Adult mouse and human organoids derived from thyroid follicular cells and modeling of Graves’ hyperthyroidism

The thyroid maintains systemic homeostasis by regulating serum thyroid hormone concentrations. Here we report the establishment of three-dimensional (3D) organoids from adult thyroid tissue representing murine and human thyroid follicular cells (TFCs). The TFC organoids (TFCOs) harbor the complete machinery of hormone production as visualized by the presence of colloid in the lumen and by the presence of essential transporters and enzymes in the polarized epithelial cells that surround a central lumen. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at comparable levels to tissue. Single-cell RNA sequencing and transmission electron microscopy confirm that TFCOs phenocopy primary thyroid tissue. Thyroid hormones are readily detectable in conditioned medium of human TFCOs. We show clinically relevant responses (increased proliferation and hormone secretion) of human TFCOs toward a panel of Graves’ disease patient sera, demonstrating that organoids can model human autoimmune disease.

Squamous Differentiation in the Thyroid: Metaplasia, Neoplasia, or Bystander?

Background. Squamous differentiation within the thyroid is seen in a variety of settings. Squamous epithelium is non-native to the thyroid, and its debated origins span reactive metaplasia and developmental/embryologic remnants. Despite a lack of clarity as to its evolution, squamous epithelium may be associated with both neoplastic and non-neoplastic processes. Methods. Thyroid pathology reports spanning a 30-year period were reviewed for terms indicating squamous features. Associated diagnostic and clinical information was collated. Results. Four hundred and twenty seven of 17,452 (2.4%) thyroid surgical pathology cases during this period utilized terminology indicating squamous differentiation including 243 malignant (58%) and 178 benign (42%) diagnoses. There were 111 (26%) primary thyroid malignancies with squamous differentiation, 116 (28%) malignancies of non-thyroid origin including local extension from nearby cancers, and 16 (4%) malignancies of uncertain primary. Most benign lesions were non-neoplastic (84%). The minor subset representing benign neoplasia was interpreted as secondary reactive changes. Conclusion. While squamous differentiation is seen routinely in the thyroid, it is most commonly reported in malignancy. For primary thyroid malignancies reported to demonstrate a squamous component, biologically aggressive tumors were overrepresented. Available evidence suggests that multiple pathways may contribute to the presence of squamous epithelium in the thyroid including metaplasia of mature follicular cells, development from established embryonic remnants, or inception in putative, incompletely characterized stem-like cells. Our retrospective review presents an institutional landscape from which further investigation into the frequency and unique histologic and molecular context of intrathyroidal squamous differentiation as a driver or terminal event in thyroid pathophysiology.

Energy drinks leading to infertility by causing cytotoxic injury of the ovaries - A randomized controlled animal trial

Aim: To observe energy drink induced ovarian cytotoxicity and possible protective effects of co-administration of omega 3 fatty acids. Methods: The experimental study comprised of 36 adult female albino rats was conducted at anatomy department, PGMI, Lahore. Animals were divided into 3 groups. Group A was control. Groups B received energy drink. Group C received omega 3 along with energy drinks once daily orally for 30 consecutive days. Rats were sacrificed under deep anesthesia on 30th day. Ovaries were extracted and sections were stained with H&E stains. Results were analyzed using SPSS. A p-value < 0.05 was considered statistically significant. Results: Animals administered with energy drink showed marked pyknosis of graafian follicular cells and oocyte nuclei when compared with control and energy dink plus omega 3 treated animals. Vacuolization of the follicular cells was also more pronounced in energy drinks treated group (p <0.001). Ovarian stroma of energy drink treated group showed marked vascular congestion and tissue infiltration as compared to the omega 3 plus energy drink treated groups.. Conclusion: Oxidative stress induced by the energy drinks is the basic cause behind the toxic change seen in ovarian histo-architecture. Omega-3 significantly decreased these detrimental effects of energy drink due to its antioxidant and anti-inflammatory action. This study can be a pioneer in guiding the population about the harmful effects of energy drinks on ovarian morphology, leading to cytotoxicity of oocyte and ultimately leading to infertility. Keywords: Energy drinks; Omega-3; Cytotoxicity; Oxidative stress; Carbonated Beverages; Infertility; Caffeine

MAPK Inhibition Requires Active RAC1 Signaling to Effectively Improve Iodide Uptake by Thyroid Follicular Cells

The Sodium/Iodide Symporter (NIS) is responsible for the active transport of iodide into thyroid follicular cells. Differentiated thyroid carcinomas (DTCs) usually preserve the functional expression of NIS, allowing the use of radioactive iodine (RAI) as the treatment of choice for metastatic disease. However, a significant proportion of patients with advanced forms of TC become refractory to RAI therapy and no effective therapeutic alternatives are available. Impaired iodide uptake is mainly caused by the defective functional expression of NIS, and this has been associated with several pathways linked to malignant transformation. MAPK signaling has emerged as one of the main pathways implicated in thyroid tumorigenesis, and its overactivation has been associated with the downregulation of NIS expression. Thus, several strategies have been developed to target the MAPK pathway attempting to increase iodide uptake in refractory DTC. However, MAPK inhibitors have had only partial success in restoring NIS expression and, in most cases, it remained insufficient to allow effective treatment with RAI. In a previous work, we have shown that the activity of the small GTPase RAC1 has a positive impact on TSH-induced NIS expression and iodide uptake in thyroid cells. RAC1 is a downstream effector of NRAS, but not of BRAF. Therefore, we hypothesized that the positive regulation induced by RAC1 on NIS could be a relevant signaling cue in the mechanism underlying the differential response to MEK inhibitors, observed between NRAS- and BRAF-mutant tumors. In the present study, we found that the recovery of NIS expression induced through MAPK pathway inhibition can be enhanced by potentiating RAC1 activity in thyroid cell systems. The negative impact on NIS expression induced by the MAPK-activating alterations, NRAS Q61R and BRAF V600E, was partially reversed by the presence of the MEK 1/2 inhibitors AZD6244 and CH5126766. Notably, the inhibition of RAC1 signaling partially blocked the positive impact of MEK inhibition on NIS expression in NRAS Q61R cells. Conversely, the presence of active RAC1 considerably improved the rescue of NIS expression in BRAF V600E thyroid cells treated with MEK inhibitors. Overall, our data support an important role for RAC1 signaling in enhancing MAPK inhibition in the context of RAI therapy in DTC, opening new opportunities for therapeutic intervention.

The intraovarian cellular origins of GDF9 and BMP15 in the mouse and aspects of their biological properties

<p>Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are both members of the TGF-ß protein superfamily and are known to be essential for normal follicular development in mammals. Several studies have highlighted the species-specific effects of BMP15 and GDF9, which could be attributed, at least in part to the differences in the follicular expression patterns and to different forms of the secreted proteins. In the mouse, GDF9 is required for follicular development, whereas BMP15 appears to be only required near ovulation with contradictory reports as to the timing of BMP15 expression. However, mouse BMP15 and GDF9 are known to have the capability of acting together synergistically. The aims of this thesis were to characterise in the mouse ovary, the expression patterns (localisation and levels) of Bmp15 and Gdf9 mRNA throughout follicular development, and to determine the peri-ovulatory expression of the corresponding proteins. In situ hybridisation and quantitative PCR analyses of ovarian samples and follicular cells collected from control and superovulated mice confirmed that Gdf9 and Bmp15 mRNA are expressed exclusively in oocytes from primary and early secondary stage follicles respectively. qPCR analysis of denuded oocytes (DO) revealed a tight correlation, and therefore co-regulation, between the expression levels of Bmp15 and Gdf9 irrespective of follicular developmental stage, with steady expression until the preovulatory LH surge when down-regulation of Bmp15 and Gdf9 occurred. Throughout the follicular developmental stages examined, Gdf9 was expressed in greater abundance relative to Bmp15, with a Bmp15:Gdf9 mRNA ratio of 1:4.12. [...] In conclusion, oocyte-derived Bmp15 and Gdf9 mRNA expression is co-regulated throughout follicular development in mice, with Gdf9 being more abundant than Bmp15, which might be an important factor in determining high ovulation quota. The expression of the target genes is down-regulated as the oocyte reaches developmental competence following the preovulatory LH surge. Protein expression data provided evidence that in vivo the immature mouse oocyte is capable of secreting all BMP15 protein forms previously detected in vitro. After the preovulatory LH surge, all visible protein forms are associated with the somatic follicular cells, in particular with the expanded cumulus mass. Of particular interest is the presence of the large protein complexes in the cumulus cell lysates, which suggests a storage and activation process involving ECM proteins, similar to the mechanism reported for other TGF-ß superfamily members, such as TGF-ß1 and myostatin. The finding that the BMP15 precursor protein is biologically active with a different activity to that of the processed mature protein form suggests that the full-length precursor protein may regulate or provide at least a portion of the biological activity of BMP15 in mice.</p>

The intraovarian cellular origins of GDF9 and BMP15 in the mouse and aspects of their biological properties

<p>Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are both members of the TGF-ß protein superfamily and are known to be essential for normal follicular development in mammals. Several studies have highlighted the species-specific effects of BMP15 and GDF9, which could be attributed, at least in part to the differences in the follicular expression patterns and to different forms of the secreted proteins. In the mouse, GDF9 is required for follicular development, whereas BMP15 appears to be only required near ovulation with contradictory reports as to the timing of BMP15 expression. However, mouse BMP15 and GDF9 are known to have the capability of acting together synergistically. The aims of this thesis were to characterise in the mouse ovary, the expression patterns (localisation and levels) of Bmp15 and Gdf9 mRNA throughout follicular development, and to determine the peri-ovulatory expression of the corresponding proteins. In situ hybridisation and quantitative PCR analyses of ovarian samples and follicular cells collected from control and superovulated mice confirmed that Gdf9 and Bmp15 mRNA are expressed exclusively in oocytes from primary and early secondary stage follicles respectively. qPCR analysis of denuded oocytes (DO) revealed a tight correlation, and therefore co-regulation, between the expression levels of Bmp15 and Gdf9 irrespective of follicular developmental stage, with steady expression until the preovulatory LH surge when down-regulation of Bmp15 and Gdf9 occurred. Throughout the follicular developmental stages examined, Gdf9 was expressed in greater abundance relative to Bmp15, with a Bmp15:Gdf9 mRNA ratio of 1:4.12. [...] In conclusion, oocyte-derived Bmp15 and Gdf9 mRNA expression is co-regulated throughout follicular development in mice, with Gdf9 being more abundant than Bmp15, which might be an important factor in determining high ovulation quota. The expression of the target genes is down-regulated as the oocyte reaches developmental competence following the preovulatory LH surge. Protein expression data provided evidence that in vivo the immature mouse oocyte is capable of secreting all BMP15 protein forms previously detected in vitro. After the preovulatory LH surge, all visible protein forms are associated with the somatic follicular cells, in particular with the expanded cumulus mass. Of particular interest is the presence of the large protein complexes in the cumulus cell lysates, which suggests a storage and activation process involving ECM proteins, similar to the mechanism reported for other TGF-ß superfamily members, such as TGF-ß1 and myostatin. The finding that the BMP15 precursor protein is biologically active with a different activity to that of the processed mature protein form suggests that the full-length precursor protein may regulate or provide at least a portion of the biological activity of BMP15 in mice.</p>

Interpretive Errors in Fine-Needle Aspiration of Thyroid: A 13-Year Institutional Experience in Lebanon

<b><i>Introduction:</i></b> Fine-needle aspiration (FNA) is a worldwide established diagnostic tool for the assessment of patients with thyroid nodules. All thyroid FNA interpretive errors (IEs) were reviewed at the American University of Beirut Medical Center over a 13-year period, in order to identify and analyze them. <b><i>Materials and Methods:</i></b> All FNAs and their corresponding pathology results are correlated yearly for quality assurance. Discrepant cases are segregated into sampling errors and IEs. All thyroid FNAs with IEs were collected from 2005 to 2017. FNA and pathology slides were reviewed by trained, board-certified cytopathologists, adhering to the latest Bethesda criteria. Reasons for erroneous diagnoses were studied. <b><i>Results:</i></b> There was a total of 11 IEs out of 340 thyroid FNAs followed by surgical resection. Five benign follicular nodules (BFNs) were misinterpreted as suspicious for carcinoma. Focal nuclear atypia in cyst-lining or follicular cells and a monotonous population of macrophages misinterpreted as Hurthle cells (HCs) were the causes of IEs in this category. Four Hashimoto’s thyroiditis (HT) cases were misinterpreted as suspicious for malignancy. Innate atypia of HCs and sampling misinterpretation were the causes of IEs in HT. One medullary and 1 follicular carcinoma were misinterpreted as suspicious for follicular neoplasm and BFN, respectively. Nine cases were better classified after review. <b><i>Conclusion:</i></b> Thyroid FNA IEs can be mitigated by meticulous screening and identification of all elements on FNA smears. Awareness of focal nuclear atypia in reactive cyst-lining and follicular cells in BFN, as well as HCs in HT, is highlighted. Adherence to The Bethesda System for Reporting Thyroid Cytopathology and consulting experienced cytopathologists significantly decrease IEs.

Primary Cilia Mediate TSH-Regulated Thyroglobulin Endocytic Pathways

Primary cilia are sensory organelles with a variety of receptors and channels on their membranes. Recently, primary cilia were proposed to be crucial sites for exocytosis and endocytosis of vesicles associated with endocytic control of various ciliary signaling pathways. Thyroglobulin (Tg) synthesis and Tg exocytosis/endocytosis are critical for the functions of thyroid follicular cells, where primary cilia are relatively well preserved. LRP2/megalin has been detected on the apical surface of absorptive epithelial cells, including thyrocytes. LRP2/megalin on thyrocytes serves as a Tg receptor and can mediate Tg endocytosis. In this study, we investigated the role of primary cilia in LRP2/megalin expression in thyroid gland stimulated with endogenous TSH using MMI-treated and Tg-Cre;Ift88flox/flox mice. LRP2/megalin expression in thyroid follicles was higher in MMI-treated mice than in untreated control mice. MMI-treated mice exhibited a significant increase in ciliogenesis in thyroid follicular cells relative to untreated controls. Furthermore, MMI-induced ciliogenesis accompanied increases in LRP2/megalin expression in thyroid follicular cells, in which LRP2/megalin was localized to the primary cilium. By contrast, in Tg-Cre;Ift88flox/flox mice, thyroid with defective primary cilia expressed markedly lower levels of LRP2/megalin. Serum Tg levels were elevated in MMI-treated mice and reduced in Tg-Cre;Ift88flox/flox mice. Taken together, these results indicate that defective ciliogenesis in murine thyroid follicular cells is associated with impaired LRP2/megalin expression and reduced serum Tg levels. Our results strongly suggest that primary cilia harbors LRP2/megalin, and are involved in TSH-mediated endocytosis of Tg in murine thyroid follicles.