scholarly journals Development of an In Vitro Human Thyroid Microtissue Model for Chemical Screening

2019 ◽  
Vol 174 (1) ◽  
pp. 63-78 ◽  
Author(s):  
Chad Deisenroth ◽  
Valerie Y Soldatow ◽  
Jermaine Ford ◽  
Wendy Stewart ◽  
Cassandra Brinkman ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Screening Program ◽  
3D Culture ◽  
Hazard Identification ◽  
Human Thyroid ◽  
Screening Assay ◽  
Thyroid Cells ◽  
Chemical Screening

Abstract Thyroid hormones (TH) are essential for regulating a number of diverse physiological processes required for normal growth, development, and metabolism. The US EPA Endocrine Disruptor Screening Program (EDSP) has identified several molecular thyroid targets relevant to hormone synthesis dynamics that have been adapted to high-throughput screening (HTS) assays to rapidly evaluate the ToxCast/Tox21 chemical inventories for potential thyroid disrupting chemicals (TDCs). The uncertainty surrounding the specificity of active chemicals identified in these screens and the relevance to phenotypic effects on in vivo human TH synthesis are notable data gaps for hazard identification of TDCs. The objective of this study was to develop a medium-throughput organotypic screening assay comprised of reconstructed human thyroid microtissues to quantitatively evaluate the disruptive effects of chemicals on TH production and secretion. Primary human thyroid cells procured from qualified euthyroid donors were analyzed for retention of NK2 homeobox 1 (NKX2-1), Keratin 7 (KRT7), and Thyroglobulin (TG) protein expression by high-content image analysis to verify enrichment of follicular epithelial cells. A direct comparison of 2-dimensional (2D) and 3-dimensional (3D) 96-well culture formats was employed to characterize the morphology, differential gene expression, TG production, and TH synthesis over the course of 20 days. The results indicate that modeling human thyroid cells in the 3D format was sufficient to restore TH synthesis not observed in the 2D culture format. Inhibition of TH synthesis in an optimized 3D culture format was demonstrated with reference chemicals for key molecular targets within the thyroid gland. Implementation of the assay may prove useful for interpreting phenotypic effects of candidate TDCs identified by HTS efforts currently underway in the EDSP.

In Vitro Culture of Human Thyroid Cells: Preliminary Findings on the Role of the Pathological Condition of the Donors

1997 ◽  
Vol 25 (2) ◽  
pp. 153-160
Author(s):  
Francesca Mattioli ◽  
Marianna Angiola ◽  
Laura Fazzuoli ◽  
Francesco Razzetta ◽  
Antonietta Martelli
Keyword(s):  
Pathological Condition ◽  
Primary Cultures ◽  
S Phase ◽  
Human Thyroid ◽  
Thyroid Cells ◽  
Toxicological Studies ◽  
Predictive Indicator

Although primary cultures of human thyroid cells are used for endocrinological and toxicological studies, until now no attention has been paid toward verifying whether the hormonal conditions to which the gland was exposed in vivo prior to surgery could influence in vitro responses. Our findings suggest that the hormonal situation in vivo cannot be used as a predictive indicator of triiodothyronine and thyroxine release and/or S-phase frequency in vitro, either with or without the addition of bovine thyrotropin.

scholarly journals Acrylamide does not induce tumorigenesis or major defects in mice in vivo

2008 ◽  
Vol 198 (2) ◽  
pp. 301-307 ◽  
Author(s):  
Ling Jin ◽  
Vanessa Chico-Galdo ◽  
Claude Massart ◽  
Christine Gervy ◽  
Viviane De Maertelaere ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Chronic Administration ◽  
Serum Levels ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Hormone Synthesis ◽  
Rat Thyroid

Chronic administration of acrylamide has been shown to induce thyroid tumors in rat. In vitro acrylamide also causes DNA damage, as demonstrated by the comet assay, in various types of cells including human thyroid cells and lymphocytes, as well as rat thyroid cell lines. In this work, mice were administered acrylamide in their drinking water in doses comparable with those used in rats, i.e., around 3–4 mg/kg per day for mice treated 2, 6, and 8 months. Some of the mice were also treated with thyroxine (T4) to depress the activity of the thyroid. Others were treated with methimazole that inhibits thyroid hormone synthesis and consequently secretion and thus induces TSH secretion and thyroid activation. These moderate treatments were shown to have their known effect on the thyroid (e.g. thyroid hormone and thyrotropin serum levels, thyroid gland morphology…). Besides, T4 induced an important polydipsia and degenerative hypertrophy of adrenal medulla. Acrylamide exerted various discrete effects and at high doses caused peripheral neuropathy, as demonstrated by hind-leg paralysis. However, it did not induce thyroid tumorigenesis. These results show that the thyroid tumorigenic effects of acrylamide are not observed in another rodent species, the mouse, and suggest the necessity of an epidemiological study in human to conclude on a public health policy.

Sensitization and desensitization of human thyroid cells in culture: effects of thyrotrophin and thyroid-stimulating immunoglobulin

1988 ◽  
Vol 119 (2) ◽  
pp. 341-349 ◽  
Author(s):  
Z. Kraiem ◽  
R. Alkobi ◽  
O. Sadeh
Keyword(s):  
Secretory Activity ◽  
Time Lapse ◽  
Human Thyroid ◽  
Thyroid Cells ◽  
Subsequent Exposure ◽  
Heterologous Desensitization ◽  
High Doses ◽  
Culture Effects

ABSTRACT Using an in-vitro system of cultured human thyroid cells and cyclic AMP (cAMP) accumulation as an index of cell stimulation, we compared TSH and thyroid-stimulating immunoglobulin (TSI) with regard to thyrocyte sensitization and desensitization. The smallest dose of TSH (0·05 mU/ml) capable of stimulating thyroid cells was the same as the minimum dose required to induce desensitization upon subsequent rechallenge with the hormone. In contrast, about 30-fold higher doses of TSI were needed to cause cell refractoriness compared with doses capable of eliciting stimulation. Moreover, significant stimulation of the thyroid with TSI was apparent much later than with TSH. A longer time-lapse was also necessary for TSI to induce densensitization. Likewise, thyrocytes recovered more slowly from TSI compared with TSH desensitization. Although at high doses TSI induced homologous desensitization, at lower doses the antibody, unlike TSH, potentiated the cAMP response to subsequent exposure to the antibody. The stimulatory doses of TSI were in the range usually encountered in active Graves' disease, which may explain why prolonged TSI in vivo sustains a hyperthyroid condition. In addition, we found that under conditions in which TSH leads to desensitization of the cAMP response, the thyroid cells maintained their responsiveness in terms of triiodothyronine secretory activity. Pre-exposure of human thyrocytes to TSI induced heterologous desensitization towards the TSH-stimulated cAMP response. Moreover, addition of the antibody to maximally desensitizing doses of TSH decreased cell sensitivity to the hormone even further. In sharp contrast, preincubation of cells with TSH, or TSH plus TSI, potentiated by four- and twofold respectively the cAMP response to subsequent challenge with TSI. Taken together, the data reveal marked differences between the action of TSH and TSI, and raise interesting questions concerning the mechanism whereby TSH potentiates the cAMP response to TSI. J. Endocr. (1988) 119, 341–349

Monoclonal Antibody-Based Screening Assay for Factor Inhibiting Hypoxia-Inducible Factor Inhibitors

2008 ◽  
Vol 13 (6) ◽  
pp. 494-503 ◽  
Author(s):  
Sang-Hyeup Lee ◽  
Jeong Hee Moon ◽  
Eun Ah Cho ◽  
Seong-Eon Ryu ◽  
Myung Kyu Lee
Keyword(s):  
Monoclonal Antibody ◽  
High Throughput Screening ◽  
Transcriptional Activity ◽  
Hypoxia Inducible Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sensitive Assay ◽  
Screening Assay ◽  
Hypoxic Conditions

The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1α (HIF-1α), and the modification abrogates the transcriptional activity of HIF-1α. Because FIH is more active on HIF-1α than prolyl hydroxylase domain proteins under hypoxic conditions, its inhibitors have potential to be developed as anti-ischemic drugs targeting normal cells stressed by hypoxia. In this study, the authors developed the first monoclonal antibody, SHN-HIF1α, specifically to Asn803 hydroxylated HIF-1α and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1α showed 740 times higher affinity to the Asn803 hydroxylated HIF-1α peptide than the unmodified one. An enzyme-linked immunosorbent assay—based system using SHN-HIF1α displayed at least 30 times more sensitivity than previous methods for screening FIH inhibitors and was easily applicable to develop a high-throughput screening system. SHN-HIF1α also showed an Asn803 hydroxylation-dependent specificity to HIF-1α in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors. ( Journal of Biomolecular Screening 2008:494-503)

IN-VITRO STUDIES OF NORMAL HUMAN THYROID CELLS: RESPONSES TO THYROTROPHIN AND DIBUTYRYL CYCLIC AMP

1977 ◽  
Vol 72 (1) ◽  
pp. 87-96 ◽  
Author(s):  
S. P. BIDEY ◽  
P. MARSDEN ◽  
J. ANDERSON ◽  
C. G. McKERRON ◽  
H. BERRY
Keyword(s):  
Cyclic Amp ◽  
Thyroid Tissue ◽  
Three Dimensional ◽  
Human Thyroid ◽  
Dibutyryl Cyclic Amp ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Normal Human

SUMMARY Follicular cells isolated from normal human thyroid tissue have been cultured for up to 140 h with bovine thyrotrophin (TSH) or dibutyryl cyclic AMP (DBcAMP). Both compounds induced marked reorganization of the cells into three-dimensional follicular structures, whilst non-supplemented cells assumed a monolayer form. Cultures treated initially with TSH or DBcAMP showed a greater iodide uptake capacity, in comparison with unsupplemented cultures, in which iodide uptake was markedly diminished after 24 h. The release of tri-iodothyronine (T3) and thyroxine (T4) into the medium was determined by radioimmunoassay. Both TSH- and DBcAMP-treated cells showed a significant increase in iodothyronine output compared with unsupplemented control cells. In contrast to the 'classical' TSH-induced depression of the T4:T3 ratio in vivo, an increase in the ratio was observed for both TSH- and DBcAMP-supplemented cells in vitro. The ratio was also significantly greater after TSH than after DBcAMP, and possible implications of this finding are discussed.

scholarly journals Reexpression of blood group ABH antigens on the surface of human thyroid cells in culture.

1982 ◽  
Vol 94 (1) ◽  
pp. 193-200 ◽  
Author(s):  
E L Khoury
Keyword(s):  
Cell Surface ◽  
Blood Group ◽  
Enzymatic Digestion ◽  
Cell Suspensions ◽  
Human Thyroid ◽  
Blood Group Antigens ◽  
Thyroid Cells ◽  
Blood Group Abh

Using indirect immunofluorescence (IFL) on viable human thyroid cultures, it has been shown that, although adult follicular cells do not express blood group ABH antigens in vivo, they invariably reexpress the corresponding antigens on the cell surface when cultured in monolayers, even for very short periods. The absence of blood group antigens on noncultured thyroid cells was confirmed by negative IFL on cell suspensions obtained after enzymatic digestion of the glands, whereas these antigens were readily demonstrable on cell suspensions obtained by trypsinization of established monolayers. The quantitative expression of ABH antigens on individual thyroid cells was variable and the cell-surface IFL pattern due to binding of blood group isoantibodies was different from that given by organ-specific thyroid autoantibodies on viable cultures. Reexpression of blood group antigens by cultured thyroid cells could not be related to the secretor status of the donors, the presence of a particular source of serum in the culture medium or cell division in vitro. After 2-3 wk in culture, thyroid cells became morphologically dedifferentiated and no longer displayed blood group antigens, though they still expressed cell-surface beta 2-microglobulin. Fibroblasts present in the primary thyroid cultures were invariably negative for ABH antigens. These results demonstrate that the surface antigenic repertoire of cultured human cells is not necessarily identical to that present on the same cells in vivo. Furthermore, the possibility that blood group natural isoantibodies bind to the cell surface must be taken into account in experiments in which cultured thyroid cells are exposed to human sera.

Local factors regulating growth and function of human thyroid cells in vitro and in vivo

2003 ◽  
Vol 213 (1) ◽  
pp. 47-58 ◽  
Author(s):  
Margaret C. Eggo ◽  
Virginia M. Quiney ◽  
Spencer Campbell
Keyword(s):  
Human Thyroid ◽  
Thyroid Cells ◽  
Local Factors ◽  
And Function

scholarly journals Diphenylpyrazole-Derived Compounds Increase Survival Time of Mice after Prion Infection

2011 ◽  
Vol 55 (10) ◽  
pp. 4774-4781 ◽  
Author(s):  
Fabienne Leidel ◽  
Martin Eiden ◽  
Markus Geissen ◽  
Hans A. Kretzschmar ◽  
Armin Giese ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Natural Infection ◽  
New Drugs ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Variant Form ◽  
Screening Assay ◽  
High Throughput Screening Assay

ABSTRACTTransmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative disorders that can be transmitted by natural infection or inoculation. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. The emergence of a variant form of CJD (vCJD), which has been associated with BSE, produced strong pressure to search for effective treatments with new drugs. Up to now, however, TSEs have proved incurable, although many efforts have been made bothin vitroandin vivoto search for potent therapeutic and prophylactic compounds. For this purpose, we analyzed a compound library consisting of 10,000 compounds with a cell-based high-throughput screening assay dealing with scrapie-infected scrapie mouse brain and ScN2A cells and identified a new class of inhibitors consisting of 3,5-diphenylpyrazole (DPP) derivatives. The most effective DPP derivative showed half-maximal inhibition of PrPScformation at concentrations (IC50) of 0.6 and 1.2 μM, respectively. This compound was subsequently subjected to a number of animal experiments using scrapie-infected wild-type C57BL/6 and transgenic Tga20 mice. The DPP derivative induced a significant increase of incubation time both in therapeutic and prophylactic experiments. The onset of the prion disease was delayed by 37 days after intraperitoneal and 42 days after oral application, respectively. In summary, we demonstrate a highin vitroefficiency of DPP derivatives against prion infections that was substantiatedin vivofor one of these compounds. These results indicate that the novel class of DPP compounds should comprise excellent candidates for future therapeutic studies.

scholarly journals Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E

2021 ◽  
Vol 22 (16) ◽  
pp. 8577
Author(s):  
Alon Ben David ◽  
Ada Barnea ◽  
Eran Diamant ◽  
Eyal Dor ◽  
Arieh Schwartz ◽  
...  
Keyword(s):  
Receptor Binding ◽  
High Throughput Screening ◽  
Lethal Dose ◽  
Screening Assay ◽  
Vitro Assay ◽  
Time To Death ◽  
High Throughput Screening Assay ◽  
Binding Inhibitors

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death (p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

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