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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.

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.

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.

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.

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.

Resveratrol is a polyphenol found in grapes and berries that has antioxidant, antiproliferative and anti-inflammatory properties. For these reasons, it is available as a dietary supplement, and it is under investigation in several clinical trials. Few data are available regarding the effects of resveratrol on thyroid function. A previous study showed that resveratrol transiently increases iodide influx in FRTL-5 rat thyroid cells. Indeed, this increase arises after short treatment times (6–12 h), and no further effects are seen after 24 h. The aim of the present study was to investigate the effects of resveratrol on iodide uptake and sodium/iodide symporter expression in thyroid cells after longer times of treatment. For this purpose, the effects of resveratrol were evaluated both in vitro and in vivo using the rat thyroid FRTL-5 cell line and Sprague-Dawley rats, respectively. In FRTL-5 cells, resveratrol decreased the sodium/iodide symporter RNA and protein expression as a function of time. Furthermore, resveratrol decreased cellular iodide uptake after 48 h of treatment. The inhibitory effect of resveratrol on iodide uptake was confirmed in vivo in Sprague-Dawley rats. This study demonstrates that with longer-term treatment, resveratrol is an inhibitor of sodium/iodide symporter gene expression and function in the thyroid. These data suggest that resveratrol can act as a thyroid disruptor, which indicates the need for caution as a supplement and in therapeutic use.

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.

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.

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 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).The 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.An Mc4 assay was used to prospectively track TSAb activity in GD patients treated with ATD over a 5-yr period.GD patients from the Chieti University participated in this study.Interventions 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.The Mc4 assay, a sensitive index of remission and recurrence, was used in this study.The 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.The Mc4 assay is a clinically useful index of remission and relapse in patients with GD. Larger studies are required to confirm these findings.

Stimulating thyrotropin receptor (TSHr) autoantibodies (TSAb) are the cause of hyperthyroidism in Graves' disease. In a patient's serum, TSAb can coexist with antagonist TSHr autoantibodies that block TSAb stimulatory activity (TSBAb); both can vary in amount and time.The objective of the study was to create a functional assay that detects only TSAb, thus having an increased accuracy for diagnosing Graves' disease.A 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.The study was conducted at the Chieti University (Outpatient Endocrine Clinic) and the University of Pisa (the Department of Endocrinology).The assay was validated using sera from 170 individuals with Graves' disease, Hashimoto's 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.Interventions included the assessment of patients' sera using human wild-type TSHr (WT-TSHr), Mc4 chimera, and binding (TRAb) assays.The Mc4 assay has the best accuracy for diagnosing Graves' disease.The 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.The Mc4 assay is a functional assay with improved sensitivity and specificity for the detection of TSAb and is clinically useful in diagnosing Graves' disease.

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.

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)