Thyroid Stimulation Blocking Antibody Research Articles

Immunotherapy with a biologically active ICAM-1 mAb and an siRNA targeting TSHR in a BALB/c mouse model of Graves' disease.

The objective was to study targeted therapies using a biologically active monoclonal antibody against intracellular adhesion molecule-1 (ICAM-1 mAb) and an siRNA targeting thyroid-stimulating hormone (TSH) receptor (TSHR) in a BALB/c mouse model of Graves' disease (GD). An improved method for establishing a stable model of GD in BALB/c mice was developed by immunization with pcDNA 3.1/TSHR 289 and electroporation (EP). The mice in which GD was successfully established were divided into a nontreated control group, which was treated with continuous immunization, and treated groups, which were treated with the siRNA and ICAM-1 mAb. Normal mice were included as a blank group. These groups were used to compare the effects of treatment with the ICAM-1 mAb and siRNA. The two novel treatments markedly improved weight loss, serum thyroxine (T4) levels, thyroid-stimulating hormone antibody (TSAb) levels, thyroid-stimulating blocking antibody (TSBAb) levels and thyroid uptake of 99mTcO4 in GD model mice. Compared with the siRNA treatment, treatment with the ICAM-1 mAb produced more obvious benefits. The differences in the posttreatment indexes between the two treatment groups were statistically significant (p < 0.05). These preliminary data suggest that both the biologically active ICAM-1 mAb and the siRNA targeting TSHR were effective. The ICAM-1 mAb exerted a better therapeutic effect than the siRNA targeting TSHR. Both treatments showed potential efficacy as novel treatments for GD and may therefore represent therapeutic options in addition to the existing drugs or interventions.

Plummer's Nails (Onycholysis) in a Thyroid-stimulation-blocking Antibody (TSBAb)-positive Patient with Hypothyroidism.

Plummer's Nails (Onycholysis) in a Thyroid-stimulation-blocking Antibody (TSBAb)-positive Patient with Hypothyroidism.

Changes of thyroid stimulating antibody and thyroid stimulating blocking antibody in treatment of anti-thyroid drugs and combining with levothyrocine to hyperthyroidism

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 χ2 test(P 0.05). 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. (Chin J Endocrinol Metab, 2017, 33: 662-667) Key words: Anti-thyroid drugs; Levothyrocine; Thyroid stimulating antibody; Thyroid stimulating blocking antibody

The Presence of Thyroid-Stimulation Blocking Antibody Prevents High Bone Turnover in Untreated Premenopausal Patients with Graves’ Disease

The Presence of Thyroid-Stimulation Blocking Antibody Prevents High Bone Turnover in Untreated Premenopausal Patients with Graves’ Disease

The Presence of Thyroid-Stimulation Blocking Antibody Prevents High Bone Turnover in Untreated Premenopausal Patients with Graves’ Disease

Osteoporosis-related fractures are one of the complications of Graves’ disease. This study hypothesized that the different actions of thyroid-stimulating hormone receptor (TSHR) antibodies, both stimulating and blocking activities in Graves’ disease patients might oppositely impact bone turnover. Newly diagnosed premenopausal Graves’ disease patients were enrolled (n = 93) and divided into two groups: patients with TSHR antibodies with thyroid-stimulating activity (stimulating activity group, n = 83) and patients with TSHR antibodies with thyroid-stimulating activity combined with blocking activity (blocking activity group, n = 10). From the stimulating activity group, patients who had matched values for free T4 and TSH binding inhibitor immunoglobulin (TBII) to the blocking activity group were further classified as stimulating activity-matched control (n = 11). Bone turnover markers BS-ALP, Osteocalcin, and C-telopeptide were significantly lower in the blocking activity group than in the stimulating activity or stimulating activity-matched control groups. The TBII level showed positive correlations with BS-ALP and osteocalcin levels in the stimulating activity group, while it had a negative correlation with the osteocalcin level in the blocking activity group. In conclusion, the activation of TSHR antibody-activated TSH signaling contributes to high bone turnover, independent of the actions of thyroid hormone, and thyroid-stimulation blocking antibody has protective effects against bone metabolism in Graves’ disease.

Growth stimulating antibody, as another predisposing factor of Graves’ disease (GD): analysis using monoclonal TSH receptor antibodies derived from patients with GD

TSH receptor antibody (TRAb) is clinically classified into thyroid stimulating antibody (TSAb) and thyroid-stimulation blocking antibody (TSBAb). Although the former is considered to cause Graves' disease (GD), its activity does not necessarily reflect hormone production and goiter size. Moreover, uptake of 99mTcO4(-), the best indicator for GD, is correlated with activity of TSH binding inhibitor immunoglobulin better than activity of TSAb. Because uptake of 99mTcO4(-) reflects thyroid volume, these observations suggest that there exist TRAb with thyrocyte growth stimulating activity (GSA) other than TSAb. In this study, we analyzed GSA of monoclonal TRAb established from patients with GD or idiopathic myxedema (IME). GSA was measured as the degree of FRTL-5 cell growth stimulated by each TRAb. The signaling pathways of the cell growth were pharmacologically analyzed. The cell growth stimulated by TSH was strongly suppressed by protein kinase A (PKA) inhibitor, but was not affected by extracellular signal regulated kinase kinase (MEK) inhibitor. Although TSAb from GD stimulated the cell growth, both inhibitors suppressed it. Surprisingly, the cell growth was also induced by TSBAb from GD and was only suppressed by MEK inhibitor. TSBAb from IME did not have GSA and attenuated the cell growth stimulated by TSH. We concluded that 1; in GD, not only TSAb but some TSBAb could stimulate thyrocyte growth. 2; TSBAb might be classified with respect to their effects on thyrocyte growth; i.e., thyrocyte growth stimulating antibody and thyrocyte growth-stimulation blocking antibody.

A 5-year-old boy with atrophic autoimmune thyroiditis caused by thyroid-stimulation blocking antibodies

A 5-year-old boy was presented for a growth disturbance, which was initially noted at 3 years of age. Endocrinological testing identified severe hypothyroidism, defined by the following levels: TSH 990.5 microU/mL, F-T3 0.26 pg/mL, and F-T4 0.09 ng/dL. Serum anti-thyroid peroxidase (TPO) antibodies were 158 IU/mL and serum thyroid-stimulation blocking antibodies (TSBab) levels were 82.1 IU/mL (normal range < 45.6). Thyroid scintigraphy with 99mTc showed markedly decreased uptake, and magnetic resonance imaging (MRI) revealed pituitary hyperplasia. He was diagnosed with atrophic autoimmune thyroiditis. His thyroid function and pituitary size normalized following thyroid hormone replacement therapy. We report a rare case of a young boy with atrophic thyroiditis caused by TSBab.

TSH-receptor Antibodies Determined by the First, Second and Third Generation Assays and Thyroid-stimulating Antibody in Pregnant Patients with Graves' Disease

The measurement of TSH receptor antibody (TRAb) has been recommended to predict the risk of neonatal hyperthyroidism (NH) in pregnant women with Graves' disease (GD). For the first generation TRAb (TRAb1) assay with commercial kit (Brahms, Berlin, Germany; or Cosmic co., Tokyo, Japan) an arbitrary limit of 40 U/l or 50% was suggested to indicate risk when measured late in pregnancy. In order to substitute TRAb1 with the second generation TRAb using porcine TSH receptor (pTRAb2) and human recombinant TSH receptor (hTRAb2) and the third generation TRAb (TRAb3) assay for this purpose, we measured TRAb in these four methods late in pregnancy in a total of 62 pregnant women with Graves' disease. The data showed that no cases with TRAb1 >50% has been missed if the TRAb1 assay was replaced by the pTRAb2, hTRAb2 or TRAb3 assay using their equivalent cut-off value of 70%, 10 IU/l, and 75%, respectively, but that an additional group of women would have been included in the risk group, especially in the TRAb3 assay. Next, the effect of maternal TRAb on thyroid function of offspring was studied in the 47 pregnant women with GD (43 with TRAb1 <50% and 4 with TRAb1 >50% during late pregnancy). In 2 women who gave birth to hyperthyroid children at days 6 and 14 of life, the maternal sera had strongly positive levels of TRAb1 (73.5% and 84.1%), pTRAb2 (84.9% and 91.5%), hTRAb2 (40.68 IU/L and 89.70 IU/L) and TRAb3 (92.1% and 93.5%) late in pregnancy, with one case displaying high positive (1114.3%) thyroid stimulating antibody (TSAb) level and the other case had moderate positive (433%) TSAb level. Of the remaining 45 women, 43 had TRAb1 <50% and the other 2 had TRAb >50% including 1 with low TSAb positive and 1 with positive thyroid stimulating blocking antibody (TSBAb) and negative TSAb; all of them gave birth to euthyroid children. Finally, a serial study regarding TRAb in 23 women with Graves' disease during pregnancy showed that TRAb1, pTRAb2, hTRAb2, TRAb3 value and TSAb level decreased significantly as pregnancy progressed. In conclusion, the present study supported TRAb as a useful marker to predict the risk of NH.

Thyroid Stimulation-Blocking Antibody and Graves' Ophthalmopathy

Thyroid Stimulation-Blocking Antibody and Graves' Ophthalmopathy

Graves' Hyperthyroidism Showing Transient Hypothyroidism during Interferon Therapy for Chronic Hepatitis Type C

We report a patient with Graves' disease in whom thyroid function was changed from initial hyperthyroidism to transient hypothyroidism and back to hyperthyroidism during interferon (IFN) therapy. A 43-year-old man was admitted to our hospital to receive IFN treatment for chronic active hepatitis (type C) in June 1998. His thyroid function was normal and testing for thyroid gland antibodies (TSH binding inhibitor immunoglobulins; TBII, anti-thyroglobulin antibodies; TgAb and anti-thyroid peroxidase antibodies; TPOAb) was negative before IFN therapy. The patient had neither history of thyroid disease nor any particular family history. He developed hyperthyroidism four months after its initiation of IFN therapy. When he was hyperthyroid, TBII, the activity of thyroid-stimulating antibodies (TSAb) and thyroid stimulation-blocking antibodies (TSBAb) were 40.2% (normal range, -15 approximately +15.0%), 1201% (normal range, <or=180%) and 52.0% (normal range, <or=45.6%), respectively. Thyroid 99mTc(technetium)-uptake ratio (Tc-UTR) was 1.07% (normal range, 0.5-3.0%). He transiently developed hypothyroidism in December 1998 and recurrent hyperthyroidism in February 1999. When he was hypothyroid, TBII, TSAb and TSBAb were 74.3%, 769% and 95.9%, respectively. To investigate the mechanism of his fluctuating thyroid status, we serially assessed the serum levels of cytokines (TNF-alpha, IFN-gamma, IL-1beta, IL-2, IL-4) and the soluble form of ICAM-1 (sICAM-1) as well as the activities of two types of TSH receptor antibodies (TRAb), TSAb and TSBAb, before and after IFN therapy. There were no characteristic changes of cytokines or sICAM-1 during the follow-up period. The transient hypothyroid state may be explained by two possible mechanisms: one may be due to the shift in the balance between the stimulating and blocking types of TRAb, and the other may be due to the complication of destructive thyroiditis that developed during IFN therapy.

TSH receptor antibodies do not alter the function of gonadotropin receptors stably expressed in eukaryotic cells.

TSH receptor (TSHr) mediates the activating action of TSH on the thyroid gland resulting in the growth and proliferation of thyrocytes and thyroid hormone production. TSHr is a major autoantigen in Graves' disease (GD) and is the target for TSHr antibodies. In GD, thyroid-stimulating antibodies (TSAb) are competitive agonists of TSH. In atrophic thyroiditis (AT), thyroid-stimulating blocking antibodies (TSHBAb) are TSH antagonists. The TSHr together with the LH receptor (LHr) and FSH receptor (FSHr) are G-protein-coupled receptors with considerable amino acid homologies in the extracellular domain. We studied the cross-reactivity of the antibodies measured in sera from patients with GD or AT on the LHr and FSHr function. We tested the activity of TSAb and TSHBAb in cell lines expressing the LHr and the FSHr. To this purpose a pSVL-FSHr construct was transfected in CHO cells and one clone was used. Twenty-eight sera from patients with GD and four from patients with AT, known to contain TSHr antibodies measured with a radioreceptor assay, were selected. TSAb and TSHBAb activities were measured in CHO cells expressing the TSHr (CHO-TSHr). TSAb and TSHBAb were then tested with the cell lines expressing the LHr and the FSHr for their ability to elicit cAMP accumulation or inhibit FSH/LH-induced cAMP production. None of the TSAb identified was able to stimulate cAMP increase in CHO-LHr or CHO-FSHr. Similarly, none of the TSHBAb was able to block the cAMP response induced by FSH or LH in the respective cell lines. Our results confirm the notion of the organ-specific nature of the TSHr antibodies.

No increase of blocking type anti-thyrotropin receptor antibodies during pregnancy in patients with Graves' disease.

Serial changes in serum levels of anti-TSH receptor antibodies were examined during and after pregnancy in six patients with Graves' disease receiving no or minimal maintenance doses of antithyroid drugs. During pregnancy, serum levels of TSH-binding inhibitory Igs (P < 0.001) and thyroid-stimulating antibodies (TSAbs) (P < 0.01) decreased gradually but increased after delivery in all patients. Activities of thyroid-stimulation blocking antibodies (TSBAbs) were lower than the cut-off value in early pregnancy, and values significantly decreased in four patients during pregnancy. The other two patients showed no significant change during pregnancy. In contrast, TSBAb levels increased significantly (P < 0.01) after delivery in all patients. We found that activities of TSH-binding inhibitory Igs, TSAb, and TSBAb decrease during pregnancy and increase after delivery, suggesting that amelioration of Graves' disease during pregnancy is induced by decrease of TSAb but not by the appearance of TSBAb.

Modulation of Fas-mediated apoptosis of human thyroid epithelial cells by IgG from patients with Graves' disease (GD) and idiopathic myxoedema.

The expression of two autoimmune thyroid diseases. GD and idiopathic myxoedema, is associated with antibodies to the thyroid-stimulating hormone (TSH) receptor. Thyroid stimulating antibodies (TSAb) in GD are TSH agonists and cause hyperthyroidism as well as goitre, whereas thyroid stimulation blocking antibodies (TSBAb) in idiopathic myxoedema are TSH antagonists and cause hypothyroidism and thyroid atrophy. We investigated the effect of antibodies to TSH receptor on Fas-mediated apoptosis of thyroid epithelial cells (thyrocytes). Human IgG was isolated from healthy donors, patients with GD and idiopathic myxoedema. Human thyrocytes were obtained from surgical specimens. Thyrocytes were cultured in the presence or absence of human IgG with or without interferon-gamma (IFN-gamma) or IL-1beta for a specified time. After incubation, we examined the level of cAMP in cultured supernatants and both Fas and Bcl-2 expression on thyrocytes. In addition, we examined anti-Fas-mediated apoptosis of thyrocytes. Fas expression on thyrocytes was significantly down-regulated by Graves' IgG and TSH, although idiopathic myxoedema IgG did not affect Fas expression on thyrocytes. Idiopathic myxoedema IgG abrogated the effect of TSH on both cAMP production and inhibition of Fas expression on thyrocytes. Treatment of thyrocytes with IL-1beta or IFN-gamma caused a marked augmentation of Fas expression on thyrocytes. The increase of Fas expression of thyrocytes induced by IL-1beta or IFN-gamma was significantly suppressed in the presence of TSH or Graves' IgG. Anti-Fas-induced apoptosis of thyrocytes was observed in thyrocytes treated with IL-1beta or IFN-gamma, but was markedly inhibited in the presence of TSH or Graves' IgG. Furthermore, idiopathic myxoedema IgG abrogated most of the inhibitory effect of TSH on Fas-mediated apoptosis of thyrocytes treated with IL-1beta or IFN-gamma. Bcl-2 expression of thyrocytes did not change after stimulation with TSH, Graves' IgG, idiopathic myxoedema IgG, IL-1beta or IFN-gamma. These results suggest that TSAb found in Graves' patients may be potentially involved in the development of goitre by inhibition of Fas-mediated apoptosis of thyrocytes. In addition, TSBAb inhibit the action of TSH and increase the sensitivity toward Fas-mediated apoptosis of thyrocytes, inducing thyroid atrophy seen in patients with idiopathic myxoedema.

Epitope mapping of tsh receptor-blocking antibodies in Graves' disease that appear during pregnancy.

Spontaneous remission of Graves' disease during pregnancy is thought to be due to a reduction of thyroid-stimulating antibody activity. We suspected, however, that a broader change in TSH receptor antibody characteristics might play an important role in modulating disease activity during pregnancy. We measured TSH binding inhibitory Ig, thyroid-stimulating antibody, and thyroid stimulating-blocking antibody activities in 13 pregnant Graves' disease patients at first, second, and third trimesters and 4 months postpartum. To measure and epitope-map thyroid-stimulating antibody and thyroid stimulating-blocking antibody activities, we used CHO cells transfected with wild-type human TSH receptor or with several TSH receptor-LH/hCG receptor chimeras: Mc1+2, Mc2, and Mc4. These chimeric cells have their respective TSH receptor residues 9-165, 90-165, and 261-370 substituted with equivalent residues of the LH/hCG receptor. Overall thyroid-stimulating antibody decreased, whereas thyroid stimulating-blocking antibody increased progressively during pregnancy. TSH binding inhibitory Ig fluctuated in individual patients, but overall the activities remained statistically unchanged. Thyroid stimulating-blocking antibody appeared in subjects who were either negative for thyroid-stimulating antibody or whose thyroid-stimulating antibody activity increased or decreased during pregnancy. Epitope mapping showed that the thyroid-stimulating antibodies were mainly directed against residues 9-165 of the N-terminus of the TSH receptor extracellular domain. All thyroid stimulating-blocking antibodies had blocking activities against residues 261-370 of the C-terminus of the ectodomain. However, the majority of the thyroid stimulating-blocking antibodies had a hybrid conformational epitope directed against N-terminal residues 9-89 or 90-165 as well. Despite a change in the activity level, we did not observe any change in the epitope of either the stimulatory or blocking Abs as pregnancy advanced. In conclusion, a change in the specificity of TSH receptor antibody from stimulatory to blocking activity was observed during pregnancy, and the appearance of thyroid stimulating-blocking antibody may contribute to the remission of Graves' disease during pregnancy.

Characterization of Thyroid-Stimulating Blocking Antibodies That Appeared During Transient Hypothyroidism After Radioactive Iodine Therapy

Hypothyroidism after radioactive iodine (RAI) therapy for Graves' disease can be transient or permanent. The cause for early transient hypothyroidism is unknown. We evaluated 11 patients who developed transient hypothyroidism within 6 months of RAI and 12 who remained euthyroid after RAI. Approximately equal numbers of patients in each group had thyroid-stimulating antibody (TSAb) that increased cyclic adenosine monophosphate (cAMP) levels in Chinese hamster ovary (CHO) cells transfected with the recombinant human thyrotropin receptor (TSHR) (WT cells). Approximately equal numbers of patients from both groups had an increase in TSAb activity post-RAI. All TSAbs had their dominant functional epitope on the N-terminus of the TSHR extracellular domain, requiring residues 90-165 for activity because they, but not TSH, completely lost stimulating activity in a receptor chimera, wherein TSHR residues 90-165 were substituted by equivalent residues of the lutropin/choriogonadotropin receptor (LH/CGR). Although equal numbers of patients in both groups had thyrotropin-binding inhibiting immunoglobulin activity (TBII), as measured by radioreceptor assay before RAI, patients with transient hypothyroidism had a surge in TBII activity and all except one became positive for thyroid-stimulating blocking antibodies (TSBAb), as measured by inhibition of TSH-stimulated cAMP from WT cells. When immunoglobulin G (IgGs) were epitope-mapped using TSHR/LH-CGR chimeras with different substitutions, 8 hypothyroid subjects had TSBAbs directed against residues 90-165 of the TSHR, as well as TSHR residues 261-370. Two had functional epitopes directed at residues 9-89 as well as TSHR residues 261-370. None of the euthyroid control patients developed TSBAbs and their TBII activity decreased post-RAI. When patients with transient hypothyroidism reverted to a euthyroid state, TSAb was still detectable in 5; however, TBII was present in all and TSBAb, although decreased, was still positive in 9. In summary, RAI therapy was associated with a change in thyroid antibody characteristics in most patients. Additionally, patients with a surge in TBII and the appearance of TSBAb developed transient hypothyroidism after RAI.

An elevation of serum immunoglobulin E provides a new aspect of hyperthyroid Graves' disease.

In hyperthyroid Graves' disease, short-term methimazole is sufficient to induce lasting remission in some patients, but even long-term treatment fails to do so in others. We have evaluated the role of autoimmune abnormalities in the helper T cell type 2 (TH2)-interleukin-13 (IL-13)-TSH receptor system in maintaining hyperthyroidism by comparing IgE levels in patients with various thyroid diseases. One hundred and ninety-three patients with hyperthyroid Graves' disease were treated with methimazole, and blood samples were obtained to measure serum levels of T4, T3, TSH, thyroglobulin, antimicrosomal antibody, TSH binding inhibitory Ig (TBII), thyroid-stimulating antibody, thyroid stimulation-blocking antibody, IgE, interferon-gamma, IL-4, and IL-13. Elevation of serum IgE (> or = 170 U/mL) was found in 35.5% of patients with hyperthyroid Graves' disease, and serum levels of T4, T:1, antimicrosomal antibody, and TBII were significantly greater in patients with IgE elevation than in those with normal serum IgE. During methimazole treatment, there was a parallel decrease in the serum T4 concentration in the presence or absence of an IgE elevation. However, there was a significantly smaller decrease in TBII in patients with elevated IgE than in those with normal IgE. As a result, the remission rate was significantly greater in patients with normal IgE than in those with IgE elevation. Serum levels of IL-13 were elevated in 64.7% of patients with IgE elevation in the absence of detectable TH1 marker, interferon-gamma. These findings suggest that in one third of patients with hyperthyroid Graves' disease, TH2 cells are stimulated and secrete excess amounts of IL-13, which subsequently stimulates B cells to synthesize more TSH receptor antibody and IgE, so that during methimazole treatment TBII decreases less in patients with IgE elevation, producing a lower remission rate.

Atrophic Autoimmune Thyroiditis with Positive Thyroid Stimulation Blocking Antibody in a Prepubertal Boy

Atrophic Autoimmune Thyroiditis with Positive Thyroid Stimulation Blocking Antibody in a Prepubertal Boy

Lack of interaction in recombinant human FSH receptor and both TSAb and TSBAb

Since cross-reactivity of TSH with the human FSH receptor has been reported, in this study we tested the effect of thyroid-stimulating antibody (TSAb) and thyroid stimulation-blocking antibody (TSBAb) on Chinese hamster ovary cells expressing human FSH receptor (CHO-hFSH-R cells). We examined the TSBAb activity of sera from hypothyroid patients who had a positive TBII to determine whether these sera also block the effect of FSH on CHO-hFSH-R cells. Although human FSH I-3 (0.25–16 ng ml ) stimulated the production of intracellular cAMP in CHO-hFSH-R cells with doseresponsive manner, neither TSAb nor TSBAb had such an effect on the cells.

Clinical Usefulness of TSAb Assay with High Polyethylene Glycol Concentrations

We previously demonstrated the stimulatory effect of polyethylene glycol (PEG) on thyroid-stimulating antibody (TSAb)-IgG-stimulated cAMP production (thyroid stimulating (TS) index) in porcine thyroid cell (PTC) assay. In the present study the clinical usefulness of the practical method using high PEG concentrations was examined. TS activity using PEG 22.5% precipitated fraction (PF) was significantly higher compared to standard TSAb activity using 12.5% PF from TSAb-positive serum, but the maximum TS activity was observed with PEG 12.5% PF + 4% PEG or PEG 22.5% PF + 2% PEG. In all cases of untreated Graves’ patients, TSAb activity determined by PEG 22.5% PF was higher compared to standard TSAb activity using PEG 12.5% PF from test serum, but the highest TSAb activity was observed by PEG 12.5% PF + 4% PEG without increased cAMP production to normal serum. TSAb was positive in 85% (40/47), 98% (46/47) and 100% (47/47) of untreated Graves’ patients by the method of PEG 12.5% PF, PEG 22.5% PF and PEG 12.5% + 4% PEG, respectively. Increased TSAb activity by PEG 12.5% PF + 4% PEG method was also observed even if the standard TSAb activity using PEG 12.5% PF method was negative in the euthyroid states of Graves’ patients during antithyroid drug therapy. The stimulatory effect of PEG on TS activity was not found in other thyroidal diseases [thyroiditis chronica (with high serum TSH), thyroid stimulation-blocking antibody (TSBAb)-positive sera (with low serum TSH), adenomatous goiter, subacute thyroiditis, and thyroid cancer]. The stimulatory effect of 5% PEG on TS activity produced directly by small amounts of Graves’ serum (50 μl) was also found, although the sensitivity was lower than with PEG-precipitated IgG from 0.2 ml serum. The clinical usefulness of the sensitive TSAb assay using PEG-precipitated IgG or direct serum assay in the presence of high PEG concentrations was demonstrated.

Polyethylene glycol augments thyroid cAMP responses by fragments from protease-digested TSAb or TSBAb-IgG

In the previous reports, we have demonstrated (1) that polyethylene glycol (PEG)(5%) augmented TSAb (thyroid stimulating antibody)-stimulated cAMP responses of porcine thyroid cells, and (2) that fragments from papain-digested TSBAb (thyroid stimulation blocking antibody) could stimulate thyroid cAMP synthesis. Thus, we studied the effect of 5 % PEG on cAMP responses stimulated by the protease-digested TSAb- or TSBAb-fragments. Stimulatory effect of 5 % PEG on cAMP production by Fab fragment (Mr 50 KDa) and the retarded fraction (Mr 20 KDa) from the gel-filtration on Sephadex G-100 using papain-digested TSAb-IgG unbound to Protein A-Sepharose was observed. Similar stimulatory effect of 5 % PEG on the second fraction (Fc with trace amounts of Fab) in the gel-filtration on Sephadex G-100 using papain digested TSAb-IgG bound to Protein A-Sepharose was observed. Stimulatory effect of PEG on the second fraction was derived from Fab fragment. PEG (5 %) also showed stimulatory effect on cAMP production by F(ab') 2 fragment (Mr 100 KDa) from the gel-filtration on Sephadex G-100 using pepsin-digested TSAb-IgG unbound to Protein A-Sepharose. PEG (5 %) augmented cAMP responses by both Fab and the retarded fractions from the gel-filtration using papain-digested TSBAb-IgG unbound to Protein A when these fractions could stimulate cAMP synthesis. In conclusion, PEG (5%) augments cAMP responses stimulated by F(ab') 2, Fab and the smaller molecular components (Mr 20 KDa) separated from protease-digested TSAb-IgG. PEG also augments cAMP responses stimulated by Fab and the smaller molecular components with thyroid stimulating activity separated from papain-digested TSBAb-IgG.