Expression Of TPO Research Articles

Abstract 3591: Upregulation of CXCL8 is an early step in the oncogenic transition of human thyroid cells by BRAFV600E

Abstract Introduction: BRAFV600E is one of the most common oncogenic mutations in human thyroid cancers, especially in papillary thyroid carcinoma (PTC). However, the early molecular changes caused by this mutation have not been studied previously in human thyrocytes in vitro. Methods: Primary cultures of cells derived from noncancerous thyroid tissue of patients (at least n=3) were infected with BRAFV600E adenovirus to mimic the mutated condition. Changes induced by the infection were measured at early times (<24h). Transcriptome analysis was performed on infected thyrocytes (n=6) to determine the highest differentially expressed genes between the control and infected groups, which were then verified at the gene and protein levels. RNAi downregulation experiments were performed to determine the cross-regulatory function of the genes. Results: The induction of BRAFV600E mutation, confirmed at the protein level, increased proliferation of the cells, and morphological changes were observed at approximately 24h post-infection. The decreased expression of thyroid-specific genes, viz. TSHR, TG, and TPO were observed after >72h post-introduction of BRAFV600E mutation. Transcriptome and protein expression analysis of BRAFV600E-positive thyrocytes showed the overexpression of chemokines CXCL8 and CXCL12, and upregulation of endothelial cell-specific molecule 1 (ESM1). These molecules are known to be associated with increased neoangiogenesis and proliferation. Functional studies showed that siRNA knockdown of CXCL8 decreased the expressions of CXCL12 and ESM1, whereas knockdown of either CXCL12 or ESM1 did not decrease the expression of CXCL8. Conclusion: Increased proliferation, de-differentiation, upregulation of chemokines, and endothelial-specific factor are effects associated with BRAFV600E-driven thyroid carcinogenesis. CXCL8 appears to play an important early regulatory role in this process. Citation Format: Rhitajit Sarkar, Priyanka Bolel, Joanna Klubo-Gwiezdzinska, Marvin C. Gershengorn. Upregulation of CXCL8 is an early step in the oncogenic transition of human thyroid cells by BRAFV600E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3591.

Contactin 6, A Novel Causative Gene for Congenital Hypothyroidism, Mediates Thyroid Hormone Biosynthesis Through Notch Signaling.

Background: Congenital hypothyroidism (CH) is the most common neonatal metabolic disorder. In patients with CH in China, thyroid dyshormonogenesis is more common than thyroid dysgenesis; however, the genetic causes of CH due to thyroid dyshormonogenesis remain largely unknown. Therefore, we aimed at identifying novel candidate causative genes for CH. Methods: To identify novel CH candidate genes, a total of 599 patients with CH were enrolled and next-generation sequencing was performed. The functions of the identified variants were confirmed using HEK293T and FTC-133 cell lines in vitro and in a mouse model organism in vivo. Results: Three pathogenic contactin 6 (CNTN6) variants were identified in two patients with CH. Pedigree analysis showed that CH caused by CNTN6 variants was inherited in an autosomal recessive pattern. The CNTN6 gene was highly expressed in the thyroid in humans and mice. Cntn6 knockout mice presented with thyroid dyshormonogenesis and CH due to the decreased expression of crucial genes for thyroid hormone biosynthesis (Slc5a5, Tpo, and Duox2). All three CNTN6 variants resulted in the blocking of the release of the Notch intracellular domain, which could not translocate into the nucleus, impaired NOTCH1 transcriptional activity, and decreased expression of SLC5A5, TPO, and DUOX2. Further, we found that DTX1 was required for CNTN6 to promote thyroid hormone biosynthesis through Notch signaling. Conclusions: This study demonstrated that CNTN6 is a novel causative gene for CH through the mediation of thyroid hormone biosynthesis via Notch signaling, which provides new insights into the genetic background and mechanisms involved in CH and thyroid dyshormonogenesis.

Inhibitory Effect of Acetaminophen on Ocular Pigmentation and its Relationship with Thyroxine in Zebrafish Embryos.

Acetaminophen (N-acetyl-p-aminophenol; APAP) is one of the most widely used analgesics. To examine the toxicity of APAP, we used zebrafish embryos as model animals to detect the effect of APAP on the thyroid system of zebrafish embryos. The zebrafish embryos were exposed to APAP from 4h post fertilization (4 hpf) until observation. The experimental results showed that APAP caused pericardial edema and decreased pigmentation in the zebrafish embryos or larvae. The APAP treatment caused a decrease in the expression of tpo and thrβ in the zebrafish at 36 and 72 hpf. The transcriptomic analysis found that APAP affected retinol metabolism, the metabolism of xenobiotics by cytochrome P450, and the tyrosine metabolism pathway. The harmful effect of APAP on zebrafish embryos might be due to its disrupting effect on the functional regulation of the thyroid hormone system.

THU237 A Case Of Primary Hypothyroidism, Dysglycemia And Epilepsy With PAX8 And SETD1A Mutation

Abstract Disclosure: S.K. Singh: None. KEYWORDS: PAX8, SETD1A, DYSGLYCEMIA INTRODUCTION: Hypothyroidism is associated with dysglycemia linked with insulin resistance and shared common genetic determinants.PAX 8 gene regulates the development of thyroid gland as well as the expression of TG,TPO and NIS genes.It impacts glucose homeostasis by favoring beta-cell survival and through thyroid hormones.Epilepsy in hypothyroid patients can be idiopathic or due to Hashimoto’s Encephalopathy (HE). Thyroid hormones can impact epileptogenesis through mitochondrial dysfunction, oxidative-stress and GABAergic transmission. THE INDEX PATIENT: A 13 year old female(weight and height age 1.5 years below her chronological age by Indian growth chart and a further lag of 18 months and 6 months, respectively by CDC chart)was diagnosed as a case of thyroprivic hypothyroidism with TSH - 100 mIU/L and positive anti-TPO antibody. She was euthyroid on 112 ug/d L-T4.She is on anticonvulsant since 12 years of age for tonic-clonic epilepsy. The MRI of brain showed focal gliosis with few small encephalomalacic streaks in right occipital lobe. On evaluation at 12.5 years of age, her fasting plasma glucose was 80 mg/dl and her HbA1c was 4.8% but 2-h post glucose was 233 mg/dl with another value of 270 mg/dl. Her father has T2DM and Hypothyroidism. Her mother is Hypothyroid. Her genetic study showed SETDIA and PAX8(-) mutations. DISCUSSION: PAX8 regulates development of thyroid gland and biosynthesis of thyroid hormones. It can impact glucose homeostasis by enhancing beta-cell survival by inhibiting apoptosis, immunomodulation, maintaining local anti-inflammatory milieu in pancreas, ensuring systemic levels of thyroid hormone to stimulate insulin secretion and protect beta-cells from environmental stressors.PAX8 mutation in a family has been shown to be associated with a spectrum of disorders encompassing Gestational Thyroid Dysfunction(GTD),Gestational Diabetes Mellitus(GDM) and T2DM.[1] Although mutation analysis was done only in the index patient, Hypothyroidism and Dysglycemia (post- glucose>200 mg/dl)in the patient and family history of Hypothyroidism and T2DM could be linked with PAX8 mutation.SETD1A mutation has been linked with early-onset epilepsy with or without developmental delay. The mutation study can aid in deciding appropriate therapy including steroids for HE. CONCLUSION: This case highlights the significance of evaluation for PAX8 mutation in hypothyroid patients, even if anti-TPO positive, particularly when there is family history of thyroid dysfunction, GDM, T2DM. It also underlies the importance of SETD1A mutation analysis in hypothyroid patients with epilepsy. Presentation: Thursday, June 15, 2023

Expression analysis of genes involved in the expansion of hematopoietic stem cells (SCF, Flt3-L, TPO, IL-3, and IL-6) in unrestricted somatic stem cells cultured on fibrin

Umbilical cord blood (UCB) transplantation is a promising therapeutic approach for patients lacking HLA-matched donors. A main limitation to the use of UCB-derived HSCs (UCB–HSCs) is the low number of transplantable cells. Novel culture strategies are being developed to increase the number of HSCs. Unrestricted somatic stem cells (USSCs) have been identified as promising stromal cells for supporting HSC expansion. The current study aimed to explore the effect of fibrin on the expression of hematopoiesis-related genes (SCF, Flt3-L, TPO, IL-3, and IL-6) in USSCs. USSCs were isolated from UCB and characterized by flow cytometry and in vitro multilineage differentiation ability. DAPI staining and the MTT assay were used to assess the effect of fibrin on USSC viability. The cell attachment was evaluated using SEM. qRT-PCR was performed to evaluate the expression of SCF, Flt3-L, TPO, IL-3, and IL-6 in USSCs cultured on 3D fibrin scaffolds. USSCs were positive for CD73, CD105, and CD166 and negative for CD45. Alizarin red and Oil red O stains confirmed calcium deposition and lipid vacuoles in USSCs. Results obtained from DAPI and MTT assays revealed a positive effect of fibrin on USSC viability. Cells cultured on fibrin express significantly higher levels of SCF and TPO compared to those grown in a 2D environment. The positive effect of fibrin on IL-6 levels was reversed. Fibrin did not affect Flt3-L expression and IL-3 mRNA expression was not detected in either group. The results of this study provide the basis for developing further research on the ex vivo expansion of HSCs with USSCs.

Intrauterine exposure to di(2-ethylhexyl) phthalate (DEHP) disrupts the function of the hypothalamus-pituitary-thyroid axis of the F1 rats during adult life.

DEHP is an endocrine disruptor widely used in the production of malleable plastics. DEHP exposure was associated with altered hypothalamic-pituitary-thyroid (HPT) axis function. Although previous studies reported deleterious effects of DEHP exposure during the intrauterine period, few studies have evaluated the direct effects triggered by this endocrine disruptor on the offspring animals' thyroid function. This study aimed to investigate the impact of intrauterine exposure to DEHP on the HPT axis function programming of the offspring animals during adulthood. Pregnant Wistar rats were orally treated with corn oil or corn oil supplemented with DEHP (0.48 or 4.8 mg/kg/day) throughout the gestational period. The offspring rats were euthanized on the 90th postnatal day. Hypothalamus, pituitary, thyroid, and liver were collected to analyze gene expression and protein content through qPCR and Western Blot. Blood was collected to determine TSH and thyroid hormone levels through fluorometric or chemiluminescence immunoassays. In the adult F1 female rats, the highest dose of DEHP decreased TSH serum levels. In the thyroid, DEHP reduced the gene expression and/or protein content of NIS, TSHR, TG, TPO, MCT8, NKX2.1, PAX8, and FOXE1. These data are consistent with the reduction in T4 serum levels of the F1 DEHP-exposed female rats. In the liver, DEHP exposure increased the mRNA expression of Dio1 and Ttr, while the highest dose of DEHP reduced the mRNA expression of Ugt1a1 and Ugt1a6. Conversely, in the F1 male adult rats, TSHB expression and TSH serum levels were increased in DEHP-exposed animals. In the thyroid, except for the reduced protein content of TSHR, none of the evaluated genes/proteins were altered by DEHP. TH serum levels were not changed in the DEHP-exposed F1 male rats compared to the control group. Additionally, there were no significant alterations in the expression of hepatic enzymes in these animals. Our results demonstrated, for the first time, that intrauterine exposure to DEHP disrupts the HPT axis function in male and female offspring rats and strongly suggest that DEHP exposure increases the susceptibility of the offspring animals to develop thyroid dysfunctions during adulthood.

Mechanism of development of residual goitre and associated thyroid disorders

In post salt iodination period, endemic goiter that generally develops for iodine deficiency is found prevalent in many regions, including India, despite adequate iodine intake. Such goiters are termed as residual goiters. Consumption of dietary goitrogens could be the possible regulator in the persistence of residual goitre and associated disorders. Residual goitres exist in Manipur, India for the consumption of bamboo shoots (BS). Therefore, to study the actions of BS on major thyroid hormone synthesizing regulatory factors at cellular and molecular levels on isolated thyrocytes conducted perusing expression of NIS, TPO, Tg and PDS proteins as well as mRNA and protein expression of NIS, TPO, Tg, PDS, MCT8 and PAX8 with and without extra iodine in in-vitro studies. Simultaneously, ROS generation in terms of H2O2 and antioxidant status, NO, LPO were assayed. Cellular and DNA damage and oxidation, including cell cycle alterations after BS exposure, were also investigated. Precursors and metabolic end products of cyanogenic glycosides on phytochemical analysis found in BS. It has pleiotropic effects on thyrocytes. It repressed the activities of Na+-K+-ATPase, TPO and D2, as well as reduced the expression of NIS, TPO, Tg, PDS proteins and MCT8 and PAX8, at mRNA level. All these effects of BS are mediated through increased ROS levels as H2O2 upsurge leading to a perturbed pro-oxidant antioxidant balance in thyrocytes associated with cellular and DNA damage and oxidation. Increased LPO and NO levels upon exposure of BS extract triggered plasma membrane and organelle damage of thyrocytes, which are major sites of NIS, TPO, PDS and MCT8, leading to thyroid disfunction. Cellular and DNA damage/oxidation was observed that leads to disruption of normal cell cycle pattern of thyrocytes which in turn causes changes in apoptotic proteins Bax, Bcl-2 and p53. These changes imply imbalance of cell death and proliferation equilibrium leading to disease conditions as found in goitre upon exposure to BS extract. Extra iodine prevents goitrogenic/anti-thyroid influences of BS on such actions to an extent. Thyroid diseases among residual goitrous population following consumption of BS evaluated by their iodine nutritional and salt iodization status, serumT3, T4 and TSH profiles, TPO- and Tg-Ab antibodies, thyroid ultrasonography, the prevalence of hypo- and hyperthyroidism and cytomorphological features. Goitrous populations are affected by subclinical and overt hypo- and hyperthyroidism with elevated thyroid autoantibodies, hypoechoic thyroid and thyroiditis. This study concludes that residual goitrous individuals are affected by different autoimmune thyroid disorders. Excess thiocyanate from dietary goitrogens of cyanogenic origin (BS) is responsible for the development of residual goitre followed by its progression towards different thyroid autoimmune disorders.

A humanized mouse model to study NK cell biology during HIV infection.

NK cells are an important subset of innate immune effectors with antiviral activity. However, NK cell development and immune responses in different tissues during acute and chronic HIV infection in vivo have been difficult to study due to the impaired development and function of NK cells in conventional humanized mouse models. In this issue of the JCI, Sangur et al. report on a transgenic MISTRG-6-15 mouse model with human IL-6 and IL-15 knocked into the previously constructed MISTRG mice. The predecessor model was deficient in Rag2 and γ chain (γc) with knock-in expression of human M-CSF, IL-3, GM-CSF, and TPO, and transgenic expression of human SIRPα. The researchers studied tissue-specific NK cell immune responses during HIV infection and clearly show that the endogenous human NK cells in the humanized mouse model suppressed HIV-1 replication in vivo. These findings provide insight into harnessing the innate immune response for clinical antiviral therapies.

In vitro effects of polychlorinated biphenyls and their hydroxylated metabolites on the synthesis and metabolism of iodothyronines in the chicken (Gallus domesticus) thyroid gland

To assess the effect of polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) on thyroid hormone [TH: thyroxine (T4) and triiodothyronine (T3)] secretion, the concentrations of iodothyronine deiodinases (DIO1, DIO2, DIO3), and mRNA expression of genes involved in TH synthesis (TSHR, NIS, TPO, TG), metabolism (DIO1, DIO2, DIO3), and transport (OATP1C1, MCT8, MCT10, LAT1), chicken thyroid explants were incubated in medium supplemented with TSH (250 mU/ml), PCB118, PCB153, 4-OH-PCB107, and 3-OH-PCB153 (0.5 × 10−8 M), and TSH together with each PCB and OH-PCB. The results of the in vitro experiment revealed that, except for 4-OH-PCB107, all applied PCBs and OH-PCBs inhibited basal and TSH-stimulated T4 secretion. Moreover, they increased basal and reduced TSH-stimulated T3 secretion. PCBs and OH-PCBs decreased the TSH-stimulated TSHR expression. Following PCB and OH-PCB exposure, significant changes in mRNA expression of NIS, TPO, and TG were observed. PCBs and OH-PCBs affected DIO1 and DIO3 transcript levels and protein abundances of each DIO. Furthermore, PCB-dependent effects on OATP1C1, MCT8, and MCT10 mRNA expression were found. In conclusion, both PCB118 and PCB153 and their OH-PCBs affect TH synthesis and deiodination processes in the chicken thyroid gland and influence TH transport across the thyrocyte membrane. In addition, the effects of PCBs and OH-PCBs depended mainly on the type of PCB congener and the exposure time. These results indicate that not only parental PCBs but also OH-PCBs are hazardous for the thyroid gland and may disrupt its endocrine function. Further studies are necessary to explain a mechanism of PCB and OH-PCB action in the avian thyroid gland.

Fructose consumption induces molecular adaptations involving thyroid function and thyroid-related genes in brown adipose tissue in rats.

The increasing incidence of metabolic diseases is in part due to the high fructose consumption, a carbohydrate vastly used in industry, with a potent lipogenic capacity. Thyroid hormones (TH) are essential for metabolism regulation and are associated with changes in body weight, energy expenditure, insulin sensitivity, and dyslipidemia. This study aimed to investigate the influence of fructose intake on thyroid function and thyroid-related genes. Male Wistar rats were divided into Control (CT, n=8) and Fructose (FT - 10% in drinking water, n=8) groups for three weeks. The FT group showed higher glycemia and serum triacylglycerol, indicating metabolic disturbances, and increased thyroid mass, accompanied by higher expression of Srebf1c and Lpl, suggesting increased lipid synthesis. The FT group also presented higher expression of Tpo and Dio1 in the thyroid, suggesting activation of the thyroid gland, but with no alterations in serum TH concentrations. Brown adipose tissue (BAT) of the FT group exhibited higher expression of Dio2, Thra, and Thrb, indicating increased T3 intra-tissue bioavailability and signaling. These responses were accompanied by increased BAT mass and higher expression of Adrb3, Pparg, Srebf1c, Fasn, Ppara, and Ucp1, suggesting increased BAT adrenergic sensitivity, lipid synthesis, oxidation, and thermogenesis. Therefore, short-term fructose consumption induced thyroid molecular alterations and increased BAT expression of thyroid hormone-related signaling genes that potentially contributed to higher BAT activity.

T cell exhaustion is associated with the risk of papillary thyroid carcinoma and can be a predictive and sensitive biomarker for diagnosis

BackgroundThe incidence of papillary thyroid carcinoma (PTC) has been steadily increasing over the past decades. Hashimoto’s thyroiditis (HT) is the most common autoimmune disease, and is related to the pathogenesis of PTC. Programmed death-1 (PD-1) is currently used for the treatment of PTC, but there are very few studies on the clinical value of PD-1 in the diagnosis and targeted therapy of PTC.MethodsThe expression of T, B, NK cells and PD-1 in the peripheral blood of 132 patients with PTC (PTC group), 48 patients with nodular goiter (NG group) and 63 healthy subjects (HP group) were detected by flow cytometry. The expression of plasma T3, T4, FT3, FT4, TSH, TGAb and TPO was detected by chemiluminescence immunoassay. Among 132 PTC, 49 PTC&HT and 83 PTC&noHT were included. Among 48 NG, 10 NG&HT and 38 NG&noHT were included. The expressions of programmed death- ligand1(PD-L1) in tumor tissues of PTC group and thyroid tissues of NG group, PD-1 and CD3 in tumor infiltration lymphocyte (TIL) were detected by immunohistochemistry.ResultsThe expression of FT3, TGAb, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ in PTC and NG was significantly higher than that in the HP group. Moreover, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ expression had significant differences between the PTC group and the NG group. In addition, the expression of TGAb, TPO, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ in PTC&HT group was significantly higher than that in the PTC&noHT group. While, the expression of B cells, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ in PTC&HT group was higher than that in NG&HT group. PD-1 showed a significant correlation with PTC lymph node metastasis. CD3+PD-1+ and CD3+CD4+PD-1+ was higher in N1 stage than in N0 stage. Immunohistochemical results showed that the expression of PD-1, CD3 and PD-L1 in PTC was significantly higher than that in NG.ConclusionsT cell exhaustion might act as a biomarker for the differential diagnosis of PTC and NG. Patients with PTC&HT have obvious T cell exhaustion and increased expression of PD-1, PD-L1.Targeting the PD-1/PD-L1 pathway could be a new approach to prevent malignant transformation from HT to PTC&HT in the future.

Generation and Transplantation of HiPSC-Derived Thyroid Follicular Cells

Abstract Despite the availability of synthetic thyroid hormone (TH) for therapeutic use, many patients with hypothyroidism do not feel well on replacement doses of TH. Indeed, hypothyroid children may develop various behavioral issues if under- or overtreated for hypothyroidism, suggesting the need of better individualized therapy. The development of stem cell-based models of thyroid replacement would greatly facilitate the treatment of post-surgical and congenital hypothyroidism and our understanding of the causes of congenital hypothyroidism. To generate thyroid follicular cells from human induced pluripotent stem cells (hiPSCs), we utilize a directed differentiation approach by activating endogenous signaling pathways we have previously identified in the mouse. We are employing a hiPSC-line with a GFP reporter targeted to the NKX2-1 locus allowing us to identify and purify cells positive for the transcription factor NKX2-1. Using these cells, we confirmed that BMP4 and FGF2 led to the induction of thyroid progenitors from foregut endoderm, expressing the four transcription factors in thyroid development NKX2-1, PAX8, HHEX and FOXE1. Subsequent differentiation of the purified, NKX2-1-positive progenitors led to follicular cell maturation, expressing genes related to TH synthesis, such as TG, TSHR, NIS (SLC5A5) and TPO. To assess the in vivo function of these hiPSC-derived thyroid follicular cells, we grafted the cells under the kidney capsule of radioablated, immunocompromised mice. First, hypothyroidism was confirmed 4 weeks post-ablation by increased TSH and low T4 levels. Next, mice received matured hiPSC-derived follicular cells, undifferentiated hiPSCs or sham surgery and were monitored for 8 weeks. In this preliminary test, no difference in TH levels was detected between the three groups. However, the grafted follicular cells formed a distinct cell population with thyroid-like histology. In order to optimize our directed differentiation protocol further and obtain a purer thyroid progenitor population, we generated a hiPSC line carrying a tdTomato reporter targeted to the PAX8 locus in addition to the NKX2-1-GFP-reporter. After directed differentiation cells sorted for NKX2-1-GFP and PAX8-tdTomato showed high induction of NKX2-1, PAX8, HHEX and FOXE1 expression, as well as expression of TG and TPO. Utilizing this double-reporter hiPSC line will enhance our ability to generate mature follicular cells for transplantation.

IL-8, MSPa, MIF, FGF-9, ANG-2 and AgRP collection were identified for the diagnosis of colorectal cancer based on the support vector machine model

ABSTRACT Colorectal cancer (CRC) is one of the most common cancer, and the early detection of CRC is essential to improve the survival rate of patients. To identify diagnostic markers for colorectal cancer (CRC) by screening differentially expressed proteins (DEPs) in CRC. The DEPs were initially obtained from 12 CRC samples and 12 healthy control samples, and verification analysis was performed in another 34 CRC samples and 34 normal controls. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment with DEPs was analyzed by the R package clusterProfiler (Version 3.2.11), and the DEP-associated protein-protein interaction (PPI) network was created from the STRING database. Additionally, Support Vector Machine (SVM) model prediction and survival analyses were conducted on the key DEPs. Preliminary screening and functional analysis showed that the DEPs mainly overrepresented in pathways such as cytokine-cytokine receptor interaction, chemokine signaling pathway, Rap1, Ras, and MAPK signaling pathways. The key DEPs, including AgRP, ANG-2, Dtk, EOT3, FGF-4, FGF-9, HCC-4, IL-16, IL-8, MIF, MSPa, TECK, TPO, TRAIL R3, and VEGF-D, were used to construct a custom chip. The drug-gene interaction network suggested that TPO was a key drug target. ROC curve showed the SVM diagnostic model with the DEPs IL-8, MSPa, MIF, FGF-9, ANG-2, and AgRP had better diagnostic performance with an AUC of 0.933. Survival analysis showed the expression of FGF9, TPO, TRAIL R3, Dtk, TECK and FGF4 were associated with prognosis. This study revealed the important serum proteins in the pathogenesis of CRC, which might serve as useful and noninvasive predictors for the diagnosis of CRC.

The molecular and gene/miRNA expression profiles of radioiodine resistant papillary thyroid cancer

BackgroundPapillary thyroid cancer (PTC) is the most frequent endocrine tumor. Radioiodine (RAI) treatment is highly effective in these tumors, but up to 60% of metastatic cases become RAI-refractory. Scanty data are available on either the molecular pattern of radioiodine refractory papillary thyroid cancers (PTC) or the mechanisms responsible for RAI resistance.MethodsWe analyzed the molecular profile and gene/miRNA expression in primary PTCs, synchronous and RAI-refractory lymph node metastases (LNMs) in correlation to RAI avidity or refractoriness.We classified patients as RAI+/D+ (RAI uptake/disease persistence), RAI−/D+ (absent RAI uptake/disease persistence), and RAI+/D- (RAI uptake/disease remission), and analyzed the molecular and gene/miRNA profiles, and the expression of thyroid differentiation (TD) related genes.ResultsA different molecular profile according to the RAI class was observed: BRAFV600E cases were more frequent in RAI−/D+ (P = 0.032), and fusion genes in RAI+/D+ cases. RAI+/D- patients were less frequently pTERT mutations positive, and more frequently wild type for the tested mutations/fusions. Expression profiles clearly distinguished PTC from normal thyroid. On the other hand, in refractory cases (RAI+/D+ and RAI−/D+) no distinctive PTC expression patterns were associated with either tissue type, or RAI uptake, but with the driving lesion and BRAF−/RAS-like subtype. Primary tumors and RAI-refractory LNMs with BRAFV600E mutation display transcriptome similarity suggesting that RAI minimally affects the expression profiles of RAI-refractory metastases. Molecular profiles associated with the expression of TPO, SLC26A4 and TD genes, that were found more downregulated in BRAFV600E than in gene fusions tumors.ConclusionsThe present data indicate a different molecular profile in RAI-avid and RAI-refractory metastatic PTCs. Moreover, BRAFV600E tumors displayed reduced differentiation and intrinsic RAI refractoriness, while PTCs with fusion oncogenes are RAI-avid but persistent, suggesting different oncogene-driven mechanisms leading to RAI refractoriness.

Crosstalk of cholinergic pathway on thyroid disrupting effects of the insecticide chlorpyrifos in zebrafish (Danio rerio)

Chlorpyrifos is a widely used organophosphate insecticide and ubiquitously detected in the environment. However, little attention has been paid to its endocrine disrupting effect to non-target organisms. In the present study, zebrafish was exposed to 13 and 65 μg/L of chlorpyrifos for 7 and 10 days to determine the induced neurotoxicity and the alteration of thyroid metabolism. The 120 h LC50 and LC10 of chlorpyrifos was estimated as 1.35 mg/L and 0.62 mg/L based on the acute embryo toxicity assay, respectively. The acetylcholinesterase (AChE) inhibitory was detected by 13 μg/L chlorpyrifos and could be reversed by the co-exposure of 100 and 1000 μg/L anticholinergic agent atropine. For thyroid hormone level, 13 and 65 μg/L of chlorpyrifos induced increased free T3 levels in 10 dpf (days post-fertilization). The expression of thyroid related genes in 7 and 10 dpf exposed zebrafish were measured by the quantitative Real-Time PCR (qRT-PCR) assay. The mRNA expression of tshba, thrb, crhb, ttr, tpo, ugt1ab and slc5a5 had significant change. However, the alterations of thyroid hormone and mRNA expression could be partly rescued by the addition of atropine. The molecular docking of chlorpyrifos and T3 to the thyroid receptor β in zebrafish using homology modelling and CDOCKER procedures shown weaker binding ability of chlorpyrifos compared to T3. Therefore, we concluded that the disturbance of thyroid signaling in zebrafish might arise from the developmental neurotoxicity induced by chlorpyrifos.

Effects of bamboo shoots (Bambusa balcooa) on thyroid hormone synthesizing regulatory elements at cellular and molecular levels in thyrocytes.

Bamboo shoots (BS) are consumed in various forms and used largely in naturopathy for curing ailments since ancient times to present days. It is eaten in South East Asian countries in several indigenous preparations. In north east India, it is consumed predominantly and used as natural cure to treat various diseases. Although known for its beneficial effects, adverse effects including goitrogenic/antithyroidal potential are emerging. Endemic goiter exists in Manipur, India even after adequate iodine intake for consumption of BS. It is thus important to study the impact of this goitrogenic food on certain thyroid hormone synthesizing regulatory factors at cellular and molecular level in thyrocytes. Phytochemical analysis of BS - Bambusa balcooa Roxb (BSBR) extract conducted. IC50 of the extract on thyrocytes in culture was determined. To study the antithyroid effects of this goitrogenic food, activity status of Na+-K+-ATPase, TPO and Deiodinase, mRNA and protein expressions of NIS, TPO and PAX8 were investigated with and without extra iodine in culture media. Simultaneously ROS generation in terms of H2O2 and antioxidant status, NO, LPO were assayed. Activities of the studied enzymes decreased depending on dose and time with increased H2O2, decreased antioxidants followed by increased NO with LPO. DNA damage and LDH also increased while mRNA and protein expression of NIS, TPO and PAX8 were downregulated. Extra iodine ameliorated all such effects partially. Bioactive constituents of the extract imbalances oxidative status of thyrocytes impairing action of hormone synthesizing elements at cellular and molecular level.

Immunocytochemical characteristics ofthyrocytes in radioiodine refractory metastases ofpapillary thyroid cancer.

The ICC research was carried out on a fine needle aspiration biopsy material of 104metastases that were found after thyroidectomy and RI therapy, i.e. in postoperative period (79- radioiodine-refractory metastases, 25- radioiodine-uptake metastases). The ICC analysis of TPO and Tg expression in PTC was performed with the use of monoclonal antibodies against TPO and Tg. RI-refractory (RIRM) and RI-uptake metastases of PTC significantly differ by the percentage of cells expressing TPO and Tg (p < 0.05, and p < 0.05respectively). The effectiveness of RI therapy was different for patients with different percentages of TPO-positive cells. A statistically significant difference was demonstrated in the expression of TPO in a punctates of RI-resistant and RI-sensitive metastatic TPC, based on which a new method for preoperative prediction of RI resistance and RI therapy efficiency was developed. It was shown that ICC determination of Tg expression in metastases is effective in preoperative monitoring of RI resistance of PTC if the part of Tg-positive cells in punctates is lower than 56%. The comprehensive study of the ICC profile of thyrocytes in RI-uptake metastases punctates allows us to develop a personified approach to prediction, monitoring and therapy of patients with PTC.

Analysis of the protective effects of γ-aminobutyric acid during fluoride-induced hypothyroidism in male Kunming mice

Context: Compounds to treat hypothyroidism in the absence of cardiac side effects are urgently required. In this regard, γ-aminobutyric acid (GABA) has gained interest due to its anti-anxiolytic, antihypertensive and antioxidant properties, and reported benefits to the thyroid system.Objective: We investigated the ability of GABA to ameliorate fluoride-induced thyroid injury in mice, and investigated the mechanism(s) associated with GABA-induced protection.Materials and methods: Adult male Kumning mice (N = 90) were exposed to NaF (50 mg/kg) for 30 days as a model of hypothyroidism. To evaluate the effects of GABA administration, fluoride-exposed mice received either thyroid tablets, or low (25 mg/kg), medium (50 mg/kg) or high (75 mg/kg) concentrations of pure GABA orally for 14 days groups (N = 10 each). The effects of low (50 mg/kg); medium (75 mg/kg) and high (100 mg/kg) concentrations of laboratory-separated GABA were assessed for comparison. Effects on thyroid hormone production, oxidative stress, thyroid function-associated genes, and side-effects during therapy were measured.Results: GABA supplementation in fluoride-exposed mice significantly increased the expression of thyroid TG, TPO, and NIS (P < 0.05), significantly improved the thyroid redox state (P < 0.05), modulated the expression of thyroid function-associated genes, conferred liver metabolic protection, and prevented changes to myocardial morphology, thus reducing side effects. Both pure and laboratory-separated GABA displayed comparative protective effects.Discussion and conclusion: Our findings support the assertion that GABA exerts therapeutic potential in hypothyroidism. The design and use of human GABA trials to improve therapeutic outcomes in hypothyroidism are now warranted.

Gestational Exposure to Di(2-ethylhexyl)phthalate Modifies the Expression Pattern of Genes Controlling Thyroid Hormone Biosynthesis in Puberal Rat Progeny

Di(2-ethylhexyl) phthalate (DEHP), a plasticizer, is known to disrupt thyroid functions but the underlying molecular mechanism remains obscure. The present study was conducted testing the hypothesis that gestational exposure to DEHP would modify the expression of specific genes controlling biosynthesis and action of thyroid hormones in the male progeny at puberal age. Pregnant rats were administered with DEHP [1, 10 and 100 mg (in olive oil)/Kg b.wt./day] from embryonic day 9 to 21 through oral route. The pups were sacrificed on post-natal day 60. Enzyme Immuno-Assay (EIA) revealed a dose-dependent decrease in serum 3,5,3’ triiodothyronine (T 3 ) and L-thyroxine (T 4 ) titres in DEHP-treated rats. Real-time RT-PCR and western blot analyses of thyroidal genes revealed decreased expression level of sodium/iodide symporter (Nis) and thyroid hormone receptor I± (TrI±) , whereas the expression of thyroid stimulating hormone receptor (Tshr) , thyroid hormone receptor I² (TrI²) and pendrin (Pds) increased. While western blot detection showed decreased expression level of thyroperoxidase (Tpo) , RTPCR data pointed out augmented expression. Western blot detection of transcriptional factors showed decreased expression levels of fork-headbox e1 (Foxe1) and hematopoietically expressed homeobox (Hhex), whereas thyroid transcription factor-1 (Ttf-1) and paired-box domain 8 (Pax8) increased. Our study demonstrates, for the first time, that gestational exposure to DEHP affects the expression of genes controlling thyroid hormone synthesis in puberal rat progeny, and the hypothyroid state in these rats may be linked to decreased expression of Nis, Tpo , Foxe1 and Hhex.

The NF-κB Family of Transcription Factors and Its Role in Thyroid Physiology.

The nuclear factor (NF)-κB signaling pathway controls a variety of important biological functions, including immune and inflammatory responses, differentiation, cell growth, tumorigenesis, and apoptosis. Two distinct pathways of NF-κB activation are known. The classical, canonical pathway is found virtually in all mammalian cells and NF-κB activation is mediated by the IKK complex, consisting of the IKK1/IKKα and IKK2/IKKβ catalytic kinase subunits and the NF-κB essential modulator (NEMO)/IKKγ protein. The NF-κB-driven transcriptional responses to many different stimuli have been widely characterized in the pathophysiology of the mammalian immune system, mainly because this transcription factor regulates the expression of cytokines, growth factors, and effector enzymes in response to ligation of cellular receptors involved in immunity and inflammation. However, an impressive literature produced in the last two decades shows that NF-κB signaling plays an important role also outside of the immune system, performing different roles and functions depending on the type of tissue and organ. In thyroid, NF-κB signaling is crucial for thyrocytes survival and expression of critical thyroid markers, including Nis, Ttf1, Pax8, Tpo, and thyroglobulin, making this transcription factor essential for maintenance of normal thyroid function.