IgG THYROTROPHIN RECEPTOR ANTIBODY ACTIVITY IN GRAVES'' DISEASE; A STUDY OF TSH AGONIST AND ANTAGONIST ACTIVITIES BY ISOELECTRIC FOCUSING

The distribution of TSH receptor antibody activity in the 7S and 19S fractions of Graves' sera has been re-evaluated. Serum fractions were obtained by gel filtration from 12 Graves' sera and assayed for TSH receptor binding activity in a radioreceptor assay. Thyroid stimulating activity was determined in a cultured porcine thyroid cell bioassay. In apparent contrast to the findings of Baker et al. (1983) TSH receptor binding activity was confined to the 7S gel filtration fraction, containing IgG, and was not detected in the 19S fraction, containing IgM. Similarly thyroid stimulating activity was detected only in the 7S fraction. 7S fractions from seven Graves' sera were fractionated by isoelectric focusing and the fractions analysed for TSH receptor binding activity and TSH agonist and antagonist activities. Five of the IgGs showed TSH agonist activity and in all five, the peak thyroid stimulating activity (measured by stimulation of cyclic AMP release from isolated porcine thyroid cells) was in fractions with a pI of between 8.0 and 9.5. In four of these five IgGs, TSH receptor binding activity showed similar isoelectric distribution to the thyroid stimulating activities. High levels of TSH receptor binding activity without associated TSH agonist or antagonist activity were however observed in some isoelectric fractions of the fifth stimulating Graves' IgG studied. All the isoelectric fractions from the fifth IgG with thyroid stimulating activities contained TSH receptor binding activity. Two of the Graves' IgGs showed TSH antagonist activity and both the TSH receptor binding and TSH antagonist activities of these IgGs showed similar isoelectric distribution with the peak activities at a pI of around 9.0. Consequently, it was not possible to separate TSH agonist or TSH antagonist activities from TSH receptor binding activity in seven Graves' sera by isoelectric focusing although in one IgG several isoelectric fractions contained isolated receptor binding activity. These findings are in keeping with the hypothesis that the biological activities of Graves' IgGs are intimately related to their ability to bind to the TSH receptor.     

The interaction between the TSH receptor and Graves' sera with TSH agonist or antagonist properties

The A subunit of the TSH receptor was prepared by reduction of human thyroid membranes with dithiothreitol, and partially purified by gel filtration. The ability of Graves' sera to inhibit TSH binding to the TSH receptor and to stimulate cyclic AMP release from isolated thyroid cells was abolished by incubation with crude and partially purified preparations of the A subunit.     

TSH RECEPTOR BINDING AND THYROID STIMULATION BY SERA FROM PATIENTS WITH GRAVES'' DISEASE

An investigation of the ability of TSH receptor antibodies to bind to the TSH receptor and stimulate thyroid function is described. Binding studies were carried out using 125I-labelled TSH and detergent solubilised porcine TSH receptors, and the parameter of thyroid stimulation employed was 125I-organification in isolated porcine thyroid cells. Different Graves' sera were found to show different dose-response relationships and three types of antibody activity were evident in the six samples studied. In two samples receptor binding and thyroid stimulating activities were approximately parallel. In two receptor binding was detectable at lower doses than stimulation while in the last two stimulation was detectable at lower doses than binding. It is proposed that these characteristics are due to different populations of receptor antibodies which exhibit different degrees of TSH agonism. Furthermore one of the reasons for the relatively poor correlation between TSH receptor binding and thyroid stimulation observed in series of individual Graves' sera may be the presence of different populations of antibodies showing different dose-response relationships with regard to binding and stimulation.     

THYROID STIMULATION BY (FAB)2 AND FAB FRAGMENTS OF TSH RECEPTOR ANTIBODY

The TSH receptor binding and thyroid stimulating properties of (Fab)2 and Fab fragments of Graves' IgG have been investigated. (Fab)2 fragments were prepared by pepsin digestion of IgG and Fab fragments by reduction of (Fab)2 or papain digestion of IgG. (Fab)2 and Fab were effective in inhibiting TSH binding to its receptor with all five patients' sera studied and both preparations stimulated cyclic AMP release from isolated thyroid cells. However Fab fragments were less active thyroid stimulators than their parent (Fab)2 in all five cases. These studies indicate that antibody divalency is not essential for thyroid stimulation by TSH receptor antibodies.     

COMPARISON BETWEEN THYROID STIMULATING AND TSH-BINDING INHIBITING IMMUNOGLOBULINS OF GRAVES'' DISEASE

In the sera of patients with Graves' disease abnormal thyroid 'stimulating' immunoglobulins have been demonstrated by in vivo and in vitro assay systems. Conflicting results have been reported when thyroid stimulating and thyrotrophin (TSH)-binding inhibiting activities have been compared. The present study was performed in forty-nine hyperthyroid Graves' patients to ascertain the relationships among TsH-binding inhibiting immunoglobulins (TBII), measured by a radio-receptor assay, thyroid stimulating antibody(TSAb), assayed by stimulation of adenylate cyclase-cAMP system in human thyroid plasma membranes, and LATS, measured by McKenzie's mouse bioassay.TBII was detected in twenty-one of forty-nine (42.9%), TSAb in thirty-five of forty-nine (71.4%) and LATS in nineteen of forty-nine (38.8%).TBII was also present in four of sixteen (25%) patients with other thyroid autoimmune disorders. When the results obtained with the different techniques were compared, correlation was found between LATS response and TSAb activity ( =0.53, P less than 0.001), while there was no correlation between TSAb and TBII activities and between LATS response and TBII activity. These data conform that TSAb is specific and sensitive marker of Graves' disease and suggest that TBII activity is not necessarily synonymous with thyroid stimulation, and could reflect a different phenomenon concomitantly produced.     

Inhibition of thyrotropin-stimulated adenylate cyclase activation and growth of rat thyroid cells, FRTL-5, by immunoglobulin G from patients with primary myxedema: comparison with activities of thyrotropin-binding inhibitor immunoglobulins

We studied the blocking type TSH receptor antibodies in 28 patients with primary myxedema and 21 patients with goitrous Hashimoto's thyroiditis by measuring the ability of their IgGs to inhibit TSH binding to its receptor, and to inhibit TSH-stimulated cAMP increase and [3H]thymidine incorporation in a rat thyroid cell line, FRTL-5. The incidences of TSH binding inhibitor immunoglobulin, thyroid stimulation inhibiting immunoglobulin and thyroid growth inhibiting immunoglobulin in patients with primary myxedema were 54.6, 75 and 65.2%, respectively, against 14.3, 0 and 17.7%, respectively, in goitrous Hashimoto's thyroiditis. The antibodies inhibited dose-dependently not only TSH stimulated but also Graves' IgG-stimulated cAMP increase and [3H]thymidine incorporation. The TSH binding inhibitor immunoglobulin activities in patients with primary myxedema were significantly correlated with both the thyroid stimulation inhibiting immunoglobulin (r = 0.665; P less than 0.01) and the thyroid growth inhibiting immunoglobulin (r = 0.618; P less than 0.01) activity. Thirteen patients whose TSH binding inhibitor immunoglobulin activities were more than 50% had both strong thyroid stimulation inhibiting immunoglobulin (75.1-100%) and thyroid growth inhibiting immunoglobulin (57.4-100%) activities. These data suggest that the vast majority of patients with primary myxedema have potent blocking type TSH receptor antibodies. These might play a role in primary myxedema causing hypothyroidism and thyroid atrophy through inhibiting TSH-stimulated cAMP generation.     

Thyroid-stimulating immunoglobulins. The related antigens

The presence of thyroid stimulating antibodies (TSAb) in patients with Graves' disease is well established. Considerable evidence has accumulated that these are antibodies to thyroid plasma membrane components related to the TSH receptor. The question of whether thyroid stimulation is mediated by a direct interaction with the TSH binding site is still debated. Recent data obtained by the use of monoclonal antibodies to the TSH receptor are consistent with the view that Graves' immunoglobulins comprise antibodies to at least two different components of the TSH receptor complex, one of which is more strictly related to the binding of TSH and the other to the transmission of the hormonal effect. The causative role of TSAb in the hyperthyroidism of Graves' disease is widely recognized. The use of human specific stimulation assays has circumvented the objection of the relatively low frequency of LATS-positive patients. Individual variations in the thyroid response may account for the lack of correlation between levels of thyroid stimulating immunoglobulins and most parameters of thyroid function. In this respect, the interference of non-stimulatory thyroid antibody and of other autoimmune mechanisms may be of importance. An important clinical implication of TSAb and TSH-binding inhibiting antibody determinations is their prognostic value in predicting the relapse of hyperthyroidism in treated patients. This clinical application has been so far limited by the technical difficulty of the assays. This emphasizes the need for a simple and reliable test, which can be used for routine measurements of TSAb.     

Stimulation of thyroidal iodothyronine 5'-monodeiodinase by long-acting thyroid stimulator (LATS)

To evaluate the effect of long-acting thyroid stimulator (LATS) on thyroid iodothyronine monodeiodinating activity, we have studied the in vitro conversion of T4 to T3 by mouse thyroid homogenate comparing tissue from LATS treated (0.1 ml LATS(+) serum, ip, for 3 days) with tissues from LATS(-) Graves' disease patients' serum or normal serum treated controls. Five out of seven LATS(+) sera were shown to stimulate the T4 5'-deiodinase significantly in mouse thyroid. There was no significant correlation between LATS titre and deiodinase activities in the different sera tested. To compare the effect of LATS and TSH (0.2 IU, ip daily), studies were carried out from 12 to 72 h. LATS had a similar latency of 12 h on the stimulation of thyroid deiodinase compared to TSH as reported earlier. However, the conversion activities reached a plateau by 12 h after LATS treatment, while it continued to rise upon daily TSH injection from 24 to 72 h. In addition, TSH caused a marked reduction of thyroid protein and an early peaking in serum T3 and T4 at 12 h, whereas LATS caused no detectable change in thyroid protein and a gradual rise in circulating T3 and T4. The kinetic analysis indicated that LATS-mediated stimulation of T4 5'-deiodinase was, similar to TSH, associated with an increase in maximum velocity (Vmax were 139, 208 and 505 pmol/mg protein/30 min respectively in control, LATS and TSH-treated animals) without a demonstrable change in the apparent Km (approximately 2.0 microM for T4). The present study demonstrated that some LATS-rich sera stimulate thyroid T4 to T3 conversion in mouse. It provides an insight into the mechanism of increased T3 secretion from Graves' thyroid glands.     

Alterations of adrenergic systems in thyroid slices from patients with Graves' disease

The responses to TSH of tissue cAMP levels in thyroid slices from patients with Graves' disease were significantly lower than those in normal thyroid slices. Conversely, tissue cAMP levels in thyroid slices from these patients were greatly increased by beta-adrenergic agonists, either isoproterenol or norepinephrine compared with those in normal thyroid slices. The elevation of cAMP levels induced by TSH in normal thyroid slices was significantly reduced by norepinephrine via alpha-adrenergic action as reported previously in canine thyroid slices, while such an elevation by TSH of cAMP levels in slices of Graves' disease thyroids was not inhibited, or rather increased by norepinephrine. These results indicate that, in addition to low responses to TSH, alpha- and beta-adrenergic systems were functionally altered in thyroid tissues of patients with Graves' disease.     

Heterogeneity of TSH receptor-binding antibodies in Hashimoto's thyroiditis and Graves' disease

The possible heterogeneity of TSH receptor antibodies in Graves' disease (GD) and Hashimoto's thyroiditis (HT) with respect to the binding site on the receptor and corresponding biological effect was studied. Employing an immunoprecipitation assay (IPA), the sera of 80% of the patients with GD (24 out of 30) and 76% of the patients with HT (16 out of 21) contained TSH receptor-binding antibodies, compared to none of the sera from 17 normal volunteers and 8 patients with nontoxic multinodular goiter. TSH inhibited immunoprecipitation by GD and HT sera. In HT sera (n = 9), but not in GD sera (n = 5), heterogeneity of the TSH-induced inhibition was observed. Four HT sera showed complete inhibition of immunoprecipitation at a saturating concentration (19.8 nM) of TSH. Five HT sera, like the 5 GD sera, showed partial inhibition of immunoprecipitation by 19.8 nM TSH. Thyroid stimulating immunoglobulins (TSI) were found in four of the five GD sera and in only one of the nine HT sera. The results suggest that different subpopulations of TSH receptor antibodies, characterized by other receptor binding sites or different affinities, are associated with autoimmune thyroid disease.     

Effects of human thyroid-stimulating hormone and immunoglobulins on adenylate cyclase activity and the accumulation of cyclic AMP in human thyroid membranes and slices.

The activation of adenylate cyclase and the accumulation of cyclic AMP resulting from the action of human thyroid-stimulating hormone (TSH), long-acting thyroid stimulator (LATS) or LATS-protector (LATS-P) have been investigated in preparations of human thyroid membranes and slices. Human TSH significantly increased adenylate cyclase activity in membranes from non-toxic goitres whereas LATS and LATS-P had no consistent effect. However, pre-incubation of goitrous membranes with LATS--immunoglobulin G inhibited the effect of TSH on adenylate cyclase. When thyroid membranes were prepared from the glands of patients with Graves's disease neither TSH nor thyroid-stimulating immunoglobulins (TSIg) stimulated adenylate cyclase significantly. Whether from non-toxic goitres or thyrotoxic tissue, the concentration of TSH needed to induce half of the maximum response was lower in thyroid slices than in membranes. Both LATS and LATS-P significantly stimulated the accumulation of cyclic AMP in slices of goitrous tissue but thyrotoxic tissue slices did not respond. In goitrous slices, submaximum concentrations of TSH and TSIg caused additive responses in the accumulation of cyclic AMP but TSIg did not increase the maximum response to TSH.     

Inhibition by immunoglobulin G of synthesis of thyroid hormone in thyroid cultures from hypothyroid patients with goitrous Hashimoto's thyroiditis

Recently, thyroid microsomal antigen was identified as thyroid peroxidase, and thyroid microsomal antibody was found to inhibit thyroid peroxidase activity in vitro. We investigated the possibility that anti-microsomal antibody inhibits the iodination of tyrosine, in vivo. Immunoglobulin G with or without anti-microsomal antibody from hypothyroid patients with goitrous Hashimoto's thyroiditis inhibited thyroid hormone synthesis in cultured slices of normal human thyroid tissue. IgGs with anti-microsomal antibody inhibited 125I thyroidal uptake and thyroid hormone synthesis stimulated by TSH more than normal IgG did. However, the same results were obtained with IgGs without anti-microsomal antibody. This effect did not involve anti-microsomal antibody, anti-thyroglobulin antibody, TSH-binding inhibitor immunoglobulin, thyroid stimulation-blocking immunoglobulin, or the cAMP level of the thyroid tissue. The ratio of organic I to inorganic I with stimulation by TSH in slices incubated with IgG from hypothyroid patients with goitrous Hashimoto's thyroiditis or normal IgG was not significantly different, but was significantly higher in slices incubated with methylmercaptoimidazole. Therefore, IgG from hypothyroid patients with goitrous Hashimoto's thyroiditis mainly suppressed 125I thyroidal uptake, rather than inhibiting thyroid peroxidase activity. In addition, this IgG was present in the serum of 11 of the 12 hypothyroid patients with Hashimoto's thyroiditis studied. This IgG may be involved in the mechanism that causes hypothyroidism in some patients with goitrous Hashimoto's disease.     

Thyroid antigen stimulates lymphocytes from patients with Graves' disease to produce thyroid-stimulating immunoglobulin (TSI).

Circulating lymphocytes from patients with Graves' disease and from control subjects were cultured in vitro alone, with normal human thyroid tissue homogenates, and with other nonthyroid human tissue homogenates. The supernatants of these cultures were assayed for human thyroid-stimulating activity by incubation with human thyroid slices in which increases in cAMP levels were then measured. Human thyroid stimulator activity was demonstrated in 16 out of 20 experiments in which lymphocytes from patients with active untreated Graves' disease (with hyperthyroidism) were cultured with normal thyroid homogenate, in 4 out of 17 experiments when control lymphocytes were similarly cultured, and in one out of 12 experiments in which the lymphocytes from the patients with Graves' disease were cultured with liver or gastric mucosa homogenate. Thyroid-stimulating activity was abolished by precipitation of the globulin from the supernatant by goat anti-human globulin serum. These results demonstrate that normal human thyroid tissue homogenates can specifically stimulate most lymphocytes from patients with Graves' disease and lymphocytes from a few normal subjects to produce human thyroid-stimulating immunoglobulins in vitro. This suggests that the human thyroid-stimulating immunoglobulins are auto-antibodies to normal thyroid constituents, but the possiblity that an antigenic change in the thyroid initiates the disease cannot be entirely excluded. The findings suggest that the prime change in Graves' disease is immunologic, perhaps a failure of immunological suppression.     

Detection and properties of TSH-binding inhibitor immunoglobulins in patients with Graves' disease and Hashimoto's thyroiditis.

TSH-binding inhibitor immunoglobulins (TBII) have been detected in patients with Graves' disease and Hashimoto's thyroiditis by using the radioreceptor assay of TSH. In untreated Graves' patients, TBII levels correlated well with thyroidal 99mTc uptake at 30 min and the grade of epithelial hyperplasia of thyroid follicles. There were many Graves' patients whose sera contained high TBII levels but no detectable bioassayable thyroid-stimulating activity (LATS), and in these patients, close correlation was observed between serum levels of TBII and bioassayable LATS-protector activity. TBII were detectable in 2 (10%) of 20 patients with Hashimoto's thyroiditis, both of whom were clinically hypothyroid. The serum or IgG fraction from one of them, however, did not contain any significant LATS, LATS-protector, or human thyroid adenylate cyclase-stimulating activity and caused inhibition of adenylate cyclase stimulation by TSH. In that patient, TBII may be acting to block TSH binding to TSH receptors, thus causing TSH unresponsiveness and hypothyroidism.     

Altered responsiveness to thyrotropin in thyroid slices of Graves' disease preoperatively treated with excess iodide.

In a previous paper, we demonstrated that the acute administration of excess iodide inhibits the adenylate cyclase-cAMP system in mouse thyroid lobes. In the present study, we examined whether presurgical therapy with stable iodide reduces the responsiveness to TSH in thyroid tissues from patients with Graves' disease. Eight patients with Graves' disease were presurgically treated with methimazole and stable iodide and six were given methimazole alone. Normal tissues from five patients with thyroid nodules were also tested. We have found that stimulation by TSH (5 and 50 mU/ml) of cAMP formation in thyroid slices from patients preoperatively treated with methimazole and iodide is significantly less than in slices from patients treated with methimazole alone. Similar observations were also made with other thyroid stimulators, such as prostaglandin E2 and 4-methylhistamine. Furthermore, thyroid slices from patients treated with methimazole alone responded to TSH to the same degree as slices of normal tissues. The data suggest that one of the reasons for the hyporesponsiveness to TSH in thyroids from patients with Graves' disease is preoperative treatment with stable iodide.     

DEMONSTRATION OF BLOCKING IMMUNOGLOBULINS G, HAVING A HETEROGENEOUS BEHAVIOUR, IN SERA OF PATIENTS WITH GRAVES'' DISEASE: POSSIBLE COEXISTENCE OF DIFFERENT AUTOANTIBODIES DIRECTED TO THE TSH RECEPTOR

Previous studies by us and others have shown that Graves' immunoglobulins G (IgGs) behaved as agonists or even antagonists of TSH. In this paper we have looked for the existence of IgG preparations without any thyroid stimulatory activity but able to significantly block the action of TSH in 128 hyperthyroid Graves' patients. The presence of TSH-binding inhibiting antibodies (TBIAb) and that of thyroid stimulating antibodies (TSAb) was evaluated by a radioreceptor assay using solubilized thyroid plasma membranes and by assaying the adenylate cyclase (AC) function of thyroid plasma membranes, respectively. Seventeen IgGs were negative for TSAb but positive for TBIAb in the screening, using only one concentration of IgG. Three kinds of activity were investigated in these IgGs at different doses: (1) TSH-binding inhibiting activity; (2) thyroid AC stimulating activity; and (3) the inhibition of TSH-induced AC stimulation. The results showed that the level of activity was not always dose-dependent. A significant (greater than 20%) inhibition of the TSH-dependent AC stimulation was present in 15 of the 17 IgGs examined: this inhibition was more elevated at lower than at higher doses in two preparations. No significant correlation was found between the three activities. In short, we have been able to demonstrate the existence of 'blocking' antibodies, apparently without any stimulatory activity, in some patients with Graves' disease. The diphasic pattern of the dose-response curves of some IgGs and the lack of correlation between the different activities can be explained by the co-existence in the sera of Graves' patients of different autoantibodies varying in concentration, binding affinity constant and intrinsic biological activity.     

Antithyrotropin antibodies in the sera of Graves' disease patients

To determine the presence and potential importance of antiidiotypic antibodies (anti-id) in the immune regulation of Graves' disease, sera from 57 patients with Graves' disease were screened before or during antithyroid therapy by enzyme-linked immunoabsorbent assay (ELISA) for presumptive anti-id, as defined by the presence of immunoglobulins (Igs) directed against TSH. The mean optical density, indicating the presence of TSH-binding antibodies, was 0.34 +/- 0.28 (+/- SD) in the sera of Graves' disease patients and 0.19 +/- 0.12 in the sera of 24 normal subjects (P less than 0.004). Control antigens (hCG and albumin) did not bind significant amounts of serum Igs. In 8 Graves' patients whose sera bound TSH, 40-80% inhibition was obtained with the addition of TSH receptor-purified IgG (approximately 1 microgram/ml) derived from a single Graves' patient's serum; no inhibition was found with normal IgG (approximately 10 micrograms/ml). Presumptive anti-id was isolated from sera of 6 Graves' patients by affinity purification with a TSH affinity column; the resultant IgG blocked immunoglobulin binding to the TSH receptor when added to the serum of the same patient from whom it had been isolated. The presence of anti-id correlated inversely with the presence of TSH receptor antibodies (r = -0.76; P less than 0.01). These studies demonstrate that 1) significant TSH binding is present in sera from Graves' disease patients, and 2) this TSH binding is specifically inhibitable by Graves' IgG, but not by normal IgG. These data support the hypothesis that TSH-binding immunoglobulins may represent anti-id that are present in Graves' disease as part of the immunological response to TSH receptor or TSH receptor antibodies. Such anti-id could modulate the expression of disease activity in Graves' disease by altering TSH receptor antibody action or production.     

An in vitro procedure for the estimation of thyroid hormone releasing factors in sera of thyrotoxic patients.

LATS containing sera and a number of Graves' disease sera stimulated T4 release from mouse thyroids in vitro as determined by RIA, thus confirming the presence of a thyroid hormone releasing factor in sera of thyrotoxic patients. The pattern of stimulation was similar to that previously shown for TSH in terms of T4 release time sequence. cAMP increase and catecholamine inhibition via alpha-adrenergic receptors. In the same in vitro system, neutralization with a human thyroid homogenate showed presence of LATS-Protector (LPA) in LATS negative thyrotoxic sera. The present study describes a simpler procedure for estimating LATS or similar activity, as compared to the McKenzie assay, and suggests identical receptor sites for TSH and other thyroid stimulators.     

T3-release from autonomously functioning thyroid nodules in vitro.

Toxic thyroid nodules have been shown to be of clonal origin. In a portion of them, point mutations affecting either the gene of the TSH receptor (TSHr) or the alpha-subunit of stimulating G-protein, consecutively leading to enhanced cAMP levels, which may enhance growth or functional activity of the thyrocyte or both, were recently found. To complement these studies, we evaluated hormone response (i.e. T3 release) in vitro from tissues derived from toxic thyroid nodules as compared directly to the surrounding paranodular tissues as well as tissues derived from euthyroid goiter and from patients with Graves' disease. Experiments were conducted in the presence and absence of bTSH or Graves' immunoglobulines. Tissues obtained during surgery were incubated over 5 h, followed by equilibrium dialysis for 24 h, and determination of free T3 in an aliquot by RIA. Basal T3 release in nodular tissues (n = 10) was significantly higher (median: 7.3 ng/l) compared to paranodular tissues (3.2 ng/l; P < 0.01), tissues derived from euthyroid goiter (1.3 ng/l; n = 12; P < 0.001) and thyroid tissues derived from patients with Graves' disease (2.5 ng/l; n = 6; P < 0.001). Upon stimulation with bTSH (1 IU/l), median T3 concentrations markedly increased to 11.5 ng/l (P < 0.05), 7.3 ng/l (P < 0.05), 4.2 ng/l (P < 0.01) and 3.2 ng/l (P = N.S.), respectively. Stimulation over basal values was 1.6-fold in nodular tissues, 2.3-fold in paranodular tissues, 3.2-fold in euthyroid goiter and 1.3-fold in Graves' disease. In toxic thyroid nodules basal hormone-releasing activities were stimulated by fifteen out of twenty (75%) Graves' sera tested. For comparison, stimulation in other tissues occurred in 45% (paranodular), 80% (euthyroid goiter) and 35% (Graves' disease), respectively. In conclusion, tissue derived from toxic thyroid nodules exhibits enhanced basal hormone release as compared to both, the surrounding paranodular tissues and tissues from euthyroid goiter in vitro, which may reflect constitutional activation of TSHr, alpha-subunit of stimulating G-protein or other so far unknown intermediate by point mutations affecting the respective genes. Hyperactivities in toxic thyroid nodules may be even further enhanced by external stimulators such as TSH or TSH receptor antibodies. The first stimulator may have clinical relevance in patients with toxic thyroid nodules and not yet suppressed TSH; the latter could play a role in the rare Marine Lenhart syndrome.     

Immunoglobulins of patients recovering from Yersinia enterocolitica infections exhibit Graves' disease-like activity in human thyroid membranes.

Substantial evidence suggests a link between infections with Yersinia enterocolitica (YE) and Graves' disease. We have now examined the sera of 72 patients recovering from YE infection for immunoglobulins that interacted with the TSH receptor in human thyroid membranes. Compared with controls, in concentrations between 1 and 4 mg/mL, patient IgG produced a significant, concentration-dependent inhibition of TSH binding (p less than 0.001) and stimulation of adenylate cyclase activity (p less than 0.005-0.05). Whereas IgG from normal individuals caused no stimulation of adenylate cyclase, IgG from controls caused some concentration-dependent displacement of TSH, as previously reported. However, IgG from convalescents of YE infections was significantly more potent than normal IgG in reducing the binding of TSH to the membrane. Thus, at each examined concentration, YE patients' IgG displaced more TSH than IgG from normal controls. For each milligram per milliliter increment of IgG in the assay, patients' IgG caused a 10.2% inhibition of TSH binding (r -0.90, p less than 0.001), significantly greater than that seen with normal IgG (p less than 0.02). The present studies provide the first demonstration that IgG of patients recovering from YE infections react with the human TSH receptor. The antibodies presumably are produced against the TSH-binding protein present in YE. However, in view of lack of evidence for thyroid dysfunction in the sera of patients recovering from yersiniosis and the presence of TSH-binding proteins in other bacteria, we postulate that infection with YE is neither necessary nor sufficient to cause thyroid autoimmune disease.(ABSTRACT TRUNCATED AT 250 WORDS)