Thyrocytes Research Articles

OR14-05 Taking Advantage Of The Interleukin-6-Biology In Differentiated Thyroid Cancer To Stimulate Sodium Iodide Symporter (NIS)-mediated Iodide Uptake In Engineered Mesenchymal Stem Cells

Abstract Disclosure: V.F. Koehler: Speaker; Self; Sanofi. L. Drago: None. M. Hageneier: None. C. Kitzberger: None. N. Schwenk: None. K. Shehzad: None. Y. Han: None. J. Nagarajah: None. P.J. Nelson: None. C. Spitzweg: None. Based on its role in mediating iodide uptake from the blood into thyroid follicular cells, the sodium iodide symporter (NIS) provides the basis for the use of radioiodine (RAI) for diagnostic imaging and therapy of differentiated thyroid cancer (DTC). The loss of functional NIS expression leads to RAI-refractory disease. The tumor-selective delivery of NIS as a transgene using mesenchymal stem cells (MSCs) represents a therapeutic strategy for RAI-refractory DTC. Interleukin-6 (IL-6) is a potent cytokine omnipresent in the inflammatory microenvironment of many solid tumors thought to sustain and promote tumor proliferation, invasion and angiogenesis, and contribute to immune escape. With the aim to selectively drive NIS-transgene expression in the context of DTC, MSCs were stably transfected with a NIS-expressing plasmid controlled by the human IL-6-promoter (IL-6-NIS-MSCs). This approach may represent a new tumor-target gene therapy for RAI-refractory DTC. To confirm the inducibility of the IL-6 promoter in IL-6-NIS-MSCs, the murine cytokines (IL-1 β, TNF-α, IFN-γ) were applied to stimulate promoter activation. The resulting functional NIS expression was analyzed by an 125Iuptake assay in vitro. The invitro IL-6 concentration in the papillary thyroid cancer cells BCPAP and K1either co-cultured with or without IL-6-NIS-MSCs was determined by ELISA. Subsequently, IL-6-NIS-MSCs were subjected to a gradient of serum free tumor cell conditioned medium (CM) and serum free unconditioned medium and its migratory response was assessed using 3D live-cell imaging migration assay.IL-6-NIS-MSCs treated with IL-1 β, IFN-γ and TNF-α revealed an increased125I uptake as compared to single stimulation studies. Asa basis for a future in vivo application, incubation of IL-6-NIS-MSC with CM of BCPAP and K1 containing diverse tumor-derived factors or directly co-cultured with these cell lines resulted in significant increase of 125I uptake as compared to untreated cells. The ELISA assay revealed high levels of IL-6 secretion in K1-CM andIL-6-NIS-MSCs co-cultured with BCPAP and K1, while a lower concentration was measured in BCPAP-CM. IL-6-NIS-MSCssubjected to a gradient between a serum free tumor cell-CM and serum free unconditioned medium, showed an increased migration towards tumor cell-CM over a period of 24 h. Taken together, our results indicate the feasibility of targeting IL-6 induction in the inflammatory-rich tumor environment of RAI-refractory DTC to re-establish functional NIS expression using engineered MSCs as delivery vehicles. These data also show us the potential of cytokine-induced promoters for driving NIS transgene expression for a novel imaging and theranostic NIS gene approach. Presentation: Friday, June 16, 2023

SAT545 Erythropoietic Thyroid Nodule in the Setting of Myelofibrosis

Abstract Disclosure: L.M. Vasquez: None. J.M. Bruder: None. M. Koops: None. J. Trejo: None. Background: Extramedullary hematopoiesis is frequently seen in non-osseous locations but not endocrine organs, this is a case of hematopoesis in a thyroid nodule. Clinical Case: A 79-year-old man with myelofibrosis diagnosed at age 66, CAD post CABG, atrial fibrillation on anticoagulation and HFrEF was referred to the endocrinology clinic due to enlarging left lobe thyroid nodule. He was diagnosed with a goiter at age 72 but denied compressive symptoms. He had no history of radiation to his neck or family history of thyroid cancer. On physical exam, the left thyroid lobe was visibly enlarged with slight right tracheal deviation without lymphadenopathy. TSH level was normal. Thyroid ultrasound demonstrated a 6.4 x 4.0 x 4.8 cm, complex cystic-solid, taller than wide nodule in the left lobe and a sub-centimeter cystic nodule in the right lobe. FNA showed hematopoietic elements, including maturing cells in three lineages of hematopoiesis. On repeat ultrasound 5 years later. The left nodule increased in size to 7.3 x 5.4 x 4.4 cm and highly suspicious (TR4 and TR3) by Ti-RADS criteria. Repeat FNA of the nodule showed many red blood cells with few hematopoietic cells, flow cytometry did not show any abnormal populations. No thyroid follicular cells were reported. Myelofibrosis was diagnosed by bone marrow biopsy many years prior. He had maintained a stable CBC without palpable spleen or hepatomegaly. Given this history without thyroid dysfunction or reported symptoms, surgery was not pursued. External beam radiation was a treatment option should the nodule cause symptoms. He continued to have mild anemia not requiring blood transfusions. He passed away 2 years later in hospice care after hospitalization for severe heart failure decompensation. Conclusion: Extramedullary hematopoiesis involving the thyroid is rare but has been reported(1). This type of extramedullary hematopoiesis. can be treated with surgery or low-dose radiation because of the radiosensitivity of hematopoietic tissue (2). This was a potential treatment modality if compressive symptoms arose, but they did not. This case illustrates a situation in which surgical intervention on highly suspicious nodules would not have been the optimal treatment choice. Reference: 1. Jan I., Weng M., Wang C., et al. Extramedullary hematopoiesis involving the thyroid: A rare cytologic finding in otherwise healthy patients and review of literature. Journal of the Formosan Medical Association. (2018) 117, 1108-1114. 2. Taher A, Vichinsky E, Musallam K et al., authors; Weatherall D, editor. Guidelines for the Management of Non-Transfusion Dependent Thalassaemia (NTDT) [Internet]. Nicosia (Cyprus): Thalassaemia International Federation; 2013. Chapter 11, EXTRAMEDULLARY HEMATOPOIESIS. Available from: https://www.ncbi.nlm.nih.gov/books/NBK190455/ Presentation Date: Saturday, June 17, 2023

FRI464 Subacute Thyroiditis Related To COVID-19 Vaccines- A Case Series

Abstract Disclosure: H. Mejia: None. S.A. Gillis Funderburk: None. S.R. Thomas: None. Background: Subacute thyroiditis (SAT) has been reported following SARS-COV-2 viral infection, as well as in recipients of the mRNA vaccines for SARS-COV-2. Patients present with palpitations, tremors and weight loss as well as low grade fevers and a tender thyroid. It is a self-limited condition that typically follows a triphasic course that may include thyrotoxicosis, hypothyroidism and return to normal thyroid function. Clinical features and sonographic findings can provide a specific diagnosis, and a radioactive iodine uptake study is often not required. Report: In 2021, 123 patients were referred to our outpatient service for evaluation of thyrotoxicosis. Among them, we identified 9 women without history of thyroid disease who presented with clinical findings of SAT with thyrotoxicosis. Patients with history of thyroid dysfunction and thyroid nodules were excluded. The symptoms began between 4-27 days after the first or second dose of either the Pfizer Bio-NTech or the Moderna COVID-19 mRNA vaccine. All tested negative for TSI and had no nodules on ultrasound. 2 patients had thyroid uptake scans which confirmed thyroiditis. All 9 patients improved with symptomatic treatment and thyroid functions normalized with time. Discussion: Since the mRNA vaccines for SARS-COV-2 became nationally available, several adverse reactions have been reported including SAT. The mechanism for post-vaccination SAT or thyroid dysfunction remains unknown. Transmembrane protease serine-2 (TMPRSS-2) and ACE-2 receptors allow the SARS-CoV-2 to enter human cells. ACE-2 and TMPRSS-2 are expressed in thyroid follicular cells, and their expression is higher than in lung cells, especially in women. These situations may explain why SARS-CoV-2 causes SAT in women more frequently. Inactive virus vaccines contain many proteins belonging to the pathogen virus and similar antigenic parts. The increased affinity of the SARS-CoV-2 towards the thyroid suggests that inactive vaccine may also affect the thyroid tissue. In the past, SAT has been reported after an inactive virus vaccine- seasonal influenza vaccine, H1N1 vaccine and hepatitis B vaccine. In addition, the COVID-19 vaccine contains aluminum hydroxide as an adjuvant. Autoimmune inflammatory syndrome induced by adjuvants (ASIA syndrome) was previously reported following various vaccines. A recent article describes a patient with COVID-19 vaccine related ASIA syndrome and SAT. In some cases, elevated proinflammatory markers and cytokines after receiving the vaccine may play a major role, as it can stimulate a significant response from our immune system. Hence, SAT should be included as a possible side effect of receiving these vaccines. Conclusion: Our case series highlights a possible relationship between the COVID-19 mRNA vaccines and subacute thyroiditis which has been previously underreported or not recognized by healthcare providers. Presentation: Friday, June 16, 2023

FRI494 Ischemic Stroke After Radioactive Iodine Therapy In Graves Disease

Abstract Disclosure: T. Rave: None. J. Giunta: None. Hyperthyroidism is defined as excess thyroid hormone, most commonly due to Graves Disease. Thiomides are first line therapy, however more definitive options are radioactive iodine (RAI) or thyroidectomy. RAI enters thyroid follicular cells and destroys the thyroid tissue with beta ray emissions. Patients with Graves disease, toxic multinodular goiter, and thyroid cancer can benefit from RAI, but it is contraindicated in pregnancy, breast feeding, and severely uncontrolled thyrotoxicosis. Early side effects are pancytopenia, thyrotoxicosis, and later complications are bone marrow suppression and cancer. There are several reports of cerebral vascular accidents (CVA) after RAI therapy, however, there is limited research demonstrating whether CVA is a direct consequence of RAI or secondary to thyrotoxicosis. This case describes a possible RAI-induced CVA. A 60-year-old female with a past medical history of type 2 Diabetes Mellitus, hypertension, obesity, and Graves disease presented with altered mental status and gait instability for 1 day. She received RAI therapy 1 day prior to symptom onset for uncontrolled Graves disease. She was previously on Methimazole and PTU, however, both were discontinued due to an intolerable rash. As a result, she was maintained on a beta blocker prior to RAI. On presentation, she was afebrile, normotensive with a heart rate of 79. She was oriented only to person and place and had an unbalanced gait. Her Burch-Wartofsky score was 30, which was concerning for thyrotoxicosis. Her TSH was undetectable and EKG showed normal sinus rhythm. She was placed on atenolol, PTU and hydrocortisone. Her CT head was negative for acute disease, however, her MRI brain diagnosed an acute ischemic stroke. During admission, she had episodes of atrial fibrillation with rapid ventricular response. This patient presented with thyrotoxicosis after RAI therapy complicated by a CVA. It is important to note that on presentation she was reliably taking a beta blocker outpatient and her EKG was normal sinus rhythm making atrial fibrillation a less likely culprit of her CVA. Research has found that hyperthyroidism alone can lead to a prothrombotic state, however, additional literature reports that both hyperthyroid and euthyroid patients have an increased risk of CVA following RAI. Whether a CVA is secondary to thyrotoxicosis or a direct effect of RAI, this potentially life-threatening complication should be discussed with patients prior to treatment. Presentation: Friday, June 16, 2023

THU656 Subacute Thyroiditis In Early Pregnancy: A Rare Case Report

Abstract Disclosure: F. El Sayed: None. D. Shelden: None. Background Subacute thyroiditis (SAT) is a self-limiting inflammatory disorder that can be caused by a viral infection and presents with neck pain, fever, and fatigue. To date, only 8 cases of SAT have been reported in pregnant women. Hyperthyroidism during pregnancy is associated with both maternal and neonatal complications. The early diagnosis and treatment of SAT during pregnancy may play a vital role in prevention of adverse fetal outcomes. Case Presentation A 37-year-old female in her 6th week was referred to the endocrinology clinic for neck pain that started two months ago following a viral sore throat. She also reported exhaustion, palpitations, and occasional dysphagia. This is her 10th pregnancy, and she reports a history of thyroid isthmic cysts with hemorrhagic degeneration drained in 2014.The patient is a former occasional smoker and denies alcohol or drug use. Family history is significant for autoimmune disease in her sibling, and type 2 diabetes mellitus in her father. On physical exam, her neck is soft, supple, with no lymphadenopathy. The thyroid gland is enlarged to approximately 40 grams and is mildly tender to palpation. Labs were significant for thyroid-stimulating hormone (TSH) of 0.01 uIU/ml (reference range 0.4-4.5 uIU/ml), serum-free triiodothyronine (FT3) of 4.7 pg/ml (reference rage 1.7-3.7 ng/dl), serum-free thyroxine (FT4) 1.6 pg/ml (reference range 0.7-1.5 pg/ml), ESR 67 mm/hr (reference range 0-18 mm/hr), CRP 52.6 mg/L(reference range <8 mg/L). Thyroid ultrasound revealed enlarged heterogeneous thyroid gland , with poorly defined hypoechoic areas. Based on patients’ symptoms, physical examination, and laboratory findings she was diagnosed with SAT. After review of prior literature and coordination with the patient’s obstetrician, she was started on prednisone 20 mg with a plan to taper by 5 mg weekly with close observation of thyroid function tests. Antibody assessment of thyroid was diffusely negative. Repeat assessment of the patient’s thyroid function tests showed improvement in tsh, FT3, and FT4 to 0.04, 2.4, and 0.9 respectively. The patient reported feeling significantly improved at the 2 week mark. Discussion and conclusion SAT during pregnancy is rare but requires careful management. To date, various treatment modalities are reported in case studies but there is a lack of consensus on medication dosing given the rarity of the presentation. The pathogenesis of SAT is still unknown but is thought to be secondary to destruction of thyroid follicular cells, resulting in the release of thyroid hormone and symptoms of thyrotoxicosis. SAT diagnosis is based on the patient's medical history, symptoms, physical and laboratory findings, and exclusion of other reasons for thyroid dysfunction. Pregnant women with hyperthyroidism need to be differentiated from Graves' disease, and monitoring of thyroid hormone levels is important for fetal outcomes. Presentation: Thursday, June 15, 2023

THU653 Subacute Thyroiditis Following Covid19 Infection

Abstract Disclosure: E. Fanous: None. J.G. Karam: None. Background: Subacute thyroiditis is a self-limiting inflammatory condition of the thyroid gland which usually occurs as a post-viral reaction, leading to the destruction of thyroid follicular cells due to infiltration of lymphocytes. During the first outbreak of COVID-19 virus, subacute thyroiditis had been reported as a complication that may occur in patients without any previous thyroid disorder [1]. Clinical Case: A 62-year-old female patient presented with few weeks history of generalized weakness, fatigue, chills, malaise, low appetite, and unintentional weight loss of around 10lbs. She stated that she feels "internal tremors" and palpitations with heart rate increasing from baseline 60 bpm to 90bpm. She had one episode of near syncope at work prompting her presentation. She reports no change in bowel movements, no heat intolerance, no fever or night sweat, no shortness of breath, no visual changes or eyes problems. Five weeks prior to presentation, the patient was diagnosed with COVID-19 infection by PCR, with a mild disease pattern that consisted of upper respiratory symptoms lasting for few days, however she continued to feel fatigue and low appetite since then, attributing her symptoms to long-term sequelae of Coivd-19. Her past medical history is remarkable for obesity, hypercholesterolemia, and two prior Covid 19 infections: in 2020, treated with monoclonal antibodies, and early 2022. The physical exam was significant for slightly tender thyroid and heart rate of 94 bpm. Thyroid function tests were consistent with hyperthyroidism; TSH<0.01mIU/ml, FT4=2.55ng/dl (0.58-1.64), T4=19.6 mcg/dl (4.4-9.5), T3=1.45ng/ml (0.72-1.35), with negative TSI, TPO AB, and TG AB. ESR was 44mm/hr, RAI uptake and scan showed very low uptake which is indicative for subacute thyroiditis. The patient was given supportive therapy. She developed mild and transient hypothyroidism at 6 weeks follow-up with TSH of 8.59 IU/ml and FT4=0.62 ng/dl (0.89-1.76) before her thyroid tests return to normal at 9 weeks with complete resolution of her symptoms. Conclusion: Prolonged diverse symptoms following Covid19 infection are often attributed to long term Covid symptoms. Health Care providers should be aware of the possibility of subacute thyroiditis following Covid 19 infection and should obtain thyroid function tests in patients who present with malaise, decreased appetite, weight loss and palpitations in the weeks following Covid 19 infection. Reference:1. Mattar SAM, Koh SJQ, Rama Chandran S, Cherng BPZ. Subacute thyroiditis associated with COVID-19. BMJ Case Rep. doi: 10.1136/bcr-2020-237336 Presentation: Thursday, June 15, 2023

SAT494 Impending Thyroid Storm during Pregnancy in a Patient with Gestational Trophoblastic Disease

Abstract Disclosure: R. Kulkarni: None. Z. Sibai: None. Background: Gestational trophoblastic disease (GTD) is a rare complication of pregnancy that arises due to abnormal proliferation of trophoblasts of the placenta. The incidence of GTD in the United States of America is about 110 to 120 per 100,000 pregnancies. Abnormal trophoblastic proliferation leads to higher than expected B-HCG levels compared to normal pregnancy. Since the beta subunit of β-hCG has similar structure to TSH, β-hCG stimulates TSH receptor and causes hyperthyroidism during pregnancy which can be asymptomatic, mild or it can lead to severe cases of thyroid storm. Clinical Case: A 19-year-old African American woman with no PMH, 8 weeks primigravida presented with complaints of nausea, abdominal pain and difficulty walking. Associated symptoms included blurry vision, excessive sweating, tremors and palpitations. Physical examination finding included sinus tachycardia and low BMIof 18.6kg/m2, bilateral hand tremors, weakness of bilateral lower extremities. Initial labs showed quantitative β-hCG level of 283,163(H) m(iU)/mL, TSH <0.010 (L) m(iU)/L , FT4 2.54 (H) ng/dL, negativeTSI and TPO Abs. Burch-Wartofsky Point Scale score was 35 suggestive of impending thyroid storm. Pelvic ultrasound showed intrauterine pregnancy with estimated gestation age of 7 weeks 3 days with findings suggestive of fetal demise. Patient underwent Dilatation and Curettage for suspected molarpregnancy. Final histopathological report of aborted specimen verified the diagnosis of partial molar pregnancy. Based on clinical history, physical finding and lab results, patient was diagnosed with gestational trophoblastic disease induced impending thyroid storm. Endocrinology initiated management with Propranolol 40 mg TID and Methimazole 10 mg daily withimprovement in her clinical symptoms in two days. One week follow up as an outpatient showed resolution of symptoms, normalization of free T4 and down trending β-hCG titers to 166 m(iU)/mL. Propranolol and methimazole were stopped. Conclusion:Gestational trophoblastic disease although a rare clinical entity, it is potentially a life-threatening condition. β-hCG is a glycoprotein, and its beta subunit is structurally similar to TSH, allowing it to bind to the TSH receptor and causing hyperstimulation of the thyroid follicular cells leading to various degrees of hyperthyroidism. GTD associated with significant high level of β-HCG, in rare cases can cause severe consequences like thyroid storm. Therefore, GTD should be considered as a differential diagnosis in patient’s presenting with sever thyrotoxicosis during pregnancy. The mainstay of management is evacuation of the molar pregnancy. In severe cases of hyperthyroidism, Beta blocker and thioamides can be considered for symptoms relief and to speed the recovery. Follow-up with serial quantitative β-hCG measurements is crucial to evaluate for persistent molar tissue. Presentation Date: Saturday, June 17, 2023

SAT572 Concomitant Metastatic Hurthle Cell Carcinoma And Rectal Cancer, Challenges In Diagnosis And Management

Abstract Disclosure: D. Khurram: None. R. Pansare: None. S.L. Mokshagundam: None. Hurthle cell carcinomas (HCC) are aggressive tumors and account for 5% of follicular cell thyroid malignancies. Tumor size, extra-thyroid extension and distant metastases indicate poor prognosis. 20-30% of HCCs have mets at initial diagnosis, frequent sites being lungs and bones. Metastatic HCCs are exceedingly difficult to manage. Detecting two primary cancers concomitantly is uncommon and poses additional challenges in diagnosis and management. We present an interesting case of HCC with mets to the manubrium, vertebrae, lungs and concomitant rectal adenocarcinoma. A 69-year-old male had MRI spine for back pain which showed multiple lytic osseous lesions within T3 and L1 vertebrae. Further imaging revealed a large lesion in the manubrium. Manubrial mass biopsy was positive for poorly differentiated carcinoma, immunohistochemical staining for CAM5.2, cytokeratin, CD 138, CD56 and TTF-1 (weak). He underwent 6 cycles of palliative chemotherapy with carboplatin and Taxol for carcinoma of unknown origin. PET scan following chemotherapy showed a large heterogenous thyroid mass with intense FDG uptake, along with a suspicious rectal mass. Left thyroid nodule FNA reported Bethesda IV follicular neoplasm, Hurthle cell type. Rectal mass biopsy reported moderately differentiated adenocarcinoma. Histology after total thyroidectomy showed unifocal oncocytic HCC, with lymphovascular invasion but no extrathyroidal extension. Molecular markers showed low TMB, stable microsatellite status, negative PDL-1, negative NTRK fusion and positive ATM E839. He received suppressive dose levothyroxine. Radio-iodine ablation was postponed owing to prioritization of radiation therapy for rectal cancer. Thyroglobulin level increased from 53 ng/ML to 216 ng/ML. He received 149 milli Curies of I131 after Thyrogen stimulation. There was mild radioiodine uptake in the thyroid bed, but the metastatic lesions were not radioiodine avid. Follow-up scans showed multiple bilateral pulmonary nodules with worsening osseous mets. Endoscopic biopsy was consistent with HCC, TTF-1 and PAX8 positive. He was started on Lenvatinib but developed diarrhea, hence switched to Pembrolizumab. Thyroglobulin level improved on Pembrolizumab but was complicated by adrenal insufficiency. For rectal cancer, he received radiation therapy and capecitabine. Our case is interesting for several reasons. Firstly, although bone mets are commonly seen in HCC, manubrial mets, especially as the presenting feature is rare. Secondly, concomitant rectal cancer delayed the treatment of the thyroid cancer. Thirdly, there is no major consensus regarding management of HCC, especially metastatic HCC. HCCs are often not radioactive iodine avid. Combination therapy with Lenvatinib and Pembrolizumab is currently under trial for aggressive thyroid malignancy. It will be interesting to follow the clinical outcomes in this individual. Presentation: Saturday, June 17, 2023

Investigation of the effects of phthalates on in vitro thyroid models with RNA-Seq and ATAC-Seq.

Phthalates are a class of endocrine-disrupting chemicals that have been shown to negatively correlate with thyroid hormone serum levels in humans and to cause a state of hyperactivity in the thyroid. However, their mechanism of action is not well described at the molecular level. We analyzed the response of mouse thyroid organoids to the exposure to a biologically relevant dose range of the phthalates bis(2-ethylhexyl) phthalate (DEHP), di-iso-decylphthalate (DIDP), di-iso-nonylphthalate (DINP), and di-n-octylphthalate (DnOP) for 24 h and simultaneously analyzed mRNA and miRNA expression via RNA sequencing. Using the expression data, we performed differential expression and gene set enrichment analysis. We also exposed the human thyroid follicular epithelial cell line Nthy-ori 3-1 to 1 µM of DEHP or DINP for 5 days and analyzed changes in chromatin accessibility via ATAC-Seq. Dose-series analysis showed how the expression of several genes increased or decreased at the highest dose tested. As expected with the low dosing scheme, the compounds induced a modest response on the transcriptome, as we identified changes in only mmu-miR-143-3p after DINP treatment and very few differentially expressed genes. No effect was observed on thyroid markers. Ing5, a component of histones H3 and H4 acetylation complexes, was consistently upregulated in three out of four conditions compared to control, and we observed a partial overlap among the genes differentially expressed by the treatments. Gene set enrichment analysis showed enrichment in the treatment samples of the fatty acid metabolism pathway and in the control of pathways related to the receptor signalling and extracellular matrix organization. ATAC-Seq analysis showed a general increase in accessibility compared to the control, however we did not identify significant changes in accessibility in the identified regions. With this work, we showed that despite having only a few differentially expressed genes, diverse analysis methods could be applied to retrieve relevant information on phthalates, showing the potential of in vitro thyroid-relevant systems for the analysis of endocrine disruptors.

Induction of epithelial-mesenchymal transition in thyroid follicular cells is associated with cell adhesion alterations and low-dose hyper-radiosensitivity.

Epithelial-mesenchymal transition (EMT) is associated with altered cellular adhesion. We previously demonstrated that cellular adhesion influences Low-dose Hyper-Radiosensitivity (HRS) in a variety of tumor cells. However, the relationship of low-dose HRS with the phenotypic plasticity incurred by EMT during the neoplastic transformation remains to be elucidated. To investigate whether acquisition of EMT phenotype during progressive neoplastic transformation may affect low-dose radiation sensitivity. Primary thyroid cells obtained from a human cystic thyroid nodule were first subjected to nutritional stress. This yielded immortalized INM-Thy1 cell strain, which was further treated with either multiple γ-radiation fractions (1.5 Gy each) or repetitive cycles of 3-methylcholanthrene and phorbol-12-myristate-13-acetate, yielding two progressive transformants, viz., INM-Thy1R and INM-Thy1C. Morphological alterations, chromosomal double-minutes, cell adhesion proteins, anchorage dependency, tumorigenicity in nude mice and cellular radiosensitivity were studied in these strains. Both transformants (INM-Thy1R, INM-Thy1C) displayed progressive tumorigenic features, viz., soft agar colony growth and solid tumor growth in nude mice, coupled with features of epithelial-mesenchymal transition and activated Wnt pathway. Incidentally, the chemical-induced transformant (INM-Thy1C) displayed a prominent HRS (αs/αr=29.35) which remained unaffected at high cell density. However, the parental (INM-Thy1) cell line as well as radiation-induced transformant (INM-Thy1R) failed to show this hypersensitivity. The study shows that induction of EMT in thyroid follicular cells may accompany increased susceptibility to low-dose ionizing radiation, which was attenuated by adaptive resistance acquired during radiation-induced transformation.

Targeting hnRNPC suppresses thyroid follicular epithelial cell apoptosis and necroptosis through m6A-modified ATF4 in autoimmune thyroid disease

Both environmental and genetic factors contribute to the etiology of autoimmune thyroid disease (AITD) including Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). However, the exact pathogenesis and interactions that occur between environmental factors and genes remain unclear, and therapeutic targets require further investigation due to limited therapeutic options. To solve such problems, this study utilized single-cell transcriptome, whole transcriptome, full-length transcriptome (Oxford nanopore technology), and metabolome sequencing to examine thyroid lesion tissues from 2 HT patients and 2 GD patients as well as healthy thyroid tissue from 1 control subject. HT patients had increased ATF4-positive thyroid follicular epithelial (ThyFoEp) cells, which significantly increased endoplasmic reticulum stress. The enhanced sustained stress resulted in cell death mainly including apoptosis and necroptosis. The ATF4-based global gene regulatory network and experimental validation revealed that N6-methyladenosine (m6A) reader hnRNPC promoted the transcriptional activity, synthesis, and translation of ATF4 through mediating m6A modification of ATF4. Increased ATF4 expression initiated endoplasmic reticulum stress signaling, which when sustained, caused apoptosis and necroptosis in ThyFoEp cells, and mediated HT development. Targeting hnRNPC and ATF4 notably decreased ThyFoEp cell death, thus ameliorating disease progression. Collectively, this study reveals the mechanisms by which microenvironmental cells in HT and GD patients trigger and amplify the thyroid autoimmune cascade response. Furthermore, we identify new therapeutic targets for the treatment of autoimmune thyroid disease, hoping to provide a potential way for targeted therapy.

Characterization of lesions of nutritional congenital goitre in cattle

There are few studies that classify and characterize the morphometric and immunohistochemical features of goitre in bovine thyroid glands (TGs). We investigated 39 bovine TGs (fetuses [9], stillbirths [18], neonates [12]) born to dams with low T4 hormone levels and no iodine supplementation and 10 (fetuses [3], stillbirths [3], neonates [4]) born to dams with normal T4 levels and supplemented with iodine. Body weight (BW), TG weight (TGW), TGW:BW ratio and histological lesions were determined. The TGs were classified histopathologically as normal gland (G0), mild goitre (G1), moderate goitre (G2) or severe goitre (G3). Various morphological and morphometric parameters were calculated from microscopic images using image analysis software. Immunohistochemistry was performed to detect proliferating cell nuclear antigen (PCNA). There were significant differences in the TGW:BW ratio among groups (P <0.05): 0.3 ± 0.1 in G0, 0.5 ± 0.3 in G1, 0.8 ± 0.3 in G2 and 1.3 ± 0.7 in G3. In G0, large homogeneous follicles with eosinophilic colloid were seen. In the groups with lesions (G1, G2 and G3), heterogeneity in follicle shape and size, height and area of thyroid follicular cells, height of thyroid follicular epithelium and PCNA immunolabelling were directly related to histopathological grade, with significant differences among groups (P <0.001), gradually increasing from G1 to G3 compared with G0. The TGW:BW ratio and histological grade were positively correlated (P = 0.008).

Structural and morphologic characteristics of nodular goiter in chronic iodine deficiency status

OBJECTIVES. Diffuse and nodular goiter are the most common thyroid gland (thyroid) diseases in children and adults living in iodine-deficient regions. Thyroid hormone potentiates progressive hypertrophy and hyperplasia of thyrocytes, with uneven proliferation of thyroid cells leading to nodule formation. Iodine deficiency, which promotes replication of thyroid follicular cells, also increases the incidence of TSH Receptor mutations, leading to receptor activation and autonomous functioning of follicles.AIM. To evaluate and describe ultrasound and cytologic thyroid nodes in the regions in Russia with proven iodine deficiency.MATERIALS AND METHODS. The study was conducted in regions with proven iodine deficiency in the Central and North Caucasian Federal Districts. Population of conditionally healthy adult volunteers (mean age — 47.5 years) was recruited as part of visiting events to screen for the occurrence of thyroid nodes.RESULTS. The results obtained indicate a high prevalence of multinodular colloid goiter in adult patients living in conditions of chronic iodine deficiency. In the Chechen Republic, the prevalence of nodular thyroid pathology was found in 64.5% (205/318) of cases, and in the Tula region — in 40.6% (116/286). In the studied sample the results of cytologic examination confirmed the presence of colloid thyroid masses in 97% (60/62) — Bethesda category II; in 3% (2/62) of patients nodular masses had Bethesda IV. Diffuse goiter prevalence in the examined population was 20.8%.CONCLUSIONS. A study indicates a high prevalence of iodine-deficient thyroid disease — multinodular colloid goiter in regions with proven chronic iodine deficiency. The wide prevalence of goiter with colloid and cystic components is confirmed by the cytologic examination with different ultrasound characteristics according to the EU-TIRADS classification. The obtained data correspond to the official statistics demonstrating high prevalence of nontoxic goiter in adults in the examined regions, which is an important argument confirming the need for systemic iodine prophylaxis.

Alterations of Bax/Bcl-2 ratio contribute to NaAsO2 induced thyrotoxicity in human thyroid follicular epithelial cells and SD rats

The environmental toxicant arsenic causes various human diseases and threatens millions of people worldwide. Recently, a limited number of studies have revealed that exposure to arsenic is associated with thyroid dysfunction, indicating its toxicological impact on the thyroid gland, however, its precise forms of damage and underlying mechanisms remain largely unknown. Here, we sought to observe the thyrotoxicity of sodium arsenite (NaAsO2) on human thyroid follicular epithelial cells (Nthy-ori 3–1) and SD rats, and explore the role of Bax/Bcl-2 ratio in the above process. Our results displayed that NaAsO2 exerted a dose-dependent inhibitory effect on the viability of Nthy-ori 3–1 cells. Alongside the increase doses of NaAsO2 exposure, morphological changes and elevated LDH levels were observed. Furthermore, apoptosis rates increased in a dose- and time-dependent manner, accompanied by a decrease in Bcl-2 and an opposite change in Bax expression. SD rats were treated with 0, 2.5, 5, and 10 mg/kg NaAsO2 for 36 weeks. Our findings revealed that NaAsO2 exposure resulted in arsenic accumulation in thyroid tissue, elevated ratio of Bax/Bcl-2, and histopathological changes of thyroid in rats, which accompanied by the decreased serum T3 and T4 levels and the increased serum TSH level. Furthermore, T3 and T4 levels were negatively correlated with Bax expression, whereas positively correlated with Bcl-2 expression. Collectively, our results suggest that NaAsO2 exposure induces cytotoxicity in Nthy-ori 3–1 cells, causes structural damages and dysfunction of thyroid in SD rats, in which the imbalance of Bax/Bcl-2 ratio may play a significant role.

Downregulation of cellular retinoic acid binding protein 1 fosters epithelial-mesenchymal transition in thyroid cancer.

Cellular retinoic acid binding protein 1 (CRABP1) participates in the regulation of retinoid signaling. Previous studies showed conflicting results regarding the role of CRABP1 in tumor biology, including protumorigenic and tumor-suppressive effects in different types of cancer. Our bioinformatics analyses suggested that CRABP1 expression was downregulated in thyroid cancer. Ectopic expression of CRABP1 in thyroid cancer cells suppressed migratory and invasive activity without affecting cell growth or cell cycle distribution. In transformed normal thyroid follicular epithelial cells, silencing of CRABP1 expression increased invasiveness. Additionally, CRABP1 overexpression was associated with downregulation of the mesenchymal phenotype. Kinase phosphorylation profiling indicated that CRABP1 overexpression was accompanied by a decrease in phosphorylation of epidermal growth factor (EGF) receptor and downstream phosphorylation of Akt, STAT3, and FAK, which were reversed by exogenous EGF treatment. Immunohistochemical analysis of our tissue microarrays revealed an inverse association between CRABP1 expression and disease stage of differentiated thyroid cancer. Taken together, our results suggest that CRABP1 expression is aberrantly lost in thyroid cancer, and this downregulation promotes the epithelial-mesenchymal transition at least partly through modulating EGF receptor signaling.

Curcumin induces mitophagy by promoting mitochondrial succinate dehydrogenase activity and sensitizes human papillary thyroid carcinoma BCPAP cells to radioiodine treatment

Thyroid cancer is one of the most common endocrine malignancies. Differentiated thyroid cancer (DTC) treatment is based on the ability of thyroid follicular cells to accumulate radioactive iodide (RAI). DTC generally has a good prognosis. However, tumor dedifferentiation or defect in certain cell death mechanism occurs in a subset of DTC patients, leading to RAI resistance. Therefore, developing novel therapeutic approaches that enhance RAI sensitivity are still warranted. We found that curcumin, an active ingredient in turmeric with anti-cancer properties, rapidly accumulated in the mitochondria of thyroid cancer cells but not normal epithelial cells. Curcumin treatment triggered mitochondrial membrane depolarization, engulfment of mitochondria within autophagosomes and a robust decrease in mitochondrial mass and proteins, indicating that curcumin selectively induced mitophagy in thyroid cancer cells. In addition, curcumin-induced mitophagic cell death and its synergistic cytotoxic effect with radioiodine could be attenuated by autophagy inhibitor, 3-methyladenine (3-MA). Interestingly, the mechanism of mitophagy-inducing potential of curcumin was its unique mitochondria-targeting property, which induced a burst of SDH activity and excessive ROS production. Our data suggest that curcumin induces mitochondrial dysfunction and triggers lethal mitophagy, which synergizes with radioiodine to kill thyroid cancer cells.

The human thyroid-derived cl-huthyrecs cell line expresses the thyrotropin (TSH) receptor and thyroglobulin but lacks other essential characteristics of thyroid follicular cells

The human thyroid-derived cl-huthyrecs cell line expresses the thyrotropin (TSH) receptor and thyroglobulin but lacks other essential characteristics of thyroid follicular cells

DBP exposure induces thyroid inflammatory impairment through activating AKT/NF-κB/NLRP3 signaling

Previous studies exhibited reproductive and neurodevelopmental toxicity in rats exposed to Di-n-butyl phthalate (DBP). However, the effects of DBP exposure on the other endocrine organ are still unclear. This study aimed to assess the impact of DBP exposure on the thyroid of male rats and the associated mechanisms. Here, rats were respectively treated with DBP at 0 (control), 50 (low dose), 250 (medium dose), or 500 (high dose) mg/kg/day dissolved in 1 ml quantity of corn oil by intragastrical administration for two weeks. The results demonstrated that the proliferation and inflammatory response changes were significantly different compared to the control. In vivo DBP is mainly converted to mono-n-butyl phthalate (MBP), an active form producing untoward reactions of DBP exposure. Therefore, for in vitro experiments, we treated the thyroid follicular epithelial cell line (Nthy-ori 3–1) in a temporal gradient using 1 mM MBP. Further in vitro studies showed that MBP exposure upregulated tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), as well as interleukin-1β (IL-1β) by activating AKT/NF-κB/NLRP3 signaling. Meanwhile, we detected that Pellino2 (Peli2) played an essential role in promoting the activation of NLRP3 inflammasome. Briefly speaking, this study confirmed that DBP exposure caused impaired thyroid structure and thyroid inflammation in male rats, which offered new views into the harm of DBP exposure on the endocrine organ.

Correction for Extrinsic Background in Raman Hyperspectral Images.

Raman hyperspectral microscopy is a valuable tool in biological and biomedical imaging. Because Raman scattering is often weak in comparison to other phenomena, prevalent spectral fluctuations and contaminations have brought advancements in analytical and chemometric methods for Raman spectra. These chemometric advances have been key contributors to the applicability of Raman imaging to biological systems. As studies increase in scale, spectral contamination from extrinsic background, intensity from sources such as the optical components that are extrinsic to the sample of interest, has become an emerging issue. Although existing baseline correction schemes often reduce intrinsic background such as autofluorescence originating from the sample of interest, extrinsic background is not explicitly considered, and these methods often fail to reduce its effects. Here, we show that extrinsic background can significantly affect a classification model using Raman images, yielding misleadingly high accuracies in the distinction of benign and malignant samples of follicular thyroid cell lines. To mitigate its effects, we develop extrinsic background correction (EBC) and demonstrate its use in combination with existing methods on Raman hyperspectral images. EBC isolates regions containing the smallest amounts of sample materials that retain extrinsic contributions that are specific to the device or environment. We perform classification both with and without the use of EBC, and we find that EBC retains biological characteristics in the spectra while significantly reducing extrinsic background. As the methodology used in EBC is not specific to Raman spectra, correction of extrinsic effects in other types of hyperspectral and grayscale images is also possible.

MiR-146b-5p regulates the scavenging effect of GPx-3 on peroxide in papillary thyroid cancer cells

BackgroundGlutathione peroxidase (GPx) is an important antioxidant enzyme in thyroid follicular cells. Reduced levels of glutathione peroxidase 3 (GPx-3) expression in papillary thyroid cancer (PTC) are associated with poor prognosis. However, the reason for the decreased expression level of GPx-3 in PTC is unclear. MethodsThe expression of GPx-3 in papillary thyroid carcinoma and adjacent normal tissue (n = 18) was detected by Western blotting. Bioinformatics was used to predict the relationship between the level of GPx-3 and gender, age, lymph node metastasis, stage, BRAFV600E mutation, and recurrence-free survival of patients. The possible upstream microRNAs of GPx-3 were analyzed by bioinformatics tools also. We verified the relationship between GPx-3 and upstream microRNA by dual luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA). ResultsThe protein level of GPx-3 decreased in PTC, and analysis of public database datasets suggests that its decreased expression may be associated with the BRAFV600E mutation. MiR-146b-5p was significantly overexpressed in PTC. The dual luciferase reporter assay verified the effect of miR-146b-5p on 3′-UTR of GPx-3 mRNA. Knockdown of miR-146b-5p in thyroid cancer cell lines TPC-1 and BCPAP increased GPx-3 expression levels, accompanied by an increase in the conversion of glutathione (GSH) to oxidized glutathione (GSSG). ConclusionsIn conclusion, the level of GPx-3 decreases in papillary thyroid carcinoma and impairs intracellular peroxide clearance, due to the inhibitory effect of miR-146b-5p. The accumulation of intracellular peroxides may contribute to the poor prognosis of thyroid cancer.