Association of CT60 cytotoxic T lymphocyte antigen-4 gene polymorphism with thyroid autoantibody production in patients with Hashimoto's and postpartum thyroiditis.

Strong genetic contribution has been demonstrated to influence the development of autoimmune thyroid disease (AITD) as well as thyroid autoantibody production. In order to assess the relation between CT60 cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphism and thyroid autoantibody production, we investigated 180 consecutive newly diagnosed patients with two forms of AITD, 105 with Hashimoto's thyroiditis (HT) and 75 with postpartum thyroiditis (PPT). We evaluated thyroid function, measured antibodies against thyroid peroxidase (TPO) and thyroglobulin (Tg), and determined CT60 CTLA-4 gene polymorphism. In HT, TPO antibody median value was significantly lower in the AA compared to the AG and GG genotypes (65, 122 and 319 U/ml, P<0.005), while the Tg antibody median value was lower in the AA compared to the AG genotype (91 and 189 U/ml, P<0.02). In PPT, the frequency of thyroid autoantibody-positive patients was higher among G-allele-carrying genotypes (P<0.04). Similar to HT, the TPO antibody median value was lower in the AA compared to the AG and GG genotypes (12, 130 and 423 U/ml, P<0.006). Hypothyroid PPT patients were more often thyroid autoantibody-positive (P<0.005) and the TPO antibody median value was higher compared to hyperthyroid PPT patients (500 and 32 U/ml, P<0.0001). The frequency of the G-allele was significantly higher among hypothyroid patients (P<0.05). Our data suggest that in both HT and PPT, the CT60 CTLA-4 gene polymorphism contributes importantly to thyroid autoantibody production. In PPT, the genotype also seems to influence thyroid function, as patients with the polymorphous allele are more prone to develop hypothyroid form of PPT.

Thyroid autoantibody production is influenced by exon 1 and promoter CTLA-4 polymorphisms in patients with Hashimoto's thyroiditis.

Strong genetic susceptibility to thyroid autoantibody (TAb) diathesis has been shown and one of the major genes involved is probably CTLA-4 gene. Our recent study of patients with Graves' disease has demonstrated that exon 1 CTLA-4 gene polymorphism influences higher TAb production. Here, we evaluated the influence of exon 1 and promoter CTLA-4 polymorphisms on TAb production in 109 newly diagnosed patients with Hashimoto's thyroiditis (HT). Serum TSH, thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TgAb) were measured. 49 A/G and -318 C/T polymorphisms were detected using polymerase chain reaction amplification of genomic DNA and restriction fragment length polymorphism analysis. Patients with AG and GG genotype had significantly higher TPOAb median values compared to patients with AA genotype (P < 0.003). Similarly, TgAb median value was significantly higher in AG patients and in the entire G-allele carrying group (P < 0.02). Compared to both T-allele carrying genotypes, CC genotype presented with significantly higher TPOAb median value (P < 0.02), whereas TgAb median values did not differ significantly between various genotypes. In conclusion, our results indicate that G allele influences higher TPOAb and TgAb production, whereas C allele affects especially TPOAb production in patients with HT. Therefore, our findings provide further evidence that CTLA-4 is a major TAb susceptibility gene.

Early changes of thyroid hormone concentrations after (131)I therapy in Graves' patients pretreated or not with methimazole.

AIM: Despite extensive use of (131)I therapy for Graves' hyperthyroidism the treatment regimen with (131)I and antithyroid drugs remain under discussion. In our prospective clinical study we followed acute thyroid hormone changes after (131)I in patients not pretreated with methimazole (MMI) and in patients with different MMI pretreatment regimens. PATIENTS, METHODS: 187 patients were treated with fixed activity of 550 or 740 MBq of (131)I. First group (71 patients) received (131)I alone. In the second group (57 patients) MMI was stopped seven days before (131)I. The third group (59 patients) received MMI until (131)I application. Initial free triiodothyronin and free thyroxin were measured in the second group 7 and 2 days before (131)I therapy and in all three groups on the day of (131)I application as well as 2, 5, 12, and 30 days afterwards. Absorbed dose was measured in each patient. RESULTS: In the non-pretreated group (131)I application was followed by a significant decrease of fT4 in 5 days and of fT3 in 2 days, higher reduction was detected in patients with higher baseline values. In MMI pretreated patients significant but clinically irrelevant increase of both thyroid hormones was detected with maximum value 7 days after discontinuation in the second group and 5 days after discontinuation in the third group. Additionally, in patients of the third group absorbed dose of (131)I was significantly lower relative to other two groups. We found no correlation between absorbed dose of (131)I and thyroid hormone changes. CONCLUSION: Our study demonstrates that (131)I application alone does not result in exacerbation of hyperthyroidism and therefore it may be considered as safe. Additionally, MMI withdrawal causes significant but clinically irrelevant elevation of thyroid hormones.

Influence of lithium on cell function in two different cell systems.

Lithium is widely used in the treatment and prophylaxis of bipolar psychiatric disorders. It accumulates in the thyroid gland and can cause goitre or thyroid dysfunction. The mechanisms of various effects of the lithium ion on thyroid cells have not been completely clarified. The aim of our work was to establish whether lithium, in the presence or absence of TSH, stimulates the synthesis of cAMP; as model systems we used a strain of rat thyroid follicular cells FRTL-5 and a line of Chinese hamster ovary fibroblasts with the human TSH receptor (CHO-R). Lithium at concentrations of 0.35 mM, 1 mM, 1.4 mM, 1.7 mM and 2 mM without TSH and at selected concentrations with TSH stimulation significantly increased cAMP synthesis in FRTL-5 and in CHO-R cells when compared with controls without lithium. These results are different from the published data, which have been unable to confirm the influence of lithium on cAMP synthesis or have even reported the inhibition of cAMP synthesis. However, in most published investigations only lithium in combination with TSH was tested. In conclusion, lithium was found to stimulate cAMP synthesis in FRTL-5 cells and in CHO-R cells.

Influence of lithium on growth and viability of thyroid follicular cells.

Lithium accumulates in the thyroid gland and can cause goiter or thyroid dysfunction. The aims of our work were: 1) to verify whether lithium stimulates proliferation of thyroid cells; as methods, the 3H-thymidine incorporation assay and the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) were used; as a model system the FRTL-5 (Fischer rat thyroid cells in low serum) cell line was selected, 2) to test whether lithium can have a cytotoxic effect on FRTL-5 cells, using the cytotoxicity assay with 51Cr release and the trypan blue exclusion method. Without TSH stimulation, lithium at 0.35-2 mM concentrations significantly increased the 3H-thymidine incorporation. A similar effect was observed in the case of the MTT assay: without TSH stimulation, lithium at 0.4-2 mM concentrations showed a significant stimulation of proliferation. Surprisingly, under TSH stimulation, lithium at the 2 mM concentration significantly inhibited proliferation of FRTL-5 cells. With the cytotoxicity assay, lithium was found to increase 51Cr release at 1.4-2 mM concentrations. Additionaly, the percentage of viable FRTL-5 cells at 0.35-2 mM concentrations of lithium was lower than in the controls without lithium. In conclusion, lithium was found to stimulate proliferation of FRTL-5 cells in conditions without TSH and, surprisingly, lithium in higher concentrations diminished proliferation of FRTL-5 cells under TSH stimulation. A cytotoxic effect of higher lithium concentrations was observed.

Early change of thyroid hormone concentration after 131I treatment in patients with solitary toxic adenoma.

AIM: In spite of extensive use of 131I for treatment of hyperthyroidism, the results of early outcome are variable. In our prospective clinical study we tested whether 131I induced necrosis causing clinical aggravation of hyperthyroidism and increasing the free thyroid hormone concentration in the serum of patients with solitary toxic adenoma not pretreated with antithyroid drugs. PATIENTS AND METHODS: 30 consecutive patients were treated with 925 MBq 131I. Serum concentration of thyrotropin (TSH), free thyroxine (fT4), free triiodothyronine (fT3), thyroglobulin (Tg), and interleukin-6 (IL-6) were measured before and after application of 131I. RESULTS: After application of 131I no clinical worsening was observed. FT4 and fT3 concentration did not change significantly within the first five days, whereas both of them significantly decreased after 12 days (p < 0.0001). Slight and clinically irrelevant increase in the level of the two thyroid hormones was observed in 9 patients. Furthermore, we observed a prolonged increase in Tg concentration and a transient increase in IL-6 concentration. CONCLUSION: Neither evidence of any clinical aggravation of hyperthyroidism nor any significant increase in thyroid hormone concentration by 131I induced necrosis of thyroid cells was found. Therefore, the application of 131I may be considered as a safe and effective treatment for patients with hyperthyroidism due to toxic adenoma.

The influence of calcium on thyroid follicular cells FRTL-5 in vitro.

The present work is based on the results of in vivo experiments on rats, which had shown that hypercalcemia had led to morphological and biochemical hyperfunction of thyroid follicular cells. The regulation of the activity of follicular cells should directly, or indirectly via paracrine action of serotonin secreted from parafollicular cells, depend on the presence of calcium ions. The effect of calcium was studied on a cell line of rat follicular cells FRTL-5 (Fischer Rat Thyroid cells in Low serum) using three methods: measuring the quantity of produced cAMP (cyclic adenosine 3',5'-monophosphate), measuring [3H]thymidine incorporation into cell DNA and transmission electron microscopy. Results show that calcium has no effect on cAMP production. Calcium at 1.3 mM, 3 mM, 10 mM, 20 mM and 30 mM concentrations increases [3H]thymidine incorporation into cell DNA when compared with controls without calcium. Calcium at the concentration of 30 mM has no effect on FRTL-5 cell morphology. TSH (thyrotropin) stimulates follicular cells; at higher extracellular concentrations (3 mM, 10 mM, 20 mM, 30 mM), calcium diminishes its effect, presumably by activation of a cAMP phosphodiesterase which disintegrates cAMP and/or by inhibition of adenyl cyclase.

The influence of serotonin on follicular cells FRTL-5 in vitro.

Serotonin is an aromatic monoamine found in parafollicular cells of rats, bats and in a human medullary thyroid carcinoma cell line. We have examined the hypothesis that serotonin stimulates follicular cells. The effect of serotonin in presence and absence of thyrotropin was studied in a cell line of rat follicular cells FRTL-5 (Fischer Rat Thyroid cells in Low serum) by measuring [3H]thymidine incorporation into cell DNA (growth assay) and by transmission electron microscopy. The cell line was used to avoid the contamination with serotonin secreted from parafollicular cells. Results show that serotonin at 3 microM, 10 microM, 30 microM, and 100 microM concentrations increases [3H]thymidine incorporation into cell DNA. Serotonin at a 1000 microM concentration reduces sharply the [3H]thymidine incorporation into cell DNA. All cytoplasmic organelles completely disappear, only a thin fibrous membrane remains. The toxic effect of serotonin is observed in the nuclei, too. As expected, thyrotropin stimulates follicular cells. Serotonin does not have any influence on that stimulative effect of thyrotropin. Rough endoplasmic reticulum consists of round vesicles, several mitochondria are present in the cytoplasm. From the surface few pseudopodia extend into the culture medium.