Thyroid-stimulating antibody mimics thyrotropin in its ability to desensitize the adenosine 3',5'-monophosphate response to acute stimulation in continuously cultured rat thyroid cells (FRT-L5).

TSH preincubation induces refractoriness to further stimulation of cAMP accumulation by the hormone in cultured thyroid cells. The question of whether the thyroid-stimulating antibodies (TSAb) present in sera of patients with Graves' disease have the same desensitizing effect as TSH was evaluated in this study using a differentiated strain of rat thyroid cells (FRT-L5) which requires TSH for growth and develops refractoriness to acute TSH stimulation of cAMP accumulation. Cells cultured for 4-5 days in medium deprived of TSH recovered responsiveness to TSH and TSAb. Refractoriness to TSH was reinduced in these cells by a 24-h preincubation with 250 microU/ml TSH. The addition of 100 microM KI or 100 microM methimazole to the medium together with TSH did not modify TSH-induced refractoriness. Four different TSAb preparations all induced refractoriness to TSH-stimulated cAMP accumulation. TSAb preincubation induced refractoriness to acute TSAb as well as TSH-stimulated cAMP accumulation. TSAb-induced desensitization was dose dependent and required prolonged (8-24 h) exposure to TSAb. Furthermore, the desensitization induced by TSAb as well as that caused by TSH were significantly inhibited by cycloheximide, indicating the need for de novo protein synthesis. In conclusion, TSAb mimics TSH in the induction of refractoriness in FRT-L5 cells.

Expression of integrins of the beta1 family in thyroid cells from patients with Graves' disease in vivo and in vitro.

The expression of the beta1 family of integrins was determined in thyroid follicular cells from patients with Graves' disease (GD). Integrin expression was quantitated by flow fluorocytometry of single cell suspensions with antibodies against the common beta1 chain and the alpha1-alpha6 subunits. Results indicated that also in thyroid glands of GD, as previously observed in nodular goiters, two follicular cell populations with different patterns of beta1 integrin expression coexist (VLAalpha3beta1 and VLAalpha1,3,5,6beta1). The VLAalpha1,3,5,6beta1 thyrocyte population in GD was more abundant than in nodular goiters, ranging from 40 to 70% of the total follicular cells and the overall expression of the beta1 integrins was a two-fold higher. In thyrocytes from patients with GD cultured in vitro, alpha3 and alpha2 expression was regulated by cell-to-cell contact as previously described in normal thyroid cells, while the expression of alpha1, alpha5 and alpha6 was quickly lost during the culture. Our data suggest that the integrin profile of the VLAalpha1,3,5,6beta1 thyrocyte population in GD is induced by micro-environmental conditions rather than being the expression of a constitutive phenotype.

Assays of TSH-receptor antibodies in 576 patients with various thyroid disorders: their incidence, significance and clinical usefulness.

UNLABELLED: The incidence and the significance of TSH-receptor antibodies in Graves' disease and in various thyroid disorders have been evaluated. TSH-binding inhibiting antibodies (TBIAb) and thyroid stimulating antibodies (TSAb) were detected in a large proportion of Graves' disease patients (TBIAb in 68.8% and TSAb in 77.8%), in a small number of patients with idiopathic myxoedema or Hashimoto's thyroiditis, and were not detected in patients with endemic euthyroid goitre, differentiated thyroid carcinoma and toxic adenoma. Furthermore, TSH-receptor antibodies were present in some patients with toxic multinodular goitre (TBIAb in 12.7% and TSAb in 15.9%). When TSH-receptor and other thyroid autoantibodies were compared, it was found that 13 of the 15 Graves' patients with negative tests for thyroglobulin and thyroid microsomal antibodies were positive for TSH-receptor antibodies. On the other hand, 9 of the 11 patients with toxic multinodular goitre who had positive TSH-receptor antibody tests, also had serum thyroglobulin and/or thyroid microsomal antibodies. No significant differences in the prevalence of TSH-receptor antibodies were found in Graves' patients irrespective of the presence of ophthalmopathy or pretibial myxoedema. Elevated TBIAb activity at the end of anti-thyroid drug treatment was found in 52.9% of Graves' patients who subsequently relapsed, while in Graves' patients in remission TBIAb was always negative. TSH-receptor antibody results were not predictive of the outcome of radioiodine treatment in Graves' disease. Finally no correlation could be found between TBIAb and TSAb in Graves' disease and Hashimoto's thyroiditis. IN CONCLUSION: the high incidence of TSH-receptor antibodies in Graves' disease confirms their pathogenetic role in the development of hyperthyroidism; TSH-receptor antibodies in Graves' disease are not significantly associated with the presence of ophthalmopathy or pretibial myxoedema; TSH-receptor antibody assays may be useful for the diagnosis of Graves' disease in the absence of other signs of autoimmunity. TBIAb seems to be a good predictor of relapse in Graves' patients treated with anti-thyroid drugs; a fraction of toxic multinodular goitre could be a nodular variant of Graves' disease.

Intercellular adhesion molecule-1 is upregulated via the protein kinase C pathway in human thyroid carcinoma cell lines.

Nonantigen specific adhesion systems lymphocyte function-associated antigen 1/intercellular adhesion molecule (LFA-1/ICAM-1) and cluster designation 2/lymphocyte function-associated antigen 3 (CD2/LFA-3) are considered a crucial step in immune-mediated cell-cell adhesion reactions. In particular, the LFA-1/ICAM-1 system is deeply involved in major histocompatibility system (MHC)-restricted and non-MHC-restricted cellular cytotoxicity of effector cells against cancer tissues. We have investigated in human thyroid carcinoma cell lines the role of the protein kinase C (PKC) pathway on ICAM-1 expression. Incubation with tissue plasminogen activator (TPA), an agonist of PKC, of two papillary (NPA and TPC-1) and one anaplastic (ARO) carcinoma cell lines induced an ICAM-1 upregulation of both protein and mRNA production. This phenomenon was dependent on RNA and protein synthesis and was inhibited by PKC antagonists such as staurosporine and H-7. A parallel increase in the soluble form of ICAM-1 followed the upregulation of cellular ICAM-1 levels induced by TPA. In conclusion, the PKC pathway is involved in the regulation of ICAM-1 expression in human thyroid carcinoma cell lines. Further studies are necessary to clarify the effects of the PKC pathway on the diffusion of thyroid tumors.

[Treatment of hot thyroid nodule with percutaneous ethanol injection. Our experience]. / Trattamento del nodulo caldo tiroideo mediante iniezione percutanea di etanolo. Nostra esperienza.

Thyroid nodule is defined as "hot" on the bous it scintigraphic appearance. It can be defined like benign nodule with autonomous functionality. On scintiscan it shows high captation because there's an increase of production and secretion of thyroid hormones with total inhibition of TSH and suppression of extranodular tissue. Generally the treatment of hot thyroid nodule was surgical or with radio-metabolic therapy. Percutaneous ethanol injection (PEI) in the treatment of hot thyroid nodules has been suggested recently. The aim of our study was to value therapeutic effects of PEI under guidance by means of ultrasound in patients with hot nodules in toxic or pretoxic phase. 36 patients with autonomous thyroid nodules of 1.8-6 cm in diameter received sterile ethanol at 95% that has been injected with a 22-gauge needle and a probe with a guide device. The administrated dose varied from 1.2 to 1.5 ml for cc of tissue in 5-12 sessions. Ethanol injection was performed in a single site in nodules with diameter < 3 cm and in double sites in nodules with diameter > 3 cm. The patients were checked after treatment and then 3 and 6 months. Our experience confirms an excellent response to PEI in these patients. In fact after therapy symptoms of hyperthyroidism and hormonal levels become normal; no patient has even reached the range of subclinical or clinical hypothyroidism. Scintigram showed that previously suppressed thyroid tissue resumed functioning; at ultrasound all nodules had shrunk: thyroglobulin levels increased during treatment because ethanol induces coagulative intranodular necrosis with release in systemic circulation of this glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

[Thyroid-stimulating immunoglobulins. The related antigens]. / Immunoglobulines thyréostimulantes. Antigènes correspondants.

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.

Role of the adenylate cyclase-cAMP system on TSH-stimulated thyroid cell growth.

TSH is a trophic factor for cultured rat thyroid cells (FRTL-5). In the present study we have investigated the mechanism by which TSH promotes cell growth and evaluated the possible role of the adenylate cyclase (AC)-cAMP system in this process. The mitogenic activity of several agents was evaluated by measuring their effect on cell number or 3H-thymidine incorporation into DNA. Forskolin and cholera toxin, two potent and specific activators of the AC, induced a dose dependent increase of 3H-thymidine incorporation. The maximal stimulation, observed at concentrations of 10 microM and 10 ng/ml, respectively, was beta 80% of that obtained with optimal concentrations of TSH. A similar effect was obtained with a Graves' IgG preparation (0.2 mg/ml) able to stimulate the thyroid AC or with 3-isobutyl-1-methyl-xanthine (IBMX, 0.5 mM), a phosphodiesterase inhibitor. 8-bromo cAMP (0.5 mM), a cAMP analog, also stimulated 3H-thymidine incorporation, and its potency was approximately 60% of that of TSH. Similar results were obtained when the mitogenic activity of these compounds was evaluated by cell number. Norepinephrine (NE, 10 microM), although devoid of AC stimulatory activity in these cells, also stimulated 3H-thymidine incorporation, but its potency was only 20-30% of that of TSH. Indomethacin (100 microM), an inhibitor of phospholipid and arachidonic acid metabolism, was able to inhibit the stimulatory effect of NE (84%), and to a lesser extent of TSH (63%) and cholera toxin, had minor effect on forskolin (24%), IBMX (16%) and Graves' IgG (8%), and no effect on 8-bromo cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Can the inhibition of prostaglandin synthesis affect the adenylate cyclase response to thyroid stimulating antibodies?

The majority of Graves' immunoglobulins have the capacity to stimulate thyroid adenylate cyclase (AC) activity in vitro. So far, the exact events leading to AC activity stimulation by thyroid stimulating antibody (TSAb) are not known. It has been suggested that TSAb activates thyroid AC through prostaglandin (PG) synthesis, implying the concept that TSH and TSAb stimulate AC in different ways. If this is so, the inhibition of PG synthesis should notably reduce the response of thyroid plasma membrane to TSAb. We have, therefore, investigated the effect of 2 inhibitors of PG synthesis on the TSAb-stimulated AC activity in a crude human thyroid plasma membrane preparation. Neither indomethacin, nor hydrocortisone, even at concentrations able to completely inhibit PG synthesis, had any significant effect on the thyroid plasma membrane response to TSAb. The results suggest, therefore, that, at least under our experimental conditions, PG does not mediate the AC activation by TSAb. Consequently, the aforementioned suggestion should not be used to claim that TSH and TSAb activate thyroid AC through different pathways. The data, however, do not exclude that TSAb may determine PG synthesis in thyroid cells through the phosphatidylinositol signal system (Pl system).

T cell subsets and thyroid-stimulating antibodies in patients with Graves' disease in clinical remission.

Patients with active Graves' disease almost constantly show phenotypic alterations of T lymphocytes, such as an increase of "activated" cells recognized by various surface markers (e.g. la antigens). Such alterations are present in a certain number of apparently cured patients. The data herein reported refer to 25 patients with Graves' disease in clinical remission, in whom we have attempted to correlate T cell subset imbalances, the presence of thyroid-stimulating antibodies (TSAb) and the outcome of the subsequent relapse. The results obtained show a significant association between TSAb and the increase of la-positive T cells: no relationship was found between TSAb and other T lymphocyte subsets. One-year clinical follow-up of the patients enabled us to see relapses of hyperthyroidism in only two patients, who had shown in the first control both TSAb positivity and increased la-positive T cells. These results, in our opinion, suggest a role of la antigens expression on T lymphocytes in the clinical course of Graves' disease.

Thyroid-stimulating immunoglobulins.

The presence of thyroid-stimulating immunoglobulins in patients with Graves' disease is well established. Considerable evidence has accumulated that these immunoglobulins are antibodies to a thyroid plasma membrane antigen whose precise nature remains to be identified. The question whether the antigen is related to the TSH receptor is still debated. The causative role of thyroid-stimulating immunoglobulins 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 long-acting-thyroid-stimulator-(LATS)-positive patients. Individual variations in the thyroid response may account for the lack of correlation between the levels of thyroid-stimulating immunoglobulins and most parameters of thyroid function. In this respect, the interference of nonstimulatory thyroid antibodies and of other autoimmune mechanisms may be of importance. An important clinical implication of thyroid-stimulating immunoglobulin determinations is their value in predicting the relapse of hyperthyroidism in treated patients. This clinical application has been so far limited by the technical difficulties of the assays. This emphasizes the need for a simple and reliable test, which can be used for routine measurements of thyroid-stimulating immunoglobulins.

Graves' disease and Turner's syndrome.

A case of a 17-year-old female with Turner's syndrome and Graves' disease is reported. The karyotype analyzed in peripheral blood lymphocytes showed a 45,X0 pattern without mosaicism. The diagnosis of Graves' disease was based on the presence of diffuse goiter and appropriate laboratory data, including elevated thyroid radioiodine uptake, increased serum thyroxine and free thyroxine index, detectable thyroid-stimulating antibody and elevated thyroid microsomal antibody titer. Hyperthyroidism was first recognized when the patient was 13-year-old and treatment wih carbimazole was instituted for 18 months. Relapse of hyperthyroidism occurred 9 months after withdrawal of therapy, and a second course of antithyroid drug treatment was given for two yr. Ovarian dysgenesis has been described with a relatively high frequency in patients with Hashimoto's thyroiditis, while the association with Graves' disease has been only occasionally encountered. This finding is surprising in view of the etiopathogenetic and genetic relationships between these two thyroid autoimmune disorders. Some possible explanations are offered to clarify this problem.

A sensitive method for assaying thyroid stimulating immunoglobulins of Graves' disease: use of the guanyl nucleotide-amplified thyroid adenylate cyclase assay.

The purpose of this study was to develop and validate a sensitive method for evaluating adenylate cyclase stimulation by thyroid-stimulating antibodies (TSAb), based on the measurement of thyroid membrane adenylate cyclase activity in the presence of a non-hydrolyzable GTP analogue, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). The addition of Gpp(NH)p (10(-5)M) produced a 10-fold increase of the sensitivity of the system for both TSH and TSAb. Immunoglobulin G preparations from sera of 30 patients with Graves' disease were tested for the adenylate cyclase stimulation either in the presence or in the absence of Gpp(NH)p: a significant stimulation was observed in 27/30 patients when the GTP analogue was added to the system, while only 20/30 patients were positive in the absence of the nucleotide. The advantage of Gpp(NH)p addition was also evident in a large series which included 57 patients with Graves' disease, 15 with Hashimoto's thyroiditis or primary myxoedema and 22 normal subjects. In fact, 88% of patients with Graves' disease resulted positive, while no significant stimulation was elicited by Hashimoto's thyroiditis, primary myxoedema and by normal immunoglobulins. The sensitivity achieved in our system which employs thyroid plasma membranes was similar to that obtained by other investigators with the use of thyroid slices or thyroid cells in primary culture. Furthermore, methods based on thyroid plasma membranes are supposed to have a better reproducibility, since the same tissue preparation, if appropriately stored, may be used in several different tests.

Simultaneous assay of thyroid adenylate cyclase- and growth-stimulating antibodies using FRTL-5 cells. Evidence suggesting their identity in patients with Graves' disease.

The relationship between thyroid growth-stimulating antibodies (TGSAb) and thyroid adenylate cyclase-stimulating antibodies (TSAb) in patients with Graves' disease is still a matter of controversy. To investigate this problem, we have developed an assay for the simultaneous measurement of TSAb and TGSAb using FRTL-5 cells. TSAb was detected by its ability to stimulate iodide (I-) uptake and TGSAb by the 3H-thymidine ([3H]-Tdr) incorporation assay. Thirty-four immunoglobulin G (IgG) preparations from patients with active Graves' disease were selected from a previous series in order to include both TSAb-negative IgGs (n = 9) and TSAb-positive IgGs (n = 25) by the cAMP stimulation assay, with a wide range of stimulatory activity. With one exception, the TSAb-positive IgGs produced a significant stimulation of I- uptake; 20 of them were also TGSAb-positive. The nine IgGs negative in the cAMP assay, were also negative in the I-uptake and the [3H]-Tdr incorporation assays. The majority of samples had a similar potency in the two assays and a significant positive correlation was found (r = 0.76; P less than 0.001). Two IgGs previously shown to inhibit TSH-stimulated adenylate cyclase in FRTL-5 cells produced an almost complete inhibition (80-90%) of both TSH- and Graves' IgG-stimulated I- uptake and [3H]-Tdr incorporation. In conclusion, using a simultaneous assay for thyroid growth and adenylate cyclase stimulation, TGSAb in Graves' patients were found only in TSAb-positive IgGs; both Graves' IgG-stimulated activities were inhibited by antibodies blocking the TSH-dependent adenylate cyclase stimulation. Our data strongly suggest that the same antibody may be responsible for both goitre and thyroid hyperfunction of Graves' disease.