Potential role of IGF-1R in the interaction between orbital fibroblasts and B lymphocytes: an implication for B lymphocyte depletion in the active inflammatory phase of thyroid-associated ophthalmopathy.

BACKGROUND: Thyroid eye disease (TED) is an inflammatory process involving lymphocyte-mediated immune response and orbital tissue damage. The anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies produced by B lymphocytes are involved in the activation of orbital fibroblasts and the inflammatory process of orbital tissue damage in TED. The purpose of this study was to explore the role of IGF-1R in the mechanistic connection between orbital fibroblasts and B lymphocytes in TED. METHODS: Orbital fibroblasts sampled from orbital connective tissues and peripheral B lymphocytes isolated from peripheral blood, which were obtained from 15 patients with TED and 15 control patients, were co-cultured at a ratio of 1:20. The level of IGF-1R expression in orbital fibroblasts was evaluated by flow cytometry and confocal microscopy. Transient B lymphocyte depletion was induced with anti-CD20 monoclonal antibody rituximab, while the IGF-1R pathway was blocked by the IGF-1R binding protein. The expression levels of interleukin-6 (IL-6) and regulated upon activation, normal T cell expressed and secreted (RANTES) in the co-culture model were quantified via ELISA. RESULTS: IGF-1R expression was significantly elevated in TED orbital fibroblasts compared to that of controls. A 24-h co-culture of orbital fibroblasts with peripheral B lymphocytes induced elevated expression levels of IL-6 and RANTES in each group (TED patients and controls), with the highest levels occurring in TED patients (T + T group). Rituximab and IGF-1R binding protein significantly inhibited increased levels of IL-6 and RANTES in the co-culture model of TED patients. CONCLUSIONS: IGF-1R may mediate interaction between orbital fibroblasts and peripheral B lymphocytes; thus, blocking IGF-1R may reduce the local inflammatory response in TED. Rituximab-mediated B lymphocyte depletion played a role in inhibiting inflammatory responses in this in vitro co-culture model, providing a theoretical basis for the clinical application of anti-CD20 monoclonal antibodies in TED.

Correlation between extraocular muscle enlargement and thyroid autoantibodies in thyroid eye disease.

PURPOSE: This study aimed to investigate the factors affecting extraocular muscle enlargement in thyroid eye disease (TED). STUDY DESIGN: Retrospective study. METHODS: The thyroid-stimulating hormone (TSH) receptor antibody (TRAb), thyroid-stimulating antibody (TSAb), antithyroid peroxidase antibody (ATPO), and antithyroglobulin antibody (ATG) levels in patients diagnosed with TED who underwent orbital magnetic resonance imaging were assessed. The control group comprised the contralateral eye of patients who underwent orbital magnetic resonance imaging (MRI) for unilateral eyelid tumors or orbital disease. The thickness of the bilateral rectus muscles and superior oblique muscles was measured on orbital MRI. Muscle enlargement was classified as unilateral/bilateral and symmetric/asymmetric. The effects of age, sex, smoking history, TSH, thyroid hormone, and thyroid autoantibodies on the muscle thickness and number of enlarged muscles were assessed by use of simple and multiple regression analyses. RESULTS: The TED and control groups comprised 41 and 44 cases, respectively. The positivity rate of TSAb in patients with TED was 92.7% higher than that of the other autoantibodies. Muscle enlargement was observed in 29 of the 41 cases (70.7%). Older age and higher TSAb levels were identified as significant factors affecting the total muscle thickness and number of enlarged muscles. Bilateral muscle enlargement and asymmetrical muscle enlargement were observed in 17 (58.6%) and 23 (79.3%) of the 29 cases, respectively. The TSAb levels and age had no significant effect on the type of muscle enlargement. CONCLUSIONS: TSAb showed significant associations with extraocular muscle enlargement. Measurement of TSAb, rather than of TRAb, may be more useful for diagnosing extraocular muscle enlargement in patients with TED.

Role of the mononuclear cell infiltrate in Graves' orbitopathy (GO): results of a large cohort study.

OBJECTIVE: The extent to which mononuclear cells and TSH-receptor autoantibodies (TRAb) contribute to Graves' orbitopathy (GO) is not completely defined. Here we investigated the relationship between the immunohistochemical phenotype of orbital infiltrating cells and GO features in a large number of patients. METHODS: We conducted an observational cohort study in 76 consecutive patients with GO (16 men and 60 women) who underwent orbital decompression over a period of 18 consecutive months. An ophthalmological evaluation was performed in all patients, as well as immunohistochemistry for CD3, CD4, CD8, CD56 (T-cell markers), CD25 (T and B-cell marker), CD20, CD19 (B-cell markers), and CD138 (plasmacell marker) in specimens collected at decompressive surgery. RESULTS: Having established cutoff values for each marker, cell infiltrates were found in 60 patients (78.9%; CD3: 39.4%, CD4 55.2%, CD8 50%, CD56: 0%, CD25: 28.9%, CD20: 51.3%, CD19: 25%, CD138: 26.3%). Eleven (14.4%) stained exclusively for CD138 (plasmacells). Patients with CD4-positive mononuclear cells had a significantly greater GO clinical activity score (CAS) (mean difference 1.07, 95% CI - 0.33 to - 1.82, P = 0.004 by univariate, P = 0.05 by multivariate analysis). CAS as well as the remaining GO features were not affected significantly by the mononuclear cell subpopulations in multivariate analyses. CONCLUSIONS: Mononuclear cell infiltrates are present in the majority of GO patients, with a small percentage represented exclusively by plasmacells. CD4 cells exert a major role on GO activity. These findings may represent a further advancement in the comprehension of GO pathogenesis.

Association of fibroblast growth factor 10 with the fibrotic and inflammatory pathogenesis of Graves' orbitopathy.

PURPOSE: The role of fibroblast growth factor (FGF) in orbital fibroblasts (OFs) is rarely known. In this study, we investigated the effect of FGF10 on fibrosis and the inflammation mechanism of Graves' orbitopathy (GO). METHODS: Orbital tissue from GO (n = 15) and non-GO (n = 15) was obtained for this study. The mRNA and protein expression levels of FGF10 and FGF receptor 2b (FGFR2b) in orbital tissue were determined by real-time polymerase chain reaction, western blot analysis, and confocal microscopy. The effects of FGF10 on transforming growth factor (TGF)-ß1 induced fibrotic proteins and interleukin (IL)-1ß- or tumor necrosis factor (TNF)-α- induced inflammatory proteins were investigated using recombinant human (rh) FGF10 and small interfering (si) RNA transfection against FGF10. RESULTS: FGF10 and FGFR2b mRNA expression levels were significantly lower in GO orbital tissues than in non-GO orbital tissues (p = 0.009 and 0.005, respectively). Immunostaining of FGF10 in orbital adipose tissues showed differences in FGF10 expression between GO and control samples. Immunostaining of FGF10 was very weak in the orbital tissues of GO patients. TGF-ß1-induced fibronectin, collagen Iα, α-smooth muscle actin protein expression in GO OFs was attenuated by rhFGF10 treatment and increased by knockdown of FGF10 via siFGF10 transfection. Similarly, IL-1ß- or TNF-α-induced IL-6, IL-8, and cyclooxygenase-2 protein production in GO OFs was either blocked by rhFGF10 treatment or further upregulated by inhibition of FGF10 via siFGF10 transfection. CONCLUSIONS: Our data demonstrate that FGF10 has beneficial effects on the inflammatory and fibrotic mechanisms of GO in primary cultured OFs, providing new insights into GO pathology and the discovery of FGF10 as a promising novel therapeutic application for the treatment of GO.

A cyclic peptide significantly improves thyroid function, thyrotropin-receptor antibodies and orbital mucine /collagen content in a long-term Graves' disease mouse model.

BACKGROUND: BALB/c mice which received long-term immunizations of adenovirus (Ad) expressing thyrotropin receptor A-subunits (TSHR) developed stable Graves' disease (GD). TSHR-derived cyclic peptide 19 (P19) was identified as effective therapy in this model. METHODS: In Ad-TSHR mice, we investigated shorter disease intervals up to 4 months for histological alterations of the orbits, fine tuning of anti-TSHR antibodies (Ab) and free thyroxine (fT4) hormone levels by using novel detection methods in an independent laboratory. Therapy (0.3 mg/kg P19 or vehicle) was given intravenously after the fourth Ad-TSHR immunization (week 11) and continued until week 19. RESULTS: Thyrotropin binding inhibitory immunoglobulins (TBII, bridge immunoassay), blocking (TBAb) and stimulating (TSAb) TSHR-Ab (both cell-based bioassays) and serum levels of fT4 were significantly elevated at week 11 in Ad-TSHR-immunized mice versus none in control mice. For the first time, TSAb, TBAb, and thyroperoxidase-Ab were detected in 17 of 19, 12/19 and 6/19 Ad-TSHR immunized mice, respectively at week 21. Also, for the first time, this study showed that P19 treatment markedly reduced serum TBII (p < 0.0001), serum fT4 (p = 0.02), and acidic mucins and collagen content in the orbital tissue of Ad-TSHR-immunized mice. CONCLUSION: P19 significantly improved thyroid function, confirming previous results in an independent second laboratory. A relevant shift of anti-TSHR antibody subpopulations in response to P19 therapy may help explain its immunological effects. Moreover, P19 exerted a beneficial effect on mucine and collagen content of orbital tissue. Hence, P19 offers a potential novel therapeutic approach for GD and associated orbitopathy.

Blocking the Thyrotropin Receptor with K1-70 in a Patient with Follicular Thyroid Cancer, Graves' Disease, and Graves' Ophthalmopathy.

Background: We report the therapeutic use of K1-70™, a thyrotropin receptor (TSHR) antagonist monoclonal antibody, in a patient with follicular thyroid cancer (FTC), Graves' disease (GD), and Graves' ophthalmopathy (GO). Methods: A 51-year-old female patient, who smoked, presented in October 2014 with FTC complicated by GD, high levels of TSHR autoantibodies with high thyroid stimulating antibody (TSAb) activity, and severe GO. K1-70 was administered at 3 weekly intervals with the dose adjusted to block TSAb activity. Her cancer was managed with lenvatinib and radioiodine therapy. Results: Following initiation of K1-70 therapy, TSAb activity measured in serum decreased and GO (proptosis and inflammation) improved. On K1-70 monotherapy during the pause in lenvatinib, several metastatic lesions stabilized while others showed progression attenuation compared with that before lenvatinib therapy. Conclusions: These observations suggest that blocking TSHR stimulation with K1-70 can be an effective treatment for GO and may also benefit select patients with FTC and GD.

Altered expression profile of BAFF receptors on peripheral blood B lymphocytes in Graves' disease.

BACKGROUND: B lymphocyte activating factor (BAFF) is a growth factor regulating B lymphocytes survival and maturation. Serum BAFF levels were elevated in patients affected with autoimmune thyroid diseases (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT). The aim of this study is to explore the association of expression levels of BAFF and its receptors with AITD. METHODS: Fifty-two GD patients, 39 Hashimoto's thyroiditis (HT) patients and 23 healthy controls (HC) were recruited in this study. Serum BAFF levels were measured by ELISA. Expression of BAFF receptors, including BAFF receptor 3 (BR3) and transmembrane activator and calcium-modulating and cyclophilin ligand interactor (TACI), on B lymphocytes were analyzed by flowcytometry. Effects of steroids on serum BAFF levels and expression of BR3 and TACI were also observed in 10 patients with Graves' orbitopathy (GO) receiving steroids therapy. RESULTS: Serum BAFF levels were significantly elevated from 0.93 ± 0.24 ng/ml in HC to 1.18 ± 0.33 ng/ml in GD (P = 0.0027) and 1.02 ± 0.24 ng/ml in HT (P = 0.0331). BR3 expression on peripheral B lymphocytes were elevated in GD (mean MFI: 4.52 ± 2.06 in GD vs. 3.00 ± 0.87 in HC, P = 0.0015), while TACI expression on peripheral B lymphocytes were decreased in GD without significance (mean MFI: 7.96 ± 4.06 in GD vs. 9.10 ± 3.37 in HC, P = 0.1285). Expression of BR3 and TACI was not changed significantly in HT patients. Steroids significantly suppressed serum BAFF concentrations (from 1.18 ± 0.27 ng/ml to 0.97 ± 0.10 ng/ml, P = 0.0364) and BR3 expression in GO patients (mean MFI from 6.26 ± 4.91 to 4.05 ± 1.58, P = 0.0083). CONCLUSIONS: Altered expression of BAFF and its receptor may mediate the autoimmunity in GD. Restoring the normal expression profile of receptors for BAFF could be a new strategy to treat GD.

Treatment of moderate to severe orbitopathy: Current modalities and perspectives.

Graves' orbitopathy (GO) is the primary cause of exophthalmos in adults. It appears in 30 to 50% of patients with Graves' disease. About 5% are moderate-to-severe cases that might be see-threatening or lead to long term disabling sequelae. Recommendations have been established in 2016 by the European thyroid association (ETA) and the European group on Grave's orbitopathy (EUGOGO), suggesting a wide use of corticosteroids in moderate to severe forms. However, disappointing results have been reported in 20 to 30% of cases. Improved understanding of pathophysiological mechanisms has allowed the use of non-specific immunomodulatory agents, currently under evaluation, and which place in the therapeutic strategy remains to be determined. Very recently, new promising therapeutic advances have emerged with the identification of new therapeutic targets, such as the TSH receptor and IGF-1 receptor complex.

Predicting the Relapse of Hyperthyroidism in Treated Graves' Disease with Orbitopathy by Serial Measurements of TSH-Receptor Autoantibodies.

The aim of this study was to investigate the potential of the new TSH-receptor antibody (TRAb) assays to predict remission or relapse of hyperthyroidism in patients with Graves' disease (GD) and Graves' orbitopathy (GO). TRAbs were measured retrospectively in sera from a cohort of GD patients with GO (n=117; remission n=38 and relapse n=79-Essen GO biobank) with automated binding immunoassays: TRAb Elecsys (Cobas Roche) and TRAb bridge assay (IMMULITE, Siemens), and the TSAb (thyroid stimulating Ab) cell-based bioassay (Thyretain, Quidel Corp.). To identify relapse risk/remission of hyperthyroidism patients were followed up at least 10 months after the end of antithyroid drug therapy (ATD) therapy. ROC plot analysis was performed to calculate cut-off levels of TRAb and TSAb for prediction of relapse and remission of hyperthyroidism. Cut-off serum levels are provided for timepoints around 3, 6, 10, and 15 months after the beginning of ATD. Repeated measurements of TRAb increase the rate of relapses predictions to 60% (Elecsys), 70% (IMMULITE), and 55% (Thyretain). Patients with remission have consistently TRAb levels below the cut off for relapse in repeated measurements. The cell-based bioassay was the most sensitive - and continued to be positive during follow up [at 15 months: 90% vs. 70% (IMMULITE) and 65% (Elecsys)]. Identification of relapsing hyperthyroidism is possible with automated immunoassays and cell-based bioassay especially with serial TRAb measurements during the course of ATD therapy. Patient who need eye surgery may profit from an early decision towards definitive treatment.

A Promising Mouse Model of Graves' Orbitopathy Induced by Adenovirus Expressing Thyrotropin Receptor A Subunit.

Background: Graves' orbitopathy (GO) is the most common and serious manifestation of Graves' disease (GD). It is characterized by orbital inflammation and tissue remodeling. Although several GO models have been reported, most lack a full assessment or mechanistic evaluation. Here, we established a promising mouse model mimicking many aspects of human GO with a frequency of 70% and characterized the key role of T cells in the progression of GO. Methods: An adenovirus expressing the human thyrotropin (TSH) receptor A subunit (Ad-TSHRA) was injected in the muscles of female BALB/C mice nine times to induce GO. At predetermined time points, histological examinations of retrobulbar tissues and thyroid glands were performed to dynamically monitor changes; serum autoantibodies and total thyroxine levels were examined to evaluate thyroid function. Flow cytometry of CD4+ T cell subgroups and RNA sequencing (RNA-Seq) of splenocytes were also performed to explore the underlying mechanism. Results: After nine injections, 7 of 10 mice challenged with Ad-TSHRA developed the orbital changes associated with GO. Seven mice manifested retrobulbar fibrosis, and four mice showed adipogenesis. Exophthalmia, conjunctival redness, and orbital lymphocyte infiltration were also observed in a subset of mice. The orbitopathy was first detected after seven injections and followed the hyperplastic change observed in thyroids after four injections. Flow cytometry revealed increased proportions of Th1 cells and decreased proportions of Th2 cells and regulatory T (Treg) cells in the splenocytes of GO mice. This change in CD4+ T cell subgroups was confirmed by orbital immunohistochemical staining. Genes involved in T cell receptor signaling, proliferation, adhesion, inflammation, and cytotoxicity were upregulated in GO mice according to the RNA-Seq; a trend of upregulation of these GO-specific genes was observed in mice with hyperthyroidism without orbitopathy after four injections. Conclusions: A GO mouse model was successfully established by administering nine injections of Ad-TSHRA. The model was achieved with a frequency of 70% and revealed the importance of T cell immunity. A potential time window from Graves' hyperthyroidism to GO was presented for the first time. Therefore, this model could be used to study the pathogenesis and novel treatments for GO.

Altered Expression of CXCL13 and Its Chemokine Receptor CXCR5 on B Lymphocytes during Active Graves' Orbitopathy.

PURPOSE: To characterize the phenotypic abnormalities of peripheral B cells in patients with Graves' orbitopathy (GO) and explore the role of chemokine CXC ligand 13 and its receptor type 5 (CXCL13/CXCR5) in relation to B-cell homeostasis using specific neutralizing antibodies. METHODS: Adults with active GO (n = 22), inactive GO (n = 28), and healthy control subjects (n = 28) were included in the study. Peripheral B cells and B-cell subsets were quantified and analyzed for CXCR5 expression by flow cytometry. The serum CXCL13 concentration was measured by enzyme-linked immunosorbent assays. For chemotactic experiments, Transwell plates were used, and migrating B cells were further analyzed by flow cytometry. RESULTS: Compared to healthy subjects, patients with active GO had a significantly higher number of CD19+ B cells and the CD19+CD27+ memory B-cell subset (P = .041 and P = .019, respectively), whereas a marginal increase in the number of these cells was found in patients with inactive GO (P = .062 and P = .087, respectively). Serum CXCL13 levels were significantly higher in patients with active GO (86.9 ± 30.4 pg/mL) than in those with inactive GO (41.7 ± 18.1 pg/mL; P < .001) and in healthy subjects (36.2 ± 7.8 pg/mL; P < .001). The increased CXCL13 concentration was positively and significantly correlated with the clinical activity score (r = 0.757, P < .001). Finally, serum from patients with active GO exerted a stronger chemotactic activity towards B cells and the CD19+CD27+ memory B-cell subset. Blocking CXCL13 or CXCR5 with neutralizing antibodies reduced B-cell migration by a mean of 20%. CONCLUSIONS: Our data suggest that aberrant CXCL13/CXCR5 expression may contribute to the deficits in B-lymphocyte homeostasis observed in active GO.

Teprotumumab: a novel therapeutic monoclonal antibody for thyroid-associated ophthalmopathy.

INTRODUCTION: Thyroid-associated ophthalmopathy (TAO) is a disfiguring, potentially blinding, and sub-optimally managed autoimmune condition. Current therapy of active TAO consists most frequently of glucocorticoid steroids, orbital radiation, or B-cell depletion; all of which are associated with substantial side effects. Teprotumumab (Tepezza) is a human monoclonal antibody against the insulin-like growth factor type I receptor (IGF-IR), recently evaluated in two clinical trials for active moderate-to-severe TAO that was recently approved by the United States Food and Drug Administration (FDA) for use in TAO. AREAS COVERED: This article reviews phase II and III placebo-controlled, double-masked, prospective, multicenter studies assessing the efficacy and safety of teprotumumab for the treatment of active, moderate-to-severe TAO. EXPERT OPINION: Teprotumumab has demonstrated substantial and rapid improvement in Clinical Activity Score and proptosis reduction in TAO compared to placebo. Subjective diplopia and quality of life were also improved in both clinical trials. Teprotumumab exhibited a favorable safety profile, with transient hyperglycemia, muscle cramps, and auditory side effects being associated with the drug; these were usually transient. The trial findings indicate that teprotumumab is a promising, potential first-line therapy for treating TAO.

Graves' disease: Clinical manifestations, immune pathogenesis (cytokines and chemokines) and therapy.

Graves' disease (GD) is characterized by thyrotoxicosis, caused by the presence of circulating thyroid stimulating antibodies (TSAb), that are determinant also in the pathogenesis of its extrathyroidal manifestations [Graves' ophthalmopathy (GO), pretibial myxedema]. T helper (Th)1 immune response prevails in the immune-pathogenesis of GD and GO, during the active phase, when Th1 chemokines, and their (C-X-C)R3 receptor, play a key role. In GD, the existing treatments are not ideal for hyperthyroidism (long-term remission with anti-thyroid-drugs only in 50% of patients; while radioiodine and surgery cause hypothyroidism). In GD, antigen-specific therapy has been recently published, with the induction of T cell tolerance via an immunization by TSH-R peptides. In GO, rituximab and drugs targeting cytokines have been evaluated. Furthermore, teprotumumab (a human monoclonal anti-IGF-1R blocking antibody) showed to be very effective in GO patients. Further researches are necessary to identify novel effective therapies targeting GD, or GO.

New insights into the pathogenesis and nonsurgical management of Graves orbitopathy.

Graves orbitopathy, also known as thyroid eye disease or thyroid-associated orbitopathy, is visually disabling, cosmetically disfiguring and has a substantial negative impact on a patient's quality of life. There is increasing awareness of the need for early diagnosis and rapid specialist input from endocrinologists and ophthalmologists. Glucocorticoids are the mainstay of treatment; however, recurrence occurs frequently once these are withdrawn. Furthermore, in >60% of cases, normal orbital anatomy is not restored, and skilled rehabilitative surgery is required. Clinical trials have shown that considerable benefit can be derived from the addition of antiproliferative agents (such as mycophenolate or azathioprine) in preventing deterioration after steroid cessation. In addition, targeted biologic therapies have shown promise, including teprotumumab, which reduces proptosis, rituximab (anti-CD20), which reduces inflammation, and tocilizumab, which potentially benefits both of these parameters. Other strategies such as orbital radiotherapy have had their widespread role in combination therapy called into question. The pathophysiology of Graves orbitopathy has also been revised with identification of new potential therapeutic targets. In this Review we provide an up-to-date overview of the field, outline the optimal management of Graves orbitopathy and summarize the research developments in this area to highlight future research questions and direct future clinical trials.

Novel therapies for thyroid autoimmune diseases: An update.

A Th1 immune-preponderance has been shown in the immunopathogenesis of autoimmune thyroiditis (AT), Graves' disease (GD) and Graves' Ophthalmopathy (GO), in which the Th1-chemokines (CXCL9, CXCL10, CXCL11), and their (C-X-C)R3 receptor, have a crucial role. Methimazole, and corticosteroids have been shown to modulate these chemokines; several efforts have been done to modulate the autoimmune reaction with other drugs, i.e. PPAR-γ, or -α ligands, or antibodies, or small molecules directed against CXCL10, or CXCR3. Antigen-specific therapy for GD, by inducing T cell tolerance through an immunization with TSH-R peptides, has been published. Drugs targeting cytokines [anti-TNFα (Etanercept), and anti-IL-6 (Tocilizumab)], and RTX (a chimeric monoclonal antibody vs. CD20) have been used in GO, with promising results. Teprotumumab (a human monoclonal anti-IGF-1R blocking antibody) has been investigated in a trial, showing it was very effective in GO patients. Still, more studies are needed for new therapies targeting autoimmune thyroid disorders.

Role of a new bioassay for thyroid-stimulating antibodies (aequorin TSAb) in Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is characterized by an autoimmune reaction against thyrotropin (TSH) receptors and is diagnosed by TSH receptor antibody (TRAb). A novel assay for thyroid-stimulating antibody (TSAb) was recently introduced using a frozen Chinese hamster ovary cell line expressing TSH receptors, cyclic adenosine monophosphate (cAMP)-gated calcium channel, and aequorin (aequorin TSAb). The aim of this study was to evaluate the role of aequorin TSAb in GO. We studied 136 Japanese patients with GO (22 euthyroid and 8 hypothyroid GO patients) at our hospital. TRAbs were estimated by first generation TRAb (TRAb 1st), second generation TRAb (hTRAb 2nd), conventional porcine TSAb, and the new aequorin TSAb assays. Aequorin TSAb, porcine TSAb, TRAb 1st, and hTRAb 2nd were positive in 125/136 (92%), 110/136 (81%), 81/130 (62%), and 93/114 (82%) patients, respectively. In patients with hyperthyroid GO, they were positive in 98/106 (98%), 96/106 (91%), 78/101 (77%), and 84/93 (90%) patients, respectively. In patients with euthyroid GO, they were positive in 19/22 (86%), 9/22 (41%), 1/21 (5%), and 6/17 (35%) patients, respectively. Aequorin TSAb levels were significantly related to TRAb 1st (r = 0.4172, p < 0.0001), hTRAb 2nd (r = 0.2592, p < 0.0001), and porcine TSAb (r = 0.4665, p < 0.0001). Clinical activity score (CAS) was significantly greater in patients with high titers of aequorin TSAb than in those with low titers. Aequorin TSAb levels were significantly related to the signal intensity ratio of the enlarged eye muscle and proptosis evaluated by MRI before steroid pulse therapy. Aequorin TSAb assay was more sensitive than the conventional assays, especially in euthyroid GO.

The Th1 chemokine MIG in Graves' ophthalmopathy.

Chemokine (C-X-C motif) ligand (CXCL)9/monokine induced by interferon (IFN)-γ (MIG) and its receptor, chemokine (C-X-C motif) receptor (CXCR)3, are involved in the pathogenesis of Graves' ophthalmopathy (GO). In tissues, recruited Type 1 helper (Th1) lymphocytes could cause an increased production of IFN-γ and tumor necrosis factor (TNF)-α, which in turn stimulate MIG secretion from these cells; all this creates an amplification feedback loop, and perpetuates the autoimmune process. In particular, MIG is secreted by fibroblasts and preadipocytes under the influence of IFN-γ. The IFN-γ induced MIG secretion in vitro, in GO fibroblasts and preadipocytes can be modulate by peroxisome proliferator-activated receptors (PPAR)-γ and PPAR-α activators. High levels of MIG in peripheral liquids could be considered as a marker of an especially Th1 orientated immune response. The presence of circulating MIG is associated with the active phase of GO. An increased concentrations of MIG in patients with GO reflect, at least in part, the orbital inflammation activity. Since MIG concentrations are significantly reduced during corticosteroids and/or radiotherapy treatments, with respect to control group and basal values of patients with GO, MIG has been proposed as a marker in the therapy choice for patients with GO. More studies are needed to investigate interactions between chemokines and cytokines in the GO pathogenesis and to evaluate whether MIG is a novel therapeutic target in these autoimmune disorders.

Differential modulation of CXCL8 versus CXCL10, by cytokines, PPAR-gamma, or PPAR-alpha agonists, in primary cells from Graves' disease and ophthalmopathy.

BACKGROUND: Thyrocytes secrete CXC chemokines, particularly (C-X-C motif) ligand (CXCL)8 and CXCL10; its physiopathological significance remains unclear. This study investigates the modulation of the secretion of CXCL8 vs. CXCL10, in human primary cells cultures of thyroid follicular cells (TFC) in Graves' disease (GD), and fibroblasts (OF) or preadipocytes (OP) from Graves' ophthalmopathy (GO). METHODS: Cells were initially incubated with different concentrations of tumor necrosis factor (TNF)α (1, 5, 10 ng/mL). Then, CXCL8 and CXCL10 were measured in the supernatants of TFC, OF or OP cells basally and after 24 h of treatment with interferon (IFN)γ (1000 IU/mL) and/or TNFα (10 ng/mL), in presence/absence of the peroxisome proliferator activated receptor (PPAR)γ agonist pioglitazone (0, 0.1, 1, 5, 10, 20 µM), or the PPARα agonist fenofibrate (5, 10, 50, 100 µM). RESULTS: CXCL8, not CXCL10, was detected in basal conditions in TFC, OF and OP. CXCL8 secretion increased dose-dependently with increasing concentrations of TNFα. CXCL10 secretion was significantly stimulated by IFNγ (P < 0.01) and not by TNFα, whereas CXCL8 was induced by TNFα (P < 0.01), and inhibited by IFNγ (P < 0.01) in TFC, OF and OP. Combining TNFα and IFNγ, the IFNγ-induced CXCL10 secretion was synergistically increased (P < 0.01) while the TNFα-induced CXCL8 secretion (P < 0.01) was reversed in all cell types. Pioglitazone had no significant effect on the secretion of CXCL8 stimulated by TNFα, while inhibited CXCL10. Fenofibrate, in presence of IFNγ plus TNFα, dose-dependently inhibited both CXCL10 and CXCL8 release. CONCLUSION: We first show that TFC, OF, and OP secrete CXCL8 and CXCL10 differentially, sustained by specific proinflammatory cytokines or their combination. This could reflect a different role of the two chemokines in the course of the disease, as CXCL10 could be associated with the initial phase of the disease when IFNγ is preponderant, while CXCL8 could be associated with a later chronic phase of the disease, when TNFα prevails.

Therapeutic Effects of Short Cyclic and Combined Epitope Peptides in a Long-Term Model of Graves' Disease and Orbitopathy.

BACKGROUND: Cyclic peptides derived from some cylindrical loops of the leucine-rich repeat domain (LRD) of the thyrotropin receptor (TSHR) have been shown to treat disease manifestations in a mouse model of Graves' disease during a long-term protocol of four-weekly immunizations with adenovirus coding for the TSHR A-subunit (Ad-TSHR289). METHODS: In a follow-up study, two additional cyclic peptides were tested, which were shortened in order to obtain additional information on the minimally involved epitopes and to enable easier production conditions. In addition, a linear peptide was tested, which mimics parts of three loops of the native TSHR LRD structure, and is potentially able to block the discontinuous epitopes of anti-TSHR antibodies. RESULTS: The novel peptides markedly reduced thyroid size, serum thyroxine levels, retro-orbital fibrosis, and tachycardia in Ad-TSHR289-immunized mice. In immunologically naïve mice, administration of the peptides did not induce any immune response. CONCLUSIONS: In summary, novel cyclic peptides mitigate many clinical findings in a mouse model of established Graves' disease and orbitopathy, and may therefore provide an additional therapeutic option compared to existing drugs or interventions.

Extraocular Muscle Enlargement and Thyroid Eye Disease-like Orbital Inflammation Associated with Immune Checkpoint Inhibitor Therapy in Cancer Patients.

PURPOSE: To describe thyroid eye disease (TED)-like orbital inflammatory syndrome in 3 cancer patients treated with immune checkpoint inhibitors. METHODS: All consecutive patients treated by the senior author who were receiving immune checkpoint inhibitors and developed TED-like orbital inflammation were included. RESULTS: Three cancer patients treated with immune checkpoint inhibitors developed orbital inflammation. The first patient was treated with a combination of a cytotoxic T-lymphocyte antigen-4 inhibitor and a programmed cell death protein 1 inhibitor and developed TED-like orbital inflammation with normal thyroid function and antibody levels. The second patient had a previous diagnosis of Graves disease without TED, and developed TED soon after initiating treatment with a programmed cell death protein 1 inhibitor. The third patient developed acute hyperthyroidism with symptomatic TED following treatment with an investigational cytotoxic T-lymphocyte antigen-4 inhibitor agent. All 3 patients were managed with either systemic steroids or observation, with resolution of their symptoms and without the need to halt immune checkpoint inhibitor treatment for their cancer. DISCUSSION AND CONCLUSIONS: TED-like orbital inflammation may occur as a side effect of immune checkpoint inhibitor therapy with anti-cytotoxic T-lymphocyte antigen-4 or anti-PD-1 inhibitors. To the best of their knowledge, this is the first reported case of TED as a result of programmed cell death protein 1 inhibitor monotherapy. All 3 patients were treated with systemic steroids and responded quickly while continuing treatment with immune checkpoint inhibitors for their cancer. With increasing use of this class of drugs, clinicians should be familiar with the clinical manifestations and treatments for this adverse reaction.