TSH Stimulation Research Articles

ZFP36L2 Role in Thyroid Functionality.

Thyroid hormone levels are usually genetically determined. Thyrocytes produce a unique set of enzymes that are dedicated to thyroid hormone synthesis. While thyroid transcriptional regulation is well-characterized, post-transcriptional mechanisms have been less investigated. Here, we describe the involvement of ZFP36L2, a protein that stimulates degradation of target mRNAs, in thyroid development and function, by in vivo and in vitro gene targeting in thyrocytes. Thyroid-specific Zfp36l2-/- females were hypothyroid, with reduced levels of circulating free Thyroxine (cfT4) and Triiodothyronine (cfT3). Their hypothyroidism was due to dyshormonogenesis, already evident one week after weaning, while thyroid development appeared normal. We observed decreases in several thyroid-specific transcripts and proteins, such as Nis and its transcriptional regulators (Pax8 and Nkx2.1), and increased apoptosis in Zfp36l2-/- thyroids. Nis, Pax8, and Nkx2.1 mRNAs were also reduced in Zfp36l2 knock-out thyrocytes in vitro (L2KO), in which we confirmed the increased apoptosis. Finally, in L2KO cells, we showed an altered response to TSH stimulation regarding both thyroid-specific gene expression and cell proliferation and survival. This result was supported by increases in P21/WAF1 and p-P38MAPK levels. Mechanistically, we confirmed Notch1 as a target of ZFP36L2 in the thyroid since its levels were increased in both in vitro and in vivo models. In both models, the levels of Id4 mRNA, a potential inhibitor of Pax8 activity, were increased. Overall, the data indicate that the regulation of mRNA stability by ZFP36L2 is a mechanism that controls the function and survival of thyrocytes.

Call for Emergency Room Guidelines to Identify Hyperthyroid Patients Prior to Contrast Imaging

Abstract Background: Thyrotoxicosis can be mistaken for conditions such as atrial fibrillation and pulmonary embolism (PE) given the nonspecific symptoms of fatigue, palpitations, and dyspnea. Patients often undergo further imaging on presentation to the emergency room (ER), many of which use iodine for contrast. This can put patients at increased risk for iodine induced hyperthyroidism and delay definitive treatment in patients with Graves’ disease, the most common cause of hyperthyroidism. Clinical Case: A 53-year-old male with history of hyperthyroidism, atrial fibrillation, and prior PE presented with palpitations to the ER. He developed worsening dyspnea on exertion and palpitations over the last three days. He was unable to afford his medications, including methimazole, for the last nine months. In the ER he was in atrial fibrillation with rapid ventricular response. Due to concern for PE, he underwent a CTA with contrast, which was negative. His physical exam was notable for a diffusely enlarged goiter. His labs showed low TSH <0.01 (norm 0.35-5.50mIU/L) and high free T4 >7.77 (norm 0.9-1.8ng/dL). TSH stimulating antibodies were elevated at 1.9 (norm <1.3 TSI index), consistent with Graves’ hyperthyroidism. Endocrinology was then consulted for severe thyrotoxicosis, initially treating the patient with PTU and propranolol. The patient was transitioned to methimazole and continued propranolol on discharge. Since he was given contrast, plan was for repeat thyroid uptake scan and iodine ablation in 3 months. However, patient was not compliant with medications, resulting in readmission for thyrotoxicosis 3 months later. Conclusion: This case highlights the impact of increased use of contrast in imaging in hyperthyroid patients. Hyperthyroid patients are at an increased risk for emboli. However, iodine can cause contrast-induced hyperthyroidism and delay definitive treatment of Graves’ disease. As almost half of thyrotoxic patients receive iodinated contrast prior to an endocrine consultation, endocrinologists should work with emergency physicians to develop a set of guidelines to identify at risk populations for hyperthyroidism (1). We advocate for urgent thyroid testing in patients with new onset atrial fibrillation, a history of Graves’ disease, specific symptoms of Graves’, or those taking thyrotoxic-inducing medications. This will assist in determining if patients should receive a prophylactic dose of anti-thyroid medication prior to iodinated contrast imaging. These guidelines can help prevent contrast induced hyperthyroidism and disruptions in treatment of Graves’ while still imaging patients for other diagnoses on the differential. Reference: (1) Giacomini A, et al. Urgent thyroid-stimulating hormone testing in emergency medicine: A useful tool? J Emerg Med. 2015;49(4):481-487.

Coexistence of Thyroid Hormone Resistance and Autoimmune Thyroid Disease: Not a Mere Coincidence

Introduction: The coexistence of thyroid hormone resistance and autoimmune thyroiditis was initially thought to be a chance event. In a large cohort study, Barcoff et al. demonstrated an increased likelihood of thyroid autoantibodies in patients with thyroid hormone resistance (RTH). We report a unique case to epitomize the coexistence of these two conditions and discuss the postulated mechanisms.Clinical Case: A 22-year-old woman with a history of Hashimoto’s thyroiditis, attention deficit hyperactivity disorder, and migraine presented to the endocrinology clinic with symptoms of weight loss, fatigue, decreased appetite, and heat intolerance for 4 months. She was diagnosed with Hashimoto’s thyroiditis at age 12 with elevated TSH only and had been on levothyroxine 25 mcg since diagnosis. Physical exam demonstrated a body mass index of 34.14 kg/m2, blood pressure of 138/91 mmHg, pulse of 77 bpm, and an enlarged palpable thyroid gland. Laboratory investigations revealed elevated thyroid peroxidase (TPO) antibody at 234 IU/mL (<9) and thyroglobulin Antibody at 3 IU/mL (<1) with elevated free T4 of 3.76 ng/dL (0.61-1.36), elevated total T4 of 21.58 mcg/dL (6.09-12.23), increased T3 uptake of 51.2% uptake (32-48.4), and upper normal TSH of 3.99 uIU/mL (0.44-5.33). Elevated free T4 was confirmed by the equilibrium dialysis method at 6.2 ng/dL (0.9-2.2) with upper normal TSH at 3.77 uIU/mL. Thyroid ultrasound demonstrated thyromegaly and hypervascularity of the gland. TSH secreting pituitary adenoma was ruled out with a normal alpha subunit of 0.2 ng/ml (0.1-0.6) and molar ratio of the alpha subunit to TSH ratio <1 along with a normal pituitary MRI. She was not taking other medications or supplements. She was diagnosed with thyroid hormone resistance and is undergoing genetic testing to differentiate the THRB genetic mutations from Non-TR-RTH. Her symptoms improved after discontinuing her levothyroxine.Clinical Lesson: Our case highlights the importance of evaluating thyroid disorders properly before starting treatment and illustrates the coexistence of autoimmune thyroiditis with thyroid hormone resistance. Barcoff et al. demonstrated that there was an increased likelihood of thyroid autoantibodies with odds ratio = 2.36 (p = 0.002) in a large patient cohort with RTH, compared to their unaffected first-degree relatives. However, since there was no correlation in increased antibody with increased age and the duration of the disease, the proposed hypothesis did not substantiate chronic TSH stimulation inducing autoimmune response. The proposed mechanism is that the elevated TSH in RTH stimulates the immune system at the TRα level which was demonstrated in murine models with increased activation of thymic function in correlation with TSH level. More research is needed to understand the underlying mechanism of their coexistence.

Radioiodine therapy of thyroid autonomy.

Radioiodine therapy (RIT) of thyroid functional autonomy (TFA) is rapidly evolving, though it has been recognized for decades as a very effective treatment of toxic nodular varieties. Indeed, TFA is a frequent cause of persistent subclinical hyperthyroidism, which should be regarded as a new metabolic syndrome, with well-established adverse cardio-vascular consequences. Sensitive TSH assays and multiparametric ultrasounds are not accurate enough to reliably diagnose TFA and identify its main variants, unifocal, multifocal (UFA/MFA) and disseminated autonomy (DISA). Modern diagnostic tools are extensively presented and rely upon Thyroid Scan imaging and quantification. A new relationship allows predicting at baseline, an excess of 123I uptake as compared to the TSH stimulation in compensated TFA. Suppressed TS are useful with either isotope, otherwise. Diagnosis of the DISA variant is presented as compared to Graves' disease. Dosimetry has some specificity in TFA work-up. Indeed, the spatial distribution of the dose is as important as the mean value itself and can be eventually controlled by adjusting the TSH level with the smart use of LT3 or antithyroid drug therapy (ATD). A review of the different ways to determine the target mass from anatomical to functional approaches is presented. Main clinical and dosimetric published results of RIT are summarized according to clinical goals. Endogenous TSH stimulation using an ATD preparation has promising results in reducing big autonomously functioning goiters. Finally, we report preliminary successful results of preventive RIT using short term LT3 suppression in compensated TFA, with low administered activities and low rate of hypothyroidism.

Recombinant human thyrotropin versus thyroid hormone withdrawal in differentiated thyroid carcinoma follow-up: a single center experience.

Our goal was to evaluate and compare the diagnostic utility of thyroid hormone withdrawal (THW) and recombinant thyroid-stimulating hormone (rhTSH) methods in detecting recurrence/persistence (R/PD) of differentiated thyroid carcinoma (DTC). The study included 413 patients with DTC who underwent total thyroidectomy and had remnant ablation. DxWBS, s-Tg levels, R/PD were evaluated retrospectively. A s-Tg level≥2 ng/mL was considered as "positive s-Tg". DxWBS and s-Tg levels were evaluated with rhTSH in 116 and THW in 297 subjects, respectively. The sensitivity and specificity of "positive s-Tg" for R/PD in THW group were 77.3% and 92.7%, with 90.3% accuracy, respectively. The sensitivity and specificity of "positive s-Tg" for R/PD in rhTSH group were 58.8% and 100% with 93.9 % accuracy, respectively. An uptake outside thyroid bed at WBS showed a sensitivity of 17.1%, specificity of 100% for R/PD with 89.4% accuracy in THW group. An uptake outside thyroid bed at WBS showed a sensitivity of 7.7%, specificity of 100% for R/PD with 88.8% accuracy in rhTSH group. Method of TSH stimulation did not influence the reliability of DxWBS. The "positive s-Tg level" had a higher sensitivity with THW when compared to rhTSH in detecting R/PD.

Stroke vs. SIADH: A case of symptomatic hyponatremia secondary to SIADH in association with COVID-19 infection

A 66-year-old male with no known past medical history presented with history of two days of throbbing frontal headache associated with nausea, vomiting and left-sided facial droop. The patient also reported dizziness, subjective chills without cough, shortness of breath, or sputum production. Laboratory findings revealed elevated inflammatory markers but normal thyroid, kidney, and liver function tests. Initial sodium level was 126 mEq/L. Urine studies showed elevated urine osmolality of 647 mOsm/Kg and urine sodium level of 175 mEq/L, both suggestive of syndrome of inappropriate secretion of antidiuretic hormone (SIADH). The TSH and ACTH stimulation tests both came back as normal. CT scan of the chest showed no evidence of lung infiltrates. Diffusion weighted MRI of the brain showed no evidence of acute or subacute CVA. SARS-CoV-2 nasal swab test was resulted positive. With these findings we diagnosed our patient with SIADH. Patient was initially started on 3% saline infusion for symptomatic hyponatremia. A fluid restricted diet was enforced. Patient was also started on salt tablets in order to increase solute intake. Sodium slowly corrected throughout the hospitalization. Of note, our patient never became hypoxic, neither did he show evidence of clinical pneumonia. A CT imaging of the chest showed no lung infiltrates, and an MRI of the brain was negative for any acute intracranial abnormalities. This case highlights the role of early recognition of SIADH in the setting of SARS-CoV-2 infection even in the absence of hypoxemia or lung pathology.

No difference in the outcome of metastatic thyroid cancer patients when using recombinant or endogenous TSH.

At present, recombinant TSH cannot be used for the treatment of metastatic differentiated thyroid cancer patients. The aim of this study was to evaluate if the type of TSH stimulation, recombinant or endogenous, had an impact on the outcome of these patients. We compared the outcome of two propensity score-matched groups of metastatic patients, stimulated by either only recombinant TSH (n = 43) or only endogenous TSH (n = 34). As expected from the matching procedure, the clinical-pathological features and the cumulative 131-I activities administered to the two groups were very similar. After 4 years of follow-up, 4% of patients were cured, 3% had biochemical disease and 93% had structural disease. However, 91% of patients obtained a clinical benefit from this therapy in terms of stabilization of the disease or complete remission or partial response. When considering the two groups separately, we did not find any difference in their outcome. When considering the response to 131-I therapy of the single type of metastases, 8% of lymph node metastases and 8% of lung metastases disappeared but none of the bone metastases. The response to 131-I therapy of the single type of metastases was similar when we looked at the two groups separately. This study shows (i) an overall clinical benefit of the 131-I therapy, since the majority of patients remained affected but with a stable disease, and (ii) that the preparation with either recombinant or endogenous TSH has no impact on the 131-I therapy efficacy and the outcome of our two groups of patients.

Quantification of the Thyroid Scan (TS) and correlation to Multiparametric Ultrasounds (MPUS): A textbook case of nuclear molecular imaging

For decades, thyroid scintigraphy (TS) has been considered an interesting tool, especially in the field of hyperthyroidism. In recent years, TS has rapidly gained importance since it provides unique molecular information that cannot be obtained by any other modality. In fact, despite a limited 6mm spatial resolution, it can highlight molecular and histo-functional changes that characterize most thyroid function disorders. However, to become such a powerful molecular image, the TS must be quantified. How much iodine is taken-up characterizes the Uptake (Up), while where iodine distributes characterizes the Spatial Targeting (ST). Methodology, results and limits of the thyroid Uptake are presented, including suppressed tests. Methods to determine the anatomical thyroid volume are revisited with special focus on planar scintigraphy. Recent developments in quantification make the 123I-TS a new molecular imaging procedure. Since 123I targets the sodium iodide symporter (NIS) and tracks the whole organification process, we derived a fundamental linear relationship between the TSH and the precocious (120–240min) Uptake (p123IUp). This relationship indicates whether the 123I input follows the physiological TSH stimulation or is predictive of a non TSH-suppressible function, whatever the imaging pattern. This allows identification of toxic or compensated (TSH>0.1mU/L) Thyroid Functional Autonomy (TFA), even at baseline. Spatial Targeting, measured with the aid of computational algorithms, provides a reproducible Spatial Targeting Index (STI). This allows estimating a functional thyroid volume, that is likely more informative than the anatomical one. Most aspects of TS quantification and the interest to compare the structure (mostly MultiParametric US) and the function (molecular 123I-TS) are presented.

PET/CT in thyroid cancer - the importance of BRAF mutations.

Thyroid cancer (TC) represents less than 1% of all newly diagnosed malignancies. In some selected cases, with a high clinical suspicion for disease but negative I-131 scan, positron emission tomography/computed tomography (PET) with F-18-Fluorodeoxyglucose (FDG) could be helpful in the detection of disease and the definition of its extent. FDG PET/CT, better if performed after TSH stimulation analogously to patient preparation done for radioiodine scintigraphy, could be useful mainly in the detection of metastatic and recurrent disease since the uptake and diagnostic sensitivity of FDG are increased by TSH stimulation. Recently, the role of oncogenic mutations in the tumorigenesis of TCs has become clearer. Among such mutations, BRAFV600E represents the most common genetic alteration. Mutated BRAF may define a more aggressive papillary carcinoma with poorer prognosis and therefore its analysis has been extensively studied as a rule-in test for thyroid carcinoma. In this paper, we try to outline the possible role of FDG PET/CT in the management of patients with TC and positive BRAF mutations and the impact that it could have on their therapeutic algorithm, in terms of thyroidectomy and radioactive iodine (RAI) therapy.

SAT-449 Factors Associated with Reduced Thyroid Hormones in Cushing Syndrome Patients Before and After Surgical Cure

Background: Hypercortisolemia adversely affects thyroid hormone secretion. We previously described the temporal pattern of thyroid function recovery in 23 patients (1). However, the factors leading to suppression and recovery of the hypothalamic-pituitary-thyroid (HPT) axis in Cushing’s syndrome (CS) are not fully understood. We performed two separate studies to investigate these factors. Methods: In study 1, we examined patients (pts, n=62) with CS who underwent curative surgery and recorded their serum morning and evening cortisol, ACTH, tumor volume and duration of symptoms and 24-hour urine free cortisol (UFC) at baseline and the morning serum free T4, TSH and T3 at six-month intervals after cure. Data were log-transformed and Pearson correlations were performed. Linear mixed models were used to study factors that predict recovery of thyroid function. In study 2, we examined the diurnal variation of TSH by performing hourly TSH measurement between 3—7 PM and 12—4 AM on a cohort of pts (n=45) before surgery. Wilcoxon Signed-Rank method was used for comparisons of mean TSH across time and Pearson correlations were performed on log-transformed data. P values <.05 were considered significant. Results: Study 1: In this larger cohort, we confirmed previous findings of suppressed or low normal fT4 and TSH values with active hypercortisolism, with normalization after cure that reflected changes in the T3:TSH, fT4:TSH and T3:fT4 ratios. There were inverse linear correlations between log10 UFC, serum AM and PM cortisol; and log10 TT3, fT4 and TSH before surgery. Independent negative prognosticators of circulating fT4 recovery included UFC greater than 1000mcg/day (nl: 3.5—45mcg/day), duration of symptoms of less than one year, and ACTH levels greater than 60pg/mL(nl: 5—45pg/mL) Study 2: The nocturnal (12 - 4AM) TSH surge was reduced, so that the difference in day and night TSH values was not statistically significant; this contrasts with the 30—50% nocturnal TSH increase above daytime values seen in healthy subjects. There was an inverse relationship between UFC and nocturnal TSH, daytime TSH and TBG values, but there was no direct relationship between UFC and percent changes in nocturnal TSH values. Conclusions: Our findings suggest that a deficit in TSH stimulation of the thyroid gland may explain the reduction in T3 and T4 levels. There is a dose-response relationship between various measures of hypercortisolemia and both thyroid hormones and the pattern of TSH secretion. Finally, the severity of hypercortisolism correlates with a longer time to recovery of the HPT axis in pts with CS after curative surgery. 1. Shekhar S et al. HPG and HPT Axes in Cushing Syndrome. J Endocr Soc, 3 S1, April May 2019

Unexpected radioactive iodine accumulation on whole-body scan after I-131 ablation therapy for differentiated thyroid cancer.

ABSTRACTWe retrospectively evaluated the frequency of unexpected accumulation of radioactive iodine on the post-therapy whole-body scan (Rx-WBS) after radioactive iodine (RAI) ablation therapy in patients with differentiated thyroid cancer (DTC). We searched our institutional database for Rx-WBSs of DTC patients who underwent RAI ablation or adjuvant therapy between 2012 and 2019. Patients with distant metastasis diagnosed by CT or PET/CT before therapy, and those had previously received RAI therapy were excluded. In total, 293 patients (201 female and 92 male, median age 54 years) were selected. Two nuclear medicine physicians interpreted the Rx-WBS images by determining the visual intensity of radioiodine uptake by the thyroid bed, cervical and mediastinal lymph nodes, lungs, and bone. Clinical features of the patients with and without the metastatic accumulation were compared by chi-square test and median test. Logistic regression analyses were performed to compare the association between the presence of metastatic accumulation and these clinical factors. Eighty-four of 293 patients (28.7%) showed metastatic accumulation. Patients with metastatic RAI accumulation showed a significantly higher frequency of pathological N1 (pN1) and serum thyroglobulin (Tg) > 1.5 ng/ml under TSH stimulation (p = 0.035 and p = 0.031, respectively). Logistic regression analysis indicated that a serum Tg > 1.5 ng/ml was significantly correlated with the presence of metastatic accumulation (odds ratio = 1.985; p = 0.033). In conclusion, Patients with Tg > 1.5 ng/ml were more likely to show metastatic accumulation. In addition, the presence of lymph node metastasis at the initial thyroid surgery was also associated with this unexpected metastatic accumulation.

Familial Longevity is Associated with an Attenuated Thyroidal Response to Recombinant Human Thyroid Stimulating Hormone.

ContextLongevity is associated with higher circulating levels of TSH in the absence of differences in circulating thyroid hormones (TH), as previously observed in F2 members of long-lived families (F2-LLS) and their partners (F2-Con). The mechanism underlying this observed difference remains unknown.ObjectiveWe hypothesized that the thyroid gland of members from long-lived families are less responsive to TSH stimulation, thereby requiring higher circulating TSH levels to maintain adequate TH levels.MethodsWe performed a case-control intervention study with a single intramuscular (gluteal) injection with 0.1 mg recombinant human TSH in a subgroup of 14 F2-LLS and 15 similarly aged F2-Con. They were followed for 4 days. No serious adverse events were reported. For analyses, we compared time trajectories of TSH and TH, and the ratio of TH to TSH using area under the curve (AUC) calculations.ResultsThe AUC free T4/AUC TSH ratio was significantly lower in F2-LLS than in F2-Con (estimated mean [95% confidence interval] 1.6 [1.2-1.9] and 2.2 [1.9-2.6], respectively, P = 0.01). The AUC thyroglobulin/AUC TSH ratio was also lower in F2-LLS than in F2-Con (median [interquartile range] 2.1 [1.4-3.6] and 3.2 [2.7-7.4], respectively, P = 0.04). We observed the same trend with the AUC free T3/AUC TSH ratio, although the difference was not statistically significant (estimated mean [95% confidence interval] 0.6 [0.4-0.7] and 0.7 [0.6-0.8], respectively, P = 0.07).ConclusionsThe present findings show that members of long-living families have a lower thyroid responsivity to TSH compared with their partners.

Preclinical studies on the toxicology, pharmacokinetics and safety of K1-70TM a human monoclonal autoantibody to the TSH receptor with TSH antagonist activity

BackgroundThe human monoclonal autoantibody K1-70™ binds to the TSH receptor (TSHR) with high affinity and blocks TSHR cyclic AMP stimulation by TSH and thyroid stimulating autoantibodies.MethodsThe preclinical toxicology assessment following weekly intravenous (IV) or intramuscular (IM) administration of K1-70™ in rats and cynomolgus monkeys for 29 days was carried out. An assessment of delayed onset toxicity and/or reversibility of toxicity was made during a further 4 week treatment free period. The pharmacokinetic parameters of K1-70™ and the effects of different doses of K1-70™ on serum thyroid hormone levels in the study animals were determined in rats and primates after IV and IM administration.ResultsLow serum levels of T3 and T4 associated with markedly elevated levels of TSH were observed in the study animals following IV and IM administration of K1-70™. The toxicological findings were attributed to the pharmacology of K1-70™ and were consistent with the hypothyroid state. The no observable adverse effect level (NOAEL) could not be established in the rat study while in the primate study it was 100 mg/kg/dose for both males and females.ConclusionsThe toxicology, pharmacodynamic and pharmacokinetic data in this preclinical study were helpful in designing the first in human study with K1-70™ administered to subjects with Graves’ disease.

Diagnostic performance of 18F-FDG-PET/CT in DTC patients with thyroglobulin elevation and negative iodine scintigraphy: a meta-analysis.

To evaluate the accuracy of 18F-FDG-PET/CT for the detection of recurrent and/or metastatic diseases in differentiated thyroid cancer (DTC) patients with thyroglobulin elevation and negative iodine scintigraphy. Whether PET/CT with TSH stimulation (sPET/CT) had better diagnostic performance than PET/CT without TSH stimulation (nsPET/CT) in this scenario was also evaluated. PubMed and Embase databases were searched for eligible studies from January 2001 to December 2018. Only studies with clearly stated reference standard (histopathology confirmation and/or clinical/imaging follow-up) were included. Publication bias was assessed by Deeks funnel plot. The pooled sensitivity, specificity, diagnostic odds ratio (DOR) and the area under the summary receiver-operating characteristics curve (AUC) for PET/CT was determined by random-effect analysis, respectively. sPET/CT and nsPET/CT were compared pairwise for all diagnostic estimate indexes using Z-test. We included 17 studies with 1195 patients in this meta-analysis. The pooled sensitivity, specificity, DOR and AUC for PET/CT on patient-based data were 0.86 (95% CI: 0.79-0.91), 0.84 (95% CI: 0.72-0.91), 31.00 (95% CI: 12.00-80.00) and 0.91 (95% CI: 0.88-0.93), respectively. There was high heterogeneity (I 2 = 80% for sensitivity, I 2 = 82% for specificity) and possible publication bias (P = 0.01). Z test did not detect statistically significant difference between sPET/CT and nsPET/CT for all the diagnostic estimate indexes (all P > 0.05). On patient-based analysis, 18F-FDG-PET/CT has high diagnostic accuracy for the detection of recurrent and/or metastatic diseases in DTC patients with thyroglobulin elevation and negative iodine scintigraphy, but existing studies were limited by high heterogeneity and possible publication bias. The diagnostic performance of sPET/CT may be not superior to nsPET/CT.

Profiling circulating microRNAs as serum biomarkers for thyroid cancer recurrence before and after recombinant human TSH stimulation

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

SAT-549 Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Primary Human Thyrocytes

Thyroid gland, as one crucial endocrine organ to synthesize and secrete thyroid hormones directly response to TSH, is indispensable to human health. The secretion of exosomes by FRTL-5 normal thyroid cell line and TPC-1 papillary thyroid cancer cell line has been verified. However, the verification and profiling of exosomes secreted by human thyroid and a possible role for exosomes in thyroid function has not been explored. To investigate whether human thyroid could secrete exosomes, normal thyroid tissue from the surgical thyroidectomy of benign follicular nodules were collected and digested. Primary thyrocytes were cultured with or without TSH, and the medium was collected. Exosomes were isolated via differential ultracentrifugation, observed under transmission electron microscopy, measured by nanoparticle tracking analysis, and confirmed by immunoblot. Further, label-free liquid chromatography (LC)-mass spectrometry (MS)/MS analysis was conducted to investigate the proteomic profiling of thyrocytes exosomes. In the present study, secreted exosomes were verified in primary human thyrocytes. Exosomes exhibited the same shape and size (102.80±36.01 vs. 95.82 ±30.10 nm, p>0.05) as control exosomes after TSH stimulation, but importantly, the total number of exosomes increased significantly after TSH stimulation for 48 h (3.46±0.56×107 vs. 1.98 ±0.20 ×107 nm, p<0.05). The mRNA level of Rab27b, the protein crucial for late endosomal formation, was also increased significantly after TSH stimulation, indicating that TSH activated the biogenesis of exosomes. Overall, 202 unique proteins were identified in thyrocytes exosomes. These included multiple proteins known to be involved in the endosomal pathway, such as annexins, and several cytoskeletal, cytoskeletal binding-proteins and motor proteins. One significant identified protein was thyroglobulin. Among the identified proteins, 77 proteins exhibited a differential expression pattern in TSH exosomes compared with the control exosomes, which mainly participate in cellular process, metabolic process, biological regulation, response to stimulus and regulation of biological process. This study provides direct evidence that human thyroid secretes exosomes. The characterization and proteomic profiling of exosome secreted by primary human thyrocytes provides potential physiological significance of exosomes in thyroid function.

SUN-576 Abrupt Switching From Hypertyroidism To Hypothyroidism With Pretibial Myxedema And Acropachy, In Graves’ Ophthalmopathy: Case Report.

Background Graves’ disease (GD) is an autoimmune thyroid disease usually associated with hyperthyroidism. There have been cases of patients switching from hyperthyroidism to hypothyroidism, and even rarer patients flipping from hypothyroidism to hyperthyroidism. However, a case of spontaneously alternating hyperthyroidism and hypothyroidism in GD is comparably an even rarer phenomenon. It is thought that the switching of stimulating TSH receptor antibodies (TSAb) and blocking TSH receptor antibodies (TBAb) has a role in alternating thyroid function. We present a case of spontaneously oscillating thyroid function, pretibial myxedema and achropachy in three months. CASE REPORT 50 y,m,active smoker(90 yr old), weight loss (12 kg),tremors, irritability,proptosis,bad sensation eyes and acropachy(FigE) hyperthyroidism and ophthalmopathy secondary GD,underwent irregular treatment with methimazole 1st y. After1y, Graves' ophthalmopathy was worsened CAS 9(FigAB),with stereotactic radiotherapy, 10sec, 20Gy,pulse methylprednisolone followed by oral prednisone and decompressive surgery, initially used methimazole 30mg/d with reduction to 10mg/d.After 3 months of corticotherapy the patient returns with complaints of intestinal constipation, asthenia, brittle nails, the appearance of pre-tibial myxedema (FigC), being confirmed hypothyroidism frankly associated with the increase of thyroid autoantibodies (tb1), thyroid USG showing parenchyma with diffuse and grossly heterogeneous ecotexture and reduced echogenicity, diffusely increased vascularization, total thyroid volume of 33.4 ml (FigD). Patient maintained ambulatory follow-up, maintaining hypothyroidism on remission with levothyroxine 6 months ago. DISCUSSION This's the first case presenting hyperthyroidism shift to abrupt hypothyroidism with myxedema and acropachy in patient with GO. GD spontaneous change from hyperthyroidism to hypothyroidism (without ablative radioactive iodine intervention or treatment with antithyroid drugs) can occur in two ways: by the unexpected development of TBI (TSH receptor blocking immunoglobulin); or by chronic lymphocytic thyroiditis surpassing the stimulatory effects of TSI (Immune Globulin stimulating TSH receptors). The shift from TSI to TBI, with changes in immunoglobulin concentration, affinity and potency, leading to TBI dominance, is usually associated with the use of DAT (antithyroid drugs) and immunosuppressive states. We describe a case of hyperthyroidism due to GD evolving with frank hypothyroidism probably related to the alternation of TSI to TBI, with a predominance of the TBI effect, with onset after immunosuppressive therapy with corticosteroids. CONCLUSIONThe occurrence of a shift of predominance of the stimulatory effect to the inhibitor on TSH receptors emphasizes the need for careful monitoring of patients with Graves' disease.

Surveillance of patients with differentiated thyroid cancer and indeterminate response: a longitudinal study on basal thyroglobulin trend.

This is a longitudinal study of retrospective data aimed at verifying whether repeated measurements of serum non-stimulated thyroglobulin (Tg) allow the prediction of persistent disease in patients with differentiated thyroid cancer (DTC) and indeterminate response. We examined 145 DTC patients with indeterminate response to therapy followed up for a median time of 68months. Tg measurements and neck ultrasound (US) were performed every 6-12months. The changes over time of repeated measurements of basal Tg were analyzed through the multilevel linear regression. Seventy (48.3%) out of 145 patients spontaneously achieved an excellent response, while persistent indeterminate response was observed in 62 (42.7%) patients. The remaining 13 (9.0%) patients had progression: 3/13 with biochemical disease and 10/13 with structural disease. Tg steadily increased in patients with progressive disease (mean percentage change + 27.1% at each follow-up visit), while Tg decreased in patients without any evidence of progression (mean percentage change - 8.8%). This different trend between the two groups was not related to either different values of median TSH at baseline (0.32 vs 0.28mIU/l, respectively) or to different trend of TSH during follow-up (p = 0.76). Basal Tg values did not increase in three out of ten patients with structural disease that was identified by neck US. The importance of the study is that, in DTC patients with indeterminate response, rising values of unstimulated Tg, independently from the basal levels, may be useful to identify patients with progressive disease. These results are also useful to avoid unnecessary TSH stimulation.

Different Thyroidal Responses to Human Chorionic Gonadotropin Under Different Thyroid Peroxidase Antibody and/or Thyroglobulin Antibody Positivity Conditions During the First Half of Pregnancy.

Thyroid peroxidase antibody (TPOAb) positivity can attenuate gestational thyroid responses to human chorionic gonadotropin (hCG) during pregnancy, whereas the effects of thyroglobulin antibodies (TgAb) remain unknown. The aim of our study was to explore the thyroid response to hCG in women with thyrotropin (TSH) levels within the method-specific reference range under different conditions of thyroid autoimmunity. The study screened 822 women at 7-20 weeks of gestation using the pregnancy-specific reference range for TSH. Serum TSH, free thyroxine (fT4), TPOAb, TgAb, and β-hCG levels were measured using electrochemiluminescence immunoassays. The enrolled pregnant women were subdivided into four subgroups based on TPOAb/TgAb positivity: co-positive for TPOAb and TgAb (group 1), isolated TPOAb positive (group 2), isolated TgAb positive (group 3), and co-negative for TPOAb and TgAb (group 4). TSH was negatively associated with hCG in all four groups (p < 0.05). fT4 was positively associated with hCG in groups 3 and 4 (p < 0.01) but not in groups 1 (p = 0.096) and 2 (p = 0.758). Group 2 was further stratified into tertiles according to TPOAb concentrations. No negative TSH/hCG association was observed in the middle- and upper-tertile groups when TPOAb were ≥53 IU/mL (p > 0.05). There was no positive fT4/hCG association in any of the three subgroups (p > 0.05). Similarly, group 3 was further stratified into tertiles according to TgAb levels. TSH was negatively associated with hCG in the lower and middle tertiles (p < 0.01), but the association was not found in the upper tertile when TgAb was ≥356 IU/mL (p = 0.191). fT4 was positively associated with hCG in the lower tertile (p = 0.027) but not in subgroups when TgAb was ≥219 IU/mL (p > 0.05). When TSH was within the pregnancy-specific reference range, high concentrations of TPOAb and TgAb attenuated the fT4 stimulation and suppression of TSH by hCG. The results imply that TgAb, in addition to TPOAb, could also interfere with thyroidal responses to hCG during the first half of pregnancy.

Defective Regulatory B Cells Are Associated With Thyroid-Associated Ophthalmopathy.

To investigate the change in IL-10-producing regulatory B cells (Breg), which suppress peripheral immune responses, in patients with thyroid-associated ophthalmopathy (TAO). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls (n = 54), patients with Graves disease (n = 26), and patients with TAO (N=125), and stimulated with CpG/CD40L. The frequency of IL-10-producing Bregs and the expression of IL-10 in response to TSH stimulation were measured by flow cytometry. CD4+ T cells were cultured with Breg-depleted PBMCs to elucidate the function of Bregs in patients with TAO. The potential immunoregulatory mechanism was also investigated by Western blot and chromatin immunoprecipitation assays. Patients with active TAO had higher baseline levels of Bregs in their peripheral blood than both healthy controls and inactive patients. TSH promoted Bregs. Bregs from patients with TAO were defective in suppressing the activation of interferon (IFN)-γ+ and IL-17+ T cells in vitro. Regulatory B cells in patients with TAO are functionally defective, suggesting that the defective Bregs might be responsible for the pathogenesis of TAO.