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In previous studies we have demonstrated that the expression of the Major Histocompatibility Complex (MHC) class I gene in thyrocytes is controlled by several hormones, growth factors, and drugs. These substances mainly act on two regions of the MHC class I promoter a “tissue-specific” region (−800 to −676 bp) and a “hormone/cytokines-sensitive” region (−500 to −68 bp). In a previous study, we have shown that the role of the “tissue-specific” region in the MHC class I gene expression is dominant compared to that of the “hormone/cytokines-sensitive” region. In the present report we further investigate the dominant role of the “tissue-specific” region evaluating the effect of thyroid stimulating hormone (TSH), methimazole (MMI), phenylmethimazole (C10), glucose and thymosin-α1. By performing experiments of electrophoretic mobility shift assays (EMSAs) we show that TSH, MMI and C10, which inhibit MHC class I expression, act on the “tissue-specific” region increasing the formation of a silencer complex. Glucose and thymosin-α1, which stimulate MHC class I expression, act decreasing the formation of this complex. We further show that the silencer complex is formed by two distinct members of the transcription factors families activator protein-1 (AP-1) and nuclear factor-kB (NF-kB), c-jun and p65, respectively. These observations are important in order to understand the regulation of MHC class I gene expression in thyroid cells and its involvement in the development of thyroid autoimmunity.

The regulation of the female reproductive system is one of the most relevant actions of thyroid hormones. Adequate thyroid hormones production is essential for normal menstrual function and fertility as well as for the successful maintenance of pregnancy. The relationship between reproductive failure and thyroid disorders is particularly relevant and attracts attention worldwide. Thyroid autoimmunity (TAI), defined by the presence of circulating antithyroid antibodies targeting thyroid peroxidase (TPOAb) and thyroglobulin (TgAb), is prevalent among women of reproductive age and is the most frequent cause of thyroid dysfunction. Several studies addressed the association between TAI, thyroid function, and fertility as well as pregnancy outcome after spontaneous or assisted conception. Infertility, miscarriages, and fetal-maternal complications are described in overt autoimmune hypothyroidism. More debatable is the role of mild thyroid dysfunction, mainly subclinical hypothyroidism (SCH), and TAI in the absence of thyroid dysfunction in infertility and reproductive outcome. Assisted reproductive technology (ART) has become an integral element of care for infertility. Women with TAI undergoing ART are of particular interest since they carry a higher risk of developing hypothyroidism after the ovarian stimulation but whether TAI, in absence of thyroid dysfunction, adversely affects ART outcome is still controversial. Likewise, the role of levothyroxine (LT4) in improving fertility and the success of ART in euthyroid women with TAI is unclear. This review discusses the role of TAI, in the absence of thyroid dysfunction, in infertility and in ART outcome.

In the past few decades, there has been a lot of interest in plant constituents for their antioxidant, anti-inflammatory, anti-microbial and anti-proliferative properties. However, concerns have been raised on their potential toxic effects particularly when consumed at high dose. The anti-thyroid effects of some plant constituents have been known for some time. Indeed, epidemiological observations have shown the causal association between staple food based on brassicaceae or soybeans and the development of goiter and/or hypothyroidism. Herein, we review the main plant constituents that interfere with normal thyroid function such as cyanogenic glucosides, polyphenols, phenolic acids, and alkaloids. In detail, we summarize the in vitro and in vivo studies present in the literature, focusing on the compounds that are more abundant in foods or that are available as dietary supplements. We highlight the mechanism of action of these compounds on thyroid cells by giving a particular emphasis to the experimental studies that can be significant for human health. Furthermore, we reveal that the anti-thyroid effects of these plant constituents are clinically evident only when they are consumed in very large amounts or when their ingestion is associated with other conditions that impair thyroid function.

The 90K protein, also known as Mac-2 BP or LGALS3BP, can activate the immune response in part by increasing major histocompatibility (MHC) class I levels. In studies on a non-immune cell model, the rat FRTL-5 cell line, we observed that transforming growth factor (TGF)-β1, like γ-interferon (IFN), increased 90K levels, despite its immunosuppressive functions and the ability to decrease MHC class I. To explain this paradoxical result, we investigated the mechanisms involved in the TGF-β1 regulation of 90K expression with the aim to demonstrate that TGF-β1 utilizes different molecular pathways to regulate the two genes. We found that TGF-β1 was able to increase the binding of Upstream Stimulatory Factors, USF1 and USF2, to an E-box element, CANNTG, at −1926 to −1921 bp, upstream of the interferon response element (IRE) in the 90K promoter. Thyrotropin (TSH) suppressed constitutive and γ-IFN-induced 90K expression by decreasing USF binding to the E-box. TGF-β1 was able to overcome TSH suppression at the transcriptional level by increasing USF binding to the E-box. We suggest that the ability of TGF-β1 to increase 90K did not result in an increase in MHC class I because of a separate suppressive action of TGF-β1 directly on the MHC class I gene. We propose that the increased levels of 90K may play a role, rather than in immune response, in the context of the TGF-β1-induced changing of the cellular microenvironment that predisposes to cell motility and cancer progression. Consistently, analyzing the publicly available cancer patient data sets cBioPortal, we found that 90K expression directly correlated with TGF-β1 and USFs and that high levels of 90K were significantly associated with increased mortality in patients affected by different types of cancer.

Background: Neonatal thyrotropin (TSH) on dried blood spot (DBS), the most common screening strategy for primary congenital hypothyroidism (CH), is influenced by numerous factors that may hinder a true CH diagnosis. A second test can thus be performed to clarify the initial findings, although its application varies among screening programs. Objectives: The aim of this study was to evaluate the effect of maternal and neonatal factors on neonatal TSH levels and offer practical screening recommendations. Methods: We retrospectively analyzed screening data of 62,132 neonates born in Abruzzo, an Italian region considered mildly iodine deficient, between 2011 and 2016. We then performed a multiple linear regression to model the relationship between TSH (the dependent variable) and 13 independent variables extracted from blood collection cards. Results: Most neonates (53,551 of 62,132, 86%) had normal TSH and no clinical indications for a second screening. A minority (1,423, 2.3%) had elevated TSH in the initial DBS, which was confirmed in 97 cases (7%) on a second screen. The remaining neonates (6,594, 10.6%) had a normal initial TSH but underwent a second test in accordance with screening protocols, and were found to have delayed TSH elevation in 23 cases (0.4%). Those 120 newborns (97 + 23), considered highly suspicious for primary CH, were referred to a pediatrician for confirmatory testing and excluded from subsequent analysis of factors influencing TSH levels. Sex (β regression coefficient, β = 1.11 female to male, 95% CI 1.09, 1.12) and age at collection (β = 0.78 day 5 to days 2–3, 95% CI 0.74, 0.83) affected neonatal TSH, suggesting the utility of specific nomograms. In addition, prematurity (β = 0.85 term to preterm, 95% CI 0.80, 0.91), dopamine use (β = 0.71, 95% CI 0.62, 0.81), and birth weight (β = 1.40 normal vs. very low, 95% CI 1.05, 1.89) strongly influenced neonatal TSH. Conclusions: Neonatal TSH is influenced by several factors supporting the delineation of local sex- and age-adjusted TSH cutoffs, and the universal adoption of a second TSH test in neonates at risk of missed primary CH diagnosis.

Resveratrol is a natural polyphenol with antioxidant, anti-inflammatory, and antiproliferative properties. We have shown previously that resveratrol decreases sodium/iodide symporter expression and iodide uptake in thyrocytes, both in vitro and in vivo. In the present study, we further investigated the effects of resveratrol, with evaluation of the expression of additional thyroid-specific genes in the FRTL-5 rat thyroid cell line: thyroglobulin, thyroid peroxidase, TSH receptor, Nkx2-1, Foxe1 and Pax8. We observed decreased expression of these genes in FRTL-5 cells treated with 10 μM resveratrol. The effects of resveratrol was further evaluated in vivo using Sprague-Dawley rats treated with resveratrol 25 mg/kg body weight intraperitoneally, for 60 days. No clinical signs of hypothyroidism were seen, although the treated rats showed significant increase in thyroid size. Serum TSH and thyroid hormone levels were in the normal range, with significantly higher TSH seen in resveratrol-treated rats, compared with control rats. Histological and immunohistochemical analyses confirmed increased proliferative activity in the thyroid from resveratrol-treated rats. These data suggest that resveratrol acts as a thyroid disruptor and a goitrogen, which indicates the need for caution as a supplement and for therapeutic uses.

Severe forms of the Coronavirus disease 2019 (COVID-19) are characterized by an enhanced inflammatory syndrome called “cytokine storm” that produces an aberrant release of high amounts of cytokines, chemokines, and other proinflammatory mediators. The pathogenetic role of the “cytokine storm” has been confirmed by the efficacy of immunosuppressive drugs such as corticosteroids along with antiviral drugs in the treatment of the severe forms of this disease. Phenylmethimazole (C10) is a derivative of methimazole with anti-inflammatory properties. Studies performed both in vitro and in vivo have shown that C10 is able to block the production of multiple cytokines, chemokines, and other proinflammatory molecules involved in the pathogenesis of inflammation. Particularly, C10 is effective in reducing the increased secretion of cytokines in animal models of endotoxic shock. We hypothesize that these effects are not limited to the endotoxic shock, but can also be applied to any disease characterized by the presence of a “cytokine storm”. Therefore, C10 may be a potential drug to be used alternatively or in association with the corticosteroids or other immunosuppressive agents in the severe forms of COVID-19 as well as other viral diseases that induce a “cytokine storm”. Preclinical and clinical studies have to be performed to confirm this hypothesis.

Nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor that is involved in inflammatory and immune responses, as well as in regulation of expression of many other genes related to cell survival, proliferation, and differentiation. In mammals, NF-κB comprises five subunits that can bind to promoter regions of target genes as homodimers or heterodimers. The most common dimer is the p50/p65 heterodimer. The several combinations of dimers that can be formed contribute to the heterogeneous regulation of NF-κB target genes, and this heterogeneity is further increased by interactions of the NF-κB dimers with other transcription factors, such as steroid hormone receptors, activator protein-1 (AP-1), and cAMP response element binding protein (CREB). In the thyroid, several studies have demonstrated the involvement of NF-κB in thyroid autoimmunity, thyroid cancer, and thyroid-specific gene regulation. The role of NF-κB in thyroid autoimmunity was hypothesized more than 20 years ago, after the finding that the binding of distinct NF-κB heterodimers to the major histocompatibility complex class I gene is hormonally regulated. Further studies have shown increased activity of NF-κB in thyroid autoimmune diseases and in thyroid orbitopathy. Increased activity of NF-κB has also been observed in thyroid cancer, where it correlates with a more aggressive pattern. Of particular interest, mutation of some oncogenes or tumor suppressor genes involved in thyroid carcinogenesis results in constitutive activation of the NF-κB pathway. More recently, it has been shown that NF-κB also has a role in thyroid physiology, as it is fundamental for the expression of the main thyroid-specific genes, such as sodium iodide symporter, thyroid peroxidase, thyroglobulin, Pax8, and TTF-1 (NKX2-1).

The literature indicates that the plasma cortisol-to-dehydroepiandrosterone-sulfate (DHEA-S) ratio is a marker of health status after menopause, when a decline in both estrogen and DHEA-S and an increase in cortisol occur. An increase in the cortisol-to-DHEA-S ratio has been positively correlated with metabolic syndrome, all-cause mortality, cancer, and other diseases. The aim of this study was to investigate the effects of a walking program on the plasma cortisol-to-DHEA-S ratio in postmenopausal women. Fifty-one postmenopausal women participated in a 13-week supervised walking program, in the metropolitan area of Pescara (Italy), from June to September 2013. Participants were evaluated in April–May and September–October of the same year. The linear mixed model showed that the variation of the log10Cortisol-to-log10DHEA-S ratio was associated with the volume of exercise (p = .03). Participants having lower adherence to the walking program did not have a significantly modified log10Cortisol or log...

Graves’ disease is the most common cause of thyrotoxicosis in women of childbearing age. Approximately 1% of pregnant women been treated before, or are being treated during pregnancy for Graves’ hyperthyroidism. In pregnancy, as in not pregnant state, thyroid-stimulating hormone (TSH) receptor (TSHR) antibodies (TRAbs) are the pathogenetic hallmark of Graves’ disease. TRAbs are heterogeneous for molecular and functional properties and are subdivided into activating (TSAbs), blocking (TBAbs), or neutral (N-TRAbs) depending on their effect on TSHR. The typical clinical features of Graves’ disease (goiter, hyperthyroidism, ophthalmopathy, dermopathy) occur when TSAbs predominate. Graves’ disease shows some peculiarities in pregnancy. The TRAbs disturb the maternal as well as the fetal thyroid function given their ability to cross the placental barrier. The pregnancy-related immunosuppression reduces the levels of TRAbs in most cases although they persist in women with active disease as well as in women who received definitive therapy (radioiodine or surgery) before pregnancy. Changes of functional properties from stimulating to blocking the TSHR could occur during gestation. Drug therapy is the treatment of choice for hyperthyroidism during gestation. Antithyroid drugs also cross the placenta and therefore decrease both the maternal and the fetal thyroid hormone production. The management of Graves’ disease in pregnancy should be aimed at maintaining euthyroidism in the mother as well as in the fetus. Maternal and fetal thyroid dysfunction (hyperthyroidism as well as hypothyroidism) are in fact associated with several morbidities. Monitoring of the maternal thyroid function, TRAbs measurement, and fetal surveillance are the mainstay for the management of Graves’ disease in pregnancy. This review summarizes the biochemical, immunological, and therapeutic aspects of Graves’ disease in pregnancy focusing on the role of the TRAbs in maternal and fetal function.

Menopause is characterized by hormonal and metabolic changes. These are linked to increased risk of cardiovascular disease, for which low blood plasma levels of high density lipoprotein (HDL) cholesterol are an independent risk factor. The present study investigated variables linked with basal plasma HDL cholesterol levels and the effects of aerobic training, on their variations, in 40 postmenopausal women.We assessed body composition, dietary habits and maximal aerobic capacity of participants. Characteristics of daily physical activity and plasma lipoproteins were measured. The women walked on 4 days/week, for 14 weeks, at moderate intensity, and they were grouped according to the resulting tertiles of basal plasma HDL cholesterol levels.Logistic regression analysis showed that waist-to-hip ratio and number of daily bouts of moderate-intensity physical activity, held for at least 10 consecutive minutes (B10m/day), are predictive variables of basal plasma HDL cholesterol levels. After the training period, the first and second tertiles increased plasma HDL cholesterol levels, while the third tertile decreased plasma HDL cholesterol levels. The tertiles showed different remodelling of spontaneous physical activity: the third tertile reduced B10m/day, while the others did not.This study provides knowledge about the relationships of plasma HDL cholesterol levels with characteristics of physical activity. Furthermore, it shows that physical exercise engagement can result in negative compensation of spontaneous physical activity that could counteract or reduce the positive effects of the aerobic training on plasma HDL cholesterol levels.

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.

Context: Graves' 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. Objective: Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. Design: We 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. Results: We 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%. Conclusion: NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.

Thyroglobulin (Tg), a major product of the thyroid gland, serves as a macromolecular precursor of thyroid hormone biosynthesis. In addition, Tg stored in the thyroid follicles is a potent regulator of thyroid-specific gene expression. In conjunction with thyroid stimulating hormone (TSH) and iodide, Tg regulates thyroid follicle function, which is the minimal functional unit of the thyroid gland. In the present study, we show that Tg stimulates growth of FRTL-5 thyroid cells in the absence of TSH, insulin and serum. Unlike TSH, Tg did not increase cellular cyclic AMP (cAMP) levels; rather, the TSH signal counteracted Tg-induced cell growth. A specific inhibitor of A-kinase, H-89, did not modulate the effect of Tg. Tg increased kinase activity of Akt to the same level as TSH, insulin and 5% serum, while LY294002 abolished Tg-induced growth. Interestingly, low Tg concentrations maximized growth-promotion activity and induction of the apical iodide transporter (PDS; SLC26A4), whereas high Tg concentrations suppressed both cell growth and the expression of thyroid-specific genes. These results suggest that a low levels of Tg in the follicular lumen might stimulates cell growth and iodide transport to accelerate the iodide organification process; however, elevated Tg levels in the follicle might then shut down all of these functions.

Quercetin is the most consumed flavonoid present in fruits and vegetables. There has been increased interest in the possible health benefits of quercetin and other flavonoids. Because it is reported that these compounds have some antithyroid properties, we were interested whether, and by what mechanism, quercetin might regulate thyroid cell growth and function. In this report we show that quercetin inhibits thyroid cell growth in association with inhibition of insulin-modulated phosphatidylinositol 3-kinase-Akt kinase activity. Furthermore, quercetin decreases TSH-modulated RNA levels of the thyroid-restricted gene sodium/iodide symporter (NIS). We associated down-regulation of NIS RNA levels with inhibition of iodide uptake at comparable quercetin concentrations and could show that the inhibitory effect of quercetin on NIS RNA levels and iodide uptake is reproduced by inhibitors of the phospholipase-A(2)/lipoxygenase pathway. The specific inhibitor of protein kinase A, H89, only partially inhibited TSH-increased NIS expression and did not reproduce the quercetin effect. The quercetin studies thus reveal that the phospholipase-A(2)/lipoxygenase pathway appears to play an important role in TSH regulation of NIS gene expression, whereas quercetin inhibition of growth appears to involve an effect on insulin/IGF-I-Akt signaling. The data raise the possibility that quercetin may be a novel disruptor of thyroid function, which has potential effects on, or use in, the therapy of thyroid diseases.

We previously reported that hormones important for the normal growth and function of FRTL-5 rat thyroid cells, TSH, or its cAMP signal plus insulin or IGF-I, could transcriptionally suppress constitutive and γ-interferon (IFN)-increased synthesis of the 90K protein (also known as Mac-2BP). Here we cloned the 5′-flanking region of the rat 90K gene and identified a minimal promoter containing an interferon response element and a consensus E-box or upstream stimulator factor (USF) binding site, which are highly conserved in both the human and murine genes. We show that suppression of constitutive and γ-IFN-increased 90K gene expression by TSH/cAMP plus insulin/IGF-I depends on the ability of the hormones to decrease the binding of USF to the E-box, located upstream of the interferon response element. This site is required for the constitutive expression of the 90K gene. Transfection with USF1 and USF2 cDNAs increases constitutive promoter activity, attenuates the ability of TSH/cAMP plus insulin/IGF-I to decrease constitutive or γ-IFN-increased 90K gene expression but does not abrogate the ability of γ-IFN itself to increase 90K gene expression.

The finding of a neck nodular lesion provides strong suspicion of primary thyroid malignancy. Direct extension into the thyroid parenchyma of carcinomas arising from pharynx, larynx, trachea or esophagus, nervous structures has been also observed in the minority of cases. The intent of our study is to present and discuss rare conditions presenting asymptomatic neck masses, with particular emphasis on pre-operative characteristics and diagnostic criteria.In our retrospective analysis, we present three cases of nodular neck lesions that have mimicked primitive thyroid pathologies at the first diagnosis.A 53-mm nodular mass in the right thyroid lobe was observed in one patient. The definitive diagnosis was Castleman's disease. The second case presented a 20-mm hypoechoic lesion in the contest of a multinodular goiter. The pre-operative suspect was thyroid carcinoma with lymphnode metastases but the definitive histology documented an 'ancient schwannoma'. A further patient presented bilateral supra-clavear and cervical lymphnodes in a multinodular goiter, initially interpreted as thyroid carcinoma with loco-regional spread. After a total thyroidectomy and cervical lymphadenectomy, the definitive histology documented foci of poorly differentiated carcinoma in cervical lymphnodes and a multinodular goiter without atypical cellularity. The patient is considered to have an occult tumor, probably arising from the breast, and she was scheduled in an oncological program.Nodular neck lesions are frequently misdiagnosed as primitive thyroid nodules in the common clinical practice. In these rare conditions, surgical exploration is advocated to reach the definitive diagnosis, to indicate the most appropriate treatment and to avoid unnecessary thyroidectomy.Ancient Schwannoma, Castleman's disease, Nodular neck lesion, Thyroid nodule.

Increased major histocompatibility complex (MHC) class I gene expression in nonimmune cell ‘target tissues’ involved in organ-specific diseases may be important in the pathogenesis of autoimmune diseases. This possibility in part evolves from studies of cultured thyrocytes where properties appear relevant to the development of thyroid autoimmune disease. In FRTL-5 rat thyroid cells in continuous culture, hormones and growth factors that regulate cell growth and function specifically decrease MHC class I gene expression. We hypothesized that this could reflect a mechanism to preserve self-tolerance and prevent autoimmune disease. The mechanisms of action of some of these hormones, namely TSH and hydrocortisone, have been already characterized. In this report, we show that IGF-I transcriptionally downregulates MHC class I gene expression and that its action is similar to that of insulin. The two hormones have a complex effect on the promoter of the MHC class I gene, PD1. In fact, they decrease the full promoter activity, but upregulate the activity of deleted mutants that have lost an upstream, tissue-specific regulatory region but still retain the enhancer A region. We show that insulin/IGF-I promotes the interactions of the p50/p65 subunits of NF-κB and AP-1 family members with these two regions, and that the tissue-specific region acts as a dominant silencer element on insulin/IGF-I regulation of promoter activity. These observations may be important to understand how MHC class I gene transcription is regulated in the cells.

Toll-like receptors (TLRs) initiate an innate immune response. TLR3 on dendritic cells recognize double-stranded (ds) RNA and then signal increases in cytokines and recognition molecules important for immune cell interactions. In this report, we demonstrate TLR3 mRNA and protein are expressed on Fisher rat thyroid cell line-5 (FRTL-5) thyroid cells and are functional because incubating cells with polyinosine-polycytidylic acid causes 1) transcriptional activation of both the nuclear factor kappaB (NF-kappaB)/Elk1 and interferon (IFN) regulatory factor-3/IFN-beta signal paths, 2) posttranscriptional activation of NF-kappaB and ERK1/2, and 3) increased IFN-beta mRNA. TLR3 can be overexpressed, along with dsRNA-dependent protein kinase, major histocompatibility complex-I or II, and IFN regulatory factor-1, by transfecting dsRNA into the cells, infection with Influenza A virus, or incubation with IFN-beta, but not by incubation with dsRNA or IFNgamma, or by dsDNA transfection. A methimazole (MMI) derivative, phenylmethimazole, to a significantly greater degree than MMI, prevents overexpression by inhibiting increased transcriptional activation of IRF-3 and of IFN-stimulated response elements, phosphorylation of signal transducers and activation of transcription (STAT-1), but not NF-kappaB activation. TLR3 can be functionally overexpressed in cultured human thyrocytes by dsRNA transfection or IFN-beta treatment. Immunohistochemical studies show that TLR3 protein is overexpressed in human thyrocytes surrounded by immune cells in 100% of patients with Hashimoto's thyroiditis examined, but not in normal or Graves' thyrocytes. We conclude that functional TLR3 are present on thyrocytes; TLR3 downstream signals can be overexpressed by pathogen-related stimuli; overexpression can be reversed by phenylmethimazole to a significantly greater extent than MMI by inhibiting only the IFN regulatory factor-3/IFN-beta/signal transducers and activation of transcription arm of the TLR3 signal system; and TLR3 overexpression can induce an innate immune response in thyrocytes, which may be important in the pathogenesis of Hashimoto's thyroiditis and in the immune cell infiltrates.

Here we report the cloning of the rat 90K, a homolog of the mouse cyclophilin C-associated protein/mouse adherent macrophage and human 90K. The protein is constitutively expressed by FRTL-5 thyrocytes, and its levels are modulated by TSH, insulin/IGF-I, and gamma-interferon. Transfection of the cells with 90K cDNA or exposure to purified 90K resulted in a significant increase of the expression of major histocompatibility complex class I but not class II antigens. An increased expression of 90K was obtained after viral infection or introduction into the cells of fragments of viral, bacterial, or mammalian double-strand polynucleotides. The increase was sequence independent, not CpG mediated, and associated with the expression of molecules characterizing antigen-presenting-cell phenotype. The present data along with results from previous studies suggest that 90K plays an important role in the maintenance of an appropriate level of immune response.

The skin is commonly affected in thyroid diseases, but the mechanism for this association is still unclear. As the skin expresses numerous neuroendocrine elements, we tested the additional cutaneous expression of mediators operating in the hypothalamic-pituitary-thyroid axis. We found significant expression of the thyroid-stimulating hormone receptor mRNA in cultured keratinocytes, epidermal melanocytes, and melanoma cells. The presence of thyroid-stimulating hormone receptor was confirmed by northern analyses and the thyroid-stimulating hormone receptor was found to be functionally active in cyclic adenosine monophosphate signal assays. Thyroid-stimulating hormone receptor expressing cells also expressed the sodium iodide symporter and thyroglobulin genes. We also found expression of deiodinases 2 and 3 (mainly deiodinase 2) in whole skin biopsy specimens, and in the majority of epidermal and dermal cells by reverse transcription-polymerase chain reaction followed by sequencing of the amplified gene segments. There was selective expression of the gene for thyroid-stimulating hormone beta; detection of the thyroid-releasing hormone gene was minimal and thyroid-releasing hormone receptor mRNA was not detected in most of the samples. Expression of functional thyroid-stimulating hormone receptor in the skin may have significant physiologic and pathologic consequences, particularly in autoimmune conditions associated with production of stimulating antibodies against the thyroid-stimulating hormone receptor. We conclude that the expanding list of neuroendocrine elements expressed in the skin supports a strong role for this system in cutaneous biology.

Transforming growth factor (TGF)-beta1-decreased major histocompatibility complex (MHC) class I gene expression in thyrocytes is transcriptional; it involves trans factors and cis elements important for hormone- as well as iodide-regulated thyroid growth and function. Thus, in rat FRTL-5 thyrocytes, TGF-beta1 regulates two elements within -203 bp of the transcription start site of the MHC class I 5'-flanking region: Enhancer A, -180 to -170 bp, and a downstream regulatory element (DRE), -127 to -90 bp, that contains a cAMP response element (CRE)-like sequence. TGF-beta1 reduces the interaction of a NF-kappaB p50/fra-2 heterodimer (MOD-1) with Enhancer A while increasing its interaction with a NF-kappaB p50/p65 heterodimer. Both reduced MOD-1 and increased p50/p65 suppresses class I expression. Decreased MOD-1 and increased p50/p65 have been separately associated with the ability of autoregulatory (high) concentrations of iodide to suppress thyrocyte growth and function, as well as MHC class I expression. TGF-beta1 has two effects on the downstream regulatory element (DRE). It increases DRE binding of a ubiquitously expressed Y-box protein, termed TSEP-1 (TSHR suppressor element binding protein-1) in rat thyroid cells; TSEP-1 has been shown separately to be an important suppressor of the TSH receptor (TSHR) in addition to MHC class I and class II expression. It also decreases the binding of a thyroid-specific trans factor, thyroid transcription factor-1 (TTF-1), to the DRE, reflecting the ability of TGF-beta1 to decrease TTF-1 RNA levels. TGF-beta1-decreased TTF-1 expression accounts in part for TGF-beta1-decreased thyroid growth and function, since decreased TTF-1 has been shown to decrease thyroglobulin, thyroperoxidase, sodium iodide symporter, and TSHR gene expression, coincident with decreased MHC class I. Finally, we show that TGF-beta1 increases c-jun RNA levels and induces the formation of new complexes involving c-jun, fra-2, ATF-1, and c-fos, which react with Enhancer A and the DRE. TGF-beta1 effects on c-jun may be a pivotal fulcrum in the hitherto unrecognized coordinate regulation of Enhancer A and the DRE.

In this study we examined the effect of the synthetic peptide thymosin-alpha1 (T(alpha)1) on MHC class I expression in FRTL-5 cells. Treatment with T(alpha)1 increased expression of MHC class I surface molecules and mRNA, which reached its peak (153 +/- 8 % of the control value) after 12 h. 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 T(alpha)1 was at the transcriptional level, and its mechanism of action is likely due to increased binding between the complex p50/fra-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 Talpha1 on the regulation of gene expression at the molecular level, and may further contribute to explaining the results obtained using Talpha1 in the control of infectious diseases and tumor growth.

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.

Aberrant expression of major histocompatibility complex (MHC) class II antigens is associated with autoimmune thyroid disease; aberrant expression duplicating the autoimmune state can be induced by interferon-gamma (IFNgamma). We have studied IFNgamma-induced human leukocyte antigen (HLA)-DR alpha gene expression in rat FRTL-5 thyroid cells to identify the elements and factors important for aberrant expression. Using an HLA-DR alpha 5'-flanking region construct from -176 to +45 bp coupled to the chloramphenicol acetyltransferase reporter gene, we show that there is no basal class II gene expression in FRTL-5 thyroid cells, that IFNgamma can induce expression, and, as is the case for antigen-presenting cells from the immune system, that IFNgamma-induced expression requires several highly conserved elements on the 5'-flanking region, which, from 5' to 3', are the S, X1, X2, and Y boxes. Methimazole (MMI), a drug used to treat patients with Graves' disease and experimental thyroiditis in rats or mice, can suppress the IFNgamma-induced increase in HLA-DR alpha gene expression as a function of time and concentration; MMI simultaneously decreases IFNgamma-induced endogenous antigen presentation by the cell. Using gel shift assays and the HLA-DR alpha 5'-flanking region from -176 or -137 to +45 bp as radiolabeled probes, we observed the formation of a major protein-DNA complex with extracts from FRTL-5 cells untreated with IFNgamma, termed the basal or constitutive complex, and formation of an additional complex with a slightly faster mobility in extracts from cells treated with IFNgamma. MMI treatment of cells prevents IFNgamma from increasing the formation of this faster migrating complex. Formation of both complexes is specific, as evidenced in competition studies with unlabeled fragments between -137 and -38 bp from the start of transcription; nevertheless, they can be distinguished in such studies. Thus, high concentrations of double stranded oligonucleotides containing the sequence of the Y box, but not S, X1, or X2 box sequences, can prevent formation of the IFNgamma-increased faster migrating complex, but not the basal complex. Both complexes involve multiple proteins and can be distinguished by differences in their protein composition. Thus, using specific antisera, we show that two cAMP response element-binding proteins, activating transcription factor-1 and/or -2, are dominant proteins in the upper or basal complex. The upper or basal complex also includes c-Fos, Fra-2, Ets-2, and Oct-1. A dominant protein that distinguishes the IFNgamma-increased lower complex is CREB-binding protein (CBP), a coactivator of cAMP response element-binding proteins. We, therefore, show that aberrant expression of MHC class II in thyrocytes, induced by IFNgamma, is associated with the induction or increased formation of a novel protein-DNA complex and that its formation as well as aberrant class II expression are suppressed by MMI, a drug used to treat human and experimental autoimmune thyroid disease. Its component proteins differ from those in a major, basal, or constitutive protein-DNA complex formed with the class II 5'-flanking region in cells that are not treated with IFNgamma and that do not express the class II gene.

Aberrant expression of major histocompatibility complex (MHC) class II proteins on thyrocytes, which is associated with autoimmune thyroid disease, is mimicked by gamma-interferon (gamma-IFN). To define elements and factors that regulate class II gene expression in thyrocytes and that might be involved in aberrant expression, we have studied gamma-IFN-induced HLA-DR alpha gene expression in rat FRTL-5 thyroid cells. The present report shows that class II expression in FRTL-5 thyrocytes is positively regulated by the class II transactivator (CIITA), and that CIITA mimics the action of gamma-IFN. Thus, as is the case for gamma-IFN, several distinct and highly conserved elements on the 5'-flanking region of the HLA-DR alpha gene, the S, X1, X2, and Y boxes between -137 to -65 bp, are required for class II gene expression induced by pCIITA transfection in FRTL-5 thyroid cells. CIITA and gamma-IFN do not cause additive increases in HLA-DR alpha gene expression in FRTL-5 cells, consistent with the possibility that CIITA is an intermediate factor in the gamma-IFN pathway to increased class II gene expression. Additionally, gamma-IFN treatment of FRTL-5 cells induces an endogenous CIITA transcript; pCIITA transfection mimics the ability of gamma-IFN treatment of FRTL-5 thyroid cells to increase the formation of a specific and novel protein/DNA complex containing CBP, a coactivator of CRE binding proteins important for cAMP-induced gene expression; and the action of both gamma-IFN and CIITA to increase class II gene expression and increase complex formation is reduced by cotransfection of a thyroid Y box protein, which suppresses MHC class I gene expression in FRTL-5 thyroid cells and is a homolog of human YB-1, which suppresses MHC class II expression in human glioma cells. We conclude that CIITA and TSH receptor suppressor element binding protein-1 are components of the gamma-IFN-regulated transduction system which, respectively, increase or decrease class II gene expression in thyrocytes and may, therefore, be involved in aberrant class II expression associated with autoimmune thyroid disease.

The major histocompatibility complex (MHC) class I gene cAMP response element (CRE)-like site, −107 to −100 base pairs, is a critical component of a previously unrecognized silencer, −127 to −90 bp, important for thyrotropin (TSH)/cAMP-mediated repression in thyrocytes. TSH/cAMP induced-silencer activity is associated with the formation of novel complexes with the 38-base pair silencer, whose appearance requires the CRE and involves ubiquitous and thyroid-specific proteins as follows: the CRE-binding protein, a Y-box protein termed thyrotropin receptor (TSHR) suppressor element protein-1 (TSEP-1); thyroid transcription factor-1 (TTF-1); and Pax-8. TTF-1 is an enhancer of class I promoter activity; Pax-8 and TSEP-1 are suppressors. TSH/cAMP decreases TTF-1 complex formation with the silencer, thereby decreasing maximal class I expression; TSH/cAMP enhance TSEP-1 and Pax-8 complex formation in association with their repressive actions. Oligonucleotides that bind TSEP-1, not Pax-8, prevent formation of the TSH/cAMP-induced complexes associated with TSH-induced class I suppression, i.e. TSEP-1 appears to be the dominant repressor factor associated with TSH/cAMP-decreased class I activity and formation of the novel complexes. TSEP-1, TTF-1, and/or Pax-8 are involved in TSH/cAMP-induced negative regulation of the TSH receptor gene in thyrocytes, suppression of MHC class II, and up-regulation of thyroglobulin. TSH/cAMP coordinate regulation of common transcription factors may, therefore, be the basis for self-tolerance and the absence of autoimmunity in the face of TSHR-mediated increases in gene products that are important for thyroid growth and function but are able to act as autoantigens.

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.

Background One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role. Objective In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock. Design Experimental animal model. Setting University laboratory. Subjects Male C57BL/6J mice weighing 18-22 g. Interventions Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation. Measurements and main results : Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages. Conclusions Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.

Increased expression of major histocompatibility complex (MHC) class-I genes and aberrant expression of MHC class-II genes in thyroid epithelial cells (TECs) are associated with autoimmune thyroid diseases. Previous studies have shown that methimazole (MMI) reduces MHC class-I expression and inhibits interferon-γ (IFN-γ or IFNG as listed in the MGI Database)-induced expression of the MHC class-II genes in TECs. The action of MMI on the MHC class-I genes is transcriptional, but its mechanism has not been investigated previously. In the present study, we show that in Fisher rat thyroid cell line 5 cells, the ability of MMI and its novel derivative phenylmethimazole (C10) to decrease MHC class-I promoter activity is similar to TSH/cAMP suppression of MHC class-I and TSH receptor genes, and involves a 39 bp silencer containing a cAMP response element (CRE)-like site. Furthermore, we show that C10 decreases MHC class-I gene expression to a greater extent than MMI and at 10- to 50-fold lower concentrations. C10 also reduces the IFN-γ-induced increase in the expression of MHC class-I and MHC class-II genes more effectively than MMI. Finally, we show that in comparison to MMI, C10 is a better inhibitor of specific protein–DNA complexes that are formed with a CRE-like element on the MHC class-II promoter. These data support the conclusion that the immunosuppressive mechanism by which MMI and C10 inhibit MHC gene expression mimics ‘normal’ hormonal suppression by TSH/cAMP.

High concentrations of iodide can induce transient, clinical improvement in patients with autoimmune Graves' disease. Previous work has related this iodide action to the autoregulatory effect of iodide on the growth and function of the thyroid; more recently, we additionally related this to the ability of iodide to suppress major histocompatibility (MHC) class I RNA levels and antigen expression on thyrocytes. In this report, we describe a transcriptional mechanism involved in iodide suppression of class I gene expression, which is potentially relevant to the autoregulatory action of iodide. Transfection experiments in FRTL-5 cells show that iodide decreases class I promoter activity and that this effect can be ascribed to the ability of iodide to modulate the formation of two specific protein/DNA complexes with enhancer A, -180 to -170 bp, of the class 1 5'-flanking region. Thus, iodide decreases the formation of Mod-1, an enhancer A complex involving the p50 subunit of NF-kappa B and a c-fos family member, fra-2, which was previously shown to be important in the suppression of class I levels by hydrocortisone. Unlike hydrocortisone, iodide also increases the formation of a complex with enhancer A, which we show, in antibody shift experiments, is a heterodimer of the p50 and p65 subunits of NF-kappa B. The changes in these complexes are not duplicated by chloride and are related to the action of iodide on class I RNA levels by the following observations. First, FRTL-5 thyroid cells with an aged phenotype coincidentally lose the ability of iodide to decrease MHC class I RNA levels and to induce changes in either complex. Second, the effect of iodide on class I RNA levels and on enhancer A complex formation with Mod-1 and the p50/p65 heterodimer is inhibited by agents that block the inositol phosphate, Ca++, phospholipase A2, arachidonate signal transduction pathway: acetylsalicylate, indomethacin, and 5,8,11,14-eicosatetraynoic acid. Interestingly, iodide can also decrease formation of the Mod-1 complex and increase formation of the complex with the p50/p65 subunits of NF-kappa B when the NF-kappa B enhancer sequence from the Ig kappa light chain, rather than enhancer A, is used as probe; and both actions mimic the action of a phorbol ester. This suggests that iodide may regulate complex formation with NF-kappa B regulatory elements on multiple genes associated with growth and function, providing a potential mechanism relating the autoregulatory action of iodide on thyroid cells and its action on class I gene expression.

Hydrocortisone decreases major histocompatibility complex (MHC) class I gene expression in rat thyroid cells and counteracts increases induced by interferons. Using FRTL-5 cells transfected with class I promoter- reporter gene chimeras, we show that hydrocortisone action is transcriptional and mediated by an element located between 180 and 170 base pairs upstream of the start of transcription. Gel shift assays reveal that hydrocortisone causes the decrease of a specific protein- DNA complex; this same complex, referred to as Mod-1, is increased by interferon. Oligonucleotide competition assays reveal that the Mod-1 complex is associated with enhancer A of the class I gene, −180 to −170 base pairs (5′-GGGGAGTCCCC-3′), immediately upstream of the interferon response element. Antibodies to fra-2, a fos family member, and to the p50, but not the p65, subunit of NF-kappa B supershift the Mod-1 complex. We suggest that hydrocortisone decreases MHC class I gene expression by reducing the formation of Mod-1, which contains both p50 and fra-2; interferon reverses the hydrocortisone effect and increases Mod-1 formation. These observations are relevant to the molecular basis of hydrocortisone therapy in autoimmune thyroid disease and to the actions of interferon to exacerbate or induce autoimmune disease.

This chapter has outlined the complex process required for thyroid growth and function. Both events are regulated by TSHR via a multiplicity of signals, with the aid of and requirement for a multiplicity of hormones that regulate the TSHR via receptor cross-talk: insulin, IGF-I, adrenergic receptors, and purinergic receptors. Cross-talk appears to regulate G-protein interactions or activities induced by TSH as well as TSHR gene expression. The TSHR structure and its mechanism of signal transduction is being rapidly unraveled in several laboratories, since the recent cloning of the receptor. In addition, the epitopes for autoantibodies against the receptor that can subvert the normal regulated synthesis and secretion of thyroid hormones, causing hyper- or hypofunction, have been defined. Studies of regulation of the TSHR minimal promotor have uncovered a better understanding of the mechanisms by which TSH regulates both growth and function of the thyroid cell. A key novel component of this phenomenon involves TSH AMP positive and negative regulation of the TSHR. Negative transcriptional regulation is a common feature of MHC class I genes in the thyroid. Subversion of negative regulation or too little negative regulation is suggested to result in autoimmune disease. Methimazole and iodide at autoregulatory levels may be important in reversing this process and returning thyroid function to normal. Their action appears to involve factors that react with the IREs on both the TSHR and the TG promoter. Too much negative regulation, as in the case of ras transformation, results in abnormal growth without function. TTF-1 is implicated as a critical autoregulatory component in both positive and negative regulation of the TSHR and appears to be the link between TSH, the TSHR, TSHR-mediated signals, TG and TPO biosynthesis, and thyroid hormone formation. Differentially regulated expression of the TSHR and TG by cAMP and insulin depend on differences in the specificity of the TTF-1 site, that is, the lack of Pax-8 interactions with the TSHR, and the IRE sites. Single-strand binding proteins will become important in determining how TSHR transcription is controlled mechanistically.

Glycoprotein hormones—lutropin/chorionic gonadotropin (LH/CG), follicle stimulating hormone (FSH), and thyrotropin (TSH)—have a high degree of primary and tertiary structure similarity, yet are target tissue specific (1–5). The structural similarities of the hormones are complemented by functional similarities (1–5). Thus, all are well recognized to activate the cAMP signal transducing system, and all modulate both growth and differentiation of their respective target tissues. It is increasingly evident, in addition, that all depend on the action of other hormones, such as insulin and insulin-like growth factor I (IGF-I), to regulate growth and differentiated function (6–8).

The present report identifies an important immunogenic region of the TSH receptor and determinants on the TSH receptor for the two types of autoantibodies seen in hyperthyroid Graves' disease and hypothyroid idiopathic myxedema, TSAbs and TSBAbs, respectively. The immunogenic domain with no important functional determinants, is contained within residues 303-382 and involves residues 352-366 in particular. There are determinants flanking the immunogenic domain on the C-terminal portion of the receptor which are the TSBAb and high affinity TSH binding sites: residues 295-306, 387-395, and tyrosine 385. Determinants on the N-terminal portion of the external domain, centered on residues 38-45, are TSAb interactions linked to low affinity TSH binding important for signal generation: threonine 40 and residues 30-33, 34-37, 42-45, 52-56, and 58-61. These determinants are conserved in human and rat receptors, are not present in gonadotropin receptors, and are each related to separate actions of TSH: binding vs. signal generation. They can, therefore, account for organ specific autoimmunity and the different disease expression effected by TSBAbs vs TSAbs, i.e. hypo- vs. hyperthyroidism, respectively. It is proposed that, in the thyroid, hormonal (TSH, insulin, hydrocortisone, IGF-I) suppression of class I genes might be one means of preserving self-tolerance in the face of the hormone action to increase the expression of tissue specific genes such as thyroglobulin and thyroid peroxidase. Inappropriately high class I expression in the thyroid, i.e. if induced by interferon, viruses, or some as yet unknown agent, would contribute to the generation of autoimmune disease. Thus, it would result in increased antigen presentation to the immune system, particularly those autoantigens increased by TSH and its cAMP signal such as thyroglobulin or thyroid peroxidase, or whose turnover is increased by TSH and its cAMP signal, such as the TSH receptor. In the case of the latter, peptide 352-366, known to be near a protease sensitive site on the receptor [41,49], would now act as a potent self-antigen and induce the formation of receptor autoantibodies. It is further proposed that methimazole and high doses of iodide are therapeutically effective agents in thyroid autoimmune disease because they, in part, decrease MHC class I gene expression. Speculation is presented which suggests that elimination of negative regulation of MHC class I and the TSH receptor is an important factor in the development of autoimmune thyroid disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Children affected with Down syndrome (DS) show deficient growth, immunodeficiency--especially concerning the T-cell population--and low plasma zinc levels. New growth charts have been recently proposed, and zinc supplementation to the diet has been reported to improve transiently the efficiency of the immune system. The aim of this study was to evaluate if in DS children zinc sulphate therapy could improve the growth rate and affect some endocrine parameters. We studied 22 patients (16 males and 6 females) who received zinc sulphate for 6 to 9 months. Fifteen of 22 patients studied reached a higher centile in their growth rate, whereas the remaining seven showed no change, at least to date. The average height velocity changed from 23.84 +/- 7.98 mm/6 months to 40.80 +/- 7.68 mm/6 months. Growth hormone serum level was 5.94 +/- 4.89 ng/ml compared with 7.49 +/- 6.75 ng/ml before and after therapy, respectively. Somatomedin serum level was 160.27 +/- 68.88 mU/ml and 205 +/- 124.07 mU/ml before and after therapy, respectively. In conclusion, zinc sulphate therapy of patients with DS affects not only the immune system, as previously reported, but can also accelerate growth.