Measurement of thyroid stimulating immunoglobulins in a new cell line transfected with a functional human TSH receptor (JPO9 cells), compared with an assay using FRTL‐5 cells

Michelangeli, V. P.; Munro, Donald; Poon, Cathy; Frauman, Albert G; Colman, Peter G

doi:10.1111/j.1365-2265.1994.tb03017.x

Cited by 44 publications

(49 citation statements)

References 25 publications

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“…The CHO bioassay is mainly used to detect plasma thyroid-stimulating antibodies (12,13). Investigation of TSH bioactivity is an option when a discrepancy between clinical or biochemical data and the plasma immunochemical TSH value is observed, probably due to variable carbohydrate structures of circulating TSH (14).…”

Section: Discussionmentioning

confidence: 99%

Plasma thyrotropin bioactivity in Down's syndrome children with subclinical hypothyroidism

Trotsenburg²,

Ris‐Stalpers³

et al. 2001

European Journal of Endocrinology

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Objective: Subclinical hypothyroidism occurs in a number of children with Down's syndrome (DS). The reason for the mildly elevated plasma thyrotropin (TSH) concentrations is not known. The present study investigated whether decreased TSH bioactivity plays a role in this phenomenon. Design: A retrospective study of plasma specimens from DS children with mildly elevated plasma TSH concentrations and thyroid hormone levels within the reference range, using a TSH receptor± adenylate cyclase mediated bioassay. Methods: Strain JP26 Chinese hamster ovary (CHO) cells, stable transfected with the human TSH receptor, were incubated with unfractionated plasma (1/10 diluted in hypotonic incubation medium) of 10 DS children with subclinical hypothyroidism and nine euthyroid children with insulindependent diabetes mellitus as controls. cAMP released in the incubation medium was measured by RIA. Mock-transfected CHO cells were used to correct for non-specific CHO response. WHO Second International Reference Preparation of human TSH was dissolved and diluted in pooled normal human plasma and simultaneously bioassayed to match patient and control results. Results: Plasma TSH levels were slightly increased in DS (mean^s.d., 6X5^1X3 mUalY reference range 0.4±4.0 mU/l). Plasma TSH levels for controls 1X3^0X4 mUal were within the reference range. Plasma thyroid hormone levels in patients and controls were normal, plasma TSH binding inhibitory immunoglobulin and thyroid peroxidase antibodies were negative. cAMP levels (corrected for non-specific CHO response) in DS patients 18X4^3X9 pmolawell and in controls 14X31 X3 pmolawell did not significantly differ from cAMP levels generated by patient-TSH equivalent TSH standards 16X3^0X9 pmolawellX Conclusions: The present results demonstrate normal TSH bioactivity in plasma of DS children, indicating that subclinical hypothyroidism in these patients is of primary (thyroidal) origin.

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Section: Discussionmentioning

confidence: 99%

Plasma thyrotropin bioactivity in Down's syndrome children with subclinical hypothyroidism

Trotsenburg²,

Ris‐Stalpers³

et al. 2001

European Journal of Endocrinology

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“…Cells were exposed to TSH at 37 C as previously described (Michelangeli et al 1994) except that tritiated cAMP was separated from other nucleotides using neutral alumina chromatography alone rather than a combination of ionexchange chromatography and neutral alumina chromatography as used by Michelangeli et al (1994). All determinations were performed in triplicate.…”

Section: Camp Assaymentioning

confidence: 99%

Differential effect of thyroid-stimulating hormone (TSH) on intracellular free calcium and cAMP in cells transfected with the human TSH receptor

Metcalfe¹,

Findlay²,

Robertson³

et al. 1998

Journal of Endocrinology

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The thyroid-stimulating hormone (TSH) binds to a receptor which activates adenylate cyclase and elevates cAMP concentration. In addition, effects of TSH on intracellular calcium and inositol phosphate accumulation have been reported. However, the mechanism of TSH-stimulated accumulation of inositol phosphates and elevation of calcium levels is unresolved. Previous work from this laboratory has shown TSH to cause acute transient increases in intracellular calcium in pig, human and FRTL-5 rat thyroid cells as well as in cells transfected with the human TSH receptor ( JPO9 cells) in some (but not all) experiments. The aim of this study was to investigate the variability of the calcium response to TSH in JPO9 cells to learn more about the nature of this calcium signal induction. Calcium responses to TSH were determined using the fluorochrome fura-2 in both monolayers of adherent cells and adherent single cells. The responses to a single addition and to repetitive additions of TSH were compared. We also determined the cAMP response to TSH using these two protocols of TSH addition.Our data show that, whereas the cAMP response to TSH is highly predictable and consistent and does not require multiple exposures to TSH, cells were unlikely to respond to TSH with an increase in calcium unless they received multiple challenges with the hormone. A single addition of 10 mU/ml TSH failed to increase calcium in any of 40 single cells examined and in only 4 of 15 monolayers of cells (27%) examined; in contrast, 10 of 12 monolayers eventually responded with an increase in calcium after multiple exposure to TSH and 18 of 67 single cells. Similar data were obtained whether calcium was measured in single cells or in populations of cells. We also demonstrated cooperativity between an adenosine derivative, N 6 -(-2-phenylisopropyl)adenosine, and TSH such that their co-administration resulted in a consistent and marked elevation in calcium levels not achieved with either agonist alone. In summary, we suggest that the coupling between the TSH receptor and the intracellular signalling system that leads to activation of intracellular calcium in JPO9 cells requires repetitive stimulation or the influence of other agonists, in contrast with the coupling between the TSH receptor and activation of the adenylate cyclase enzyme.

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“…Furthermore, the characteristics of FRTL-5 cells still vary among the laboratories, so that comparison of the results obtained at various institutions is quite difficult. Third, patients' IgGs do not always react in the same way with rat TSH receptors as with human TSH receptors [7][8][9], so that the use of the cell line with human TSH receptors is better than that with rat TSH receptors. …”

Section: Thyroid-stimulatingmentioning

confidence: 99%

Detection of Thyroid-Stimulating Antibody Using Frozen Stocks of Chinese Hamster Ovary Cells Transfected with Cloned Human Thyrotropin Receptor.

1997

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Abstract.To measure thyroid-stimulating antibody (TSAb) in sera from patients with Graves' disease, we developed a new assay system with using frozen stocks of CHO-K1 cells. CHO-K1 cells trasfected with cloned thyrotropin (TSH) receptor on a 96 well plate were frozen in Cell BankerTM and stored at -70 °C . Three days before the assay, they were thawed in the culture medium and allowed to grow in a monolayer until use. The medium was replaced with medium containing IgGs from the patients, then after 2 h, it was collected and concentrations of adenosine 3'-5'-cyclicmonophosphate (cAMP) were measured.This method is sensitive enough to detect TSAb and it is simpler and easier than the methods which use FRTL-5 cells.

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Measurement of thyroid stimulating immunoglobulins in a new cell line transfected with a functional human TSH receptor (JPO9 cells), compared with an assay using FRTL‐5 cells

Cited by 44 publications

References 25 publications

Plasma thyrotropin bioactivity in Down's syndrome children with subclinical hypothyroidism

Plasma thyrotropin bioactivity in Down's syndrome children with subclinical hypothyroidism

Differential effect of thyroid-stimulating hormone (TSH) on intracellular free calcium and cAMP in cells transfected with the human TSH receptor

Detection of Thyroid-Stimulating Antibody Using Frozen Stocks of Chinese Hamster Ovary Cells Transfected with Cloned Human Thyrotropin Receptor.

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