Fabrizio Monaco

Thymosin‐α1 regulates MHC class I expression in FRTL‐5 cells at transcriptional level

In this study we examined the effect of the synthetic peptide thymosin‐α1 (Tα1) on MHC class I expression in FRTL‐5 cells. Treatment with Tα1 increased expression of MHC class I surface molecules and mRNA, which reached its peak (153u2004± 8u2009% of the control value) after 12u2004h. Chloramphenicol acetyltransferase (CAT) analysis, following transfection with a plasmid containing the regulatory sequence of MHC class I (or its deletion derivatives) with the CAT reporter gene, and electrophoretic mobility shift assay experiments demonstrated that the action of Tα1 was at the transcriptional level, and its mechanism of action is likely due to increased binding between the complex p50u2009/u2009fra‐2 and the enhancer A sequence of the 5 flanking region of a swine class I gene (PD1). An increase in the expression of MHC class I surface molecules was also observed by flow cytometry in murine and human tumor cell lines and in primary cultures of human macrophages. This study shows for the first time an effect of Tα1 on the regulation of gene expression at the molecular level, and may further contribute to explaining the results obtained using Tα1 in the control of infectious diseases and tumor growth.

A New Small-Molecule Antagonist Inhibits Graves' Disease Antibody Activation of the TSH Receptor

CONTEXTnGraves disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD.nnnOBJECTIVEnOur objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon.nnnDESIGNnWe aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA.nnnRESULTSnWe tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%.nnnCONCLUSIONnNCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.

Zinc Sulfate Supplementation Improves Thyroid Function in Hypozincemic Down Children

In subjects affected by trisomy 21 (Down syndrome), hypothyroidism is the most common endocrinological deficit. Plasma zinc levels, which are commonly detected below the normal range in Down patients, are related to some endocrinological and immunological functions; in fact, zinc deficiency has been shown to impair immune response and growth rate. Aims of this study were to evaluate (1) the role of zinc deficiency in subclinical hypothyroidism and (2) thyroid function changes in Down children cyclically supplemented with zinc sulfate. Inverse correlations have been observed between age and triiodotironine (T3) and between zinc and thyroid-stimulating hormone (TSH); higher TSH levels have been found in hypozincemic patients at the beginning of the study. After 6 mo of supplementation, an improvement of thyroid function (TSH levels: 3.96 ± 1.84 vs 2.64 ± 1.33 mUI/mL basally and after 6 mo, respectively) was observed in hypozincemic patients. In the second cycle of supplementation, a similar trend of TSH was observed. At the end of the study, TSH significantly decreased in treated hypozincemic subjects (4.48 ± 1.93 vs 2.96 ± 1.20 mUI/mL) and it was no longer different in comparison to normozincemic patients. We suggest zinc supplementation to the diet in hypozincemic Down children as a simple and useful therapeutic tool.

Role of smoking in goiter morphology and thyrotropin response to TRH in untreated goitrous women

Three groups of women were evaluated for TSH response to TRH and for goiter morphology by means of thyroid ultrasonography: group A = goitrous nonsmokers; group B1 = goitrous moderate-smokers; group B2 = goitrous heavy-smokers. They were compared with a control group (group C) made up nongoitrous, nonsmoking women. The size of the goiter was not correlated with the daily consumption of cigarettes, even though in heavy smokers a nodular goiter was prevalent as shown by ultrasonography. The serum values of TT3 showed significant differences between nonsmokers and heavy smokers (p< 0.005), whereas the serum values of TT4 and of basal TSH showed no statistically significant differences. On the contrary, the TSH response to TRH showed a significant difference between heavy and nonsmokers (p < 0.05). In conclusion, it has been demonstrated that goitrous cigarette heavy smokers show: i) A prevalence of statistically significant nodular goiter; ii) A significantly higher TT3 serum levels; iii) A significantly higher reof TSH to TRH. These data suggest that cigarette smoking favors the development of nodular goiter and can involve the central regulation of the hypothalamus-pituitary-thyroid interaction.

THYROID FUNCTION AND PLASMA SELENIUM IN CHRONIC UREMIC PATIENTS ON HEMODIALYSIS TREATMENT

It has been shown recently that Selenium (Se), an essential trace element for humans, is involved in the regulation of thyroid function, since the enzyme that catalyzes the liver conversion of the thyroid hormone T4 to the more active form T3 is a selenoenzyme. In chronic uremic patients, low blood Se levels as well as thyroid function abnormalities are often found. The present study was carried out to verify whether any correlation exists between Se levels and thyroid function, and to evaluate possible changes in hormonal pattern during Se supplementation in 10 chronic uremic patients on hemodialysis (HD) treatment. Se was supplemented orally as sodium selenite over six consecutive months. Basic plasma Se levels were significantly lower in patients than in normal controls. Right from the start of Se supplementation, plasma Se concentration promptly normalized and leveled off in the normal range throughout the study. Significant increase of FT3 and reduction of TSH levels were detected during Se supplementation. In Se-supplemented patients, a significant direct correlation was also found between reverse T3 (rT3) and TSH, and a significant inverse correlation was found between Se and TSH. Our results suggest that Se deficiency in chronic uremic patients represents a factor influencing the thyroid function and that the Se status should be determined in the evaluation of thyroid metabolism in these patients.

A TSHR-LH/CGR Chimera that Measures Functional Thyroid-Stimulating Autoantibodies (TSAb) Can Predict Remission or Recurrence in Graves' Patients Undergoing Antithyroid Drug (ATD) Treatment

CONTEXTnA functional thyroid-stimulating autoantibodies (TSAb) assay using a thyroid-stimulating hormone receptor chimera (Mc4) appears to be clinically more useful than the commonly used assay, a binding assay that measures all the antibodies binding to the thyroid-stimulating hormone receptor without functional discrimination, in diagnosing patient with Graves disease (GD).nnnOBJECTIVEnThe objective of the study was to investigate whether an Mc4 assay can predict relapse/remission of hyperthyroidism after antithyroid drug (ATD) treatment in patients with GD.nnnDESIGNnAn Mc4 assay was used to prospectively track TSAb activity in GD patients treated with ATD over a 5-yr period.nnnSETTING AND PATIENTSnGD patients from the Chieti University participated in this study.nnnINTERVENTIONSnInterventions included the assessment of patients sera using the Mc4 assay, the Mc4-derivative assay (Thyretain), and a human monoclonal thyroid-stimulating hormone receptor antibody, M22 assay.nnnMAIN OUTCOME MEASURESnThe Mc4 assay, a sensitive index of remission and recurrence, was used in this study.nnnRESULTSnThe TSAb levels significantly decreased only in the remitting group as evidenced by Mc4 assay values at the end of ATD (0.96 ± 1.47, 10.9 ± 26.6. and 24.7 ± 37.5 arbitrary units for the remitting, relapsing, and unsuspended therapy groups, respectively). Additional prognostic help was obtained by thyroid volume measurements at the end of treatment. Although not statistically significant, the Mc4 assay has a trend toward improved positive predictive value (95.4 vs. 84.2 or 87.5%), specificity (96.4 vs. 86.4 and 90.9%), and accuracy (87.3 vs. 83.3 and 80.9%) comparing the Mc4, Thyretain, and M22 assays, respectively. Thyretain has a trend toward improved negative predictive value (82.6 vs. 81.8 and 76.9%) and sensitivity (80 vs. 77.8 and 70%) comparing Thyretain, Mc4, and M22 assays, respectively.nnnCONCLUSIONnThe Mc4 assay is a clinically useful index of remission and relapse in patients with GD. Larger studies are required to confirm these findings.

The flavonoid quercetin inhibits thyroid-restricted genes expression and thyroid function.

Quercetin is the most abundant flavonoid present in a broad range of fruit and vegetables. Furthermore, quercetin is available as dietary supplements that are based on its antioxidant, antiproliferative and anti-inflammatory properties. However, concerns have been raised about the potential toxic effects of excessive intake of quercetin, and several studies have demonstrated that flavonoids, included quercetin, can interfere with thyroid function. In a previous report, we showed that quercetin inhibits thyroid-cell growth and iodide uptake. The latter effect was associated with down-regulation of sodium/iodide symporter gene expression. In the present study, we have evaluated the effects of quercetin on the expression of other thyroid-restricted genes, and we show that quercetin decreases the expression of the thyrotropin receptor, thyroid peroxidase and thyroglobulin genes. We further investigated the inhibitory effects of quercetin on thyroid function in vivo through evaluation of radioiodine uptake in the Sprague-Dawley rat, which was significantly decreased after 14 days of quercetin treatment. These data confirm that quercetin can act as a thyroid disruptor, and they suggest that caution is needed in its supplemental and therapeutic use.

A TSHr-LH/CGr Chimera that Measures Functional TSAb in Graves' Disease.

CONTEXTnStimulating thyrotropin receptor (TSHr) autoantibodies (TSAb) are the cause of hyperthyroidism in Graves disease. In a patients serum, TSAb can coexist with antagonist TSHr autoantibodies that block TSAb stimulatory activity (TSBAb); both can vary in amount and time.nnnOBJECTIVEnThe objective of the study was to create a functional assay that detects only TSAb, thus having an increased accuracy for diagnosing Graves disease.nnnDESIGNnA TSHr chimera (Mc4) that retains an agonist-sensitive TSAb epitope but replaces a TSBAb epitope was stably transfected in cells to establish the Mc4 assay.nnnSETTINGnThe study was conducted at the Chieti University (Outpatient Endocrine Clinic) and the University of Pisa (the Department of Endocrinology).nnnPATIENTSnThe assay was validated using sera from 170 individuals with Graves disease, Hashimotos thyroiditis, and nonautoimmune hyperthyroidism and normal subjects from Chieti University. A second blinded study evaluated sera from 175 patients with autoimmune thyroid disease (mainly Graves disease) from the University of Pisa.nnnINTERVENTIONSnInterventions included the assessment of patients sera using human wild-type TSHr (WT-TSHr), Mc4 chimera, and binding (TRAb) assays.nnnMAIN OUTCOME MEASURESnThe Mc4 assay has the best accuracy for diagnosing Graves disease.nnnRESULTSnThe Mc4 assay has a better diagnostic accuracy than WT-TSHr and second-generation TRAb assays. Indeed, the sensitivity of the WT-TSHr, TRAb, and Mc4 assays was 97.3, 86.5, and 100%, respectively, whereas the specificity was 93.1, 97, and 98.5%, respectively.nnnCONCLUSIONnThe Mc4 assay is a functional assay with improved sensitivity and specificity for the detection of TSAb and is clinically useful in diagnosing Graves disease.

High glucose levels increase major histocompatibility complex class I gene expression in thyroid cells and amplify interferon-γ action

Increased major histocompatibility complex (MHC) class I gene expression in target tissues may be relevant to the pathogenesis of autoimmune diseases. In this study, we questioned whether high glucose levels might increase MHC class I levels and thereby contribute to autoimmune complications. We used thyrocytes in continuous culture, because there is an increased incidence of autoimmune thyroiditis in type 2 diabetics and because transcriptional regulation of MHC class I is well studied in these cells. Northern analysis and flow cytometry showed that 20 and 30 m MD -glucose up-regulated MHC class I expression and that the glucose effect was additive to and independent of interferon-. The effect was specific, because L-glucose did not modify class I expression. The glucose acted transcriptionally, requiring both enhancer A and a cAMP-response element-like element located in the hormone-sensitive region of the MHC class I 5flanking region. These elements are different from those activated by interferon-. High glucose levels increase formation of the MOD-1 complex with enhancer A; MOD-1 is a heterodimer of fra-2 and the p50 subunit of NF-B. Both TSH and insulin are required for full expression of the glucose activity in thyrocytes. The glucose effect is partially blocked by wortmannin, suggesting involvement of the PI3K signal system. The data support the possibility that high serum glucose levels in type 2 diabetic patients may increase MHC class I levels in target tissues and contribute to autoimmune complications of the disease. (Endocrinology 143: 1008 –1017, 2002)

Gestational Diabetes and Thyroid Autoimmunity

Background. About 10% of pregnancies are complicated by previously unknown impairment of glucose metabolism, which is defined as gestational diabetes. There are little data available on prevalence of thyroid disorders in patients affected by gestational diabetes, and about their postgestational thyroid function and autoimmunity. We therefore investigated pancreatic and thyroid autoimmunity in gestational diabetic patients and in women who had had a previous gestational diabetic pregnancy. Methods. We investigated 126 pregnant women at the time of a 100-g oral glucose tolerance test: 91 were classified as gestational diabetics, and 35 were negative (controls). We also studied 69 women who had delivered a baby 18–120 months prior to this investigation and who were classified at that time gestational diabetics (38 women) or normally pregnant (31 women; controls). Results. Our data show no differences for both thyroid function and prevalence of autoimmune disorders during pregnancy; however, a significant increase in thyroid autoimmunity was seen in women previously affected by gestational diabetes. This increased prevalence of thyroid autoimmunity was not associated with the development of impaired glucose metabolism after pregnancy. Conclusions. Our data suggest that maternal hyperglycemia is a risk factor for the development of thyroid autoimmunity, a conclusion that should now be confirmed in a larger cohort of patients.