A case of neuroblastoma in DICER1 syndrome: Chance finding or noncanonical causation?

DICER1 syndrome is an inherited disorder associated with at least a dozen rare, mainly pediatric-onset tumors. Its characterization remains incomplete. Some studies suggested that neuroblastoma (NB) may be involved in this syndrome. Here, we describe the case of a 14-year-old female presenting with a multinodular goiter (MNG) and a collision tumor composed of NB and cystic nephroma (CN). She is a carrier of a deleterious germline mutation in exon 23 of DICER1 and harbored different somatic mutations in the CN and MNG. However, no second hit was found in the NB, questioning its status as a DICER1-related tumor.

Protein kinase Akt activity in human thyroid tumors.

We studied the expression and activation of the main effector protein kinase of phosphatidylinositol-3-kinase cascade (PI3K) ­ Akt in conventionally normal tissues, benign and highly differentiated (with and without metastases) human thyroid tumors. There was a difference in the Akt1 amount in tumor tissue compared with normal tissue in papillary carcinomas and tissue of multinodular goiter. Akt expression both in tumor and conventionally normal tissues of follicular adenoma was significantly lower than in follicular carcinoma. The lowest level of Akt expression was observed in tissues of multinodular goiter. Total activity of all three isoforms of Akt1/2/3 was lower in tumors compared to conventionally normal tissue. Thus, Akt activity (according to Thr308 phosphorylation) is not associated with proliferative processes in the tumor tissue of the thyroid. Apoptosis level detected in these tissues was not associated with the protein kinase activity either. Possible mechanisms of signaling cascade PI3K/Akt inhibition in thyroid tumors are discussed.

Influence of thyroid disorders on the kidney expression and plasma activity of aminopeptidase A.

OBJECTIVE: Thyroid disorders may affect blood pressure and renal function modifying factors of the plasmatic and kidney renin-angiotensin system such as aminopeptidase A (AP A) that metabolizes angiotensin II to angiotensin III. We investigated the expression of AP A in the kidney, as well as its enzymatic activity in the plasma of euthyroid, hyperthyroid, and hypothyroid adult male rats. METHODS: Hyperthyroidism was induced by daily subcutaneous injections of tetraiodothyronine. Hypothyroid rats were obtained by administration of methimazole in drinking water. Expression of AP A was determined by Western blot analysis. Plasma AP A activity was measured fluorometrically using glutamyl-ß-naphthylamide as substrate. RESULTS: While hyperthyroid rats exhibited lower levels of plasma AP A activity than controls, the kidney of hyperthyroid animals expressed significantly higher AP A than controls and hypothyroid animals. CONCLUSIONS: A discrepancy between the high expression of AP A in kidney of hyperthyroid rats and the low activity of AP A measured in plasma and kidney of hyperthyroid animals was found. The posttranslational influence of environmental biochemical factors may be in part responsible for that divergence.

Abnormalities of apoptosis of the thyroid gland cells from extratumoral microfollicular tissue.

AIM: The aim of this study was to evaluate the frequency of existence of thyroid extratumoral normo- and microfollicular tissue in patients with thyroid carcinoma and peculiarities of apoptosis in mentioned tissue. MATERIALS AND METHODS: Using samples of normo- and microfollicular thyroid tissue it was determined the content of fragmented DNA and intensity of stimulated internucleosomal DNA fragmentation; activities of caspase-3 and cysteine lysosomal cathepsins. RESULTS: It was found that normofollicular tissue is observed more often in patients with nodal euthyroid goiter but microfollicular tissue is more common for patients with carcinoma. Extratumoral microfollicular tissue was found in the thyroid of patients above 50 years old mostly, and more rarely in young ones. The fragmented DNA concentration in microfollicular tissue was lower by a factor of 3.5 and intensity of stimulated internucleosomal DNA fragmentation was also decreased. Activity both of cathepsin B in lisosomes and caspase-3 in lysates of such tissue was also decreased. CONCLUSIONS: The decrease of intensity of spontaneous apoptosis and the absence of its modulation/induction following proapoptotic factors in extratumoral microfollicular thyroid tissue may be considered as a respond of the thyroid gland tissue to an existence of carcinoma.

Investigation of the Mg-HCO3⁻-ATPase activity of thyroid tissue cells under various pathologies.

Distribution of the Mg²âº-dependent, HCO3⁻-stimulated ATPase (HCO3⁻-ATPase) was investigated in sub-cellular fractions of the tissue of the human thyroid gland. It was found that especially high enzymatic activity is characteristic of mitochondria and the endoplasmic reticulum. Correlation between various pathologies of the thyroid and HCO3⁻-ATPase activity was demonstrated. The activity was evaluated using the difference between active and passive ATPase. During development of the various pathologies, the sub-cellular fractions of the gland showed uneven alterations of HCO3⁻-ATPase activity. Intriguingly, the enzyme kinetic parameters are also changed, i.e. in the different pathologies both enzyme activity and its affinity towards bicarbonate ions are altered. This raises the question whether pathology is caused by, or results in, changes in the enzyme at the molecular level.

Positive correlation between type 1 and 2 iodothyronine deiodinases activities in human goiters.

Type 1 (D1) and 2 (D2) iodothyronine deiodinases are selenocysteine-containing enzymes that catalyze the deiodination of T4 to T3 in the thyroid and in peripheral tissues. Despite their importance to the plasma T3 pool in human beings, there are few studies about their behavior in human thyroids. In order to better understand iodothyronine deiodinase regulation in the thyroid gland, we studied thyroid tissue samples from follicular adenoma (AD, n = 5), toxic diffuse goiter (TDG, n = 6), nontoxic multinodular goiter (NMG, n = 40), papillary thyroid carcinoma (PTC, n = 8), and surrounding normal tissues (NT, n = 7) from 36 patients submitted to elective thyroidectomy. D1 and D2 activities were determined by quantification of the radioiodine released by ¹²5I-rT3 or ¹²5I-T4 under standardized conditions, and expressed as pmol rT3 deiodinated per minute and mg protein (pmol rT3 min⁻¹ mg⁻¹ ptn) and fmol T4 deiodinated per minute and mg protein (fmol T4 min⁻¹ mg⁻¹ ptn), respectively. D1 activity detected in TDG and AD tissues were significantly higher than in NT, PTC or NMG samples. D2 activity was also significantly higher in TDG and AD samples than in PTC, NMG, or NT. There was great variability in D1 and D2 enzymatic activities from distinct patients as well as from different areas from the same goiter. There was a positive correlation (P < 0,0001, r = 0.4942) between D1 and D2 activities when all samples were taken into account, suggesting that-in the thyroid-these two iodothyronine deiodinases may have related regulatory mechanisms, even if conditioned by other as yet unknown factors.

Cytophotometric expression of Caspase-3 in papillary thyroid carcinoma in nodular goiter colloid.

OBJECTIVE: To describe quantitative cytophotometric expression of the marker caspase-3 in colloid goiter and in papillary carcinoma of the thyroid, comparing the immunoexpression between them. METHODS: An immunohistochemistry study has been made on the protein caspase-3 in 17 of paraffin blocks of papillary carcinoma of the thyroid and 20 cases of colloid goiter, using SAMBA 4000 ® (System of Microscopic Analysis of Automatic Search), aiming analysis of the two variables: marker index and optical density. RESULTS: Statistic analysis indicated a significant difference for marker index between colloid goiter and papillary carcinoma, being this difference larger in the carcinoma. Significant difference in optical density hasn't been found. For colloid goiter, the estimated correlation coefficient between the marker index and optical density was 0,72, indicating the rejection of the null hypothesis (p <0,001), affirming that positive and significant association exists between them. For the papillary carcinoma of the thyroid, the same correlation was 0,34 and so, it is not possible to affirm that there's an association between them. CONCLUSION: For colloid goiter there is a positive and significant association between the two variables - marker index and optical density -, while for the papillary carcinoma, this is not proved. The quantitative analysis for caspase-3 demonstrates that apoptosis is larger in the papillary carcinoma of the thyroid than in colloid goiter.

Expressão citofotométrica da Caspase-3 no carcinoma papilífero da tireóide e no bócio colóide / Citophotometric expression of Caspase-3 in papillary thyroid carcinoma in nodular goiter colloid

OBJETIVO: Descrever a expressão citofotométrica quantitativa do marcador caspase-3 no bócio colóide e no carcinoma papilífero da tireóide e comparar a imunoexpessão entre as doenças. MÉTODOS: Realizou-se estudo imunoistoquímico da proteína caspase-3 em 17 blocos de parafina de carcinoma papilífero da tiróide e 20 de bócio colóide, através do sistema SAMBA 4000 - (Sistema de análise microscópica de busca automática), objetivando-se analisar duas variáveis: índice de marcagem e densidade óptica. RESULTADOS: Houve diferença significativa quanto ao índice de marcagem da caspase-3, entre o bócio colóide e o carcinoma papilífero, sendo maior no carcinoma, e não foi encontrada diferença significativa quanto à densidade óptica. Para o bócio colóide, o coeficiente de correlação estimado entre o índice de marcagem e a densidade óptica foi igual a 0,72, indicando assim, a rejeição da hipótese nula (p <0,001), afirmando-se que existe associação positiva e significativa entre o índice de marcagem e a densidade óptica da caspase-3. Para o carcinoma papilífero da tiróide, o coeficiente de correlação estimado entre o índice de marcagem e a densidade óptica 3 foi de 0,34. O resultado do teste estatístico indicou que não se pode afirmar que existe associação entre esses parâmetros. CONCLUSÃO: Para o bócio colóide existe associação positiva e significativa entre as duas variáveis, índice de marcagem e a densidade óptica da caspase-3, enquanto que para o carcinoma papilífero não existe essa associação. O estudo comparativo entre a análise quantitativa da caspase-3, demonstrou que a apoptose é mais evidente no carcinoma papilífero do que no bócio colóide.

OBJECTIVE: To describe quantitative cytophotometric expression of the marker caspase-3 in colloid goiter and in papillary carcinoma of the thyroid, comparing the immunoexpression between them. METHODS: An immunohistochemistry study has been made on the protein caspase-3 in 17 of paraffin blocks of papillary carcinoma of the thyroid and 20 cases of colloid goiter, using SAMBA 4000 ® (System of Microscopic Analysis of Automatic Search), aiming analysis of the two variables: marker index and optical density. RESULTS: Statistic analysis indicated a significant difference for marker index between colloid goiter and papillary carcinoma, being this difference larger in the carcinoma. It hasn't been found significant difference in optical density. For colloid goiter, the estimated correlation coefficient between the marker index and optical density was 0,72, indicating the rejection of the null hypothesis (p <0,001), affirming that positive and significant association exists between them. For the papillary carcinoma of the thyroid, the same correlation was 0,34 and so, it is not possible to affirm that there's an association between them. CONCLUSION: For colloid goiter there is a positive and significant association between the two variables - marker index and optical density -, while for the papillary carcinoma, this is not proved. The quantitative analysis for caspase-3 demonstrates that apoptosis is larger in the papillary carcinoma of the thyroid than in colloid goiter.

Increased lipid peroxidation and impaired enzymatic antioxidant defense mechanism in thyroid tissue with multinodular goiter and papillary carcinoma.

OBJECTIVES: We aimed to evaluate the oxidant/antioxidant status of thyroid tissue in patients with multinodular goiter, papillary carcinoma and to compare with their nonpathologic tissues. METHODS: We studied 41 patients with multinodular goiter who underwent surgical treatment. The patients were divided into three groups according to clinical diagnosis. Malondialdehyde, selenium, total superoxide dismutase and glutathione peroxidase of thyroid tissue samples were determined in 14 toxic multinodular goiters, 18 non-toxic multinodular goiters, and 9 papillary carcinomas. RESULT: Superoxide dismutase and glutathione peroxidase and selenium were found lower but malondialdehyde was higher in both nodule and cancerous tissues compared with those of control ones. The level of malondialdehyde in non-toxic multinodular goiters group was higher than toxic multinodular goiters group in nodule tissues. CONCLUSIONS: It can be stated that the lipid peroxidation is increased and enzymatic free radical defense system was significantly impaired in patients with both multinodular goiters and papillary carcinomas.

Pendred syndrome in two Galician families: insights into clinical phenotypes through cellular, genetic, and molecular studies.

CONTEXT: We studied two families from Galicia (northwest Spain) with Pendred syndrome (PS) and unusual thyroid phenotypes. In family A, the proposita had a large goiter and hypothyroxinemia but normal TSH and free T3 (FT3). In family B, some affected members showed deafness but not goiter. OBJECTIVE: Our objective was to identify the mutations causing PS and molecular mechanisms underlying the thyroid phenotypes. INTERVENTIONS: Interventions included extraction of DNA and of thyroid tissue. PATIENTS: Propositi and 10 members of the two families participated in the study. MAIN OUTCOME MEASURES: Main outcome measures included SLC26A4 gene analysis, deiodinase activities in thyroid tissue, and c.416-1G-->A effects on SLC26A4 splicing. In addition, a primary PS thyrocyte culture, T-PS2, was obtained from propositus B and compared with another culture of normal human thyrocytes, NT, by Western blotting, confocal microscopy, and iodine uptake kinetics. RESULTS: Proposita A was heterozygous for c.578C-->T and c.279delT, presented with goiter, and had normal TSH and FT3 but low FT4 attributable to high type 1 and type 2 iodothyronine deiodinase activities in the goiter. Propositus B bore c.279delT and a novel mutation c.416-1G-->A; some deaf relatives were homozygous for c.416-1G-->A but did not present goiter. The c.279delT mutation was associated with identical haplotype in the two families. T-PS2 showed truncated pendrin retained intracellularly and high iodine uptake with low efflux leading to iodine retention. CONCLUSIONS: c.279delT is a founder mutation in Galicia. Proposita A adapted to poor organification by increasing deiodinase activities in the goiter, avoiding hypothyroidism. Lack of goiter in subjects homozygous for c.416-1G-->A was due to incomplete penetrance allowing synthesis of some wild-type pendrin. Intracellular iodine retention, as seen in T-PS2, could play a role in thyroid alterations in PS.

[Cytofluorymetric evaluation of antigen regions of human thyroid peroxidase in patients with Graves' disease and non-toxic multinodular goiter using mouse monoclonal antibodies]. / Ocena cytofluorymetryczna wybranych regionów antygenowych ludzkiej tyreoperoksydazy u pacjentów z choroba Gravesa-Basedowa oraz z wolem wieloguzkowym nietoksycznym z zastosowaniem mysich przeciwcial monoklonalnych.

The aim of this study was to estimate expression of surface antigen regions of TPO (thyroid peroxidase: #1, #18, #30, #64 epitopes) on thyroid cells in 15 patients with non-toxic multinodular goiter (NTMG) and 15 patients with Graves' disease (GD). The thyrocytes were identified by indirect method: in first stage we added mouse monoclonal autoantibodies specific for TPO regions and in second stage we conjugated this complex with rabbit anti-mouse antibodies IgG (Fab')2 with FITC. All investigations were performed by flow cytometry using apparatus Coulter EPICS XL. The percentages of thyroid cells with expression of antigen regions of TPO 1, 18, 30, 64 were measured in relationship to the responsible anti-TPO concentrations: 50, 100, 200, 400, 800, 1600 microg/ml. The analysis of the expression of epitope #64 TPO revealed insignificantly increased percentages of thyroid cells in patients with GD (73% vs 45%, ns) in comparison to NTMG at anti-TPO antibody concentration of 1600 microg/ml. In addition, we observed that reduction concentration of anti-TPO antibodies leads to the decreased percentage of thyroid cells with antigen region #64 expression. In patients with GD percentage of this cells was significantly higher (48% vs 7% p<0.019, 29% vs 56% p<0.05) in compared to the percentage of thyroid cells from patients with NTMG at concentration of 200-800 microg/ml anti-TPO antibodies. Analysis of epitopes #1 and #18 shown higher percentage of thyroid cells in GD (25% vs 20%, ns for #1 epitope) and (25% vs 13%, ns for #18 epitope) in comparison to the patients with NTMG at concentration 1600 microg/ml of anti-TPO antibodies. The percentages of thyrocytes with epitopes #1 and #18 were decreased in relation to the reduction of anti-TPO concentrations. However, in all our patients epitope #30 TPO was found only in 8% thyroid cells. We conclude that in young patients thyroid immune and nonimmune diseases predispose to elevated expression of TPO epitopes (#1, #18, #64) which suggested increase stimulation and activation of thyroid cells during inflammatory reaction within thyroid gland. Furthermore, dominance expression of #64 TPO epitope in Graves' patients which recognized B domain could be a useful marker of activity of immune process in concentration between 200-800 microg/ml of TPO antibodies.

Application of mouse monoclonal antibodies for identification of antigen regions of human thyroid peroxidase in adolescents with Graves' disease and non-toxic multinodular goiter by flow cytometry.

Thyroid peroxidase (TPO) is the major thyroid autoantigen recognized by serum autoantibodies from patients with Graves' disease (GD) or Hashimoto's thyroiditis directed to two immunodominant conformational regions termed A and B. The epitopes of human TPO have been defined using a panel of mouse monoclonal antibodies (mAbs). The aim of this study was to estimate the expression of chosen surface antigen regions of TPO (1, 18, 30, 64 epitopes) on thyroid cells in 15 patients with non-toxic multinodular goiter (NTMG) and 15 patients with GD. The thyrocytes were identified by indirect method: in the first stage we added mouse monoclonal autoantibodies specific for TPO regions and in the second stage we conjugated this complex with rabbit anti-mouse antibodies IgG (Fab')(2) with FITC. All investigations were performed by flow cytometry using Coulter EPICS XL apparatus. The percentages of thyrocytes with expression of epitopes 1, 18, 30, 64 TPO were measured in relation to the respective anti-TPO concentrations: 50-1600 microg/ml. The analysis of epitopes located in immunodominant regions (IDR) of TPO revealed higher percentages of thyrocytes in cases with GD in comparison to NTNG. The most predominant difference was observed for mAb 64 epitope (48 vs 7%, p < 0.019; 39 vs 5%, p < 0.017) at the concentration of 100-200 microg/ml mAbs. The expression of 18 epitope on thyrocytes was also statistically higher in Graves' patients than in the NTMG (14 vs 6%, p < 0.025) at concentration of 400 microg/ml mAbs. However, this expression was much less pronounced. In all the cases, the percentages of thyrocytes with epitopes 1 and 30 were in low detection (8-15% of positive cells). In conclusion, our findings suggest that the elevated expression of TPO epitopes 18 and 64 in young patients with thyroid autoimmune diseases increase stimulation and activation of thyroid cells during inflammatory reaction within the thyroid gland. In addition, predominant expression of 64 TPO epitope that recognizes B domain in GD patients could be a useful marker of the immune process in the thyroid gland.

Association of serum paraoxonase activity with insulin sensitivity and oxidative stress in hyperthyroid and TSH-suppressed nodular goitre patients.

OBJECTIVE: Low serum paraoxonase (PON) activity is thought to be a risk factor for the development of atherosclerosis. The present study was designed to evaluate PON1 activity and its relationship with preatherosclerotic markers such as lipid peroxidation and insulin resistance in hyperthyroid patients before and after propylthiouracil (PTU) treatment and in subjects with iatrogenic subclinical hyperthyroidism. PATIENTS AND DESIGN: Twenty patients with hyperthyroidism, 20 patients with euthyroid multinodular goiter (MNG) and 20 age- and sex-matched healthy controls were enrolled in the study. Insulin sensitivity index, PON activity and lipid peroxidation were measured at baseline and 2 months after achieving euthyroidism or subclinical hyperthyroidism. Levothyroxine was given as a part of TSH suppression therapy in multinodular goitre patients. MEASUREMENTS: Insulin sensitivity was determined by an oral glucose tolerance test (OGTT) based on the insulin sensitivity index (ISI) formula, serum paraoxonase activity was determined with a spectrophotometric method. Lipid peroxidation was measured by the formation of thiobarbituric acid reactive substances (TBARs). RESULTS: ISI was significantly lower in the hyperthyroid group than baseline levels in MNG patients and controls (P < 0.001). While ISI increased after treatment in the hyperthyroid group (P < 0.01), it significantly decreased with L-T4 treatment in the MNG group (P < 0.01). Serum paraoxonase activity was significantly lower in the hyperthyroid group before treatment than baseline and final measurements of other groups (P < 0.05). While PON activity increased after restoration of the euthyroid state in the hyperthyroid group (P < 0.05), it decreased with L-T4 treatment in the MNG group (P < 0.05). Lipid peroxidation was significantly higher in hyperthyroid group compared to baseline levels of other groups (P < 0.05). It decreased after treatment in the hyperthyroid group (P < 0.05) but a significant increase was observed following L-T4 treatment in the MNG group (P < 0.05). Serum paraoxonase activity was found to be negatively correlated with serum TT4 (r = -0.32, P = 0.003), TT3 levels (r = -0.31, P = 0.004), TBARs levels (r = 0.32, P = 0.003) and positively correlated with ISI (r = 0.35, P = 0.001) and high-density lipoprotein (HDL) cholesterol levels (r = 0.35, P = 0.0011) in the hyperthyroid and MNG groups. CONCLUSIONS: Iatrogenic thyroid hormone excess seems to mimic the effects of endogenous thyroid hormone excess on paraoxonase activity, insulin sensitivity and oxidative stress. These findings suggest that TSH suppression with levothyroxine may increase oxidative stress and LDL oxidation and thereby promote atherogenesis.

Increased thymidine kinase activity in human thyroid toxic adenomas: effects of exposure to epidermal growth factor in vitro.

UNLABELLED: The activity of thymidine kinase (TK-EC 2.7.1.21)--an enzyme functioning as a part of the pyrimidine salvage pathway of DNA synthesis--is closely related to growth processes. The aim of the study was to measure TK activity in homogenates of human thyroid tissue of the following types: non-toxic nodular goiter (NTNG)--macroscopically unchanged tissue, non-toxic adenoma (NTA), and toxic adenoma (TA) (obtained from patients, who--before the surgery--had been treated with thyrostatic drugs for thyrotoxicosis). Thyroid tissue was obtained from female patients subjected to subtotal thyroidectomy at the Department of Endocrine Surgery, Medical University of Lódz. Thyroid homogenates were incubated in the presence of epidermal growth factor (EGF), used in five different concentrations (0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml). TK activity was estimated by chromatographic measurements of the amount of the main reaction product--deoxythymidine monophosphate. RESULTS: 1) We did not observe any significant difference between TK activity in the homogenates of the thyroid tissue collected from NTNG and NTA; TK activity was clearly higher in the homogenates of adenomatous tissue, collected from the patients with TA; 2) EGF increased TK activity in the homogenates of the macroscopically unchanged tissue, collected--during surgery--from the patients with NTNG, as well as in homogenates of thyroid tissue from NTA; 3) In case of hyperactive thyroid tissue, obtained from TA, EGF tended to increase TK activity, however, without any statistical differences. Our results confirm TK increased activity in hyperactive thyroid tissue. At the same time, the obtained data suggest a certain role of EGF in goiter formation in humans.

[Plasminogen activators of urokinase and tissue types and their inhibitor (PAI-1) in cytosol fraction in thyroid diseases]. / Aktivatory plazminogena urokinaznogo i tkanevogo tipov i ikh ingibitor (PAI-I) v tsitozol'noi fraktsii pri zabolevaniiakh shchitovidnoi zhelezy.

Enzyme immunoassay (ELISA) was used to estimate the levels of plasminogen activators of urokinase (uPA) and tissue (tPA) types and one of their inhibitors (PAI-I) in the cytosolic fraction of the thyroid in 129 patients with malignant and benign tumors and various non-cancer diseases of the gland. Tumors from patients with thyroid cancer displayed the lowest levels of tPA and the highest levels of uPA and PAI-I, while those from patients with benign thyroid diseases, including adenoma, had high concentrations of tPA and relatively low levels of uPA and PAI-I in the tissue of the diseased organ. At the same time, the lowest levels of uPA and PAI-I were found in patients suffering from toxic goiter with and without adenomatosis. In terms of uPA and PAI-I levels, patients with nodular colloidal goiter were intermediate between those with toxic goiter and adenoma, on the one hand, and those with thyroid cancer, on the other.

Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase (ThoX, LNOX, Duox) genes and proteins in human thyroid tissues.

The large homolog of NADPH oxidase flavoprotein LNOX2, and probably LNOX1, are flavoproteins involved in the thyroid H(2)O(2) generator. Western blot analysis of membrane proteins from normal human thyroid, using antipeptide antibodies, indicated that LNOX1,2 are 164-kDa glycoproteins and that N-glycosylated motifs account for at least 10-20 kDa of their total apparent molecular mass. Northern blot analysis of 23 different human tissues demonstrated that LNOX2 messenger RNA (mRNA) is strongly expressed only in the thyroid gland, although blast analysis of expressed sequence tags databases indicated that LNOX genes are also expressed in some nonthyroid cells. We investigated LNOX1,2 gene and protein expressions in normal and pathological human thyroid tissues using real-time kinetic quantitative PCR and antipeptide antibodies, respectively. In normal tissue, LNOX1,2 are localized at the apical pole of thyrocytes. Immunostaining for LNOX1,2 was heterogeneous, inside a given follicle, with 40-60% of positive follicular cells. Among normal and pathological tissues, variations of LNOX1 and LNOX2 mRNA levels were parallel, suggesting a similar regulation of both gene expressions. Whereas LNOX mRNAs seemed slightly affected in benign disease, the expression of protein was highly variable. In multinodular goiters, 40-60% of cells were stained. In hypofunctioning adenomas, LNOX immunostaining was highly variable among follicles, whereas sodium/iodide (Na+/I-) symporter immunostaining was decreased. In hyperfunctioning thyroid tissues, only few cells (0-10%) were weakly stained, whereas sodium/iodide symporter staining was found in the majority of follicular cells. In conclusion, LNOX proteins are new apical glycoproteins with a regulation of expression that differs from other thyroid markers.

Telomerase activity and telomere length in thyroid neoplasia: biological and clinical implications.

Despite several recent studies, the biological status and clinical relevance of telomerase expression in tumours derived from the thyroid follicular cell remain controversial. This study has analysed a series of normal, benign, and malignant thyroid samples using two novel approaches: the use of purified epithelial cell fractions to eliminate false-positives due to telomerase-positive infiltrating lymphocytes; and the simultaneous measurement of telomere length to provide a clearer interpretation of telomere dynamics in thyroid neoplasia. The data obtained support the prediction that the epithelial component of non-neoplastic thyroid and of follicular adenomas is telomerase-negative, any positive results being explicable by lymphocyte infiltration. In contrast, many malignant tumours, both follicular and papillary, were telomerase-positive. However, serial dilution of extracts indicated a wide spectrum of activity in these cancers, possibly related to variation in the proportion of telomerase-positive cells. Furthermore, an unexpectedly high proportion were telomerase-negative, a finding which was not explicable by technical problems such as TRAP (telomeric repeat amplification protocol) assay sensitivity. Many of these apparently telomerase-negative tumours had abnormally long telomeres. Correlation of telomerase and telomere length data suggests that thyroid cancers fall into three biological groups: telomerase-positive lesions, consistent with the conventional model of telomere erosion followed by telomerase reactivation; telomerase-negative tumours, which maintain telomere length by a mechanism independent of telomerase; and telomerase-negative tumours which are still undergoing telomere erosion and may therefore be composed of mortal cancer cells. From a clinical standpoint, it is concluded that telomerase detection on unfractionated tissue, such as fine needle aspirates, is of no value as a marker of malignancy in follicular lesions, due to both low sensitivity and specificity.

Erythrocyte, plasma, and serum antioxidant activities in untreated toxic multinodular goiter patients.

Erythrocyte, plasma, and serum antioxidant activities were studied in patients with newly diagnosed and untreated toxic multinodular hyperthyroid goiter and compared to healthy control subjects. Erythrocyte antioxidant enzyme activities, glutathione, malondialdehyde, and ceruloplasmin levels were significantly increased, whereas serum vitamin E, plasma vitamin C, and selenium levels were decreased in hyperthyroid patients compared to control subjects. The findings show that untreated toxic multinodular goiter causes profound alterations in components of the antioxidant system in erythrocytes indicative of increased oxidative stress. Taken together, these data suggest that hyperthyroid patients may benefit from dietary supplements of antioxidants.

Cathepsin B activity and protein levels in thyroid carcinoma, Graves' disease, and multinodular goiters.

Cathepsin B (CB) is involved in the hydrolysis of thyroglobulin (Tg) and thought to be regulated by thyroid stimulating hormone (TSH) in the normal thyroid. Our analyses of 91 thyroid tissues from 71 patients with Graves' disease (GD), multinodular goiter (MNG), papillary carcinoma (PC), or follicular carcinoma (FC), demonstrated a 2-fold increase in expression of CB in GD and an average increase of 1.5-fold in MNG (varying from 10-fold below normal to 6-fold above normal in MNG nodules), as might be predicted by the altered functional status of thyroid follicular cells in those diseases. However, CB activity was not downregulated in conjunction with the known "blocking effect" of malignancy on many thyroid functions, but rather increased on average 9-fold in papillary carcinomas (n = 33), and also showed a marked increase in 2 follicular carcinomas. Activity measurements were confirmed by CB protein detection on Western blot with moderately increased CB protein levels demonstrated in GD, variable expression in nodules of MNG, and markedly increased protein expression in carcinomas. In all diseased states, increased protein was detected primarily as overexpression of the 27 kd heavy chain of 2-chain mature CB and less frequently as overexpression of 31 kd single-chain mature CB. However, an additional 35 kd protein form was noted in 3 of 9 PCs, 1 of 2 FCs, and 1 of 4 GD cases but in none of 10 MNG cases. In conjunction with elevated CB activity plus additional protein bands on Western blots, altered patterns of CB immunohistochemical staining were observed, irrespective of the type of thyroid disease, suggesting certain common changes in CB expression, posttranslational processing, and vesicular trafficking. In summary, GD and MNG thyroid tissues demonstrated altered CB expression in keeping with predicted functional changes in thyroid follicular cells, while increased CB expression in carcinomas indicated a more pathological role for CB in thyroid cancers, possibly related to the processes of invasion or metastasis.

Immunohistochemical assessment of peroxidase-like immunoreactivity in the thyroid gland and its correlation with biochemical assay.

BACKGROUND: Peroxidase content has been recently evaluated in normal thyroid and in different thyroid disorders by biochemical, histochemical, ultrastructural and immunocytochemical methods. Nevertheless immunocytochemical detection of thyroid peroxidase in thyroid samples conventionally processed for histology has never been done using a commercially available antibody, neither its correlation with the biochemical activity on adjacent samples. METHODS: In this study we have analyzed normal thyroid tissue (3 patients), follicular adenoma (2 patients) and multinodular goiter (2 patients) conventionally processed for histology and stained by immunocytochemistry (Avidin Biotin System) using a polyclonal (rabbit) antibody for horseradish peroxidase (Serotec). Biochemical assay was performed on adjacent samples according to Hosoya method. RESULTS: Normal thyroid showed peroxidase immunoreactivity in the majority of follicular cells; neoplastic cells of adenomas were variably stained. Biochemical assay showed positive correlation with ICC ranging from 20.4 micrograms/mg/prot a in multinodular goiter to 42.12 in normal thyroid, up to 122 of follicular adenoma. CONCLUSIONS: Peroxidase content in the thyroid gland may be of clinical interest in several thyroid diseases, and in this study we have demonstrated that thyroid peroxidase can be detected by ICC in routinely processed thyroid samples using a commercially available antibody.