Thyrotropin Binding Inhibitor Research Articles

MON-263 Graves’ Disease Presenting As Chronic Diarrhea In A Toddler

Background: Hyperthyroidism is uncommon in the pediatric population and is particularly rare in patients less than 4 years of age with a reported incidence of 1 per 1,000,000 (1). The most common cause of hyperthyroidism in patients beyond infancy remains to be Graves’ disease, and its presentation may vary by age. We report a case of a toddler with Graves’ disease who presented with chronic diarrhea. Clinical Case: The patient is an 18 month old boy who initially presented to urgent care at the age of 16 months with diarrhea and emesis. He was diagnosed with viral gastroenteritis and sent home. He continued to have intermittent emesis and persistent diarrhea that did not improve with a change in milk formulation. He was seen by pediatric gastroenterology where GI work-up was negative. He presented to his pediatrician 2 months later for re-evaluation. His growth chart was notable for a 1.2 kilogram weight loss in 3 weeks and growth acceleration of 10.2 cm in 6 months (20.4 cm/yr). Laboratory work-up showed a suppressed TSH of <0.05 uU/ml. He was referred to pediatric endocrinology. The patient was born full term with no prenatal or perinatal complications. He had been gaining weight appropriately until about 16 months of age. He was not taking any medications or supplements. There was no family history of autoimmune, gastrointestinal, or thyroid diseases. At his initial endocrine visit, his examination was notable for normal heart rate and blood pressure, mild bilateral exophthalmos, and a prominent palpable thyroid gland. There was no lid lag, tremors, or tongue fasciculations. Reflexes were brisk. The remainder of his exam was normal. Further labs revealed free T4 >6 ng/dl, total T4 29.7 mcg/dL (normal 6-13.2), total T3 > 651 ng/dl. Thyroid stimulating immunoglobulin was >700% and thyrotropin binding inhibitor was 61% (normal <16%). The diagnosis of Graves’ disease was made and the patient was started on methimazole 5 mg daily. His symptoms improved after 1 week of starting treatment with less frequent diarrhea, and weight gain. Repeat labs demonstrated improving free T4 4.5 ng/dl (n 0.8-1.7) and total T3 420 ng/dl (n 80-200). He continues to have close follow up in our clinic. Clinical Lessons: We present the diagnosis of Graves’ disease in one of the youngest patients to be reported. Though Graves’ disease continues to be the most common cause of hyperthyroidism in toddlers, other causes should be considered in this age range. This case highlights the spectrum of the presentation of hyperthyroidism and impresses upon the provider to consider hyperthyroidism as a cause for unexplained chronic diarrhea in a young pediatric patient. Reference:(1) Ho, YH et al. A 3-year-old girl with Graves’ disease with literature review. Ann Pediatr Endocrinol Metab. 2014 Sep; 19(3): 154-158.

Blockade of Costimulation Between T Cells and Antigen-Presenting Cells: An Approach to Suppress Murine Graves' Disease Induced Using Thyrotropin Receptor-Expressing Adenovirus

Immune responses require costimulatory interactions between molecules on antigen-presenting cells and T cells: CD40 binding to CD40 ligand and B7 binding to CD28. Graves' hyperthyroidism is induced in BALB/c mice by immunization with thyrotropin receptor (TSHR) A-subunit adenovirus (Ad-A-subunit). We attempted to modulate Ad-A-subunit-induced Graves' disease using adenoviruses expressing costimulation "decoys": CD40-IgG-Fc (CD40-Ig) to block CD40:CD40-ligand interactions and CTLA4-Fc (CTLA4-Ig) to prevent B7:CD28 binding. Unexpectedly, coimmunizing mice with Ad-A-subunit and excess control adenovirus (1:10 Ad-A-subunit:Ad-control) reduced TSHR antibody levels (thyrotropin binding inhibition [TBI]). Furthermore, only 15% of mice developed hyperthyroidism versus 75% using the same Ad-A-subunit dose (10(8) particles) without Ad-control. This effect was related to the dose of control adenovirus but not to the adenovirus insert, the timing or immunization site. Increasing the Ad-subunit dose (10(9) particles) and decreasing the control adenovirus dose (10:1 Ad-A-subunit:Ad-control) induced high TBI levels and 80% of mice were hyperthyroid. Coimmunization with Ad-CD40-Ig (but not Ad-CTLA4-Ig) reduced the incidence of hyperthyroidism to 40%. Using appropriate controls and adenovirus ratios, our data suggest the importance of CD40:CD40-ligand interactions for inducing Graves' hyperthyroidism by Ad-A-subunit. Furthermore, our observations emphasize the potential pitfalls of non-specific inhibition by coimmunization with two adenovirus species.

A direct binding assay for thyrotropin receptor autoantibodies.

There is, at present, no assay in clinical use for the direct assay of autoantibody binding to the thyrotropin receptor (TSHR). We now describe a direct thyrotropin receptor autoantibody binding assay (DTAb) using a secreted form of the TSHR ectodomain (TSHR-289) without the need for antigen purification. The assay compensates for the low TSHR autoantibody concentration in serum by capturing a relatively large amount of patient immunoglobulin G (IgG) on high-capacity beads, a reversal of standard methods that typically first immobilize antigen. TSHR-289 captured by Graves' IgG was detected in a colorimetric reaction using a biotinylated murine monoclonal antibody to the poly-histidine tail engineered into the antigen. By this approach, sera from 11 normal individuals provided a mean optical density (OD) value of 0.20 +/- 0.08 SD (range 0.06-0.33). Of 38 sera from unselected patients with a history of Graves' disease (untreated and treated), 29 (76%) generated OD values > 0.37 (2 SD above the mean for the normal sera), the highest being OD 1.38. Surprisingly, 3 of 13 (23%) sera from TPO autoantibody-positive patients with Hashimoto's thyroiditis also provided values > 2 SD above the normal sera. The extent of direct autoantibody binding to the TSHR correlated closely with the thyrotropin binding inhibition (TBI) values (r = 0.881; p < 0.001). One serum was clearly positive in only the direct binding assay and another in only the TBI assay. The data obtained with the direct binding assay correlated less well with the thyroid-stimulating antibody (TSAb) assay (r = 0.582; p < 0.001). In summary, we describe a new direct DTAb assay that correlates more closely with the TBI than with the TSI assays. Future studies in a large series of clinically defined patients will be needed to evaluate the clinical utility of the DTAb assay.

An elevation of stem cell factor in patients with hyperthyroid Graves' disease.

Graves' disease is an autoimmune disorder characterized by the presence of antibodies against thyrotropin receptor (TRAb). Stem cell factor (SCF), derived from bone marrow, is known to promote lymphohematopoiesis. To investigate the relation between the alteration in plasma levels of SCF, thyroid hormone status, and TRAb measured by thyrotropin binding inhibition (TBI), 13 untreated, 21 treated, and 4 relapsed hyperthyroid Graves' disease patients, 21 patients with Hashimoto's thyroiditis, 6 patients with subacute thyroiditis, and 11 control subjects were examined. In untreated hyperthyroid Graves' disease patients, serum levels of thyroxine (T4) and triiodothyronine decreased rapidly by methimazole treatment, and TBI decreased progressively, but variably. Simultaneously, the elevated plasma levels of SCF decreased gradually and progressively. The plasma levels of SCF correlated curvilinearly with the serum levels of T4. In 4 patients with relapsed hyperthyroid Graves' disease, TBI was marginally positive in 3 patients and negative in 1, but plasma levels of SCF were elevated significantly in all 4 patients. In patients with subacute thyroiditis and Hashimoto's thyroiditis with or without T4 replacement, plasma levels of SCF did not differ from that of controls. These findings indicate that the elevation of plasma levels of SCF relates to the longstanding thyrotoxic state and that short-term thyrotoxicosis does not significantly affect plasma levels of SCF. It remains to be determined whether the elevation in plasma levels of SCF is induced by excess thyroid hormone, reflecting the hypermetabolic state, or whether the elevation of plasma levels of SCF contributes to stimulation of lymphocytes producing TRAb.

Simple radioimmunological assay of anti-alpha-galactosyl antibody (anti-Gal): application to autoimmune thyroid diseases and thyroid-associated ophthalmopathy.

Two radioimmunological techniques for the measurement of anti-alpha-galactosyl (anti-Gal) antibodies were developed and we demonstrated that this antibody is present in the serum of most untreated patients with Graves' disease, thyroid-associated ophthalmopathy (TAO) and autoimmune thyroiditis. Thyrotropin-binding inhibiting immunoglobulins (TBII) and Thyroid Stimulating Antibodies (TSAb) are frequently found in serum of patients with Graves' disease but not with autoimmune thyroiditis. Although TBII and anti-Gal were found sometimes simultaneously in the same serum samples, we demonstrated that anti-Gal could be present in the absence of TBII and reciprocally.

Hyperthyroidism in Growth Hormone Deficient Children Treated with Human Growth Hormone

Two children with growth hormone (GH) deficiency developed hyperthyroidism during the course of their therapy with human GH (hGH). The first patient, who had been irradiated for panhypopituitarism due to suprasellar germinoma, was treated with corticosteroid, 1-deamino-8-D-arginine-vasopressin (DDAVP), l-thyroxine (T4) and GH. Eight years after the initiation of therapy, hyperthyroidism developed without any specific symptoms. Laboratory examination revealed elevated thyroid hormone, positive thyrotropin-binding inhibiting immunoglobulin (TBII) and antimicrosomal antibody. The second patient, with optic nerve atrophy treated with GH and l-T4, developed hyperthyroidism after 10 years of therapy. This patient also showed elevated thyroid hormone, positive anti-thyroglobulin antibody, anti-microsomal antibody and TBII. After being treated with mercazol, the thyroid function of both patients became normal.Although diabetes mellitus (DM) and hypothyroidism are occasionally associated with GH treatment, the development of hyperthyroidism has rarely been reported. This hyperthyroidism associated with GH treatment may occur by chance. However, some other factors, which produce autoimmune antibodies, may also involve the development of hyperthyroidism. These two cases caution us that hyperthyroidism can develop even in a patient with secondary hypothyroidism during treatment with GH and l-T4.

Induction of Thyrotropin Receptor (TSH-R) Autoantibodies and Thyroiditis in Mice Immunized with the Recombinant TSH-R

Two groups of 10 Balbc by Jico mate mice were immunised on days 0, 15 and 35, with the extra cellular domain (ECD) of the human thyrotropin receptor (TSH-R) expressed as a fusion protein in bacteria (group 1) or with the maltose binding protein (MBP) fusion partner alone (group 2). Blood was obtained on days 0, 22, 32, 42 and 49 and samples from the individual animals pooled for each group. Serum and immunoglobulin (IgG) preparations were tested, using CHO cells expressing the human TSH-R (JP26 and JP09) for thyroid stimulating (TSAb); thyroid blocking (TBAb) and thyrotropin binding inhibiting (TBII) activities. Neither serum nor IgGs were found to contain TSAb at any time point. TBII activity was present in the serum of both groups on day 32 and in group 1 only on day 49; when the test was performed on IgGs, only the MBP-ECD day 49 preparation remained significantly positive for TBII (p < 0.005). Significant TBAb activity was present in both the serum and IgG of group 1 day 49 (p < 0.005) and to a lesser extent on 42 (p < 0.02). Following the second immunisation (day 15) both groups and had decreased circulating T4 levels (p < 0.05) when compared with day 0 in each case. Group 2 were unaffected by the third immunisation on day 35 but the MBP-ECD group again had significantly decreased T4 levels (p < 0.02) compared with MBP day 49 and (p < 0.03) when compared with MBP-ECD day 0. Histological examination of thyroids from group 1 animals revealed extensive vascularisation and an atypical lymphoblastoid infiltration which was not observed in control mice. These preliminary results indicate that care is required in interpreting data since a nonreceptor antigen was shown to decrease circulating thyroxine and serum from these animals had apparent TBII like activity. However, the results obtained with the IgGs suggest that receptor autoantibodies can be induced by immunising with the human TSH-R, in addition, the immunised mice show histological evidence for the development of thyroiditis.

The presence of higher levels of thyroglobulin, but not thyroid autoantibodies, in the thyroid vein in Graves' disease.

To investigate the possible major source of thyroid autoantibodies production, blood samples were obtained from thyroid vein, jugular vein and peripheral vein during subtotal thyroidectomy in 12 patients with Graves' disease (11F, 1M; age 16-39 yr). Among them, 7 were treated preoperatively by methimazole, 4 by iopanoic acid and 1 by propranolol. All blood samples were assayed for thyroglobulin (Tg), thyrotropin binding inhibition immunoglobulin (TBII), antithyroglobulin antibody (TgAb) and antimicrosomal antibody (McAb). Tg, a native product of thyroid gland, was markedly elevated in the thyroid veins, over 4 to 6 folds that of jugular veins or peripheral veins. However, the level of thyroid autoantibodies including TBII, TgAb and McAb in the thyroid veins were not significantly different from that in the jugular or peripheral veins. Our preliminary data suggest that it is thyroglobulin, and not thyroid antibodies that is present at higher level in the thyroid vein than the periphery.

Inhibition of thyrotropin binding to receptor by synthetic human thyrotropin beta peptides.

In order to study the structure and function relationships of the thyrotropin (TSH)-specific beta-subunit, we produced 11 synthetic overlapping peptides containing the entire 112-amino acid sequence of human beta TSH and tested them for activity in TSH radioreceptor assay using both human and porcine thyroid membranes. Synthetic peptides representing four regions of the beta-subunit demonstrated the ability to inhibit binding of 125I-bovine TSH to crude thyroid membranes. The peptide representing the -COOH terminus of the subunit (beta 101-112) possessed highest binding activity, inhibiting binding of labeled TSH with an EC50 of 80 microM. The remaining active peptides were: beta 71-85 (104 microM), beta 31-45 (186 microM), beta 41-55 (242 microM), and beta 1-15 (331 microM). Specificity of the binding activity was shown by the inability of the peptides representing the remainder of the subunit to inhibit binding of label and by the inability of any of the peptides to inhibit binding of 125I-epidermal growth factor to the same thyroid membranes. The low affinity of the peptides as compared with native hormone is in agreement with previous studies of synthetic alpha-subunit peptides and, further, suggests that the interaction of beta TSH with receptor is multifaceted, requiring cooperative binding of these sites for the observed high affinity of the whole hormone. These studies are in agreement with previous predictions of active regions by chemical modification but add two regions to the list, showing the utility of the synthetic peptide strategy in the study of peptide hormone structure-activity relationships.

Juvenile Graves disease: Usefulness and limitations of thyrotropin receptor antibody determinations

Thyrotropin receptor antibodies (TRAb) were measured in serum from 49 patients with active and inactive juvenile Graves disease, and the results were compared with values from control subjects and patients with Hashimoto disease. With a thyrotropin binding inhibition immunoglobulin (TBII) assay, TRAb were found in serum from 25 (93%) of 27 patients with untreated, active Graves disease. The TRAb values remained positive in 20 (72%) of 29 patients during the first 6 months of antithyroid therapy and in 13 (54%) of 24 patients during the second 6 months. After discontinuation of antithyroid therapy, 15 patients experienced 18 episodes of relapse of thyrotoxicosis; during relapse TRAb values were positive in all but one patient. Among 19 patients who remained clinically and biochemically euthyroid after cessation of antithyroid therapy, TRAb were not detected in 28 (78%) of 36 serum specimens. Of the eight positive values from six patients, no antiidiotypic antibodies were found, and thyroid stimulating immunoglobulins (TSI) were not detected in four specimens. The TRAb determination correctly predicted the subsequent clinical course in 26 (72%) of 36 patients. Furthermore, TRAb values and results of triiodothyronine suppression tests were in agreement in 27 of 36 patients, or 75% of the time. TSI were present in serum from only 19 (73%) of 26 patients with active disease; however, TSI were negative in all patients with inactive Graves disease. During the management of Graves disease, TRAb measurements by TBII determinations are valuable in the diagnosis of active disease, assist in the decision to discontinue antithyroid drug therapy, and are useful as the T3 suppression test to predict the clinical course of the disease. The TSI measurement is most useful to determine the activity of the disease when TRAb values are positive in a euthyroid patient.

Interaction of the thyrotropin receptor on rat FRTL-5 thyroid cells with thyrotropin and a thyrotropin-stimulating autoantibody from Graves' patients

FRTL-5 rat thyroid cells were either surface-labeled with 125I or biosynthetically labeled with [ 3H]N-acetylglucosamine, solubilized by lithium diiodosalicylate and immunoprecipitated after sequential exposure to bovine thyrotropin and anti-bovine thyrotropin. Autoradiography of polyacrylamide gels run under denaturing conditions and in the presence of a reducing agent revealed two prominant bands with approximate molecular weights of 66–70 kDa and 47 kDa. Immunoprecipitation of the same radiolabeled and solubilized membrane preparations with a Graves' disease IgG having thyroid stimulating but no thyrotropin-binding inhibiting activity revealed only one major band, migrating near the 47 kDa component reactive with thyrotropin. No bands were immunoprecipitated in control incubations using normal human IgG or substituting radiolabeled, solubilized membranes from a rat thyroid cell line with no thyrotropin receptor activity. Thin layer chromatography of Folch extracts of the [ 3H]-N-acetylglucosamine-labeled immunoprecipitates obtained by either procedure indicated that a specific thyroid ganglioside was coprecipitated with the immunoprecipitated proteins in both cases.

Methodological aspects and clinical results of an assay for thyroid-stimulating antibodies: correlation with thyrotropin binding-inhibiting antibodies.

Several methodological aspects concerning the measurement of thyroid-stimulating antibodies (TSAb) were examined using a 5000-g particulate fraction from human thyroid tissue. TSH displayed maximal stimulation at 32 C. An ATP-regenerating system had no influence at ATP concentrations greater than 0.67 mM. Theophylline did not influence cAMP concentrations in the incubation medium after stimulation with increasing dosages of TSH, suggesting the absence of active phosphodiesterases in the membrane preparations. Sera dialyzed against Tris-HCl were as suitable as immunoglobulin G preparations for determination of TSAb. One hundred and nineteen patients with ophthalmic Graves' disease, 42 of whom were hyperthyroid at the time of study, were investigated for TSH binding-inhibiting antibodies ( TBIAb ) and TSAb. In 76% of these patients, concordance between the presence and absence of both activities was found independent of whether they were untreated, treated, or in remission. When gradations of potency in the two systems were compared, linear regression revealed a correlation coefficient of 0.59. The coefficient correlation increased when TBIAb and TSAb were each determined in a single assay using identical thyroid membrane preparations. In four other patients, both TSAb and TBIAb were measured repeatedly during the course of the disease. In three patients, both activities were parallel, whereas in one patient, dissociation of the two activities was found. The data suggest the presence of antibodies with varying stimulating and binding-inhibiting properties. However, in hyperthyroidism of Graves' disease, both activities usually were present in similar quantities.

Immunoglobulin G inhibitor of thyroid-stimulating antibody is a cause of delay in the onset of neonatal Graves' disease.

Studies were carried out with the serum IgG from a mother and her two children who developed neonatal Graves' disease several weeks after birth. The maternal IgG: (a) stimulated the human thyroid in vitro, but maximal stimulation was found only with dilution of the IgG; (b) was very potent in the long-acting thyroid stimulator (LATS)-protector assay, but only when an inhibitor of the system was diluted out; (c) inhibited a standard preparation of LATS in the mouse bioassay; (d) was biphasic in the thyrotropin-binding inhibition (TBI) assay, i.e., enhanced binding at low concentrations of IgG and inhibited binding at high levels. Enhancement in the TBI assay was found only with particulate preparations of human thyroid membranes as receptor and not when that material was solubilized, nor with guinea pig fat cell membranes as receptor. Serial blood samples from the second child were obtained at birth and until 3 mo of age. In the thyroid slice (cyclic AMP) assay system there was a negative dose-response relationship in testing IgG until age 45 d when it became positive, coinciding with the clinical recognition that hyperthyroidism had developed. The data are compatible with a concept that this mother's IgG contained thyroid-stimulating antibody (TSAb) and another moiety that inhibited TSAb through an action on the thyroid cell membrane, thus delaying the onset of hyperthyroidism in the neonate until the inhibiting IgG was metabolically cleared to an ineffective concentration.

Thyroid-stimulating antibody (TSAb) of Graves' disease

The current knowledge of thyroid-stimulating antibody (TSAb) and its significance in Graves' disease is reviewed under 4 headings. 1) Methods of assay; these are categorized as thyroid-stimulation or thyrotropin-receptor-modulation type methods. The latter are convenient but non-specific and the former are inconvenient but specific. The use of guinea pig fat cell membranes as a source of receptor for thyrotropin may improve the specificity of the thyrotropin-binding inhibition (TBI) system. 2) Immunochemistry of TSAb; evidence for the restricted heterogeneity, or oligoclonality, of the antibody as it occurs in some sera, viz. selected for the very high titer, includes a relatively constant pI on isoelectric focussing, restriction to IgG1 and having only λ or k as the light chain. 3) Are antibodies other than TSAb pathogenic in hyperthyroidism? data are provided indicating the presence in one serum of an antibody that inhibits the action of TSAb in vitro . Clinically this novel antibody caused delayed onset of neonatal hyperthyroidism in 2 children. The prevalence of the antibody and its general clinical significance are unknown, but ways of testing for its presence are reviewed. 4) Clinical significance of the assay of TSAb; TSAb occurs in at least 90% of patients but should not be necessary for the diagnosis of Graves' disease. Its persistence at the end of a course of antithyroid drugs predicates relapse; a high level on first diagnosis may forecast such persistence and be an indication for ablative therapy for hyperthyroidism. A high level of TSAb in the third trimester of pregnancy is a reliable index of neonatal hyperthyroidism. It should be recognized that there is a marked tendency for TSAb values to fall throughout the course of pregnancy.

Role of phospholipids in the structure and function of the thyrotropin receptor.

Phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine interact with 125I-thyrotropin and inhibit its binding to thyroid plasma membranes; phosphatidylcholine is not similarly effective. The interaction has been monitored by column chromatography on Sephadex G-100 which shows, for example, that 125I-labeled thyrotropin forms an adduct with phosphatidylinositol but not with phosphatidylcholine. Formation of the 125I-labeled thyrotropin-phosphatidylinositol adduct is dependent on the phosphatidylinositol concentration but can be reversed by both unlabeled thyrotropin and excess membranes. The efficacy of the phospholipid interaction and the phospholipid inhibition of thyrotropin binding to thyroid membranes is paralleled by changes in fluorescence and fluorescence polarization imposed on the 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of thyrotropin. These changes are reversed by unlabeled thyrotropin but not by prolactin, placental lactogen, or growth hormone; similar changes are not observed when phospholipids are incubated with dansylated growth hormone, prolactin, and placental lactogen. Monovalent potassium, sodium, and lithium salts neither prevent nor reverse the formation of the phospholipid-dansyl-thyrotropin adduct; these results contrast with the effects of the same salts on the formation of ganglioside adducts with dansyl-thyrotropin. Despite their ability to interact witw 125I-thyrotropin in solution, neither phosphatidylinositol, phosphatidylserine, nor phosphatidylethanolamine, when incorporated in a liposome, binds the 125I-labeled ligand. These same phospholipids have no effect on ganglioside binding of 125I-labeled thyrotropin when gangliosides are incorporated in a liposome. These phospholipids do, however, modulate the expression of the glycoprotein component of the thyrotropin receptor when it is imbedded in a liposome. The phosphatidylinositol in this case serves as a negative modulator, both by decreasing the incorporation of the glycoprotein component of the receptor into the liposome and by inhibiting the binding activity of the glycoprotein component which is incorporated. Speculation is offered as to a possible role of the phospholipids in the message transmission process which would be consistent with current studies demonstrating a direct interaction of acidic phospholipids with thyrotropin. The effect of phospholipids on liposomes containing the glycoprotein component of the thyrotropin receptor raises the possibility that phospholipids and, in particular, phosphatidylinositol, may also play a role in regulating the insertion and expression of this receptor component in thyroid plasma membranes.

Serum protein inhibition of thyrotropin binding to human thyroid tissue.

We used a modification of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum. TBI activity was found in both gamma-globulin and alpha-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic gamma-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a gamma-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH, receptor antibody in the serum of patients with Graves' disease.

Inhibition of thyrotropin binding to human thyroid plasma membranes by thyroid hormones and “reverse triiodothyronine”

Triiodothyronine, reverse triiodothyronine and thyroxine were found to inhibit 125I labelled thyrotropin binding to human thyroid plasma membranes in vitro. Both the thyrotropin binding and the effect of the above iodoamino-acids on this binding were pH, temperature and time dependent, 50% inhibition of thyrotropin binding was observed at 2×10−7M concentration of reverse triiodothyronine or thyroxine and at 1.1 × 10−6M concentration of triiodothyronine. The kinetic studies of thyrotropin binding revealed that the maximal capacity of receptor sites for the pituitary hormone is unaffected by the presence of thyroid hormones. On the other hand the association and dissociation constants for thyrotropin binding changed when iodoaminoacids were present in the incubation medium /Ka 8.13 × 107M−1 vs 1.6 × 108M−1 and Kd 1.14 × 10−8M vs 4.55 × 10−9M respectively, depending on the pH/. The double reciprocal plots showed competitive mechanism of inhibition. The present study suggest that triiodothyronine, reverse triiodothyronine and thyroxine are able to modify the thyrotropin binding to membrane receptors.

Thyroglobulin Inhibition of Thyrotropin Binding to Thyroid Plasma Membrane*

The effect of thyroglobulin (TG) on binding of TSH to thyroid plasma membranes was studied in vitro. Human and bovine thyroid plasma membranes have specific binding sites for bovine [125I]TSH. The binding of [125I]TSH is inhibited by the addition of purified TG (100 ng/microgram/ml). The inhibitory mechanism appears to be noncompetitive when subjected to Lineweaver-Burk analysis. Incubation of TG with TSH did not show an interaction, as assessed by sucrose gradient centrifugation. Plasma membranes prepared from human thyroid tissue have specific binding sites for human TG, as shown by [125I]TG binding assay. The TG binding was not affected by adding low concentrations of unlabeled bovine TSH. In the presence of very high concentrations of TSH, TG binding was increased. Hemoglobin, beta-lactoglobin, and ovalbumin did not have an inhibitory effect on [125I]TSH and [125I] TG binding to membrane preparations. Both [125I]TSH and [125I]TG binding were inhibited by 10 mM neuraminic acid. These results suggested that 1) TG released from thyroid gland may have a regulatory effect on TSH binding to its specific receptor, and 2) there are specific binding sites for TG on the thyroid plasma membrane.