Immunohistochemical localization of galectin-3 in malignant and benign human thyroid tissue.

BACKGROUND: Galectin-3 is an endogenous beta-galactoside binding lectin with putative roles in development, immunomodulation, transformation and metastasis. The purpose of this study was to analyze galectin-3 expression in a series of human thyroid neoplastic lesions. METHODS: A total of 76 cases, including 47 specimens of thyroid malignancies, 14 follicular adenomas and 15 specimens of normal thyroid tissue, were analyzed immunohistochemically using a monoclonal antibody to galectin-3 and avidin-biotin-peroxidase complex (ABC) method. RESULTS: The immunohistochemical staining results showed galectin-3 expression in neoplastic cells of all 20 cases of papillary carcinoma, 11 out of 15 follicular carcinomas, both oxyphilic carcinomas, all 10 anaplastic carcinomas and 5 out of 14 follicular adenomas. Galectin-3 localization was mostly cytoplasmic, but also membraneous or nuclear in some cells. Follicular cells in normal thyroid tissue were negative. CONCLUSIONS: The results of this study indicate that galectin-3 gene is expressed at the protein level in most thyroid carcinomas and some adenomas. Galectin-3 expression was not clearly correlated with histopathological aggressiveness, dedifferentiation state or determination of malignancy of the follicular tumour. The role of galectin-3 in thyroid tumour biology remains to be elucidated.

[Effects of amiodarone on the thyroid gland: appearance of microlesions in follicles of hyperplastic rat thyroid after chronic amiodarone treatment]. / Dejstva amiodarona na tireoidnu zlezdu--pojava mikrolezija u folikulima hiperplasticne tireoidne zlezde pacova posle hronicnog tretmana amiodarom.

Using the rat and guinea-pig as an experimental animal model, the effect of amiodarone (Cordarone) on normal of hyperplastic thyroid was investigated in eu- or hypothyroid animals. Following short- or long-term treatments with different doses of amiodarone (5-50 mg/day) and amiodarone-equivalent amounts of stable iodine, serum thyroid hormones were assayed, followed by determination of thyroid uptake of radioiodine, the weight of the thyroid gland and its histological structure. Having received amiodarone chronically, euthyroid rats showed decreaed levels of serum T3 and T4, in contrast to euthyroid guinea-pigs in which no appreciable differences between amiodarone-treated and untreated animals were observed. However, the weight of the thyroid decreased in both species after amiodarone treatment. A similar effect was seen after the treatment with excessive iodine, but only in euthyroid rats, while the guinea-pigs showed increased gland weight and activation of the follicular epithelium, as seen on histological sections. These differences obeserved in thyroid glands of rats and guineapigs were probably associated with the facts that the normal dietary iodine intake was lower in guinea-pigs and their thyroid glands were more sensitive to excess of iodine than that of rats. After chronic amiodarone treatment, hypothyroid rats with hyperplastic thyroids showed a much greater increase of serum T4 than the control rats, while the T3 increase was appreciably smaller. After several months (5 and 6.5) of amiodarone treatment, the appearance of various lesions was observed in the thyroid follicular epithelium of these animals. These microlesions probably resulted in a direct toxic effect of amiodarone on the hyperplastic gland. The results of the present investigation suggest that amiodarone effects on the thyroid may not be associated with excessive iodine exclusively, but also with the specific effects of amiodarone on this gland.

[Effects of amiodarone on the thyroid gland in euthyroid and hypothyroid animals]. / Efekti amiodarona na tireoideju eutireoidnih i hipotireoidnih zivotinja.

Amiodarone is a benzofurane derivative which contains an appreciable amount of iodine (37%). It is used in cardiology as an antianginal and antiarrhythmic drug. Using guinea-pigs and rats as the animal model systems, the effects of amiodarone on normal and hyperplastic thyroid glands were investigated in eu- and hypothyroid animals. After the amiodarone treatment, considerable differences were observed in the levels of thyroid hormones and in the structure of the gland which proved dependent not only on the length of treatment, but also on animal species and the functional status of the gland before treatment. At extended amiodarone application, the rats with hyperplastic thyroid developed microlesions in epithelium of some follicles.

Altered terminal glycosylation of thyroglobulin in papillary thyroid carcinoma.

Samples of thyroglobulin (Tg) were isolated from specimens of differentiated thyroid carcinoma of the papillary type and from normal adjacent glandular tissue, and the content of sialic acid was estimated. Also the in vitro incorporation of 14C-sialic acid, in the form of both CMP (cytidine 5'-monophospho-)--activated and non-activated N-acetyl-neuraminic acid, into Tg of malignant and morphologically normal thyroid. The sialic acid content of Tg preparations from papillary thyroid carcinomas varied considerably (0.27-0.92 mg/100 mg Tg). In six cancerous Tg samples the content of sialic acid was markedly lower than that in Tg from the corresponding apparently normal thyroid tissue (0.71:1.11 mg per 100 mg Tg). In addition, in comparison with the control, the incorporation of non-activated 14C-sialic acid into Tg of malignant thyroid tissue was considerably lower (-41%). However, the incorporation of CMP-activated 14C-sialic acid into cancerous Tg was greater than into Tg of morphologically unchanged tissue of the same gland (+29%). The reduced content and incorporation rate of sialic acid into Tg of differentiated thyroid carcinoma is probably the consequence of disturbances in terminal glycosylation of the Tg molecule in malignantly transformed thyroid tissue. The enhanced incorporation of CMP-sialic acid into cancerous Tg suggests that Tg sialylation in carcinoma is probably altered in the sialic acid activation phase.

Long-term follow up of serum thyroglobin levels and its clinical implications in subjects after surgical removal of "cold" thyroid nodule.

Increased thyroglobulin (Tg) serum level in females (n = 49) suffering from scintigraphically "cold" thyroid nodule (395 +/- 168, micrograms/l, mean +/- SD) is significantly higher (p less than 0.001) in comparison with the data obtained from euthyroid healthy persons (28 +/- 10, n = 15) or those diagnosed with scintigraphically functional thyroid nodule (72 +/- 14, n = 15). No correlation between Tg and TSH serum levels has been seen before surgical removal of "cold" nodule (rxy = +0.34). In 49 out of 75 cases (65.3%) struma colloids were confirmed pathohistologically. After surgery, "cold" thyroid nodule was removed, serum Tg level decreased progressively within several days and three months afterwards, Tg level in most patients was mainly within the normal range for a healthy subject. The positive correlation between Tg and TSH was established three months after surgical removal of the nodule (rxy = +0.90). An elevation of serum Tg level was found in 16 out of the 49 patients during different postoperative periods (from 2 to 9 years). Under thyroxine treatment, in most of these patients a significant decrease of serum Tg level was observed. However, in a few treated patients, no change in Tg level was seen. Further clinical control in thyroxine-nonsuppressive patients discovered a compensatory hypertrophy of thyroid tissue and the appearance of a new thyroid nodule later. Present data and clinical experience suggest that long-term follow-up of serum Tg level in patients after surgical removal of "cold" thyroid nodule, brings about to early diagnosis of thyroid tissue postoperative hypertrophy before the appearance of thyroid gland nodule relapse.

[Iodine levels in salt for human consumption and aspects of iodine prophylaxis in Yugoslavia]. / Sadrzaj joda u soli za ljudsku upotrebu i neki aktuelni aspekti jodne profilakse u nasoj zemlji.

Iodine content of iodinated salt intended only for human consumption was eyamined in samples from all domestic manufacturers (salt mines in: Tuzla, Pag, Ulcinj, Ston, Nin, Seca-Portoroz). Sampling was made from commercially available packs (1 kg) or from food industries (large packs). It was established that iodine content of examined samples (57) varied considerably and ranged from 1.8 to 12.2 mg iodine/kg salt. Iodinated salt from the Tuzla and Pag salt mines contained iodine within the prescribed limits in both small and large packs. Similar finding of iodine content was in the small packs of the Ulcinj salt mines, however, their large packs (10 kg) had a markedly low level of iodine (1.8-4.7 mg). Lower iodine content was also noted in salts (1 kg) from the Nin, Ston and Seca-Portoroz mines. Of the 57 examined samples in 70% iodine content was lower than the prescribed dose (7.6 mg). In addition, some mines had low iodine content in all of the 100% of their respective samples. Ever since iodine prophylaxis was introduced in Yugoslavia a number of factors influenced the decrease of iodine intake by the organism to a considerable degree. The results of our investigations and some epidemiological data indicate that the issue of increasing the amount of iodine from the present 7.6 to 15 mg iodine/kg salt should be reconsidered. Besides, it is necessary to arrange the systematic quality control of iodinated salt.

[Changes in serum thyroglobulin levels during the TRH test in euthyroid female patients with diffuse and nodular goiters]. / Promena koncentracije tireoglobulina u serumu u toku TRH testa kod eutiroidnih bolesnica sa difuznom i nodusnom strumom.

Concentrations of thyroglobulin (Tg) in the sera of 48 euthyroid females of different ages suffering from diffuse and nodular goitre during TRH test, were determined. The aim of this study was to examine the correlation between the increased TSH levels and Tg, and thyroid hormones in the serum in dependence of pathomorphological thyroid status and the patients' ages. TSH maximal level reached (+314%) compared with the basal level 25 minutes after TRH injection, while max Tg level was found 60 minutes after TRH in 15 female patients, medium age 41 (range:33-48), with diffusely enlarged thyroid gland. Concentrations of Tg and T4 in the serum of patients increased at the same time, although no statistical difference for T4 was noted. In 23 females, aged over 60 years (range: 65-80), suffering from euthyroid diffuse goitre, maximal TSH level was achieved later, i.e. 60 minutes after TRH injection, while max Tg was found 90 min after TRH. If we compare Tg serum levels in geriatric females with the data obtained in the younger group, we will note that the increase of Tg concentrations was significantly lower and delayed in elderly subjects (p less than 0.001). Data on TSH-and Tg-increase during TRH test in 10 females suffering from solitary, functioning nodular goitre, were very similar to findings obtained in 15 female patients with diffusely enlarged thyroid (25 minutes after TRH max TSH + 205%, and max Tg level + 198%). The obtained results indicate that Tg serum levels were increased after TRH injection in 48 euthyroid females.(ABSTRACT TRUNCATED AT 250 WORDS)

Occurrence of antithyroxine antibodies in rabbits immunized with iodine-poor human thyroglobulin.

Thyroglobulin (Tg) is a large glycoprotein with polymorphic structure and its heterogeneity has been demonstrated by many investigators. In order to obtain appropriate antibodies against human Tg which appear in the circulation of patients with thyroid carcinoma, a number of rabbits (19) were immunized with poorly iodinated h-Tg (0.05%). During the period of immunization the level of T3 and T4 was followed in sera, as well as the titre of anti-h-Tg antibodies. The production of antibodies against h-Tg was observed in all immunized rabbits (8, 13, 16, and 54 weeks from the first immunization). Titres of anti-h-Tg antibodies in sera at the 16 and 54-week bleeding were very high (Ka = 2.0 X 10(10) M-1). A few immunized rabbits were found to have a very low concentration of serum T4 (determined by RIA-PEG method). Sera from these animals contained antibodies against T4, but not against T3. Their identification and characterization was performed by a radioimmunological method. In summary, our results show that after immunization of rabbits with low-iodinated, i.e. hormone-poor human Tg, antibodies against thyroxine can be produced. However, occurrence of anti-thyroxine antibodies in some immunized rabbits indicates that the immunogenicity of hormone residues in poorly iodinated h-Tg is much lower than in normal iodinated molecule.

Enhanced acid protease activity of lysosomes from papillary thyroid carcinoma.

In vitro lysosomal acid protease activity was studied in human papillary thyroid carcinoma (n = 13). As a control, morphologically normal thyroid tissue from the same patient was used in each individual case of carcinoma. Although a marked variation may be observed between individual cases, each examined papillary thyroid carcinoma showed significantly greater activity of acid proteases, both per unit weight of wet thyroid tissue and per unit of lysosomal proteins, in comparison to the corresponding control (range, 24%-248%). In conclusion, it is suggested that enhanced proteolytic activity of lysosomal acid proteases in papillary carcinoma is probably a result of disturbance in catabolic degradation of the thyroglobulin molecule in malignantly transformed thyroid tissue.

Reduced acid protease activity in vitro of lysosomes from thyroid gland of rats chronically treated with propylthiouracil and perchlorate.

We have recently demonstrated that proteolytic activity of lysosomal acid proteases from papillary carcinoma is significantly higher than in morphologically normal thyroid tissue. In the present study the activity of lysosomal acid proteases from parenchymatous proliferated thyroid epithelium, induced by action of antithyroid substances, has been examined in an in vitro system using 125I-labelled rat thyroglobulin as a substrate. Thyroid lysosomes were isolated from rats treated chronically for 3-4 weeks with propylthiouracil (PTU, 0.1% in drinking water) and perchlorate (NaClO4, 200 mg/rat/day) by centrifugation between 800 and 20,000 x g. It was observed that, in contrast to human malignant thyroid tissue, the proteolytic activity of lysosomal acid proteases from antithyroid substance-induced hyperplastic goitre was markedly reduced in comparison with control thyroid tissue (29-50%). Since reduced activity of total lysosomal proteases was found both per unit of wet weight thyroid tissue and per unit of lysosomal proteins, the results suggest that changes in lysosomal enzymes may probably have more quantitative than qualitative nature.

Reduced proteolytic activity in vitro of lysosomes isolated from "cold" thyroid nodule.

The proteolytic activity of lysosomes isolated from solitary "cold" thyroid nodule and morphologically normal perinodular thyroid tissue was examined in parallel in an in vitro system using 125I-labelled rat Tg as substrate. Lysosomes were isolated by centrifugation of the tissue homogenates between 800 and 20,000 x g. The optimal proteolytic activity of acid proteases from human thyroid tissue was observed between pH 3.6 and 4.8. The evident differences in the proteolytic activity of lysosomal acid proteases between nodular and perinodular tissues were observed. It was found that the activity of lysosomal proteases from "cold" nodule in both the same amount of wet weight thyroid tissue and the same amount of lysosomal proteins was significantly lower (29-54%) than in the morphologically normal perinodular tissue used as a control. The reduced proteolytic activity of lysosomes from patients with "cold" nodule provides further confirmation on the low metabolic activity of thyrocytes in non-functioning thyroid nodules.

Some characteristics of soluble thyroid proteins in human fetus during morphogenesis of follicular structure.

Soluble thyroid proteins of the human fetus were studied in parallel with the formation of the follicular structure of the gland using polyacrylamide disc electrophoresis and a radioimmunological method for measuring thyroglobulin (Tg). The study covered 24 fetuses obtained after sectio parvae performed at 12-28 weeks of gestation for socio-medical reasons. Before the first trimester of gestation the human fetal thyroid, which did not have an organized follicular structure contained a significant amount of the 12S protein fraction, but only trace amounts of Tg. In the cells of a gland of afollicular structure immunofluorescent material was confirmed by reaction with anti-Tg antibodies. Later, with the formation of follicles and the appearance of follicular colloid, the absolute and relative amounts of Tg in the fetal gland increased, while the 12S subunit decreased. The Tg content in the fetal thyroid was positively correlated with the total weight of the gland, i.e. gestational age. The T4 and T3 contents in Tg of human fetal thyroids (20-24 weeks) amounted to 0.54 +/- 0.39 and 0.12 +/- 0.09 mole/Tg mole, respectively. The 27 S iodoprotein was not found in any of the extracts of fetal thyroids with formed follicles and considerable amounts of follicular colloid. The finding of the 12 S protein in the human thyroid during early fetal life, i.e. in the prefollicular phase, implies a low rate of aggregation of Tg subunits (12 S). The absence of the 27 S iodoprotein from the fetal thyroid with organized follicular structure suggests the yet incomplete morphological and biochemical maturation of the fetal gland.

Depletion and reaccumulation of follicular thyroglobulin (TG) in guinea-pig thyroid gland after short- and long-term administration of thyrotropin (TSH).

The relationships among the content and composition of soluble iodoproteins in thyroid gland, the serum Tg concentration and the morphological structure of gland were investigated in guinea-pigs during short-term and long-term TSH administration (2 I.U. per day for 1 to 28 days). A significant decrease in the content of Tg in the gland (from 6.50 mg to 1.32 mg/100 mg tissue), disappearance of 12 S iodoprotein, hypertrophy and hyperplasia of the follicular cells were observed in guinea-pigs treated with TSH up to 7 days. Later on, after depletion of follicular colloid, reaccumulation of colloid in the preexisting and newformed follicles (a marked increase of Tg and 12 S protein) and partial involution of the structural changes occurred. The serum Tg concentration increased during the whole period of TSH treatment (mean values from 100 to 500 micrograms/l). A marked positive correlation between serum Tg concentration and thyroid weight was found. Serum T3 and T4 concentrations increased transiently with maximal values on the 1st day (T3, from 0.86 to 3.26 nmol/l, T4, from 44 to 138 nmol/l) and decreased thereafter. The results imply that different regulatory mechanisms exist which control thyroid hormone secretion and transfer of Tg from the gland into blood circulation. The serum Tg level is directly dependent on the total bulk of thyroid tissue, but is considerably less dependent on the structure of the gland. The reaccumulation of colloid (Tg) in thyroid follicles of guinea-pigs treated with TSH is a result of the reduction of the stimulatory effect of TSH on the process of Tg degradation during chronic hormone application. Several different factors may be responsible for the appearance of refractoriness or desensitization of the thyroid to prolonged exogenous TSH stimulation.

Serum T4, T3, r-T3, TSH and Tg levels in cord blood and maternal circulation at the time of delivery.

The concentrations of T4, T3, r-T3, TSH and Tg were determined in parallel in maternal serum at the time of delivery and in cord blood. The serum T3 concentration in cord blood was significantly lower than that in the serum of the mother. However contrary to T3, r-T3 in cord serum was significantly higher than in maternal serum. Also, the level of TSH in cord blood was considerably higher than in maternal serum. The concentrations of total T4 in maternal and cord serum did not differ markedly, even though the values (mean and individual) were somewhat lower in the cord serum. The concentrations of Tg in cord and maternal serum varied widely, although the mean value for Tg in cord serum was somewhat higher than in maternal serum. No correlation was found between Tg concentration and TSH level in cord or maternal serum.

Susceptibility to proteolysis of thyroglobulin from rats and guinea-pigs treated with excess iodide.

The effect of excess iodide on proteolysis of in vivo 125I-labelled thyroglobulin (Tg) from rats and guinea-pigs was investigated in vitro using preparations of thyroid lysosomes and exogenous proteases (Pronase). Lysosomes were obtained by centrifugation of pig thyroid homogenates. This lysosomal preparation induced maximal proteolysis of Tg at pH 3.6 to 4.6 and was stimulated by KCl. Treatment of rats with excess iodide for up to 28 days had no effect on the proteolysis of Tg by lysosomes or Pronase. Decreased resistance of Tg to proteolysis by lysosomes and pronase was observed in guinea-pigs treated with excess iodide for 3 days, whereas Tg from guinea-pigs treated for longer time periods had increased resistance to proteolysis. This difference in the susceptibility to proteolysis of Tg provides an explanation why long-term administration of excess iodide is goitrogenic in guinea-pigs but not in rats.