Tocilizumab improves clinical outcome in patients with active corticosteroid-resistant moderate-to-severe Graves' orbitopathy: an observational study.

Background: Graves' orbitopathy (GO) is an autoimmune disorder affecting the orbital fat and muscles. A significant role of IL-6 in the pathogenesis of GO has been described and tocilizumab (TCZ), an IL-6 inhibitor targeting IL-6R has been given in some patients. The aim of our case study was to evaluate the therapeutic outcome of TCZ in non-responders to first line treatments with corticosteroids. Methods: We conducted an observational study of patients with moderate to severe GO. Twelve patients received TCZ in intravenous infusions at a dose of 8mg/kg every 28 days for 4 months and followed up for additionally 6 weeks. The primary outcome was improvement in CAS by at least 2 points, 6 weeks after the last dose of TCZ. Secondary outcomes included CAS <3 (inactive disease) 6 weeks after TCZ last dose, reduced TSI levels, proptosis reduction by > 2mm and diplopia response. Results: The primary outcome, was achieved in all patients 6 weeks after treatment course. Furthermore all patients had inactive disease 6 weeks after treatment cessation. Treatment with TCZ reduced significantly median CAS by 3 units (p=0.002), TSI levels by 11.02 IU/L (p=0.006), Hertel score on the right eye by 2.3 mm (p=0.003), Hertel score on the left eye by 1.6 mm (p=0.002), while diplopia persisted in fewer patients (25%) after treatment with TCZ (not statistically significant, p=0.250). After treatment with TCZ, there was a radiological improvement in 75% of patients, while 16.7% showed no response, and in 8.3% of patients deterioration was established. Conclusion: Tocilizumab appears to be a safe and cost effective therapeutic option for patients with active, corticosteroid-resistant, moderate to severe Graves' orbitopathy.

Characterizing the Interplay of Lymphocytes in Graves' Disease.

Graves' disease (GD) is a thyroid-specific autoimmune disease with a high prevalence worldwide. The disease is primarily mediated by B cells, which produce autoantibodies against the thyroid-stimulating hormone receptor (TSHR), chronically stimulating it and leading to high levels of thyroid hormones in the body. Interest in characterizing the immune response in GD has motivated many phenotyping studies. The immunophenotype of the cells involved and the interplay between them and their secreted factors are crucial to understanding disease progression and future treatment options. T cell populations are markedly distinct, including increased levels of Th17 and follicular helper T cells (Tfh), while Treg cells appear to be impaired. Some B cells subsets are autoreactive, and anti-TSHR antibodies are the key disease-causing outcome of this interplay. Though some consensus across phenotyping studies will be discussed here, there are also complexities that are yet to be resolved. A better understanding of the immunophenotype of Graves' disease can lead to improved treatment strategies and novel drug targets.

TSAb Modulates Microglia M1 Polarization via Activation of the TSHR-Mediated NF-κB Signaling Pathway.

INTRODUCTION: Thyrotropin receptor-stimulating antibody (TSAb) is a pathogenic antibody in the serum of patients with Graves' disease. The binding of TSAb to thyroid-stimulating hormone receptor (TSHR) in non-thyroid tissue may be associated with the occurrence and development of Graves' disease-related complications. However, only few studies have been conducted on the effects of TSAb on the brain, and the pathogenesis of acute hyperthyroidism myopathy (ATM) is unclear. Therefore, this study aimed to explore the effect of TSAb on the polarization of BV-2 cells in the brain and its possible mechanism and provide a basic experimental basis for ATM. METHODS: BV-2 cells were treated with different concentrations of TSAb. The relative survival rate of BV-2 cells was determined using the CCK-8 assay; the migration ability of BV-2 cells was detected using the Transwell migration assay; and the expression levels of M1/M2 polarization markers (CD86, inducible nitric oxide synthase [iNOS], CD206, and arginase 1 [Arg-1]), TSHR, tumor necrosis factor-alpha (TNF-α), and nuclear factor-kappa B (NF-κB) protein in BV-2 cells were measured using WB. RESULTS: Compared with the negative control group, the proliferative activity of BV-2 cells was significantly increased in the 20, 50, and 100 ng/mL TSAb groups, and the migration ability of BV-2 cells was significantly enhanced in the 50 and 100 ng/mL TSAb groups. The expression levels of M1 polarization markers (CD86 and iNOS), TSHR, TNF-α, and NF-κB protein in BV-2 cells treated with 50 and 100 ng/mL TSAb for 24 h were significantly upregulated, whereas those of M2 polarization markers (CD206 and Arg-1) significantly decreased. CONCLUSIONS: TSAb can induce abnormal activation of microglia, polarize to the M1 phenotype, and promote the inflammatory cascade reaction, in which TSHR plays a key role in NF-κB activation and proinflammatory cytokine release.

Consistency Between Thyrotropin Receptor Antibody (TRAb) and Thyroid-Stimulating Antibody (TSAb) Levels in Patients with Graves Disease.

OBJECTIVE: To investigate the consistency between thyrotropin receptor antibody (TRAb) and thyroid-stimulating antibody (TSAb) levels in patients with Graves disease (GD). METHODS: We performed a cross-sectional observational study to recruit eligible patients with GD who visited the outpatient endocrinology clinic for the purpose of evaluating the consistency between their TRAb and TSAb levels. Our cohort included 28 men and 99 women. RESULTS: The median levels of TRAb and TSAb were 5.65 IU/L and 3.76 IU/L, respectively, in the enrolled patients with GD. The levels of TRAb (5.03 vs 8.42 IU/L; P = .008) and TSAb (2.69 vs 5.37 IU/L; P = .008) in patients with adequate thyroid regulation were all lower than those in patients with inadequate thyroid regulation. CONCLUSIONS: Although TRAb is closely related to TSAb, we observed high heterogeneity of TRAb due to relatively low consistency between the levels of the 2 antibodies.

The role of thyroid-stimulating hormone and thyroglobulin antibody in abnormally invasive placenta.

PURPOSE: Thyroid hormones and antibodies are known to participate in angiogenesis and invasion and also thyroid hormone receptors are expressed in the placenta. We aimed to evaluate the relationship of serum levels of thyroid-stimulating hormone (TSH), thyroid hormones (TH), and anti-thyroid antibodies with abnormally invasive placenta (AIP). We also aimed to investigate whether they are related with cesarean hysterectomy and massive blood transfusion need in AIP cases. METHODS: A total of 88 pregnant patients were enrolled in this prospective case-control study (30 with AIP, 28 with non-adherent placenta previa totalis (PPT) and 30 controls). Serum TSH, thyroid hormone [T3 (triiodothyronine) and T4 (thyroxine)] and thyroid antibodies against thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) levels were studied in maternal serum at initial admission to our Perinatology Unit (at early third trimester). The factors associated with increased risk of AIP, cesarean hysterectomy, massive blood transfusion, and adverse perinatal outcomes were evaluated with multiple logistic regression analysis. Adjusted odds ratios and 95% confidence intervals were also calculated. RESULTS: Serum TSH and TgAb levels were significantly lower in the AIP group than both PPT and control groups (p = .01, p < .001 and p < .001, p < .001 respectively). Decreased serum levels of TSH (<2.16 mIU/L) and TgAb (<2.70 mIU/L) levels and high previous cesarean section rates were found to be independently associated with AIP in pregnant women with PPT (OR: 0.4, 95% CI: 0.1-0.9; p = .04, OR: 0.7, 95%CI: 0.4-1.3, p = .02 and OR: 0.1, 95% CI: 0.1-0.5, p = .01). Decreased serum TSH and TgAb levels were found to be independently associated with an increased rate of cesarean hysterectomy and massive blood transfusion in AIP cases (OR: 3.7, 95% CI: 1.4-9.8; p = .01, OR: 1.8, 95% CI: 1.1-3.1; p = .03 and OR: 2.6, 95% CI: 1.0-6.5; p = .05, OR: 2.2, 95% CI: 1.1-4.1 p = .02). Decreased TSH and TgAb serum levels were also found to be independently associated with adverse perinatal outcomes in AIP cases (OR: 3.4, 95% CI: 1.3-11.0; p = .01 and OR: 1.978, 95% CI: 2-3.6; p = .03). CONCLUSION: Decreased serum TSH and TgAb levels, and previous history of cesarean section were all found to be significantly associated with AIP in cases with PPT. We suggest that maternal serum TSH and TgAb levels can provide additional contribution to obstetric Doppler ultrasound in the diagnosis of AIP and thus can reduce the risks of unplanned cesarean hysterectomy in cases with PPT.

Sight-Threatening Graves' Ophthalmopathy during the Third Trimester of Pregnancy: A Case Report.

Dysthyroid optic neuropathy is a severe manifestation of Graves' ophthalmopathy that can result in permanent vision loss. We report a 37-year-old pregnant woman with Graves' ophthalmopathy which was deteriorated to dysthyroid optic neuropathy in the third trimester of pregnancy. Diplopia, bilateral eye lid retraction, lid edema and proptosis were observed in the 29th week of gestation. Thyroid-stimulating hormone (TSH) level was decreased with a normal level of free triiodothyronine (FT3) and an upper normal level of free thyroxine (FT4). Anti-TSH receptor antibodies (16.2 IU/L, reference range < 2.0 IU/L) and thyroid stimulating antibody (4,443%, reference range < 120%) were positive. Magnetic resonance imaging (MRI) demonstrated a significant enlargement of the extraocular muscles with a high signal intensity on T2-weighted image. She was diagnosed as Graves' ophthalmopathy and subclinical hyperthyroidism, and followed without treatment. In the 34th week of gestation, the symptom of color vision abnormality appeared, suggesting dysthyroid optic neuropathy. She delivered a female infant during the 36th week of gestation. Four days after delivery, she had a spontaneous orbital pain. MRI showed that the extraocular muscles were more enlarged than the findings in the 29th week of gestation. FT3 and FT4 levels were mildly elevated. Dysthyroid optic neuropathy was diagnosed. She was treated with methylprednisolone pulse therapy and retrobulbar injections of betamethasone valerate, and the ocular symptoms improved. The present case shows that the glucocorticoid therapy performed one week after delivery is effective against Graves' ophthalmopathy which was deteriorated to dysthyroid optic neuropathy during the third trimester of pregnancy.

Analytical performance evaluation of thyroid-stimulating hormone receptor antibody (TRAb) immunoassays.

BACKGROUND: Thyroid-stimulating hormone receptor (TSHR)-activating autoantibodies stimulate thyroid growth and hormone synthesis/secretion, causing hyperthyroidism of Graves' disease (GD). TRAb measurement helps diagnose GD and is an important first test in evaluating hyperthyroidism according to the recent American Thyroid Association guidelines. We compared the performance of the BRAHMS TRAK Kryptor (Thermo Scientific) and Roche cobas TRAb immunoassays for use in GD. METHOD: Method comparison (n = 40) and clinical agreement were assessed between the Kryptor, cobas e411, and cobas e601. The analytical performance of Kryptor and cobas e411 were assessed for within- and between-day imprecision across 20 days, linearity, functional assay sensitivity (FAS), dilution recovery, and cut-off verification. RESULTS: The Kryptor, e411, and e601 TRAb immunoassays correlated well (r > 0.95, overall percent agreement = 0.95, Cohen's kappa = 0.90). With a total allowable error of 20%, percent bias was within 13%, which was minimally negative at <20 IU/L, but highly positive (33%-34%) >20 IU/L. The Kryptor, but not e411, was linear across the claimed analytical measuring range (AMR). The claimed functional assay sensitivity (FAS), which was close to the clinical GD cut-off 1.8 IU/L, was verified for Kryptor and e411. CONCLUSION: Overall, our evaluation demonstrates acceptable comparability between TRAb immunoassays with in-house imprecision up to 13% and 10% on Kryptor and e411, respectively. While Roche has preferable calibration frequency and on-board reagent stability, both platforms demonstrate acceptable imprecision using patient samples at their claimed FAS, which is important for GD diagnosis. Diluted results (using a negative patient pool as diluent) exhibits proportional positive bias on the Kryptor relative to the Roche methods.

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.

Analytical and clinical performance of newly developed immunoassay for detecting thyroid-stimulating immunoglobulin, the Immulite TSI assay.

Graves' disease (GD) is caused by autoantibodies against the thyrotropin receptor (TRAb), and among the three types of TRAbs, only the stimulating type (TSI) is known to be associated with GD. In this study, we evaluated the analytical performance of a new fully automated chemiluminescent TSI immunoassay, namely, the Immulite TSI assay, and compared the diagnostic efficacy of the assay with the Elecsys Anti-TSH receptor (TSHR) assay. Precision was evaluated using two levels of quality control reagents, and linearity was evaluated across the expected analytical measurement range (0.18-37.35 IU/L) at five levels using clinical samples. A comparative evaluation between the two assays was performed using 187 clinical samples, and the concordance of qualitative results was also assessed. The repeatability and total imprecision (% coefficient of variation) of the Immulite TSI assay were 3.19% and 3.46% at 0.93 IU/L, and 3.76% and 5.42% at 19.3 IU/L, respectively. The linearity of this assay ranged from 0.16 to 6.17 IU/L. A high degree of correlation was observed between quantitative values from each assay (correlation coefficient = 0.819). Moderate agreement between methods was observed with an overall qualitative agreement of 93.0%. Among 13 cases with discordant qualitative results, the Immulite TSI assay generated more favorable results consistent with clinical diagnoses of patients than the Elecsys Anti-TSHR assay. The Immulite TSI assay showed reliable analytical performance and good correlation with the Elecsys Anti-TSHR assay and we expect this method will be helpful for clinicians to evaluate patients with hyperthyroidism.

Possible participation of colloid antigen 2 and abhormone (IgG with hormone activity) for the etiology of Graves' disease.

The theory that antibody (Ab) directed against the TSH receptor (TSHR) (TSHRAb) is the causal factor of Graves' disease seems unlikely. Corticosteroids have not had a curative effect on the hyperthyroidism of Graves' disease despite their effectiveness for other autoimmune diseases. Two kinds of TSHRAb, thyroid-stimulating Ab (TSAb) and thyroid-blocking Ab (TBAb), are known as causal factors of hyperthyroidism and hypothyroidism, respectively. Previously, we reported that TSAb may be thyroid stimulating animal IgG-like hormone and TBAb may be the precursor of TSAb. In this paper we suggested that TBAb (precursor) converts to TSAb (active form) via the action of the protease, colloid antigen 2 (CA2). We speculate that the conversion of TBAb to TSAb is controlled by two factors: the protease and an anti-protease Ab. When anti-protease Ab levels are high, the patient exhibits hypothyroidism due to the increase in TBAb levels caused by neutralization of the protease. When anti-protease Ab levels are negative, the patient's hypothyroidism disappeared by the negative serum TBAb due to increased protease. An immunoglobulin G (IgG) with enzyme activity is known as an abzyme, which may be an undeveloped form. IgG with hormone activity may be likewise called an abhormone, which could also be an undeveloped form. The tumor marker CEA is a known member of the IgG supergene family. Many ancestral versions of proteins may have been produced as an IgG form. Possible participation of colloid antigen 2 and abhormone for the etiology of Graves' disease is suggested.

Antithyroid Drugs Inactivate TSH Binding to the TSH Receptor by their Reducing Action.

Backgroud and Objective: Antithyroid drugs (ATDs) [methylmercaptoimidazole (MMI) and propylthiouracil (PTU) ] are used to treat hyperthyroidism in Graves' disease. The effect of ATDs and reducing agents (mercaptoethanol, dithiothreitol and cysteine) on bovine (b) TSH binding to human (h) and porcine (p) TSH receptor (R) was examined. METHODS AND RESULTS: (1) ATDs was pre-incubated with hTSHR coated tube for 1- 4 h, washed free of ATDs, and then 125I-bTSH binding to hTSHR after 1 h incubation was examined. MMI (10-40 mM) decreased 125I-bTSH binding in a dose-dependent manner and binding decreased proportionally as preincubation time increased from 1 to 4 h. PTU (10mM) slightly decreased binding, When reducing agents were pre-incubated with hTSHR for 2 h, 125I-bTSH binding similarly decreased. (2) Porcine thyroid membrane was pre-incubated with both agents for 2 h. Then, the washed or unwashed membrane was incubated with 125I-bTSH for 1 h. 125I-bTSH binding in both methods decreased. (3) When the effect of ATDs or reducing agents on the biological activity of 125I-bTSH and thyroid stimulating antibody (TSAb) was examined after gel-filtration of 125I-bTSH- and TSAb- treated with both reagents for 1 h, no inactivation was observed. (4) ATDs showed similar reducing action as reducing agents because iodine (I+) was reduced to I- by ATDs. CONCLUSION: ATDs inactivate the TSH-binding site of TSHR by reduction, although ATDs do not inactivate bTSH and TSAb activity. This suggests that TSAb would not stimulate the thyroid due to the inactivation of the TSHR when ATDs are administered to patients with Graves' disease.

Changes of thyroid stimulating antibody and thyroid stimulating blocking antibody in treatment of anti-thyroid drugs and combining with levothyrocine to hyperthyroidism / 中华内分泌代谢杂志

Objective Changes of thyroid stimulating antibody(TSAb) and thyroid stimulating blocking antibody(TSBAb) in the treatment of anti-thyroid drugs(ATDs), and the effect of ATDs combining with levothyrocine(LT4) on TSAb and TSBAb were analyzed. Methods Using recombinant Trxfus. TSHRn protein and Trxfus. TSHRc protein as antigens, and TSH receptor antibody(TRAb)-N(TSAb binding hot spots), TRAb-C(TSBAb binding hot spots)in the serum of thyroid disease patients were measured with ELISA. The changes of TRAb-N, TRAb-C over 36 months in 117 TRAb-N positive Graves′ patients with hyperthyroidism were analyzed retrospectively. In the course of treatment, 41 cases as A group with ATDs and LT4 treatment, 76 cases as B group with only ATDs, The changes of TRAb-N and TRAb-C were observed in the two groups. Results (1)According to the change of TRAb-N, 117 TRAb-N positive Graves′ patients with hyperthyroidism were different. In group Ⅰ, 10 patients continued to have persistently positive TSAb and continued to have hyperthyroidism, remission rate 0%. In group Ⅱ, 17 patients showed complicated TRAb-N changes, 12 of 17 patients got relapse, 5 of 17 patients got remission, remission rate 29.4%. And in group Ⅲ, with TRAb-N dropping gradually, 15 of 89 patients got relapse, 74 of the 89 patients got remission, remission rate 83.1%. Three groups were significantly different with x2 test(P0.05). Conclusion TSAb and TSBAb can be used to document TRAb-function, which is significant for us to predict the changes of thyroid function. During ATDs treatment, the temporary early low-dose application of LT4 did not significantly affect TSAb and TSBAb.

Possible role of autoimmunity in patients with premature ovarian insufficiency.

BACKGROUND: To evaluate the involvement of immune abnormality in patients with idiopathic premature ovarian insufficiency (POI). In addition to the known etiology, autoimmune disorders may be a pathologic mechanism for POI. MATERIALS AND METHODS: Our study was a prospective controlled trial. Twenty women with POI, reasons other than autoimmune excluded, were enrolled in this study. The control group consisted of 17 healthy women. In both groups, family and personal history were taken and the levels of follicle stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, prolactin, anti-Müllerian hormone, inhibin B, antithyroglobulin and antithyroid peroxidase antibodies were determined. Antiovarian antibodies and subpopulations of peripheral blood T-lymhocytes were also determined. RESULTS: Participants in the study group exhibited hypergonadotropichypogonadism, while high levels of follicle stimulating hormone and low levels of inhibin B and anti-Müllerian hormone were observed. In 16 (80%) patients, POI was associated in their personal and familial history with another autoimmune disease. Fifty percent of patients presented highly elevated antithyroid antibodies. The lymphocyte subset, especially B cells, was significantly higher (p=0.014), and peripheral regulatory lymphocytes CD25+ high were significantly lower (p=0.015) in the study group than in the control group. Anti- ovarian antibodies were detected in 20% of patients with POI. CONCLUSION: We presume that the presence of anti-ovarian antibodies together with abnormalities of cellular immunity may in some cases potentially represent the involvement of an autoimmune mechanism in idiopathic POI.

Related factors in predicting relapse of Graves' disease after treatment with antithyroid drugs / 中华内分泌代谢杂志

Objective To explore the value of thyroid-stimulating antibody(TSAb) and degree of goiter in predicting the outcome of Graves'disease after antithyroid drug treatment. Methods Seventy-one patients with Graves'disease were given antithyroid drugs for (2. 8±1. 4)years and then followed up for(22±6.0)months.Finally,age,gender,thyroid function,TSAb and goiter size at the time of drug withdrawal were compared between the relapsed and relieved groups. TSAb was measured in all patients by using HEK-hTSHR cells. Results Eleven of 71 patients relapsed during the follow-up after drug withdrawal. The relapse rate (42. 9% ,6/14)in patients with positive TSAb was significantly higher than that (8.8%, 5/57) in patient with negative TSAb (X2 = 9.97, P<0.01). The relapse rates in patients with normal size thyroid, Ⅰ degree goiter,Ⅱ degree goiter were 6.25%, 12.2%,35.7% respectively. TSAb activity, positive rate and goiter size of the relapsed patients at the time of drug withdrawal were significantly higher than those of relieved patients (P<0.05 or P<0. 01). Conclusion TSAb activity and goiter size at the time of drug withdrawal are two effective prognostic markers of relapse in Graves' disease treated with antithyroid drugs.

Correlation between the Thyroid Associated Ophthalmopathy and Thyroid Function State

PURPOSE: To determine the prevalence of thyroid-associated ophthalmopathy (TAO) and the correlation between TAO and the thyroid function state. METHODS: 40 patients with TAO were selected among 2,000 patients of thyroid disease between September 1, 1995 and December 31, 2000, and classified by the Van Dyk's ''NOSPECS and RELIEF'' classification. T3, T4, TSH and Thyroid stimulating antibody (TSAb) levels were examined before and after treatment of TAO and thyroid disease. Relationship between TAO and thyroid function state was evaluated by using paired t-test and logistic regression test. RESULTS: The prevalence of TAO was about 2% in thyroid disease patients. Clinical manifestations of TAO were eyelid retraction (75%), exophthalmos (62.5%), soft tissue periorbital swelling (42.5%), diplopia and extraocular muscle restriction (20%), keratopathy (2.5%) and optic neuropathy (2.5%). Thyroid function state decided by T3, T4 and TSH level had no correlation with the improvement of TAO (p<0.05). But, TSAb level in improved TAO group (n=8) significantly decreased after treatment of TAO and thyroid disease (p<0.05). CONCLUSIONS: Thyroid function restoration may not help to improve the course of TAO. But, low TSAb level can be thought as a significant marker in improvement of TAO.

The signal pathways involved in the effects of thyroid stimulating antibody on the secretory function of thyrocytes / 中国实用内科杂志

Objective To investigate the signal pathways involved in the effects of thyroid stimulating antibody(TSAb)on the secretory function of cultured thyrocytes.Methods (1)With the method of enzyme-linked immunosorbent assay (ELISA),the PKA and PKC activities were observed.(2)Inhibitors or activators of protein kinases (PKA and PKC) were used to activate or block the signal pathways and the secretion of T3 measured by radioimmunoassay.Results (1)TSAb activated both PKA and PKC in thyrocytes in a dose-,time-dependent manner(.P.

A Case of Neonatal Graveses Disease / 대한소아내분비학회지

Neonatal Gaves disease is a relatively rare condition due to transplacental passage of Thyroid-stimulating antibody(TSAb) from a mother with active or inactive Graveses disease or autoimmune thyroiditis. A 11-day-old female newborn was referred to our department of pediatrics from a local clinic because of low level T4(3.55microg/dl) concurrent with high level TSH (501.74uIU/ml) on the 5th day neonatal metabolic screening. But, our repeated laboratory data showed very high serum T4(59.6microg/dl), T3(1,600ng/dl), suppressed TSH(0.43uIU/ml), and the presence of TSH receptor antibody. Her mother was treated with propylthiouracil(PTU) for Graves disease during pregnancy. Therefore, we thought it was a delayed-onset neonatal hyperthyroidism, because the fetal thyroid gland was initially suppressed by antithyroid drug taken during pregnancy. After initiating antithyroid drug therapy for the hyperthyroid nature, TSH levels became elevated again, while thyroid hormone levels decreased. Maternal and infant blood samples at the 23th day after birth were examined for serum autoantibodies directed towards the TSH receptor(Thyrotropin-binding inhibitory immunoglobulin:TBII, Thyroid-stimulating antibody:TSAb, Thyroid-stimulating blocking antibody:TSBAb) and high levels of TBII and TSAb were detected. About 2 months after birth, TBII and TSAb decreased within normal limit, and then we could stop antithyroid medication in safety. We report here a case of neonatal Graveses disease with very high level of T4 and T3, but firstly presented as hypothyroid nature on neonatal screening because of the maternally transferred antithyroid drug, PTU.

A Case of Neonatal Hyperthyroidism with Unilateral Ear Anomaly

Neonatal hyperthyroidism is a very rare disorder occurring typically in the offspring of patients with Graves' disease or chronic thyroiditis. It is caused by the transplacental passage of thyroid stimulating antibodies (TSAb) from the mother to the fetus. There has been few reports of neonatal hyperthyroidism associated with congenital anomalies. We experienced a case of neonatal hyperthyroidism with unilateral microtia and agenesis of external auditory canal in a female neonate born to a mother who had euthyroid but with a thyroid stimulating antibody. The patient was presented with unusual alertness, tachycardia, tachypnea, watery diarrhea, periorbital swelling and exophthalmos. Diagnosis was made by thyroid function tests and TSAb. She was treated with Lugol solution, PTU and propranolol. New she is 6 months old and in good condition with no symptoms of hyperthyroidism.

Difference of Thyroid Stimulating Antidody Activities Measured in Chinese Hamster Ovary Cells Stably Transfected with Human TSH Receptor and in FRTL-5 Cells in Graves Disease and Its Clinical Correlations / 대한내분비학회지

Background : Thyroid stimulating antibodies result in the development of hyperthyroidism and goiter in Graves disease. However, thyroid stimulating antibody activities do not correlate with the clinical features in many patients with Graves disease. The purpose of this study is to address this discrepancy between thyroid stimulating antibody activities and clinical features of Graves patients. Methods: We measured thyroid stimulating antibody activities simultaneously using human TSH receptor transfected Chinese hamster(hTSHR-CHO) cells and rat thyroid(FRTL-5) cells in 57 untreated patients with Graves disease, and compared their activities with clinical features including thyroid hormone levels. Results : The detection rate of thyroid stimulating antibody measured by hTSHR-CHO cells was 90% in 57 untreated Graves patients and it was higher than that measured by FRTL-5 cells. Thyroid stimulating antibody activity by hTSHR-CHO cells was significantly correlated with that by FRTL-5 cells(r=0.5, p<0.001), however, 18 of 57(32%) patients showed marked discrepancy of thyroid stimulating antibody activity between in hTSHR-CHO and FRTL-5 systems. Thyroid stimulating antibody activity measured by hTSHR-CHO cells was significantly correlated with serum total T3, free T4 levels, and goiter size but not 99mTc-thyroid uptake. On the other hand, thyroid stimulating antibody activity measured by FRTL-5 cells was significantly correlated with goiter size and 99mTc-thyroid uptake but not thyroid hormone levels. The difference between function and goiter size with respect to thyroid stimulating antibody measurement in two cells system is, nevertheless, particularly evident in the free T4/goiter ratio in patients with high hTSHR-CHO and low FRTL-5 cell assay values. Conclusion: These findings suggest that thyroid stimulating antibodies in Graves disease are heterogeneous population in terms of responses to different origin of cells. Further, thyroid stimulating antibody activities measured by FRTL-5 cells tend to correlate better with goiter size and Tc-thyroid uptake, whereas thyroid stimulating antibody activities measured by hTSH-CHO cells correlate better with thyroid hormone levels.

Thyroid Stimulating Antibody Assay with Chinese Hamster Ovary Cells Expressing Human Thyroid Stimulating Hormone (TSH) Receptor; Optimization of Assay Condition / 대한내분비학회지

We investigated the optimal condition of thyroid stimulating antibody(TSAb) assay using Chinese hamster ovary cells transfected with cDNA of human TSH receptor(TSHr-CHO) stably expressing functional TSH receptors. The extracellular cAMP responses of TSHr-CHO cells to the stimulation of bTSH or Graves' IgG were observed in three different incubation media. Stimulation indices of extracellular cAMP were higher when sucrose containing NaCl-free isotonic Hank's balanced salt solution(HBSS)(media A)was used as incubation media than those of NaCl-free hypotonic HBSS(media B) or those of NaCl containing isotonic HBSS(media C). The incubation of TSHr-CHO cells in media B caused marked increase in the basal cAMP level without concomittant fold-increase in the stimulated cAMP level at various doses of bTSH and Graves' IgG. Decreasing the stimulation indices of extracellular cAMP, use of media B failed to detect TSAb activities in two TSAb-positive Graves' IgG tested. In case of media C, extracellular cAMP responses are poor at 0.001 and 0.1U/L of bTSH and at all doses of Graves' IgG tested(0.5, 1, 5g/L). The incubation of TSHr-CHO cells in media B caused significant increase in the number of trypan blue-stained, nonviable cells(5.7+-1.5, 7.6+-1.9 and 8.5+-1.6% at 1, 2 and 3h of incubation, respectively; p<0.01) comparing to those incubated in media A or media C(about 2-3% in both media). Those decrease in the viability of TSHr-CHO cells when incubated in hypotonic incubation media may explain the decrease in the stimulation index of extracellular cAMP with the use of media B in contrast to the case of FRTL-5 cells. TSAb assay of 87 consecutive fresh Graves' patients with TSHr-CHO cells using media A detected TSAb activities in 90%(78 patients) of them, and moreover TSAb activities showed significant positive correlation with the pre-treatment serum T_3 and free T_4 levels of those patients. We conclude that TSAb assay with TSHr-CHO cells is a sensitive and physiologically relevant assay system to measure TSAb activities merely through measurements of extracellular cAMP provided that the cells are incubated in NaCl-free isotonic incubation media.