorized by the disease phenotype or their function. In the group of immune regulatory genes, HLA-DR3 was shown long ago to be associated with GD and HT, and the presence of arginine at position 74 of the HLA-DRβ chain is now known to be the specific sequence variant conferring risk. Three additional susceptibility genes related to immune regulation have also been revealed—CTLA-4, CD40, and PTPN22, as well as two thyroid-specific genes: thyroglobulin and the TSH receptor (TSHR). While CTLA-4, PTPN22, and thyroglobulin confer susceptibility to both GD and HT, CD40 and TSHR are specific for GD.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)