Thyroid Growth Stimulating Immunoglobulins Research Articles

Assessment of thyroid growth stimulating activity of immunoglobulins from patients with autoimmune thyroid disease by cytokinesis arrest assay.

To develop a novel bioassay for the assessment of thyroid cell growth stimulating activity using cytochalasin B (CB) and to test immunoglobulins (IgGs) from patients with autoimmune thyroid diseases. The assay is based on the principle that growing cells during incubation with CB show an increased number of nuclei in a cell (N/C index), since CB, at appropriate concentrations, is known to inhibit cytoplasmic cleavage without affecting nuclear mitosis. The N/C index represents potential DNA production while cells are incubated with CB. FRTL-5 thyroid cells were incubated with various thyroid stimulators in TSH-free medium containing 2 mg/J CB for 3 days. After the incubation, the cells were harvested in trypsin/EDTA to obtain single cell suspension, fixed, dropped onto a glass slide, stained and observed under a microscope to determine the N/C index. Bovine TSH at 10(-3)-1.0 U/I, forskolin at 1x10(-7)-10(-5) mol/l, cholera toxin at 10x10(-5)-10(-3) mg/l, or (Bu)2cAMP at 1 x 10(-5)-10(-3) mol/l increased the N/C index up to approximately 2.0 in a dose-dependent manner. IgGs not only from 27 patients with untreated goitrous Graves' disease but also from 14 patients with goitrous Hashimoto's thyroiditis elicited an increase in the N/C index, which exceeded the mean + 2 S.D. of the values for 17 normal subjects (mean +/- S.D., 1.063 +/- 0.014). Four patients with primary myxedema displayed a normal N/C index. In Graves' disease, the N/C index did not correlate significantly with thyroid stimulating antibodies (TSAb) activities but did correlate significantly with estimated goiter size (P < 0.05). IgGs containing blocking-type TSH-receptor antibodies inhibited the TSH- or Graves IgG-stimulated increase in N/C index almost completely, but did not influence the stimulatory effect of IgG from two patients with Hashimoto's thyroiditis. We have developed a sensitive and simple assay for thyroid growth stimulating activity by using CB, and found that all tested patients with goitrous Graves' disease and goitrous Hashimoto's thyroiditis have thyroid growth stimulating immunoglobulins whose activity does not correlate with TSAb.

The effect of treatment with levothyroxine or iodine on thyroid size and thyroid growth stimulating immunoglobulins in endemic goitre patients

We assessed the effect of levothyroxine or iodine on thyroid size and on thyroid growth stimulating immunoglobulins in endemic goitre patients. Levothyroxine or iodine was given orally in an open randomized prospective study (100 and 200 micrograms respectively). Thirty-seven euthyroid patients with diffuse iodine deficiency goitres and thyroid growth stimulating immunoglobulins were studied. Thyroid size, thyroid growth stimulating immunoglobulins (mitosis arrest assay), basal TSH, free T3, free T4, thyroid anti-microsomal antibodies, antithyroglobulin antibodies, anti-TSH receptor antibodies and urinary iodine excretion were measured. Thyroid size decreased significantly in both groups, in the levothyroxine group more than in the iodine treated group. Thyroid growth stimulating immunoglobulins levels also decreased significantly in both groups. Between groups there was no statistically significant difference. A statistically significant correlation between thyroid growth stimulating immunoglobulins reduction profiles and goitre size reduction could not be established. TSH levels became suppressed in the levothyroxine group while the T4 values rose; in the iodine treated group TSH levels stayed constant as did T4. None of the patients developed thyroid microsomal or thyroglobulin auto-antibodies and/or hyperthyroidism during the treatment. Levothyroxine as well as iodine was effective in reducing thyroid size as well as thyroid growth stimulating immunoglobulins levels in endemic goitre patients. Since in both groups TSH levels were not related to thyroid size reduction, other factors than TSH suppression must be responsible for the observed thyroid size reduction. Iodine itself by virtue of its antiproliferative action on thyrocytes may have had a direct action on the goitre reduction during iodine treatment; however, the levothyroxine dose, containing less iodine, had a similar effect. A complicated picture hence emerges with regard to factors involved in the shrinkage of iodine deficiency goitre during thyroxine or iodine therapy. These findings indicate that TSH and thyroid growth promoting immunoglobulins are not the only influences on the size of endemic goitres, although it cannot be excluded that these two factors contribute to influence the pathogenetic process.

Induction of c-fos and c-myc mRNA expression by immunoglobulin G from patients with Graves’ disease in thyrotrophin-dependent rat thyroid cell line (FRTL5)

The present study was conducted to evaluate effects of autoantibodies in patients with Graves' disease on induction of c-fos and c-myc mRNA expression in rat thyroid cell line (FRTL5). IgG fractions were isolated from 11 patients with Graves' disease, and six healthy subjects, with protein A-Sepharose. FRTL5 cells which had been grown to subconfluency and deprived of TSH for a week were exposed to the IgG for an hour. Expression of c-fos and c-myc mRNAs was examined by the Northern blot method using nick-translated v-fos and c-myc probes. C-fos and c-myc transcripts were induced by IgGs from two patients with Graves' disease, which displayed much higher activities in assays for TSH binding inhibitor immunoglobulins, thyroid stimulating antibodies and thyroid growth-stimulating immunoglobulins, assessed by measuring inhibition of 125I-TSH binding to the TSH-receptor, cAMP production and 3H-thymidine incorporation in FRTL5 cells, respectively, compared with those in the remaining patients. The induction of c-fos and c-myc mRNAs by IgG from a patient with Graves' disease was suppressed by preincubation with IgGs from two patients with primary myxoedema who were known to have a blocking type TSH-receptor antibody. These data suggest that the binding of the antibodies to the TSH-receptor followed by cAMP production is related to the induction of c-fos and c-myc mRNAs and, thus, to the growth of FRTL5 cells. To our knowledge, this is the first report demonstrating that autoantibodies induce proto-oncogene mRNA expression.

Thyroid autoimmunity in endemic goitre caused by excessive iodine intake.

The pathophysiology of endemic goitre caused by excessive iodine intake is not well defined. By interacting with the immune system, iodine excess may trigger the development of autoimmune thyroid disease such as lymphocytic Hashimoto's thyroiditis (LT). In an attempt to examine this further, we compared the presence of thyroid autoantibodies in 29 goitrous children, from an iodine excess area, and in 26 healthy children, from an iodine sufficient area, of north central China. Serum was tested for antimicrosomal (MAb), anti-thyroglobulin (TgAb), second colloid antigen antibodies (CA2-Ab) and TSH binding inhibitory immunoglobulins (TBII). Affinity chromatographically purified IgG was tested for thyroid growth-stimulating activity (TGI) by two different methods: a sensitive cytochemical bioassay (CBA) using guinea-pig thyroid explants and a mitotic arrest assay (MAA) employing a continuous rat thyroid cell line (FRTL-5). We found no increased prevalence of LT in patients with endemic iodine goitre. The levels of MAb, TgAb and CA2-Ab did not differ significantly between the two groups of children. Further, TBII were not present in either group. Thyroid growth-stimulating immunoglobulins (TGI) were the major autoantibodies found in children with goitres caused by iodine excess. In the CBA, 12 of 20 (60%) goitrous children and 0 of 12 (0% P less than 0.05) healthy children were positive for TGI. Similar results were found in the MAA, and a good correlation between results of the CBA and MAA was found (P = 0.003). Maximal TGI activity in dose-response CBA showed a good relation with clinical goitre size (r = 0.63; P less than 0.05) indicating a possible pathophysiological role for these antibodies. We conclude that endemic iodine goitre is not associated with Hashimoto's lymphocytic thyroiditis. Nevertheless, autoimmune growth factors such as TGI may play a primary role in the pathogenesis of thyroid growth in this condition.

Evidence that thyroid growth promoting activity of immunoglobulin preparations is due to contamination with EGF.

Immunoglobulin (IG) preparations may be contaminated with growth factors. Therefore, we investigated whether the growth promoting activity in IG preparations (thyroid growth stimulating immunoglobulins = TGI) from patients with sporadic goitre may be caused by contaminating EGF (epidermal growth factor). EGF in sera as well as in indifferently prepared IG of patients with recurrent goitre (n = 23), Graves' disease (n = 19) and normals (n = 17) was determined by EGF receptor assay. Comparatively, the ability for stimulating thyroid cell growth was determined in these IG preparations (2 mg/ml). EGF in ammoniumsulphate (AS) precipitates was about 2-fold higher than serum EGF. The growth promoting activity of indifferent IG preparations correlated with the EGF content. After additional purification on protein A-sepharose, neither EGF, nor a growth promoting activity was found in these IG preparations. We therefore conclude, that the growth promoting activity of crude IG preparations may be due to a contamination with EGF.

Stimulation of thyroid cell growth by thyrotropin and epidermal growth factor in isolated porcine thyroid follicles

[3H]thymidine incorporation during culture of thyroid follicles isolated from porcine glands (Schatz et al. 1983, 1986) can be taken for estimating thyroid growth stimulating immunoglobulins (TGI) (Drexhage et al. 1980; Chiovato et al. 1983; Valente et al. 1983). In this study we wanted to characterize our assay system in order to answer the question whether TSH itself was responsible for stimulation of [3H]thymidine incorporation observed when using TSH as standard in our experiments. Furthermore, the effect of TSH and epidermal growth factor (EGF) (Eggo et al. 1983; Westermark et al. 1985) should be tested both without and with anti-TSH serum or anti-EGF serum present in the medium.

Pathophysiological and clinical implications of thyroid growth-stimulating immunoglobulins: evidence for their intrathyroidal production.

Thyroid growth-stimulating immunoglobulins are responsible for goitre formation not only in Graves' and Hashimoto's disease but also (partly) in simple goitre, especially in recurrent cases. TGI are antibodies, the TSH receptor, however, appears not to be the antigenic site in simple goitre. In our own studies two TGI assay systems ([3H]thymidine incorporation into isolated porcine thyroid follicles and into FRTL-5 cells) gave comparable results. TGI was found in 20 out of 72 patients with simple goitre (27%) coming from an endemic goitre area (biased by selection, i.e. certainly including sporadic forms) and in 10 out of 26 patients (38%) with recurrent goitre. TGI levels were higher in thyroidal venous blood compared to cubital blood (simultaneously drawn during surgery) indicating an intrathyroidal production of TGI. Thyroid growth-blocking antibodies as estimated in the FRTL-5 cell assay in one of our patients might be of significance not only in primary myxoedema but also for congenital hypothyroidism.

Workshop on thyroid growth-stimulating immunoglobulins A report

Workshop on thyroid growth-stimulating immunoglobulins A report

Evidence for intrathyroidal production of thyroid growth-stimulating immunoglobulins

Evidence for intrathyroidal production of thyroid growth-stimulating immunoglobulins

Further evaluation of an immunoprecipitation assay for TSH-receptor autoantibodies in Graves' disease

In ten patients with untreated Graves' disease, quantitative titers of TSH receptor antibodies, as measured by a recently developed immunoprecipitation assay (IPA), were correlated with results obtained in three other methods and with the severity of the hyperthyroidism, as assessed by thyroid function tests. Nine patients were positive in the IPA, six in the TBII (TSH-binding inhibitor immunoglobulins), six in the TSI (thyroid-stimulating immunoglobulins), and seven in the TGI (thyroid-growth stimulating immunoglobulins) assay. Three patients were positive in all four assays. No correlation was found between the IPA values and the results obtained in the TBII, TSI, or TGI assays. There was a modest correlation between the TBII and TSI assays ( r = .74, p < 0.02). There was a modest but significant correlation between the IPA titers of TSP receptor antibodies and serum T 3 concentration, both before treatment ( r = .63, P < 0.05) and during treatment (n = 5; r = .84, P < 0.05). No correlation between the severity of the hyperthyroidism and TBII, TSI, or TGI assays was observed. Finally, TGI did not correlate with goiter size as estimated by palpation. These results suggest that the IPA may be useful in monitoring the immunologic activity of TSH receptor antibodies in patients with Graves' disease. They suggest further that the IPA detects a number of different subpopulations of TSH receptor antibodies, including TSI, TBII, and perhaps TGI. This property may be particularly useful in screening for monoclonal TSH receptor antibodies.

The prevalence of immunological abnormalities in endemic simple goitre.

Thyroid growth stimulating immunoglobulins microsomal antibodies and antibodies against thyroglobulin were determined in patients with simple goitre (n = 20) and controls (n = 6) living in an iodine deficient area. In addition, lymphocytic infiltration of thyroid tissue, the amount of the various lymphocyte subsets (Leu 4+, Leu 3a+, and Leu 2a+ T-cells as well as B1+ B cells) in the thyroid gland, as well as the expression of the histocompatibility antigen HLA-DR on thyrocytes and intrathyroidal T-lymphocytes were examined. Goitrous patients were subdivided into two groups according to their individual iodine supply estimated by iodine excretion values, and immunological parameters were compared between patients with low (group A, iodine excretion less than 70 micrograms/24 h) and with higher (group B, iodine excretion greater than 100 micrograms/24 h) iodine supply. Thyroid growth stimulating immunoglobulins and antithyroid antibodies were equally prevalent in the two patient groups, but were absent in controls. Lymphocytic infiltration of thyroid tissue was present to a comparable extent in patients of groups A and B, but to a distinctly lower degree in control persons. Intrathyroidal T-lymphocyte subsets did not differ between patients and controls. B-lymphocytes, germinal centres as well as DR+ thyrocytes were detected in goitrous patients of both groups, but never in control persons. Thus, immunological abnormalities frequently occur in patients with simple goitre and do not depend upon individual iodine supply.

Thyroid growth stimulating immunoglobulins (TGI) are not TSH-receptor antibodies

Thyroid growth stimulating immunoglobulins (TGI) are not TSH-receptor antibodies

Assay for thyroid growth stimulating immunoglobulins: stimulation of [3H]thymidine incorporation into isolated thyroid follicles by TSH, EGF, and immunoglobulins from goitrous patients in an iodine-deficient region.

Estimations were carried out of [3H]thymidine incorporation during culture of isolated porcine thyroid follicles in the presence of standard TSH or epidermal growth factor (EGF) both alone and together with anti-TSH or anti-EGF serum, and of immunoglobulins fractionated from the sera of patients from our endemic goitre area with iodine deficiency. A significant stimulation of thymidine incorporation was exerted by TSH when thyroglobulin was present in the culture medium, and also by EGF. TSH stimulation was abolished by anti-TSH serum, in contrast to the effect of EGF. Anti-EGF serum, on the other hand, blocked the effect of EGF, but not that of TSH. The sera of 20 out of 72 patients (27%) with euthyroid goitre (not operated on) and of ten out of 26 patients (38%) with recurrent euthyroid goitre contained immunoglobulins stimulating thyroid growth (TGI). There was no correlation of TGI with other thyroid antibodies. TGI have to be regarded as a pathogenetic factor of euthyroid goitre also in patients from an endemic region especially in recurrent cases.

Autoimmunity and the pathogenesis of localized thyroid autonomy (Plummer's disease).

The pathogenesis of Plummer's disease (localized thyroid autonomy) is incompletely known. It has been shown that a normal thyroid follicle can harbour cells with widely different properties, and hyperactive follicles may arise from cells with both high replicating and hormonogenic potencies under the influence of chronic mild stimulation. We now find thyroid growth-stimulating immunoglobulins (TGI) to be present in low or intermediate titre in the serum of 7 of 9 patients with Plummer's disease. Haemagglutinating antibodies against thyroid microsomal antigen were present in low titre in two of these as well as in three of 21 other patients with this disorder. Two of the patients had a suppressible goitre in addition to the autonomous nodule. One of these is described in more detail. The possibility is discussed that autoimmunity may play a pathogenic role in Plummer's disease. TGI, in relatively low titres as found here, could exert the chronic mild stimulation supposed to be the prime event in the generation of hyperactive follicles. Whether autonomy is intrinsically present in these follicles or triggered by stimulation, remains to be established. Hyperthyroidism supervenes only when the mass of autonomous cells surpasses a certain limit. It appears that these patients with Plummer's disease should be included in the multidimensional spectrum of autoimmune thyroid disease.

Further studies on thyroid growth-stimulating immunoglobulins in euthyroid nonendemic goiter.

Sixty-two consecutive patients with sporadic euthyroid goiter (57 women and 5 men) from noniodine-deficient areas, including 15 patients with diffuse goiter, 39 patients with multinodular goiter, and 8 patients with a single nodule, were studied for the presence of serum thyroid growth-stimulating immunoglobulins (TGI) by the ultrasensitive cytochemical bioassay based on DNA cytophotometry (Feulgen-cytochemical bioassay). Using strictly specified conditions, 43 patients (67%) were positive. Values tended to be high in diffuse goiter, nodular goiters reccuring after partial thyroidectomy, and those with recent growth. Thirty-seven individual immunoglobulin (Ig)-rich fractions obtained by ammonium-sulfate precipitation from 20 normal subjects, 13 atrophic thyroiditis patients, and 4 dyshormonogenetic goiter patients were tested similarly, and only 3 gave positive growth assays. These results lend further support to our concept that a majority of patients with sporadic nontoxic simple goiters have a variant of thyroid autoimmune diseases separate from lymphocytic thyroiditis. With regard to assays thought to reflect activities of TSH receptor antibodies, none of 20 tested Ig preparations stimulated thyroid cAMP production. The TSH binding inhibition assay gave weak positive activity, but failed to correlate with either TGI or TSH unresponsiveness to TRH. These findings suggest that sporadic goiter TGI is not directed to the TSH-binding site. Dose-response studies performed with Igs of patients with nontoxic goiters and with human TSH standard and goitrous hyperthyroid Graves Igs as controls all revealed bell-shaped responses. Similar maximal values were reached regardless of the growth stimulus applied. However, approximately 10 times more Ig was needed to reach maximal responsiveness in sporadic goiter than in goitrous Graves' disease (i.e. 125-500 micrograms vs. 15-125 micrograms Ig/ml culture fluid). The optimal dose of human TSH ranged from 0.01-1.0 microU/ml. The assays in the present series of the 62 consecutive patients with nontoxic diffuse or nodular goiter were all carried out with a fixed amount of 125 micrograms Ig/ml and considering a value above 5% of cells in the S-phase as a positive assay. Some Ig preparations negative for TGI at this concentration may contain TGI when tested using other doses, and these are a prerequisite to assess the potency of growth antibodies in individual patients.

Hyperthyroidism following primary hypothyroidism in association with polyendocrine autoimmunity

A 37 year old male with a strong family history of autoimmune disease presented with typical symptoms of hyperthyroidism. He had exophthalmos but no goitre. Hyperthyroidism was confirmed by failure of 131I neck uptake to suppress after 7 days treatment with triiodothyronine. Six years previously a diagnosis of primary hypothyroidism has been made. At diagnosis of hyperthyroidism, thyroglobulin antibodies, thyroidal microsomal antibodies and thyroid stimulating immunoglobulins were detected. The absence of thyroid growth stimulating immunoglobulins and presence of immunoglobulins blocking TSH-induced growth may account for the absence of goitre throughout. HLA -B8, -B, -DR3 and -DR4 genotypes, low C4 complement concentrations and islet cell autoantibodies were detected at the time of diagnosis and 1 year later diabetes mellitus developed.

Measurement of thyroid growth-stimulating immunoglobulins in patients with goitrous thyroid disease

Measurement of thyroid growth-stimulating immunoglobulins in patients with goitrous thyroid disease

Estimation of thyroid growth stimulating immunoglobulins by 3H-thymidine incorporation into isolated porcine thyroid follicles

Estimation of thyroid growth stimulating immunoglobulins by 3H-thymidine incorporation into isolated porcine thyroid follicles

Thyroid growth stimulating immunoglobulins in patients of an iodine-deficient goiter region estimated by 3H-thymidine incorporation into isolated porcine thyroid follicles

Thyroid growth stimulating immunoglobulins in patients of an iodine-deficient goiter region estimated by 3H-thymidine incorporation into isolated porcine thyroid follicles

In vitro generation of NADPH as an index of thyroid stimulating immunoglobulins (TGI) in goitrous disease.

A quantitative cytochemical assay for the measurement of total NADPH formation in guinea pig thyroid tissue following incubation with thyroid stimulators was validated and applied to the measurement of such stimulators in IgG concentrates prepared from human plasma. Pentose shunt enzyme activity was not uniformly distributed in thyroid tissue but this could be overcome and NADPH generation in such tissue used to assess accurately thyroid stimulators if a sufficient number of thyroid cells were measured. Specificity studies showed that antiserum to human IgG significantly diminished the NADPH generating capacity of IgG concentrates prepared in plasma from goitrous patients while antiserum to h-TSH had no such effect. The measurement of thyroid growth stimulating immunoglobulins (TGI) depended not only on the amount of NADPH generated but also on the IgG concentration at which maximum responses occurred. TGI present in goitrous Graves' disease were 10 times more potent than those present in euthyroid goitre, while such stimulators when present in toxic nodular goitre appeared to possess an intermediate potency. This finding, when taken together with the demonstration that TSH stimulation decreased intercellular differences in pentose shunt activity, provides experimental evidence for the hypothesis that an acute intense growth stimulus affecting all thyroid cells produces the diffuse hyperplasia characteristic of Graves' disease. In contrast, the weaker stimulus observed in nodular goitre may, over a period of time, result in the asymmetric hyperplasia commonly observed in this condition.