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.

Comparison of a Novel Homogeneous Cyclic Amp Assay and a Luciferase Assay for Measuring Stimulating Thyrotropin-Receptor Autoantibodies.

OBJECTIVE: Stimulating thyrotropin-receptor antibodies (TSAb) cause Graves' disease (GD). We tested a novel homogeneous fluorescent 3',5' cyclic adenine monophosphate (cAMP) assay for the detection of TSAb in a bioassay. METHODS: Chinese hamster ovary (CHO) cell lines expressing either a chimeric (MC4) or wild-type (WT) TSH-R were incubated with the adenyl cyclase activator forskolin, a human TSAb monoclonal antibody (M22), and with sera from GD patients. Intracellular cAMP levels were measured using a Bridge-It® cAMP assay, and the results were compared with a luciferase-based bioassay. RESULTS: Both cell lines were stimulated with forskolin concentrations (0.006-200 µM) in a dose-dependent manner. The linear range in the MC4 and WT cells was 0.8-25 and 3.1-50 µM, respectively. Levels of cAMP and luciferase in forskolin-treated MC4 and WT cells were positively correlated (r = 0.91 and 0.84, both p < 0.001). The 50% maximum stimulatory concentration of forskolin was more than 16-fold higher for the CHO-WT cells than the CHO-MC4 cells in the cAMP assay and 4-fold higher in the luciferase assay. Incubation of both cell lines with M22 (0.006-50 ng/mL) resulted in a dose-dependent increase in cAMP levels with linear ranges for the MC4 and WT cells of 0.8-12.5 and 0.2-3.125 ng/mL, respectively. Comparison of cAMP and luciferase levels in M22-treated MC4 and WT cells also showed a positive correlation (r = 0.88, p < 0.001 and 0.75, p = 0.002). A positive correlation was also noted when using patient samples (r = 0.96, p < 0.001) that were all TSH-R-Ab binding assay positive. CONCLUSION: The novel, rapid, simple-to-perform cAMP assay provides TSAb-mediated stimulatory results comparable to a luciferase-based bioassay.

TSH RECEPTOR ANTIBODIES: RELEVANCE & UTILITY.

Objective: Antibodies (Abs) to the thyrotropin (TSH) receptor (TSH-R) play an important role in the pathogenesis of autoimmune thyroid disease (AITD). We define the complex terminology that has arisen to describe TSH-R-Abs, review the mechanisms of action of the various types of TSH-R-Abs, and discuss significant advances that have been made in the development of clinically useful TSH-RAb assays. Methods: Literature review and discussion. Results: TSH-R-Abs may mimic or block the action of TSH or be functionally neutral. Stimulating TSH-R-Abs are specific biomarkers for Graves disease (GD) and responsible for many of its clinical manifestations. TSH-R-Abs may also be found in patients with Hashimoto thyroiditis in whom they may contribute to the hypothyroidism of the disease. Measurement of TSH-R-Abs in general, and functional Abs in particular, is recommended for the rapid diagnosis of GD, differential diagnosis and management of patients with AITD, especially during pregnancy, and in AITD patients with extrathyroidal manifestations such as orbitopathy. Measurement of TSH-R-Abs can be done with either immunoassays that detect specific binding of Abs to the TSH-R or cell-based bioassays that also provide information on their functional activity and potency. Application of molecular cloning techniques has led to significant advances in methodology that have enabled the development of clinically useful bioassays. When ordering TSH-R-Ab, clinicians should be aware of the different tests available and how to interpret results based on which assay is performed. The availability of an international standard and continued improvement in bioassays will help promote their routine performance by clinical laboratories and provide the most clinically useful TSH-R-Ab results. Conclusion: Measurement of TSH-R-Abs in general, and functional (especially stimulating) Abs in particular, is recommended for the rapid diagnosis, differential diagnosis, and management of patients with Graves hyperthyroidism, related thyroid eye disease, during pregnancy, as well as in Hashimoto thyroiditis patients with extra-thyroidal manifestations and/or thyroid-binding inhibiting immunoglobulin positivity. Abbreviations: Ab = antibody; AITD = autoimmune thyroid disease; ATD = antithyroid drug; cAMP = cyclic adenosine 3',5'-monophosphate; ELISA = enzyme-linked immunosorbent assay; GD = Graves disease; GO = Graves orbitopathy; HT = Hashimoto thyroiditis; MAb = monoclonal antibody; TBAb = thyrotropin receptor blocking antibody; TBII = thyroid-binding inhibiting immunoglobulin; TSAb = thyrotropin receptor-stimulating antibody; TSB-Ab or TRBAb = thyrotropin receptor-stimulating blocking antibody; TSH = thyrotropin; TSH-R = thyrotropin receptor.

A Novel Long-Term Graves' Disease Animal Model Confirmed by Functional Thyrotropin Receptor Antibodies.

INTRODUCTION: A novel long-term murine model for Graves' disease (GD) using repeated, long-term immunizations with recombinant adenovirus expressing the extracellular A-subunit of the human thyrotropin receptor (Ad-TSHR) was applied to evaluate the functional anti-TSHR-antibody (TSHR-Ab) profile. METHODS: BALB/c mice received 7 immunizations with either 1010 plaque-forming units of Ad-TSHR or control Ad-GFP. Naïve (nonimmuized native) mice were also studied. Three 3-weekly immunizations were followed by 4-weekly boosts until the 7th immunization. Blocking (TBAb) and stimulating (TSAb) TSHR-Ab were measured with bioassays. Assay cut-offs for TBAb/TSAb were at 34% inhibition and a specimen-to-reference ratio (SRR) of 140%. RESULTS: Nineteen (8 Ad-TSHR-, 4 Ad-GFP-immunized, and 7 native) mice were investigated. All native mice were negative for TSHR-binding inhibitory immunoglobulins (TBII) prior to immunization. Native and Ad-GFP mice were negative in weeks 17 and 27 for TBII and TBAb/TSAb. In native mice, the free thyroxine (fT4) levels (median [25th percentile; 75th percentile]) were in the upper normal range (1.2 ng/mL [1.1; 1.6]) prior to immunization, at weeks 17 (2.2 ng/mL [2.1; 2.4]) and 27 (1.4 ng/mL [1.1; 1.7]), respectively. In contrast, in Ad-TSHR-immunized mice, fT4 values were markedly increased at weeks 17 (4.4 ng/mL [3.9; 6]) and 27 (4.5 ng/mL [4.2; 6]) compared to those in Ad-GFP mice (2 ng/mL [1.8; 2.1] and 1.4 ng/mL [1.1; 1.6]), respectively (p = 0.0008, p = 0.001). In contrast, at week 17, in Ad-TSHR mice, the mean TBII, TBAb, and TSAb levels were 40 IU/L (40; 40); 62% inhibition (38; 69), and 116% SRR (97; 185), respectively; at week 27, they were 40 IU/L (39; 40); 65% inhibition (34; 80) and 95% SRR (63; 187), respectively. Three serum samples from Ad-TSHR mice (38%) demonstrated dual TBAb/TSAb positivity. CONCLUSIONS: TBAb/TSAb were highly prevalent in Ad-TSHR-immunized mice, thus confirming the successful establishment of a novel, long-term murine model for GD. All TBAb- and TSAb-positive Ad-TSHR-immunized mice were TBII-positive. Thus, the binding immunoassay did not differentiate between TSHR-Ab functionality.

Comparison of a Bridge Immunoassay with Two Bioassays for Thyrotropin Receptor Antibody Detection and Differentiation.

A rapid and fully automated chemiluminescent immunoassay for the detection of thyrotropin receptor autoantibodies (TSHR-Ab) based on a bridge technology was compared with two bioassays that measure either stimulating (TSAb) or blocking (TBAb) antibodies for the detection and differentiation of TSHR-Ab. A total of 229 patients with various thyroid disorders [151 with Graves' disease (GD), 35 with Hashimoto's thyroiditis (HT), 32 with nodular goiter, and 11 with thyroid cancer] were included. The bridge immunoassay was performed according to the manufacturer's instructions (cut-off>0.55 IU/l). TSAb and TBAb were measured with reporter bioassays. Blocking activity was defined as percent inhibition of luciferase expression relative to induction with bovine TSH alone (cut-off>34% inhibition). TSAb was reported as percentage of specimen-to-reference ratio (> 140 SRR%). The 3 TSHR-Ab assays were negative in all patients with benign euthyroid nodular goiter and differentiated thyroid cancer. In contrast, in all patients with GD, irrespective of the disease duration, TSHR-Ab positivity was present in 127 of 151 (84%) and 140 (93%) for the bridge assay and TSAb bioassay, respectively (p<0.001). Fifteen of 151 (10%) GD samples were positive in the TSAb bioassay but negative in the bridge assay. The bridge assay and the TSAb bioassay correlated positively (r=0.39, p<0.0001) in patients with GD. Both assays detected TSHR-Ab in all ten untreated hyperthyroid patients with GD. In GD patients with a duration of less than six months, 27/29 (93%) and 28 (97%) were TSHR-Ab positive with the bridge and TSAb bioassay, respectively. In comparison, TSHR-Ab were present in two of 35 (6%) and five (14%) HT patients with the bridge and TSAb bio-assay, respectively. TSHR blocking antibodies were present in one (3%) patient with HT and in two (1%) patients with GD; these two GD patients were also bridge assay positive but TSAb bioassay negative. In conclusion, the bridge immunoassay and both bioassays are highly sensitive for the detection of TSHR-Ab. The bridge assay is, however, also positive in the presence of TSHR blocking antibodies detected in a TBAb bioassay.

Thyroid Stimulating Hormone Receptor Antibodies in Thyroid Eye Disease-Methodology and Clinical Applications.

BACKGROUND: Thyroid stimulating hormone receptor antibodies (TSHR-Ab) cause autoimmune hyperthyroidism and are prevalent in patients with related thyroid eye disease (TED). PURPOSE: To provide a historical perspective on TSHR-Ab and to present evidence-based recommendations for clinical contemporary use. METHODS: The authors review the recent literature pertaining to TSHR-Ab in patients with TED and describe the various immunoassays currently used for detecting TSHR-Ab and their clinical applications. RESULTS: We provide a historical summary and description of the various methods used to detect TSHR-Ab, foremost, the functional TSHR-Ab. Increasing experimental and clinical data demonstrate the clinical usefulness of cell-based bioassays for measurements of functional TSHR-Ab in the diagnosis and management of patients with autoimmune TED and in the characterization of patients with autoimmune-induced hyperthyroidism and hypothyroidism. Thyroid stimulating hormone receptor antibodies, especially the functional stimulating antibodies, are sensitive, specific, and reproducible biomarkers for patients with autoimmune TED and correlate well with clinical disease activity and clinical severity. Unlike competitive-binding assays, bioassays have the advantage of indicating not only the presence of antibodies but also their functional activity and potency. CONCLUSIONS: Measurement of TSHR-Ab (especially stimulating antibodies) is a clinically useful tool for the management of patients with TED.

Mycophenolate plus methylprednisolone versus methylprednisolone alone in active, moderate-to-severe Graves' orbitopathy (MINGO): a randomised, observer-masked, multicentre trial.

BACKGROUND: European guidelines recommend intravenous methylprednisolone as first-line treatment for active and severe Graves' orbitopathy; however, it is common for patients to have no response or have relapse after discontinuation of treatment. We aimed to compare the efficacy and safety of add-on mycophenolate to methylprednisolone in comparison with methylprednisolone alone in patients with moderate-to-severe Graves' orbitopathy. METHODS: MINGO was an observer-masked, multicentre, block-randomised, centre-stratified trial done in two centres in Germany and two in Italy. Patients with active moderate-to-severe Graves' orbitopathy were randomly assigned to receive intravenous methylprednisolone (500 mg once per week for 6 weeks followed by 250 mg per week for 6 weeks) either alone or with mycophenolate (one 360 mg tablet twice per day for 24 weeks). The prespecified primary endpoints were rate of response (reduction of at least two parameters of a composite ophthalmic index [eyelid swelling, clinical activity score, proptosis, lid width, diplopia, and eye muscle motility] without deterioration in any other parameter) at 12 weeks and rate of relapse (a worsening of symptoms that occurred after a response) at 24 and 36 weeks. Rates of response at week 24 and sustained response at week 36 were added as post-hoc outcomes. Prespecified primary outcomes and post-hoc outcomes were assessed in the modified intention-to-treat population (defined as all patients assigned to treatment who received at least one infusion of methylprednisolone, when outcome data were available), and safety was assessed in all patients who received at least one dose of study drug. This trial is registered with the EU Clinical Trials Register, EUDRACT number 2008-002123-93. FINDINGS: 164 patients were enrolled and randomised between Nov 29, 2009, and July 31, 2015. 81 were randomly assigned to receive methylprednisolone alone and 83 to receive methylprednisolone with mycophenolate. In the intention-to-treat population at 12 weeks, responses were observed in 36 (49%) of 73 patients in the monotherapy group and 48 (63%) of 76 patients in the combination group, giving an odds ratio (OR) of 1·76 (95% CI 0·92-3·39, p=0·089). At week 24, 38 (53%) of 72 patients remaining in the monotherapy group and 53 (71%) of 75 patients remaining in the combination therapy group had responded to treatment (2·16, 1·09-4·25, p=0·026). At week 24, relapse occurred in four (11%) of 38 patients in the monotherapy group and four (8%) of 53 patients in the combination group (OR 0·71, 0·17-3·03, p=0·72). At week 36, relapse occurred in an additional three (8%) patients in the monotherapy group and two (4%) patients in the combination group (0·65, 0·12-3·44, p=0·61). At week 36, 31 (46%) of 68 patients in the monotherapy group and 49 (67%) of 73 patients in the combination group had a sustained response (OR 2·44, 1·23-4·82, p=0·011). 23 patients had 24 serious adverse events, with 11 events in ten patients in the combination group and 13 events in 13 patients in the monotherapy group. Mild and moderate (grade 1-2) drug-related adverse events occurred in 16 (20%) of 81 patients receiving monotherapy and 21 (25%) of 83 patients receiving combination therapy (p=0·48). INTERPRETATION: Although no significant difference was seen in the rate of response at 12 weeks or rate of relapse at 24 and 36 weeks, post-hoc analysis suggested that addition of mycophenolate to treatment with methylprednisolone improved rate of response to therapy by 24 weeks in patients with active and moderate-to-severe Graves' orbitopathy. FUNDING: Novartis, Germany.

United States and European Multicenter Prospective Study for the Analytical Performance and Clinical Validation of a Novel Sensitive Fully Automated Immunoassay for Calcitonin.

BACKGROUND: The objective of this study is the validation and proof of clinical relevance of a novel electrochemiluminescence immunoassay (ECLIA) for the determination of serum calcitonin (CT) in patients with medullary thyroid carcinoma (MTC) and in different diseases of the thyroid and of calcium homeostasis. METHODS: This was a multicenter prospective study on basal serum CT concentrations performed in 9 US and European referral institutions. In addition, stimulated CT concentrations were measured in 50 healthy volunteers after intravenous calcium administration (2.5 mg/kg bodyweight). RESULTS: In total, 1929 patients and healthy controls were included. Limits of blank, detection, and quantification for the ECLIA were 0.3, 0.5, and 1 ng/L, respectively. Highest intra- and interassay coefficients of variation were 7.4% (CT concentration, 0.8 ng/L) and 7.0% (1.1 ng/L), respectively. Medians (interval) of serum CT concentrations in 783 healthy controls were 0.8 ng/L (<0.5-12.7) and 3 ng/L (<0.5-18) for females and males, respectively (97.5th percentile, 6.8 and 11.6 ng/L, respectively). Diagnostic sensitivity and specificity were 100%/97.1% and 96.2%/96.4%, for female/males, respectively. Patients (male/female) with primary hyperparathyroidism, renal failure, and neuroendocrine tumors showed CT concentrations >97.5th percentile in 33%/4.7%, 18.5%/10%, and 8.3%/12%, females/males, respectively. Peak serum CT concentrations were reached 2 min after calcium administration (161.7 and 111.8 ng/L in males and females, respectively; P < 0.001). CONCLUSIONS: Excellent analytical performance, low interindividual variability, and low impact of confounders for increased CT concentrations in non-MTC patients indicate that the investigated assay has appropriate clinical utility. Calcium-stimulated CT results suggest good test applicability owing to low interindividual variability.

Expression of the tetraspanin family members Tspan3, Tspan4, Tspan5 and Tspan7 during Xenopus laevis embryonic development.

Tetraspanins comprise a large family of integral membrane proteins involved in the regulation of cell adhesion, migration and fusion. In humans it consists of 33 members divided in four subfamilies. Here, we examined the spatial and temporal gene expression of four related tetraspanins during the embryonic development of Xenopus laevis by quantitative real-time PCR and in situ hybridization: Tspan3 (encoded by the gene Tm4sf8 gene) Tspan4 (encoded by the gene Tm4sf7), Tspan5 (encoded by the gene Tm4sf9) and Tspan7 (encoded by the gene Tm4sf2). These genes appeared first in the vertebrates during the evolution and are conserved across different species. In humans, they were associated with several diseases such as sclerosis, mental retardation and cancer; however their physiological role remained unclear. This work provides a comprehensive comparative analysis of the expression of these tetraspanins during the development of X. laevis. The more closely related tetraspanins Tspan3, Tspan4 and Tspan7 exhibited very similar spatial expression patterns, albeit differing in their temporal occurrence. The corresponding transcripts were found in the dorsal animal ectoderm at blastula stage. At early tailbud stages (stage 26) the genes were expressed in the migrating cranial neural crest located in the somites, developing eye, brain, and in otic vesicles. In contrast, Tspan5 appeared first at later stages of development and was detected prominently in the notochord. These data support close relatedness of Tspan3, Tspan4 and Tspan7. The expression of these tetraspanins in the cells with a high migratory potential, e.g. neural crest cells, suggests their role in the regulation of migration processes, characteristic for tetraspanin family members, during development. Similarity of the expression profiles might indicate at least partial functional redundancy, which is in concordance with earlier findings of tissue-limited or absent phenotypes in the knock-down studies of tetraspanins family members performed.