Rapamycin improves Graves' orbitopathy by suppressing CD4+ cytotoxic T lymphocytes.

CD4+ cytotoxic T lymphocytes (CTLs) were recently implicated in immune-mediated inflammation and fibrosis progression of Graves' orbitopathy (GO). However, little is known about therapeutic targeting of CD4+ CTLs. Herein, we studied the effect of rapamycin, an approved mTOR complex 1 (mTORC1) inhibitor, in a GO mouse model, in vitro, and in patients with refractory GO. In the adenovirus-induced model, rapamycin significantly decreased the incidence of GO. This was accompanied by the reduction of both CD4+ CTLs and the reduction of orbital inflammation, adipogenesis, and fibrosis. CD4+ CTLs from patients with active GO showed upregulation of the mTOR pathway, while rapamycin decreased their proportions and cytotoxic function. Low-dose rapamycin treatment substantially improved diplopia and the clinical activity score in steroid-refractory patients with GO. Single-cell RNA-Seq revealed that eye motility improvement was closely related to suppression of inflammation and chemotaxis in CD4+ CTLs. In conclusion, rapamycin is a promising treatment for CD4+ CTL-mediated inflammation and fibrosis in GO.

Translocated HMGB3 is involved in papillary thyroid cancer progression by activating cytoplasmic TLR3 and transmembrane TREM1.

The family of high mobility group box (HMGB) proteins participates in various biological processes including immunity, inflammation, as well as cancer formation and progression. However, its role in thyroid cancer remains to be clarified. We performed quantitative RT-PCR (qRT-PCR), western blot, enzyme-linked immunosorbent, immunohistochemistry, and immunofluorescence assays to evaluate the expression level and subcellular location of HMGB3. The effects of HMGB3 knockdown on malignant biological behaviors of thyroid cancer were determined by cell proliferation assays, cell cycle and apoptosis assays, and transwell chamber migration and invasion assays. Differential expression genes (DEGs) altered by HMGB3 were analyzed using the Ingenuity Pathway Analysis (IPA) and TRRUST v2 database. HMGB3 correlated pathways predicted by bioinformatic analysis were then confirmed using western blot, co-immunoprecipitation, dual-luciferase reporter assay, and flow cytometry. We found that HMGB3 is overexpressed and its downregulation inhibits cell viability, promotes cell apoptosis and cell cycle arrest, and suppresses cell migration and invasion in thyroid cancer. In PTC, both tissue and serum levels of HMGB3 are elevated and are correlated with lymph node metastasis and advanced tumor stage. Mechanistically, we observed the translocation of HMGB3 in PTC, induced at least partially by hypoxia. Cytoplasmic HMGB3 activates nucleic-acid-mediated TLR3/NF-κB signaling and extracellular HMGB3 interacts with the transmembrane TREM1 receptor in PTC. This study demonstrates the oncogenic role of HMGB3 cytoplasmic and extracellular translocation in papillary thyroid cancers; we recommend its future use as a potential circulating biomarker and therapeutic target for PTC.

REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway.

Persistent acinar to ductal metaplasia (ADM) is a recently recognized precursor of pancreatic ductal adenocarcinoma (PDAC). Here we show that the ADM area of human pancreas tissue adjacent to PDAC expresses significantly higher levels of regenerating protein 3A (REG3A). Exogenous REG3A and its mouse homolog REG3B induce ADM in the 3D culture of primary human and murine acinar cells, respectively. Both Reg3b transgenic mice and REG3B-treated mice with caerulein-induced pancreatitis develop and sustain ADM. Two out of five Reg3b transgenic mice with caerulein-induced pancreatitis show progression from ADM to pancreatic intraepithelial neoplasia (PanIN). Both in vitro and in vivo ADM models demonstrate activation of the RAS-RAF-MEK-ERK signaling pathway. Exostosin-like glycosyltransferase 3 (EXTL3) functions as the receptor for REG3B and mediates the activation of downstream signaling proteins. Our data indicates that REG3A/REG3B promotes persistent ADM through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway. Targeting REG3A/REG3B, its receptor EXTL3, or other downstream molecules could interrupt the ADM process and prevent early PDAC carcinogenesis.

Dickkopf (Dkk)-2 is a beige fat-enriched adipokine to regulate adipogenesis.

In the past decades, remarkable efforts have been made to unravel the regulation of adipose tissue metabolism, given the increasing prevalence of obesity and its huge impact on human health. Wnt signaling pathway is closely involved in this entity. As extracellular inhibitors to Wnt signaling, secreted protein Dickkopfs (Dkks) may be potential targets to combat obesity and related metabolic disorders. In this study, we showed that Dkk2 was a beige fat-enriched adipokine to regulate adipogenesis. Dkk2 was strikingly expressed in beige fat depot compared to classic white, brown, and subcutaneous fat. Dkk2 treatment inhibited adipogenesis in 3T3-L1 pre-adipocytes, C3H10T1/2 mesenchymal stem cells, and primary bone marrow mesenchymal stromal cells. Activation of the master adipogenic factor PPARγ by the synthetic Thiazolidinedione ligand rosiglitazone largely rescued the inhibition of adipogenesis by Dkk2. Furthermore, adenoviral overexpression of Dkk2 in the liver to mimic its gain-of-function showed minimal effect on whole-body metabolism. These results collectively suggest that Dkk2 is a first-in-class beige fat adipokine and functions mainly through a paracrine manner to inhibit adipogenesis rather than as an endocrine factor. Our findings aid a better understanding of beige fat function and regulation and further, provide a potential therapeutic target for treating obesity.

A Promising Mouse Model of Graves' Orbitopathy Induced by Adenovirus Expressing Thyrotropin Receptor A Subunit.

Background: Graves' orbitopathy (GO) is the most common and serious manifestation of Graves' disease (GD). It is characterized by orbital inflammation and tissue remodeling. Although several GO models have been reported, most lack a full assessment or mechanistic evaluation. Here, we established a promising mouse model mimicking many aspects of human GO with a frequency of 70% and characterized the key role of T cells in the progression of GO. Methods: An adenovirus expressing the human thyrotropin (TSH) receptor A subunit (Ad-TSHRA) was injected in the muscles of female BALB/C mice nine times to induce GO. At predetermined time points, histological examinations of retrobulbar tissues and thyroid glands were performed to dynamically monitor changes; serum autoantibodies and total thyroxine levels were examined to evaluate thyroid function. Flow cytometry of CD4+ T cell subgroups and RNA sequencing (RNA-Seq) of splenocytes were also performed to explore the underlying mechanism. Results: After nine injections, 7 of 10 mice challenged with Ad-TSHRA developed the orbital changes associated with GO. Seven mice manifested retrobulbar fibrosis, and four mice showed adipogenesis. Exophthalmia, conjunctival redness, and orbital lymphocyte infiltration were also observed in a subset of mice. The orbitopathy was first detected after seven injections and followed the hyperplastic change observed in thyroids after four injections. Flow cytometry revealed increased proportions of Th1 cells and decreased proportions of Th2 cells and regulatory T (Treg) cells in the splenocytes of GO mice. This change in CD4+ T cell subgroups was confirmed by orbital immunohistochemical staining. Genes involved in T cell receptor signaling, proliferation, adhesion, inflammation, and cytotoxicity were upregulated in GO mice according to the RNA-Seq; a trend of upregulation of these GO-specific genes was observed in mice with hyperthyroidism without orbitopathy after four injections. Conclusions: A GO mouse model was successfully established by administering nine injections of Ad-TSHRA. The model was achieved with a frequency of 70% and revealed the importance of T cell immunity. A potential time window from Graves' hyperthyroidism to GO was presented for the first time. Therefore, this model could be used to study the pathogenesis and novel treatments for GO.

Effect of Dihydrotestosterone on CostimulatoryMolecules in a Mouse Model of Graves' Disease.

Objective Graves' disease is the most common autoimmune thyroid disease and its prevalence and clinical manifestations are disparate between females and males. Costimulatory molecules play an essential role in regulating autoimmune responses. The objective of this study was to determine if expression of inhibitory molecules was correlated with treatment by dihydrotestosterone (DHT) in an in vivo BALB/c mouse model of experimental autoimmune Graves' disease.Methods Female BALB/c mice were immunized three times with thyroid stimulating hormone receptor A-subunit encoded by adenovirus to establish a Graves' disease model. Three different doses of DHT or a matching placebo were administered by implantation of slow-release pellets a week before the first immunization. Four weeks after the third immunization, the mice were euthanatized, and then the spleen and thymus were removed. Total thyroxine and free thyroxine levels in serum of mice were detected using a radioimmunoassay kit. Real-time polymerase chain reaction was performed to estimate the expression of costimulatory molecules in lymphocytes from the spleen and thymus. Flow cytometry was used to analyze the percentage of CD4+ T cells in splenic lymphocytes. Quantitative data were compared with unpaired t-tests. Correlation between two variables was analyzed using Analysis of Variance.Results Treatment with DHT can dramatically reduce total thyroxine and free thyroxine levels. Higher expression of programmed death-1 was found in the spleen of Graves' disease mice receiving 5 mg of DHT treatment (0.635±0.296 vs. 0.327±0.212; t=2.714, P=0.014), similarly, T-cell immunoglobulin domain and mucin domain 3 (TIM-3) in both the spleen (1.004±0.338 vs. 0.646±0.314; t=2.205, P=0.022) and the thymus (0.263±0.127 vs. 0.120±0.076; t=3.221, P=0.004) also increased after 5 mg of DHT treatment compared with the parallel placebo model mice. Moreover, the percentage of CD4+ T cells declined in the splenic lymphocytes of Graves' disease mice treated with 5 mg of DHT (19.90%±3.985% vs. 24.05%±2.587%; t=2.804, P=0.012). A significant negative association was observed between expression of TIM-3 in the spleen and serum levels of total thyroxine (r=-0.7106, P=0.014) as well as free thyroxine (r=-0.6542, P=0.029).Conclusion This study demonstrates that DHT can ameliorate experimental autoimmune Graves' disease, which may occur by up-regulating expression of programmed death-1 and TIM-3 and inhibiting development of CD4+ T cells.

[Epithelial-mesenchymal transition induces cancer stem cell generation in human thyroid cancer cells in vitro].

OBJECTIVE: Epithelial-mesenchymal transition (EMT) has been implicated in the initiation and conversion of early stage tumors into invasive malignancies and is associated with the "stemness" of cancer cells. The present study was designed to identify whether EMT induces cancer stem cell generation and tumor progression in human thyroid cancer cells in vitro. METHODS: FTC133 cells, as EMT-negative cells, were used for EMT induction by hypoxia-inducible factor-1α (HIF-1α) transfection. And EMT features were then examined by Western blot, immunofluorescent staining, invasion and proliferation assays. Moreover, stem-like side population (SP) cells were sorted with flow cytometry from FTC133 cells before and after EMT. The proportion of SP was compared and stemness, self-renewal and tumorigenicity in vitro were identified in SP cells. RESULTS: Overexpression of HIF-1α induced FTC133 cells to undergo EMT. And it down-regulated epithelial marker E-cadherin, up-regulated mesenchymal marker vimentin and caused nucleus translocation of ß-catenin and highly invasive and metastatic properties. Most importantly, the induction of EMT promoted proportion of stem-like side population cells (0.70% vs 0.03%, P < 0.05) with higher sphere formation and clone forming capability in contrast to non-side population cells. CONCLUSIONS: EMT can induce cancer stem cell generation and tumor progression in thyroid cancers. Further understanding the role of EMT and cancer stem cells in cancer progression may reveal new preventive and therapeutic targets for thyroid cancers.

Epithelial-mesenchymal transition triggers cancer stem cell generation in human thyroid cancer cells.

Increasing evidence has shown that cancer stem cells or tumor initiating cells are the 'root cause' of malignant cancers. However, the exact origin of cancer stem cells still remains obscure in thyroid research. EMT has been implicated in the initiation and conversion of early-stage tumors into invasive malignancies and is associated with the stemness of cancer cells. Based on these facts, a new hypothesis was suggested that EMT induces cancer stem cell generation and tumor progression in human thyroid cancer cells in vitro. In the present study, FTC133 cells identified as EMT-negative cells were used for EMT induction by HIF­1α transfection. Overexpression of HIF-1α induced FTC133 cells to undergo EMT, downregulated the epithelial markers E-cadherin, upregulated the mesenchymal marker vimentin, and associated with highly invasive and metastatic properties. Most importantly, the induction of EMT promoted the stem-like side population cell proportion in the FTC133 cells. These results indicate that EMT induction promotes CSC traits and cell proportions in the thyroid cancer cells, which implies that EMT could induce cancer stem cell generation and tumor progression in thyroid cancers. Further understanding of the role of EMT and cancer stem cells in cancer progression may reveal new targets for the prevention or therapy of thyroid cancers.

Expression and significance of IGF-1 and IGF-1R in thyroid nodules.

There are limitations in the diagnosis of thyroid nodules, especially follicular lesions, and their pathogenesis remains unclear. Insulin-like growth factor-1 (IGF-1) has been implicated in tumor cell apoptosis, transformation, invasion, and metastasis; however, its role in thyroid nodules is undetermined. The aim of this study is to investigate the relationship between expression of IGF-1 and thyroid nodule, and evaluate the role of IGF-1 in differential diagnosis and pathogenetic function of benign and malignant thyroid nodules. Sixty-two paraffin-embedded thyroid tissues from patients with thyroid nodules were selected, including 18 follicular adenomas (FA), 17 nodular goiters, 13 papillary thyroid carcinomas (PTC), 2 follicular thyroid carcinomas, and 12 normal controls. IGF-1 and IGF-1R protein and mRNA expression was detected by immunohistochemistry (IHC) and real-time quantitative PCR (qRT-PCR), respectively. Grouping comparisons were employed among the above groups based on the clinical and pathological subtypes. IGF-1 and IGF-1R expression was significantly higher in FA, nodular goiters, and PTC than that in the controls (all P < 0.01). Similarly, IGF-1 mRNA expression was also significantly higher in FA (P < 0.05), nodular goiters (P < 0.01), and PTC (P < 0.01) compared with the controls. IGF-1R mRNA expression was also significantly higher in FA, nodular goiters, and PTC (all P < 0.01) compared with the controls. Compared with FA and nodular goiters, PTC showed significantly higher IGF-1 and IGF-1R protein (P < 0.01, P < 0.05, respectively) and mRNA expression (P < 0.01, P < 0.05, respectively). IGF-1 probably plays an important role in the genesis and development of certain solid cold thyroid nodules, including PTC, nodular goiters, and FA. Detection of IGF-1 and IGF-1R expression in thyroid tissues by IHC or qRT-PCR is hard to distinguish malignant from benign lesions.

Prognostic factors for differentiated thyroid carcinoma and review of the literature.

OBJECTIVE: Prognostic factors of differentiated thyroid carcinoma (DTC) are analyzed to justify the diagnostic and therapeutic modalities of DTC in current practice. METHODS: Patients undergoing curative resection for histologically diagnosed DTC (n = 150) were consecutively enrolled, and the clinical and pathological data were retrospectively reviewed. RESULTS: The DTC patient cohort consisted of 113 females (75.3%; mean age at the time of onset, 40.1 ± 12.0 yr) and 37 males (24.7%; 47.5 ± 16.2 yr). The pathological types of DTC included papillary thyroid carcinoma (n = 131, 87.3%) and follicular thyroid carcinoma (n = 19, 12.7%). The follow-up period ranged from 4.2 to 31 years, in which period 140 (93.3%) patients survived, 30 (20.0%) patients relapsed, and 10 (6.7%) patients died of DTC. Surgical procedures used for the curative resection consisted of near-total or subtotal thyroidectomy (n = 83, 55.3%), partial thyroidectomy (n = 64, 42.7%) and total thyroidectomy (n = 3, 2.0%). Out of those patients undergoing concomitant lymph node dissection (n = 63, 42.0%), 45 patients (71.4%) had detectable lymph node metastases. Postoperatively, 12 patients (8.0%) received external beam radiotherapy, 16 patients (10.7%) received chemotherapy, 37 patients (24.7%) received 131I therapy, and 66 patients (44.0%) received additional long-term L-T4 or thyroid hormone treatment. Age of onset, tumor size at initial visit, and rate of early metastasis showed statistically significant differences between the mortality group and the survival group (P <0.05) and between the recurrence group and the recurrence-free group (P <0.05). CONCLUSIONS: Age, tumor size at initial visit, and early metastasis are prognostic factors for DTC, requiring a stratified management in clinical practice.

[An exploration of induction methodology and experimental duration of Graves disease animal model].

OBJECTIVE: To compare the efficacy of Graves disease animal models induced by thyroid stimulating hormone receptor (TSHR) plasmid DNA (pcDNA3.1-TSHR) and by TSHR A subunit recombinant adenovirus (Ad-TSHR289), and to investigate the influence of duration for preparing animal model induced by Ad-TSHR289 on Graves hyperthyroidism and its related indices. METHODS: The plasmid group and the adenovirus group were set up respectively. The plasmid group: 21 female BALB/c mice were randomly divided into model group (n = 12) and control group (n = 9). The model group were injected intradermally with pcDNA3.1-TSHR 50 µg, once every 3 weeks, totally 3 times. Then 4 weeks after the last immunization, the mice were euthanized to obtain blood for testing TSHR antibody (TRAb), total T(4), and thyroid tissue for histological examination. The controls were injected with the same dose of pcDNA3.1 in the same way. The adenovirus group: 52 female BALB/c mice were divided into 10-week model group (n = 8), 14-week model group (n = 10) and 18-week model group (n = 8), and the respective controls (n = 8, n = 10, n = 8) were set up. All model groups were injected intramuscularly with Ad-TSHR289, three times at three weekly intervals. Then the mice were euthanized at 4, 8 and 12 weeks to test TRAb, total T(4) level and to observe the change of thyroid histology. The controls were treated with the same dose of Ad-lacz in the same way. Another 8 mice were scheduled to test the dynamic variation of TRAb before and after the 3 times immunization. RESULTS: In the plasmid model group, only two of 12 mice developed weak antibody responses against TSHR, and no elevated total T(4) level and no hyperplasia changes of thyroid were observed. In the 10-week model group, all mice had high level TRAb [(807.65 ± 136.33) U/L], Six-eighths mice had hyperthyroidism exhibited hyperplasia changes. In the 14-week model group, the TRAb level [(650.12 ± 192.88) U/L] and the incidence of hyperthyroidism (3/10) were lower than those in 10-week group. Histologically, the degree of thyroid hyperplasia lightened to a small extent, but its positive rate did not decline. In the 18-week model group, only 2 of 8 mice displayed slightly elevated TRAb level, and no mice showed increased total T(4) level. Additionally, thyroid tissues of 2 mice were mildly abnormal. Compared with the model groups at different time, the change of antibody levels of the mice for TRAb dynamic observation exhibited the similar trend. CONCLUSIONS: Being good at repeatability and high incidence of hyperthyroidism, the animal model of Graves disease induced by Ad-TSHR289 is still an ideal research tool presently. The duration of model can be maintained 18 weeks, and 10 weeks is the best period to sustain characteristic of Graves disease.

[Beneficial effects of retinoic acid on in vitro invasiveness of human thyroid carcinoma cell lines].

OBJECTIVE: To investigate the anti metastatic potential of retinoic acid as an important determinant of metastatic behavior in thyroid carcinoma and understand the role of invasion associated proteins. METHODS: Differentiated thyroid carcinoma cell lines FTC-133 and XTC.UC1, anaplastic thyroid cancer cell lines C643 and HTH74 were studied. All cell lines were cultured with all-trans-RA (ATRA) or solvent ethanol. The in vitro invasion and adhesion potency were studied by transwell experiment and short-term adhesion assay. The functions of invasion associated proteins, urokinase type plasminogen activator (uPA), uPA receptor (uPAR), MMP2 and E-cadherin were investigated by semi-quantitative RT-PCR and Western blot. RESULTS: In vitro invasion assay revealed that ATRA treatment could reduce the invasive potency in all the thyroid cancer cell lines. On Day 5 of ATRA treatment, the numbers of cells that migrated through extracellular matrix were as follows, in contrast to control group, FTC-133: 91±9 vs 118±10, C643: 92±17 vs 164±21, HTH74: 87±18 vs 169±15, and XTC.UC1: 95±23 vs 136±15, respectively (all P<0.05). Short term adhesion assay suggested that ATRA increases cancer cell adhesion to extracellular matrix (ECM) in C643, HTH74 and XTC.UC1. RT-PCR and Western blot both revealed diminished expression of uPAR in all four carcinoma cell lines. In C643 and HTH74 cell lines, the expression of uPA was reduced and the expression of E-Cadherin was increased; whereas the MMP2 expression was not significantly down-regulated in ATRA treated group. In ATRA treated FTC-133 and XTC.UC1 cell lines, MMP2 expression was decreased, but no significant changes in uPA and E-Cadherin expression were observed. CONCLUSION: The present study demonstrates the influence of ATRA on two important determinants of metastatic behavior ("de adhesion" and proteolysis) in thyroid carcinoma cell lines.

[Effect of palmitate on proliferation and apoptosis of INS-1 pancreatic beta-cell].

AIM: To investigate the effect of saturated fatty acid palmitate (PA) on cell viability of MIN6 beta-cell and the possible cell cycle pathways affected by PA. METHODS: MIN6 cells were synchronized at G(0); phase by serum deprivation for 36 h, and further MIN6 cells were exposed to different concentrations of PA(0.25-1.0 mmol/L, 45 min-24 h)compared with control cell treated with BSA.Cell viability was assessed by MTT colorimetric assay. The cell cycle was measured by FACS analysis, and cell cycle proteins were further detected using Western blot. RESULTS: (1)PA significantly affected the cell viability of MIN6 cells. (2)The G(0);/G(1); cell cycle arrest (n=6, P<0.05) also induced by PA, whereas MIN6 cells in S and G(2);/M phase were decreased. (3) For cell cycle proteins, PA treatment caused significant reductions in cyclin D1 and CDK4 levels, which was consistent to the cell cycle delay. CONCLUSION: The findings thus suggest that increased PA directly affects pancreatic beta-cell proliferation, possibly by reducing the levels of cyclin D1/CDK4 in the cells, which results in arresting in progression through the G(1); phase to the S phase of the cell cycle.

[Surgical treatment of hyperthyroidism].

OBJECTIVE: In order to explore the management of peri-operation and the therapeutic effect in the surgical treatment of hyperthyroidism. METHODS: Fifty five cases of hyperthyroidism were undergone near-total thyroidectomy, during the operation recurrent laryngeal nerve was exposed, and the parathyroid was found with microscope when necessary. The third rank of inferior thyroid arteries were ligated to guarantee the blood supply for parathyroid. RESULTS: All cases underwent near-total thyroidectomy. There was no mortality, and no permanent recurrent laryngeal nerve palsy occurred, and no permanent hypoparathyroidism, and no recurrent hyperthyroidism. Follow-up was carried out 16 months to approximately 5 years after near-total thyroidectomy patients, Hypothyroidism occurred in 15 cases (57.7%), serum calcium levels were 2.15-2.45 mmol/L. CONCLUSIONS: Special attention should be given to the management of peri-operation, the above the method can prevent operative complication in the surgical treatment of hyperthyroidism, with excellent result.