Comparison of M22-based ELISA and human-TSH-receptor-based luminescence assay for the measurement of thyrotropin receptor antibodies in patients with thyroid diseases.

Previously, a new procedure for measuring serum TSH receptor autoantibodies (TRAb) was reported in which the autoantibodies inhibit binding of a human monoclonal thyroid stimulating antibody M22 to TSHR-coated ELISA plate wells (TRAb ELISA). The aim of the present study was to evaluate the clinical performance of this assay in comparison to the second generation TRAb assay (TRAb LIA) based on the recombinant human TSH-receptor and chemiluminescence technology (TRAb LIA). Among the 158 patients, 84 patients suffered from Graves' disease (GD), 34 patients had Hashimoto's thyroiditis (HT), and 40 patients had euthyroid nodular thyroid disease (NTD) without signs of autoimmunity. TRAb measurements were performed according to the manufacturer's instructions. Out of 84 GD patients, 80 (95.2%) were TRAb positive as detected by the TRAb LIA. One GD patient had TRAb values within the grey zone (1.0-1.5 IU/l). All patients with HT and NTD were negative except in 6 (8.1%) cases whose TRAb values were within the grey zone. On the basis of the recommended cutoff value (TRAb 1.0 IU/l), the TRAb ELISA found 78 of 84 (92.9%) GD patients to be TRAb positive. None of the patients with HT, but two cases (5.0%) with NTD were TRAb positive. The diagnostic sensitivity of the TRAb LIA and TRAb ELISA assays was 95.2 and 92.9%, while the specificity was 100% and 97.3%, respectively. There was a close correlation (r=0.968, p<0.0001) between both assays in 84 patients with GD. Additionally, the between-run imprecision close to the cutoff limit was assessed. The calculated between-run coefficient of variation (CV) of the TRAb ELISA was 28.2% at the recommended cutoff value of 1.0 IU/l. Due to the evaluated imprecision data we propose a higher cutoff value correlating with a between-run CV of 20% (functional assay sensitivity). Our results indicate that due to a worse imprecision the TRAb ELISA has a slightly lower sensitivity and specificity compared to the TRAb LIA assay. These findings suggest that the M22 monoclonal antibody-based TRAb ELISA is not as reliable as other second generation TRAb assays in the diagnosis of Graves' diseases.     

Levels of autoantibodies against human TSH receptor predict relapse of hyperthyroidism in Graves' disease.

The aim of this study was to evaluate the ability of the more sensitive second-generation TSH receptor (TRAb) assay to predict recurrent Graves' disease (GD) vs. remission depending on TRAb levels. 93 patients with active GD were included in the study. By using a cut-off limit of 1.0 IU/l, all 93 patients were positive for TRAb (median: 4.6 IU/l) at the time of their first visit (single point measurement in median 5.1 months after initial diagnosis). Subsequently, 33 patients went into remission and were euthyroid during follow-up (median follow-up: 21.7 months), whereas 60 patients did not go into remission or developed relapse over the following 24 months. Median TRAb levels in the group of remission were significantly (p < 0.0001) lower than TRAb values in the relapse group (2.1 compared to 8.6 IU/l). Applying ROC plot analysis to compare different TRAb thresholds, a cut-off of 10 IU/l was established. Here, the specificity for relapse was 97 % as only 1 of 29 patients with TRAb values above 10 IU/l went into remission during follow-up, whereas all other 28 patients developed a relapse (positive predictive value for relapse: 96.4 %). In contrast, TRAb values lower than 10 IU/l had no impact on the prediction of remission. In conclusion, our data clearly indicate that TRAb measurement is useful for identifying patients that will not benefit from long-term antithyroid drug treatment.     

Second-generation thyrotropin receptor antibodies assay and quantitative thyroid scintigraphy in autoimmune hyperthyroidism.

Graves' disease (GD) is characterized by circulating TSH receptor antibodies (TRAb), and so-called hyperthyroid autoimmune thyroiditis (H-AIT) generally shows negative TRAb results with first-generation assays. However, a positive titer was observed in up to 13% of patients with euthyroid or hypothyroid autoimmune thyroiditis (AIT) by second-generation TRAb assays, and a larger increase is expected in hyperfunctioning forms. A thyroid 99mTc-pertechnetate uptake (TcTUs) cutoff of 2% previously was shown to accurately discriminate between GD and H-AIT. Here we evaluated the relationship between second-generation TRAb assays and TcTUs in 139 patients with untreated autoimmune hyperthyroidism. An increase in TRAb levels was found in 114 of 139 patients (82%). All patients with TcTUs >2% and 66% of those with lower values had positive TRAb measurements. When the cutoff was increased to 5.9 U/l, positive TRAb occurred in 92.6% of those having higher TcTUs and in 1.4% of those with lower TcTUs. TRAb levels significantly increased in patients with TcTUs higher than 2% as compared with those having lower values, while no differences occurred in patients with TcTUs ranging from 0 to 1%, from 1.1 to 1.5%, or from 1.6 to 2%. A significant relationship between TRAb and TcTUs (as well as fT3, fT4, and thyroid volume) was found. TcTUs, fT4, fT3, and thyroid volume significantly increased in patients with positive TRAb (cutoff 1.5 U/l) compared with those testing negative. Our data indicate that a large proportion of patients with hyperthyroidism and hypoechoic thyroid, including those previously diagnosed as having H-AIT, actually have circulating TRAb. TRAb levels predicate the degree of iodine uptake (as measured by TcTUs) and thyroid hyperfunction. The role of baseline TRAb measurement by second-generation assays to predict patients' outcome needs to be further evaluated and compared with TcTUs in prospective studies.     

Distinction between autoimmune and non-autoimmune hyperthyroidism by determination of TSH-receptor antibodies in patients with the initial diagnosis of toxic multinodular goiter.

Distinguishing Graves' disease (GD) from a toxic multinodular goiter (TMG) subgroup with a diffuse but uneven Tc-distribution depends on the diagnostic power of the TSH-receptor antibody (TRAb) determination. Bioassays using CHO cell lines expressing the hTSH-receptor or a new TBII assay, which uses the hTSH-receptor as an antigen (DYNOTEST TRAK human, Brahms, Germany), showed a higher sensitivity for the detection of TRAbs in patients with GD than assays using solubilized porcine epithelial cell membranes. The aim of this study was to investigate whether the new Dynotest TRAK human assay has an increased sensitivity to distinguish GD from non-autoimmune hyperthyroidism. Therefore, we examined 21 consecutive patients with the initial diagnosis of TMG for thyroid-stimulating antibodies (TSAbs, JP26 cell assay) and TBII with the new highly sensitive Dynotest TRAK human (Brahms, Germany). The initial diagnosis of TMG was based on suppressed TSH and a patchy Tc-uptake of more than 1 % and less than 7 % or TSH of more than 0.3 mIE/l with a patchy Tc-uptake of more than 1.5 % and less than 7 % and negative TBII values in a displacement assay using solubilized porcine epithelial cell membranes (TRAK, Brahms, Germany). 11 sera from these 21 patients showed TSAb activity. Furthermore, 10 of these 11 TSAb-positive sera were also positive in the Dynotest TRAK human assay, whereas one serum sample was borderline positive. TSAb activity and inhibition of (125)I-bTSH binding in the Dynotest TRAK human assay correlated well (r = 0.7). Therefore, 11 of the 21 investigated patients initially classified as TMG actually had GD, which was undetectable using the porcine TBII assay. In conclusion, TSAbs or TRAbs detected with the Dynotest TRAK human have the highest diagnostic power to differentiate GD from TMG. Because of the less cumbersome assay technique, the Dynotest TRAK human measurements should be obtained for all patients with non-typical TMG to differentiate GD from non-autoimmune hyperthyroidism in order to select the appropriate therapy for these patients.     

Improved prediction of relapse of Graves' thyrotoxicosis by combined determination of TSH receptor and thyroperoxidase antibodies.

BACKGROUND: Recently, we and others have demonstrated that high levels of auto-antibodies to the human TSH-receptor (TRAb) predict relapse of hyperthyroidism in Graves' disease (GD). Our objective was to extend the outcome of the prediction by combining TRAb with thyroperoxidase antibody (TPO-Ab) measurement. PATIENTS AND METHODS: One hundred and thirty-one GD patients (118 females, 13 males) were analysed, of whom 94 patients (71.8%) had relapse, whereas 37 (28.2%) went into remission. Second generation TRAb and TPO-Ab assays were performed in GD patients with relapse and remission in mean 4.3 months after initial diagnosis. RESULTS: The mean anti-TPO-Ab levels were similar in all patients with relapse and remission. However, there was a steady decline from 4047 U/ml to 530 U/ml in the remission group that correlated positively with TRAb values (>2 to >10 IU/l). The relapse group remained at consistently high levels. The positive predictive value (PPV) for relapse in patients with TRAbs >6 IU/l and anti-TPO-Abs >5000 U/ml was 100, whereas TRAbs >6 IU/l and anti-TPO-Abs >500 U/ml were associated with a PPV of 93.7 up to 96 (p=0.008). These Ab constellations accounted for about one third of all GD patients. For patients with TRAbs between >2 and <6 IU/l the PPV was 66.7-90.0. CONCLUSION: Our follow-up analysis indicates that the prediction of relapse of GD can be improved by a combined measurement of TRAb and TPO-Ab. In patients with moderately increased Abs, determined about 6 months after initial diagnosis, an ablative therapy can be approached without delay.     

Does autoantibody-negative Graves' disease exist? A second evaluation of the clinical diagnosis.

Advanced technical methods are essential for accurate diagnosis of Graves' or Basedow's disease (GD). Inadequate methods may lead to a false diagnostic conclusion. We have analyzed the clinical features and methodology aspects of cases diagnosed as GD with negative findings for TSH receptor autoantibodies. The initial diagnosis was based on clinical findings (patient record, hypermetabolic state, goiter palpation) and laboratory testing (fT4 and TSH). From a total of 255 newly registered patients with GD, fifty-one (20%) were negative in a conventional porcine TBII assay. All fifty-one patients were retested with 131I or 99mTc uptake tests, thyroid scintigraphy, and a second-generation TBII assay. Results disclosed twenty-one cases (8.3%) with diagnosis other than GD: ten cases of autonomous hyperthyroidism (Plummer's disease), seven cases of painless thyroiditis and four cases of euthyroid endocrine ophthalmopathy. All twenty-one patients remained negative in the second-generation TBII assay. Measurement by second-generation TBII assay was performed on the remaining thirty patients initially found negative for TBII. As a result of this reevaluation, only 234 of the original 255 patients had GD. Of those, 231 (204 according to porcine plus 27 according to human TRAb assay) had detectable TBII (98.7%). This investigation stresses the problem of correct diagnosis and the methodological limitations in the assessment of laboratory parameter validity in GD. Based on this work, TSH receptor autoantibody-negative GD is extremely rare.     

Binding, stimulating and blocking TSH receptor antibodies to the thyrotropin receptor as predictors of relapse of Graves' disease after withdrawal of antithyroid treatment.

TSH-receptor autoantibodies (TRAbs) are a valuable diagnostic tool for confirming a diagnosis of Graves' disease (GD). While there is evidence that high TRAb levels are associated with relapse of GD, whether a discrimination of TRAb into stimulating (TSAb) and blocking (TBAb) autoantibodies would benefit the clinician in terms of outcome prediction remains unclear. To address this issue, we have determined TRAb, TSAb and TBAb levels in serum samples of ninety-six euthyroid patients with GD taken four weeks after antithyroid drug withdrawal (ATDT). Forty-seven patients (49 %) underwent relapse of GD within two years. Amongst those, forty-one (87 %) had been positive for TRAb and thirty-five (74 %) for TSAb after treatment. All patients except one were negative for TBAb. The correlation between TRAb and TSAb in those treated GD patients was relatively weak (r = 0.268, p < 0.001). Based on a cut-off limit of 1.5 IU/l, the positive and negative predictive values with respect to prediction of relapse were too low for any clinical relevance (TRAb: 49 % and 54 %; TSAb: 51 % and 55 %). However, when a cut-off level above 10 IU/l was used, the positive and negative predictive values increased to 83 % and 62 %. The additional measurement of TSAb or TBAb in those samples after therapy did not add additional information, even at higher decision thresholds. In conclusion, differentiation of TRAb into TSAb and TBAb is of no help in the prediction of relapse of GD in euthyroid patients at the end of ATDT, and only high TRAb levels are associated with relapse.     

Thyroid autoantigens and their relevance in the pathogenesis of thyroid autoimmunity

Humoral and cellular immune responses are both involved in autoimmune disorders of the thyroid gland. In the last five years, new substantial data have been obtained on the nature and the expression of thyroid cell surface autoantigens and on the demonstration of the functional heterogeneity of autoantibodies to the thyroid stimulating hormone (TSH) receptor. In the present report, attention will be mainly focused on recent studies carried out in our laboratory. The main autoantigens so far identified include the 'microsomal' antigen, thyroglobulin and the TSH receptor. For many years the 'microsomal' antigen (M) was considered a poorly characterized constituent of the cytoplasm of the thyroid cell. In the last five years, several lines of evidence were provided indicating that M is also well represented on the surface of the follicular cell and is identical to thyroid peroxidase (TPO). The use of anti-TPO monoclonal antibodies, presently available, have confirmed this antigenic identity. Microsomal (anti-TPO) antibodies are very useful markers of autoimmune thyroid disorders and are generally present in Hashimoto's thyroiditis, idiopathic myxedema and Graves' disease. TSH receptor antibodies (TRAb) are present in the sera of patients with Graves' disease. TRAb are able to stimulate thyroid adenylate cyclase and also to mimic TSH in its thyroid growth stimulation. Thus, these antibodies may have a pathogenetic role in goiter formation and in thyroid hyperfunction in Graves' disease. TRAb were also shown to inhibit both TSH binding to its receptor and TSH-stimulated adenylate cyclase activity. Recently TRAb, which inhibited TSH-stimulated adenylate cyclase activity, were found in idiopathic myxedema patients and may be responsible for impairment of thyroid function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of TSH binding to chicken thyroid by some cases of LATS (long acting thyroid stimulator)

TSH receptor antibody (TRAb) activity using chicken thyroid receptor (c-TRAb) and porcine thyroid receptor (p-TRAb) was determined by the incubation of 125I-bovine TSH with each receptor. Both c-TRAb and p-TRAb activity in LATS positive and negative Graves' sera were compared. 15 out of 39 LATS positive sera and 4 out of 46 LATS negative sera had positive c-TRAb activity. On the other hand, all LATS positive sera and 33 out of 46 LATS negative sera had positive p-TRAb activity. No relationship between c-TRAb and p-TRAb activity was observed, and there was also no correlation between c-TRAb and LATS activity. Changes in c-TRAb, p-TRAb and LATS activity in the clinical course of patients with Graves' disease were examined. These activities were parallel in some cases, but in others they were not. A weak c-TRAb activity was observed in 4 out of 29 Hashimoto's disease, but all cases with thyroid cancer and subacute thyroiditis showed no activity. Sera with positive c-TRAb activity did not stimulate chicken thyroid in chick bioassay. These results suggest that some cases of TRAb in Graves' disease (mainly LATS) inhibit TSH binding to chicken thyroid receptor (non-mammalian species) in the same way as mammalian thyroid, but may not have any stimulatory action on thyroid hormone synthesis. It is interesting to note that TRAb including LATS have the similar effect on TSH receptor even in nonmammalian species.     

Thyrotropin receptor non-mediated thyroid stimulating immunoglobulin in Graves' disease.

There exists a consensus that hyperthyroid Graves' disease is caused by thyrotropin receptor (TSH-R) autoantibodies. To test the possibility that the TSH-R is the sole antigen for thyroid stimulating antibodies (TSAb), we compared bioactivities of Graves' IgGs between non-thyroid mammalian cells transfected with human TSH-R cDNA and the reference thyroid bioassay. A Graves' IgG with TSH-binding inhibitor immunoglobulin (TBII) activity (89%) markedly stimulated cAMP formation in both CHO-K1 cells transfected with TSH-R cDNA (340 microU/ml of TSH equivalent) and rat thyroid cells, FRTL-5, (410 microU/ml of TSH equivalent). In contrast, a TBII negative (-1.5%) IgG from another patient with Graves' disease showed a strong thyroid stimulating activity (87 microU/ml of TSH equivalent) when FRTL-5 cells were used for the assay. But no stimulating activity was observed in this IgG when CHO-K1 cells transfected with TSH-R cDNA were used, suggesting a possible existence of TSH-R non-mediated thyroid stimulating immunoglobulin in some cases of Graves' disease.     

Detecting TSH-receptor antibodies with the recombinant TBII assay: technical and clinical evaluation.

We evaluated the technical robustness of the new commercial TBII assay using human recombinant TSH-R, and describe its use for the clinician in the routine laboratory. The human recombinant TSH-R assay (DYNOtest TRAK human) was compared to a conventional TBII assay (TSH-REZAK). Specificity was adjusted at 99.1% for both assays by ROC plot analysis including 113 healthy individuals. Sensitivity in 115 patients with active Graves' Disease (GD) was 98.2% for the DYNOtest TRAK human compared to 68.4% for the TSH-REZAK (p<0.0001). Comparison of the ROC-calculated cut off confirmed the recommended cut-off for the DYNOtest TRAK human, since 11% inhibition of tracer equals 1 IU/L, which is recommended as the grey zone. At the recommended cut-off (2 IU/L, 22% inhibition), the sensitivity is still 93.9% with 100% specificity. The ROC plot-derived cut-off of the TSH-REZAK (4.4%, 2 to 10 U/L) is below the grey zone of 10-15 U/L. At the recommended cut off of 15 U/L, the sensitivity is 43.0% with a specificity of 100%. Both assays showed a good correlation (r = 0.82, p < 0.0001); however, assay comparison revealed a constant bias in favour of the DYNOtest TRAK human. Applying the ROC plot-derived cut-off of 11 % inhibition (1 IU/L) for the DYNOtest TRAK human, we found 15 of 50 patients with autoimmune thyroiditis (AIT) and 6 of 23 patients with goitre (all < 1.5 IU/L). These patients would have been missed using the recommended 2 IU/L. The difference in sensitivity between the DYNOtest TRAK human and the TSH-REZAK was highly significant in the GD group, but not in other groups, indicating that the DYNOtest TRAK human has a higher sensitivity for GD without compromising specificity. In summary, the proposed high sensitivity of the new TBII assay using human recombinant TSH-R could be confirmed with the commercial product. This method offers a clear advantage over conventional TBII assays to confirm or exclude the diagnosis of GD. The recommended cut-off is very stringent, and until we have more information on the clinical relevance of low-level TBII between 1 and 1.5 IU/L, those patients should be monitored for the development of autoimmune thyroid disease.     

Exceptional hyperthyroidism and a role for both major histocompatibility class I and class II genes in a murine model of Graves' disease

Autoimmune hyperthyroidism, Graves' disease, can be induced by immunizing susceptible strains of mice with adenovirus encoding the human thyrotropin receptor (TSHR) or its A-subunit. Studies in two small families of recombinant inbred strains showed that susceptibility to developing TSHR antibodies (measured by TSH binding inhibition, TBI) was linked to the MHC region whereas genes on different chromosomes contributed to hyperthyroidism. We have now investigated TSHR antibody production and hyperthyroidism induced by TSHR A-subunit adenovirus immunization of a larger family of strains (26 of the AXB and BXA strains). Analysis of the combined AXB and BXA families provided unexpected insight into several aspects of Graves' disease. First, extreme thyroid hyperplasia and hyperthyroidism in one remarkable strain, BXA13, reflected an inability to generate non-functional TSHR antibodies measured by ELISA. Although neutral TSHR antibodies have been detected in Graves' sera, pathogenic, functional TSHR antibodies in Graves' patients are undetectable by ELISA. Therefore, this strain immunized with A-subunit-adenovirus that generates only functional TSHR antibodies may provide an improved model for studies of induced Graves' disease. Second, our combined analysis of linkage data from this and previous work strengthens the evidence that gene variants in the immunoglobulin heavy chain V region contribute to generating thyroid stimulating antibodies. Third, a broad region that encompasses the MHC region on mouse chromosome 17 is linked to the development of TSHR antibodies (measured by TBI). Most importantly, unlike other strains, TBI linkage in the AXB and BXA families to MHC class I and class II genes provides an explanation for the unresolved class I/class II difference in humans.     

The Diagnostic Utility of Color Doppler Ultrasonography,TC -99 Pertechnetate Uptake,and TSH-Receptor Antibody for Differential Diagnosis of Graves’ Disease and Silent Thyroiditis:A Comparative Study

ObjectiveThe differential diagnosis of Graves disease (GD) and silent thyroiditis (ST) is important for the selection of appropriate treatment. To date, no study has compared the diagnostic utility of color Doppler ultrasonography (CDUSG), Tc-99m (technetium-99m) pertechnetate uptake, and thyroid-stimulating hormone (TSH)-receptor antibody (TRAb) for the differential diagnosis of these two conditions. In the present study, we compared the diagnostic utility of inferior thyroid artery (ITA) peak systolic and end diastolic velocities (PSV and EDV) measured by CDUSG, Tc-99m pertechnetate uptake, and TRAb for differential diagnosis of GD and ST.MethodsA total of 150 subjects with GD, 79 with ST, and 71 healthy euthyroid controls were included in the study. Diagnoses of GD and ST were made according to patient signs and symptoms, physical examination findings, the results of TRAb and Tc-99m pertechnetate uptake, and follow-up findings. All subjects underwent CDUSG for the quantitative measurement of ITA blood-flow velocities.ResultsThe mean ITA-PSV and EDV in patients with GD were significantly higher than in ST patients. In receiver operating characteristic analysis, the sensitivity/ specificity of the 30 and 13.2 cm/s cutoff values of the mean ITA-PSV and EDV for discrimination of GD from ST were 95.3/94.9% and 89.3/88.6%, respectively. The sensitivity/specificity of the 1.0 international unit (IU)/L and 3% cutoff values of the TRAb and Tc-99m pertechnetate uptake analyses were 93.0/91.0% and 90.7/89.9%, respectively.ConclusionThe measurement of ITA-PSV by CDUSG is a useful diagnostic tool and is a complementary method to the TRAb and Tc-99m pertechnetate uptake methods for differential diagnosis of GD and ST. (Endocr Pract. 2014;20:310-319)     

Augmentative effect of polyethylene glycol, polyvinyl alcohol and dextran on thyroid-stimulating antibody stimulated cAMP production in FRTL-5 cells and CHO cells expressing human TSH receptor

Previously we reported the augmentative effect of nonionic hydrophilic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA) and dextran on thyroid-stimulating antibody (TSAb) activity in porcine thyroid cell assays. We examined whether a similar phenomenon occurs in FRTL-5 thyroid cells and CHO cells expressing the human (h) TSH receptor (CHO-hTSHR cells). As with porcine thyroid cells, PEG 22.5% precipitated crude IgG from serum of patients with Graves' disease, significantly increased cAMP production as compared with PEG 12.5% precipitated crude IgG in both FRTL-5 cells and CHO-hTSHR cells. PEG 5% augmented purified-TSAb-IgG-stimulated cAMP production in both cell assays. TSAb activities and positivity by the direct assay using whole serum (0.05 ml) in the presence of 5% PEG in untreated Graves' patients were significantly increased as compared with the absence of 5% PEG. The augmentative effects of PVA 10% or dextran T-70 10% on TSAb-IgG-stimulated cAMP production were also observed in both cell assays. PVA 10% did not augment TSH-stimulated cAMP production in spite of weak augmentation by dextran 10% in both cell assays. Lack of the augmentative effects of PEG 5%, PVA 10% and dextran 10% on cAMP produced by GTPgammaS, forskolin and pituitary adenylate cyclase activating polypeptide was observed in both cell assays. The augmentative effects of these polymers in both cell assays similar to porcine thyroid cells suggest that there is no apparent species specificity among human, porcine and rat thyroid cells as far as TSH receptor linked cAMP production in cell membranes existed.     

Study on the Relationship Between TSHR Gene and Thyroid Diseases

Thyroid stimulating hormone receptor (TSHR) is thought to play a critical role in the pathogenesis of certain thyroid diseases, including Graves' disease (GD), multinodular thyroid goiter (MTG), and Hashimoto's thyroiditis (HT). In order to understand whether single nucleotide polymorphisms in the TSHR gene contribute to thyroid diseases, we have conducted a case-control study in which, we examined 8 TSHR gene single-nucleotide polymorphisms in introns 1, 4, 5, 6 and exons 7 and 8, respectively, among patients with thyroid diseases. These included one family with GD (3 patients and 9 healthy members); 60 patients with familiar thyroid diseases (30 with GD, 20 with MTG, and 10 with HT patients), 48 sporadic patients with GD and 96 healthy control individuals. Direct sequencing of all 10 exons and part of introns of TSHR gene, in these patients as well as healthy controls revealed eight polymorphisms. A novel polymorphism in exon 8 AGA(Arg) → CGA(Arg). However, there were no significant differences between patients and controls in the incidence of these polymorphisms. These results suggest that the polymorphisms (polymorphism in intron 1 at 81 bp upstream of exon 2; polymorphism in intron 4 at 135 bp upstream of exon 5; polymorphism in intron 4 at 365 bp upstream of exon 5; polymorphism in intron 5 at 69 bp upstream of exon 6; means polymorphism in intron 6 at 13 bp downstream of exon 6; polymorphism in intron 6 at 187 bp upstream of exon 7; E7+16: polymorphism in 16 bp of exon 7; polymorphism in 40 bp of exon 8) of the TSHR gene may not contribute to the pathogenesis of thyroid diseases.     

Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1 receptors: a tale of two antigens implicated in Graves' disease

Thyroid-stimulating hormone receptor (TSHR) plays a central role in regulating thyroid function and is targeted by IgGs in Graves' disease (GD-IgG). Whether TSHR is involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the orbital manifestation of GD, remains uncertain. TSHR signaling overlaps with that of insulin-like grow factor 1 receptor (IGF-1R). GD-IgG can activate fibroblasts derived from donors with GD to synthesize T cell chemoattractants and hyaluronan, actions mediated through IGF-1R. In this study, we compare levels of IGF-1R and TSHR on the surfaces of TAO and control orbital fibroblasts and thyrocytes and explore the physical and functional relationship between the two receptors. TSHR levels are 11-fold higher on thyrocytes than on TAO or control fibroblasts. In contrast, IGF-1R levels are 3-fold higher on TAO vs control fibroblasts. In pull-down studies using fibroblasts, thyrocytes, and thyroid tissue, Abs directed specifically against either IGF-1Rbeta or TSHR bring both proteins out of solution. Moreover, IGF-1Rbeta and TSHR colocalize to the perinuclear and cytoplasmic compartments in fibroblasts and thyrocytes by confocal microscopy. Examination of orbital tissue from patients with TAO reveals similar colocalization to cell membranes. Treatment of primary thyrocytes with recombinant human TSH results in rapid ERK phosphorylation which can be blocked by an IGF-1R-blocking mAb. Our findings suggest that IGF-1R might mediate some TSH-provoked signaling. Furthermore, they indicate that TSHR levels on orbital fibroblasts are considerably lower than those on thyrocytes and that this receptor associates with IGF-1R in situ and together may comprise a functional complex in thyroid and orbital tissue.     

弥漫性毒性甲状腺肿患者血清高半胱氨酸蛋白61及Fractalkine水平的表达及其临床意义

摘要 目的：探讨弥漫性毒性甲状腺肿（GD）患者血清高半胱氨酸蛋白61（CYR61）、Fractalkine水平的表达及其临床意义。方法：选取2018年3月～2021年10月河北省邯郸市中心医院收治的57例GD患者作为研究组。另取同期健康体检者50例。采集所有受试者的静脉血,检测血清CYR61、Fractalkine水平,甲状腺功能及甲状腺自身抗体相关指标。采用Pearson检验分析GD患者血清CYR61、Fractalkine水平与甲状腺功能及甲状腺自身抗体相关指标的相关性。结果：研究组血清CYR61、Fractalkine水平均高于对照组,差异均有统计学意义（均P＜0.05）。研究组血清游离三碘甲腺原氨酸（FT3）、游离四碘甲腺原氨酸（FT4）均高于对照组,而促甲状腺激素（TSH）水平低于对照组,差异均有统计学意义（均P＜0.05）。研究组抗甲状腺球蛋白抗体（TGAb）、甲状腺过氧化物酶抗体（TPOAb）及促甲状腺激素受体抗体（TRAb）水平均高于对照组,差异均有统计学意义（均P＜0.05）。经Pearson相关性分析发现,GD患者血清CYR61、Fractalkine水平与FT3、FT4、TGAb、TPOAb、TRAb水平均呈正相关,而与血清TSH水平呈负相关（均P＜0.05）。结论：GD患者血清CYR61、Fractalkine水平异常高表达,且与患者甲状腺功能及甲状腺自身抗体有关。     

A case of Graves' disease with false hyperthyrotropinemia who developed silent thyroiditis.

We encountered a patient who developed silent thyroiditis during the course of Graves' disease. The diagnosis of silent thyroiditis was made on the basis of a low thyroidal 131I uptake, no response to the thyrotropin releasing hormone (TRH) test, and subsequent hypothyroidism despite the presence of high titers of thyrotropin (TSH) receptor antibody (TRAb) and thyroid stimulating antibody (TSAb). The patient, in addition, had a discrepancy between serum TSH and thyroid hormone values. This was due to the presence of interfering substances that react to mouse IgG in the sera since serum TSH levels were decreased in a dose dependent manner by the addition of increasing amounts of mouse IgG to the sera. It should therefore be noted that silent thyroiditis can develop in patients with Graves' disease. Furthermore, clinicians should be aware that two-site immunoassay kits that use mouse monoclonal antibodies are subject to interference by some substances, possibly antibodies which react to mouse IgG.     

Similar triiodothyronine releasing activity of thyroid stimulating antibodies in human and porcine thyroid slices

In an approach to addressing species specificity of thyroid stimulating antibodies (TSAb) stimulation of T3 release by Graves' sera was comparatively studied in human and porcine thyroid slices. A high sensitivity and specificity was found for the T3 bioassay independently on the use of human or porcine thyroid. Moreover, activity indices of the individual sera in both tissues were significantly correlated to each other and to circulating hormone levels in untreated disease. In conclusion, we suppose a lack of functionally relevant differences between target antigens, brought about probably by the TSH receptor itself and other membrane components, in human and porcine thyroid. Thus, for clinically applicable T3 releasing bioassay porcine thyroid may be alternatively used. In addition, this bioassay renders the advantage of reflecting the activity of disease.