Evaluation of galectin-8 expression in thyroid tumors.

The expression of galectin-8 (gal-8) has been shown to be altered during neoplastic transformation of certain cell types. This is the first study aimed to analyze the expression of this protein in normal and pathological human thyroid tissue. A total of 41 archival thyroid tissue samples (5 follicular adenomas, 31 papillary carcinomas, 5 follicular carcinomas) together with 36 adjacent hyperplastic or normal thyroid tissues were analyzed by immunohistochemistry. Galectin-8 was expressed in the majority of papillary carcinomas (27/31; 87%). Positive but weaker staining was also found in some of the follicular thyroid carcinomas (2/5; 40%) and adenomas (2/5; 40%). This protein was not detectable in five normal thyroid tissue samples, whereas hyperplastic areas adjacent to tumor were weakly positive in 9 out of 31 cases (29%). High gal-8 immunostaining in papillary thyroid carcinoma indicates that gal-8 may potentially serve as a marker of papillary thyroid carcinoma. However, it does not seem to be helpful in the differential diagnostics of follicular carcinoma and adenoma. Further studies are required to determine biological functions and molecular mechanisms underlying the increased expression of gal-8 protein in thyroid lesions, particularly, in papillary thyroid carcinoma.

Changes in the balance between proliferation and apoptosis during the progression of malignancy in thyroid tumours.

The aim of this study was to gain better insight into molecular changes which reflect disturbances in the balance between proliferation and apoptosis during progression of thyroid malignancy from papillary microcarcinoma (PMC) via clinically manifest papillary carcinoma (PTC) to anaplastic carcinoma (ATC). The apoptosis related molecules (Bcl-2, Bax) and proliferation related marker (PCNA) were analysed immunohistochemically in 120 archival cases comprising PMC (n=34), PTC (n=52) and ATC (n=34). In addition, in situ apoptotic cell death was analysed by the TUNEL method. The average Bcl-2 staining score did not differ between PMC and PTC (p>0.05), but was significantly lower in ATC (p<0.05).The Bax score was higher in PTCs and ATCs than in PMCs (p<0.05). Due to these changes, the Bcl-2/Bax ratio showed a marked decrease from PMC to ATC (p<0.05), while proliferation activity increased significantly from PTC to ATC (p<0.05). Despite high Bax expression, the rate of apoptotic cell death was low in the investigated carcinomas, especially in ATC, i.e. the increase in proliferative activity was not counterbalanced with appropriate cell death. Differences were found in the expression of apoptotic molecules (Bcl-2 and Bax), their ratio (Bcl-2 /Bax) and in the rate of apoptotic cell death and proliferative activity between PMC, PTC and ATC, indicating that disturbances in the balance between apoptosis and proliferation, in favour of the latter, occur gradually during the progression of malignancy in thyroid tumours.

Unilateral and bilateral congenital sensorineural deafness in client-owned pure-breed white cats.

BACKGROUND: Congenital sensorineural deafness has been reported frequently in experimental mixed-breed white cats but there is a paucity of data on occurrence of deafness in client-owned pure-breed white cats. OBJECTIVE: To describe hearing status in client-owned pure-breed white cats. ANIMALS: Eighty-four pure-breed client-owned cats with white coat color of 10 registered breeds presented for routine hearing evaluation before breeding (1995-2008). METHODS: Hearing was assessed by click-evoked brainstem auditory evoked response. RESULTS: Overall deafness prevalence was 20.2%; 9 cats (10.7%) were bilaterally deaf and 8 cats (9.5%) were unilaterally deaf. There was no association between sex and deafness status (P= .85). Deafness status was associated with iris color (P= .04). CONCLUSIONS AND CLINICAL IMPORTANCE: Congenital sensorineural deafness frequently occurs in pure-breed cats with white coat color. Unilateral sensorineural deafness was as common as bilateral deafness.

Efficacy of a single oral dose of a live bivalent E. coli vaccine against post-weaning diarrhea due to F4 and F18-positive enterotoxigenic E. coli.

F4- and F18-positive enterotoxigenic E. coli strains (F4-ETEC and F18-ETEC) are important causes of post-weaning diarrhea (PWD) in pigs. F4 (antigenic variant ac) and F18 (ab and ac) fimbriae are major antigens that play an important role in the early stages of infection. Herein, the efficacy of a live oral vaccine consisting of two non-pathogenic E. coli strains, one F4ac- and one F18ac-positive, was evaluated using F4ac-ETEC and F18ab-ETEC challenge models. A randomized, masked, placebo-controlled, block design, parallel-group confirmatory study with two different vaccination-challenge intervals (7 and 21 days) was conducted for each challenge model. The vaccine was administered in one dose, to ≥18-day-old piglets via drinking water. Efficacy was assessed by evaluating diarrhea, clinical observations, weight gain and fecal shedding of F4-ETEC or F18-ETEC. Anti-F4 and anti-F18 immunoglobulins in blood were measured. The vaccination resulted in significant reductions in clinical PWD and fecal shedding of F4-ETEC and F18-ETEC after the 7- and 21-day-post-vaccination heterologous challenges, except for after the 21-day-post-vaccination F4-ETEC challenge, when the clinical PWD was too mild to demonstrate efficacy. A significant reduction of mortality and weight loss by vaccination were observed following the F18-ETEC challenge. The 7-day protection was associated with induction of anti-F4 and anti-F18 IgM, whereas the 21-day protection was mainly associated with anti-F4 and anti-F18 IgA. The 7-day onset and 21-day duration of protection induced by this vaccine administered once in drinking water to pigs of at least 18days of age were confirmed by protection against F4-ETEC and F18-ETEC, and induction of F4 and F18-specific immunity. Cross protection of the vaccine against F18ab-E. coli was demonstrated for both the 7- and 21-day F18-ETEC challenges.

Coexpression of functionally active receptors for thyrotropin and platelet-derived growth factor in human thyroid carcinoma cells.

In the present study we show the simultaneous expression of functionally active receptors for TSH and platelet-derived growth factor (PDGF) in a newly established human anaplastic thyroid carcinoma cell line, HTh 74. In Northern blot analysis of RNA extracted from HTh 74 cells a low expression of both TSH and PDGF receptor messenger RNA was found. These observations in conjunction with the fact that the cells contain cytokeratin clearly demonstrate that the cells are bona fide epithelial thyroid cells. Stimulation of HTh 74 cells with TSH led to a concentration-dependent increase in cAMP formation, showing a functional activity of the TSH receptors. Northern blot analysis, immunoprecipitation, immunofluorescence staining, and binding experiments showed the presence of both alpha- and beta-type PDGF receptors in the HTh 74 cells. The functional activity of the PDGF receptors was demonstrated by ligand-induced internalization of the receptors and PDGF-induced growth of the HTh 74 cells. The significance of the expression of PDGF receptors on thyroid epithelial cells is not clear. However, it might reflect the gain of a new growth stimulatory pathway participating in the transformation of the epithelial thyroid cells. Alternatively, the PDGF receptors may be remnants from an immature progenitor cell from which the undifferentiated carcinoma has evolved.

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.

Thyroid hormone synthesis and storage in the thyroid gland of human neonates.

The aim of this study was to evaluate the morphological and biochemical maturation of the thyroid gland in human neonates. The mean iodine concentration in the thyroid gland of very premature infants (less than 32 weeks gestational age, 0-3 days survival, n = 12) was significantly lower than in the older group (34-41 weeks gestational age, 0-30 days survival; n = 15; p < 0.05). For the whole group of neonates there was a statistically significant linear correlation between duration of life, i.e. gestational age and survival, and iodine concentration (r = 0.64, p < 0.01). Although there was wide dispersion of the results the same tendency was seen for thyroglobulin (Tg) concentration in the thyroid gland (r = 0.52, n = 21; p < 0.05). Comparative histological examination of the fetal thyroids gave results in accordance with the biochemical data as intrafollicular colloid appeared to be more abundant in more mature thyroids. The iodine content in Tg was found to be 0.63 +/- 0.22% in very preterm neonates and was slightly but not significantly lower than that found in the thyroids of the older group (0.82 +/- 0.14%; p = 0.055). The content of T4 and T3 per Tg molecule in the neonates was related to the iodine content. The differences in mean values of T4/Tg and T3/Tg molar ratios between the two groups were not significant: T4: 2.8 +/- 1.8 mol/ mol, T3: 0.29 +/- 0.12 mol/mol in very preterm neonates; and T4: 3.5 +/- 0.7 mol/mol, T3: 0.34 +/- 0.09 mol/mol in the older group. These results offer useful information for further analysis of the development of thyroid function in the human neonate.

Changes in the balance between proliferation and apoptosis during the progression of malignancy in thyroid tumours.

The aim of this study was to gain better insight into molecular changes which reflect disturbances in the balance between proliferation and apoptosis during progression of thyroid malignancy from papillary microcarcinoma (PMC) via clinically manifest papillary carcinoma (PTC) to anaplastic carcinoma (ATC). The apoptosis related molecules (Bcl-2, Bax) and proliferation related marker (PCNA) were analysed immunohistochemically in 120 archival cases comprising PMC (n=34), PTC (n=52) and ATC (n=34). In addition, in situ apoptotic cell death was analysed by the TUNEL method. The average Bcl-2 staining score did not differ between PMC and PTC (p>0.05), but was significantly lower in ATC (p<0.05).The Bax score was higher in PTCs and ATCs than in PMCs (p<0.05). Due to these changes, the Bcl-2/Bax ratio showed a marked decrease from PMC to ATC (p<0.05), while proliferation activity increased significantly from PTC to ATC (p<0.05). Despite high Bax expression, the rate of apoptotic cell death was low in the investigated carcinomas, especially in ATC, i.e. the increase in proliferative activity was not counterbalanced with appropriate cell death. Differences were found in the expression of apoptotic molecules (Bcl-2 and Bax), their ratio (Bcl-2 /Bax) and in the rate of apoptotic cell death and proliferative activity between PMC, PTC and ATC, indicating that disturbances in the balance between apoptosis and proliferation, in favour of the latter, occur gradually during the progression of malignancy in thyroid tumours.

The efficacy of the thyroid peroxidase marker for distinguishing follicular thyroid carcinoma from follicular adenoma.

AIM: Expression of thyroid peroxidase (TPO) in the thyroid gland tissue is well known as a sensitive marker of the thyroid malignancy. We have evaluated immunohistochemical assay of TPO for distinguishing follicular thyroid carcinoma from follicular adenoma. MATERIALS AND METHODS: Sections of formalin-fixed tissues obtained from 92 patients with thyroid tumors (52 follicular carcinomas and 40 follicular adenomas including the Hurthle cell type) were analyzed using a monoclonal antibody (TPO mAb 47) and the avidin-biotin peroxidase complex immunohistochemical technique. Lesions with staining of more than 80% of the follicular cells/specimen were considered benign, while less than 80% were considered malignant. RESULTS: TPO immunostaining correlated with the histopathological diagnosis in 24/40 cases of follicular adenomas and 41/52 cases of follicular carcinomas, giving a specificity of 60% and a sensitivity of 79%. CONCLUSION: These results suggest that immunohistochemical assay of TPO expression has limited value for the differential diagnosis of follicular thyroid carcinoma from thyroid follicular adenoma.

Galectin-3 expression in papillary microcarcinoma of the thyroid.

AIMS: Galectin-3 is a beta-galactoside binding protein, recently recognized as a promising molecular marker of thyroid malignancy. As reported in several studies, galectin-3 is highly expressed in papillary thyroid carcinoma, but its expression has not been investigated in papillary microcarcinoma, which is a variant of papillary thyroid carcinoma. METHODS AND RESULTS: Using a monoclonal antibody to galectin-3 and the avidin-biotin-peroxidase complex (ABC) immunohistochemical technique, we analysed galectin-3 expression in 63 cases of papillary microcarcinoma. The results showed immunohistochemical reactivity for galectin-3 in 51 (80.9%) cases. Intensity of staining varied from strong or moderate to weak. Galectin-3 localization was mostly cytoplasmic, but also membranous or nuclear in some cells. Immunohistochemical expression of galectin-3 was not found in 12 (19.1%) cases. Most galectin-3 negative microcarcinomas (10/12) were of the non-classical type, i.e. without papillary architecture. Neither the frequency nor the intensity of a positive reaction was related to tumour size. CONCLUSIONS: Galectin-3 gene is expressed at the protein level in most papillary microcarcinomas, although with slightly lower frequency than that reported for clinically evident papillary thyroid carcinoma. The presence of galectin-3 in clinically silent microcarcinomas may indicate that galectin-3 is not related to growth or aggressiveness of papillary thyroid microcarcinomas but rather plays some other role in thyroid tumour biology.

Galectin-3 and proliferating cell nuclear antigen (PCNA) expression in papillary thyroid carcinoma.

BACKGROUND AND AIM: To examine the relationship between galectin-3 and cell proliferation in thyroid tumor tissue. Galectin-3, a beta-galactoside binding protein, has recently been recognized as a promising molecular marker of thyroid malignancy, due to its high expression in thyroid carcinomas and absence from normal or benign thyroid tissue. However, its exact role in thyroid tumor biology is still unknown. PATIENTS AND METHODS: We examined the relationship between galectin-3 and cell proliferation by comparative immunostaining for galectin-3 and proliferating cell nuclear antigen (PCNA) in paraffin-embedded tissues from 126 cases of papillary thyroid carcinoma. RESULTS: Positive cytoplasmic immunostaining for galectin-3 was found in 115 (91.3%) cases. Nuclear staining for PCNA was detectable in 93 (74.4%) cases. A low level of PCNA staining (less than 10% positive cells) was found in 36 (28.6%) cases, moderate staining for PCNA (more than 10% but less than 30% positive cells) in 35 cases (27.8%), while highly increased PCNA expression (more than 30% positive cells) was found in 32 (25.4%) cases. Moderate or strong galectin-3 expression, found in 99 cases, was associated with highly increased PCNA staining in 28.3% of them but with no detectable PCNA expression in 24.3% of them. CONCLUSION: These results suggest that overexpression of galectin-3 is not clearly related to proliferative activity of papillary thyroid carcinoma cells as assessed by PCNA immunostaining.

[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.

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.

Galectin-3 and carcinoembryonic antigen expression in medullary thyroid carcinoma: possible relation to tumour progression.

AIMS: Galectin-3 is a beta-galactoside binding protein involved in multiple biological processes through interactions with complementary glycoconjugates. We analysed the expression and coexpression of galectin-3 and carcinoembryonic antigen (CEA), one of the putative galectin-3 ligands, in medullary thyroid carcinoma (MTC). METHODS AND RESULTS: An immunohistochemical study using monoclonal antibodies was performed on paraffin sections of 20 cases of sporadic MTC comprising 10 cases without and 10 cases with lymph node metastases at the time of surgery. CEA expression was found in all tumours, distributed predominantly in the cytoplasm and occasionally at the cell surface. In the majority of cases (18/20) moderate to strong intensity of staining was found in most of the cells. Positive cytoplasmic staining for galectin-3 was found in 16/20 cases, but varied in intensity and distribution from weak/focal (7/16) to moderate (7/16) or strong (2/16). More intense staining for galectin-3 was mainly associated with MTC cases involving lymph node metastases. Eight out of these 10 cases showed moderate to strong galectin-3 expression concomitant with CEA expression throughout the tumour tissue. CONCLUSIONS: These findings suggest that galectin-3 might play a role in the pathobiology of MTC. Simultaneous expression of galectin-3 and CEA in the same tumour cells at an advanced stage of MTC indicates the possibility of their autocrine cooperation during tumour progression.

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.

[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.

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.

Differential expression of galectin-3 in papillary projections of malignant and non-malignant hyperplastic thyroid lesions.

Galectin-3 is a a beta-galactoside binding protein recently proposed to be a promising presurgical molecular marker for distinguishing benign from malignant thyroid neoplasms. We analyzed galectin-3 expression immunohistochemically in papillary areas of hyperplastic lesions of benign thyroid tissue in comparison with malignant papillary projections of papillary thyroid carcinoma (PTC). A monoclonal antibody to galectin-3 and ABC immunohistochemical technique were used to evaluate galectin-3 expression in 26 cases of benign papillary hyperplasia (8 cases of hyperplastic adenoma, 8 cases of hyperplastic colloid goiter, 10 cases of Graves disease) in comparison with 25 cases of PTC. Immunohistochemical results showed no reactivity for galectin-3 in papillary areas of benign hyperplastic lesions. Strong cytoplasmic galectin-3 immunoreactivity was found in all 25 cases of PTC. These results show that galectin-3 expression is a feature of malignant papillary projections but not of benign papillary hyperplasia. Thus, the immunohistochemical evaluation of galectin-3 might contribute to differential diagnosis between malignant and benign thyroid lesions with papillary projections.