Biomedical subjects

P A Ealey

Publications and source records attributed to P A Ealey.

At least 19 recordsLinked to original sources

PubMed1996

An investigation into the lability of the bioactivity of human growth hormone using the ESTA bioassay.

We compared the bioactivity attributable to human growth hormone (hGH) in serum samples, determined at the time of their collection, with that after storage for 2-18 months at -20 degrees C. The samples were obtained from volunteers and patients who underwent provocative tests of hGH secretion, and the bioactivity was determined in the ESTA bioassay, which is based upon the use of Nb2 cells. We report that, in some subjects, the bioactivity of samples collected at the response peaks deteriorated on storage for as little as 2 months. The decrease in hGH bioactivity was systematic in that it consistently declined so as to approach the values initially determined by an immunoassay (Hybritech IRMA). This differential lability was a characteristic of the peak samples, and was not observed for either samples collected before and after the peaks of hGH secretion or for purified preparations of hGH which were subjected to a range of freeze/thaw and storage regimens. We suggest that this unusual lability is indicative of transient shifts in the spectrum of the variants of hGH which are present in the circulation following stimulation by provocative agents. This study emphasises the need to minimise the risk of introducing storage artefacts in investigations into the responses of hGH to provocation.

Adult↗

PubMed1995

The development of an eluted stain bioassay (ESTA) for human growth hormone.

The basic characteristics of MTT-formazan production by both quiescent Nb2 cells and those activated by fetal calf serum or human growth hormone (hGH) are described. These characteristics are exploited for the development of an MTT-ESTA bioassay for purified preparations of lactogens such as growth hormone. The resulting in vitro bioassay is sensitive and precise, with a detection limit of about 0.05 mU hGH/l (19 ng/l) and a within-assay imprecision of 2.5% in the presence of 0.3 mU hGH/l (114 ng/l). When utilizing quiescent Nb2 cells for bioassays, large magnitudes of response are observed. The major component of the response is clearly derived from metabolic activation of the cells, rather than increased cell proliferation. The response was abolished by anti-human growth hormone. Delayed addition of the latter demonstrated that the presence of the hormone is required for the entire 96 h of the recommended bioassay incubation period to obtain the maximum response. At high doses, the dose-response relationship reaches a prolonged plateau which covers 4 orders of magnitude of incremental hormone concentrations. A decline in response is observed at the highest dose tested, 10(6) mU hGH/l (385 mg/l). This auto-inhibition is consistent with recent reports of a reduction in response due to stoichiometric blockade of sequential receptor dimerisation which is crucial for activation of both somatogenic and lactogenic receptors by hGH.

Animals↗

PubMed1993

The development and characterization of an eluted stain assay (ESTA) for the insulin-like growth factors.

An Eluted Stain Assay System (ESTA) has been adapted for the bioassay of the insulin-like growth factors, IGF-I and IGF-II. This ESTA is based on the Fischer Rat Thyroid cell line FRTL-5 which was grown as uniform, adherent microcultures on 96-well microtitre plates. The cells were stimulated with the growth factors for 48 h in hormone and serum free conditions. Responses were determined by the addition of the tetrazolium salt MTT which was reduced to a purple formazan product in a dose related manner. This was directly eluted from the cells and measured with a microtitre plate reader. The signal generated was solely dependent on metabolic activation of the cells, since no increase in cell numbers was detected during the bioassay. The advantages of using this method are its sensitivity, precision, specificity, rapidity and high sample throughout. This bioassay, which is based on a colorimetric method, is technically convenient compared with other systems including the earlier cytochemical bioassays and the radioisotopic methods. We have demonstrated that this MTT ESTA provides a useful method for the study of complex interactions between IGF-I and IGF-II and their related binding proteins and that IGF bioactivity in serum may also be investigated using this ESTA bioassay.

Adult↗

PubMed1990

Thyroid-stimulating antibody activity between different immunoglobulin G subclasses.

To investigate the distribution of thyroid-stimulating antibody (TSAb) activity between IgG subclasses, sera from 11 patients with Graves disease (including the National Institute of Biological Standards and Control (NIBSC) Research Standard, long acting thyroid stimulator-B) were fractionated by chromatography on affinity columns of monoclonal IgG subclass antibodies or protein A to deplete all but a single subclass. The resulting fractions were 98% or more pure for a single subclass. In all 11 patients, TSAb activity appeared to be confined to the IgG1 fraction as determined by cAMP production on addition of the fractions to the FRTL-5 rat thyroid cell line. In all of eight specimens from seven patients so tested, the whole serum activity was recovered in the IgG1 fraction, after adjusting for the recovery of the isotype from the column. TSAb activity in one serum comprised both lambda and kappa light chains but was IgG1 restricted. This IgG subclass restriction was not found when the same fractions were tested for thyroglobulin, microsomal/thyroid peroxidase, or tetanus toxoid antibody activity. Together with previous results showing marked restriction of both light chain usage and isoelectric point of TSAb, these results support the idea that Graves' disease may be the result of an oligo- or possibly monoclonal response at the B cell level.

Cyclic AMP↗

PubMed1988

An improved metaphase index assay for detecting thyroid growth stimulators using FRTL-5 thyroid cells cultured on a microtitre plate.

The metaphase index assay (MIA) for thyroid growth stimulators, as originally described, used FRTL-5 thyroid cells cultured in Bellco culture chambers and glass microscope slides. The metaphases were observed using the nuclear strain aceto-lacto orcein. However the surface properties of the glass proved to be variable and so polystyrene microscope slides were substituted. The aceto-lacto orcein stain was found to be unsuitable for use with polystyrene because of the solvents and mountant used. Therefore combinations of various other nuclear stains and mounting media were tested. The Giemsa stain, which was found to be the most satisfactory, could be applied to FRTL-5 cells maintained on the large variety of plastic supports now available for tissue culture, e.g., 96 well microtitre plates. This permitted the design of an MIA which is much more convenient, robust and economical in its use of clinical samples. The results with seven IgG preparations derived from the sera of patients with a variety of thyroid disorders are presented. In its revised form, the metaphase index assay provides a rapid screening assay for thyroid growth stimulators, such as autoantibodies and TSH.

Azure Stains↗

PubMed1988

Kappa/lambda immunoglobulin distribution in Graves' thyroid-stimulating antibodies. Simultaneous analysis of C lambda gene polymorphisms.

From patients with untreated Graves' disease 11 sera showing high cAMP release in the FRTL-5 cell assay were studied for relative proportions of kappa or lambda Ig molecules showing cAMP releasing activity. Immunoabsorption of gamma-globulins was performed using monoclonal murine anti-kappa or anti-lambda antibodies linked to cyanogen bromide-activated sepharose. Specific kappa- or lambda-adsorbed fractions were also eluted from immunoabsorbents using chaotrophic thiocyanate buffers and equilibrated with pH 7.4 low salt buffer by dialysis. Immunoabsorption and elution experiments showed that five Graves' sera contained predominant cAMP-releasing activity within lambda Ig fractions, whereas two Graves' sera showed predominant cAMP-releasing activity in kappa Ig fractions. Four sera showed cAMP release approximately equally divided between kappa and lambda Ig both after immunoabsorption and specific anti-kappa or anti-lambda eluates were studied. C lambda genotypes were examined by Southern blotting and restriction fragment length polymorphism analysis of Eco RI-digested genomic DNA from 158 patients with Graves' disease in parallel with 112 normal controls and 29 patients with autoimmune hypothyroidism. Notable shifts in proportions of 8/8 and 18/18 genotypes were present when Graves' patients were compared with normal controls. Allelic frequencies and ratios of genotype 8 to 18 were significantly different (P less than 0.05) when Graves' patients were compared either to normal controls or to patients with autoimmune hypothyroidism.

Absorption↗

PubMed1988

ESTA: a bioassay system for the determination of the potencies of hormones and antibodies which mimic their action.

A bioassay system named ESTA (eluted stain assay) has been developed to measure hormones and antibodies which mimic their action. It is derived from approaches used for cytochemical bioassays. Unlike the latter which use tissue segments or sections, ESTA is based upon uniform microcultures of target cells maintained in microtitre plates. Direct elution of the cytochemical stain from these microcultures into the wells of the microtitre plates permits rapid quantification with a microtitre plate reader. We describe ESTA systems for GH, prolactin, thyroid stimulators and human chorionic gonadotrophin which utilize the reduction of a tetrazolium salt to a formazan by intracellular dehydrogenase as the cytochemical system. These provide examples of ESTA systems in which the assay signal depends solely upon an increase in cell number in response to the hormone, or in which there is additional enzymic amplification.

Animals↗

PubMed1987

Thyroid-stimulating hormone stimulates increases in inositol phosphates as well as cyclic AMP in the FRTL-5 rat thyroid cell line.

Studies were conducted to determine whether thyroid-stimulating hormone (TSH; thyrotropin), a hormone known to increase cytosol concentrations of cyclic AMP, also stimulates the formation of inositol phosphates in thyroid cells. TSH and noradrenaline both stimulated [3H]inositol phosphate formation in a concentration-dependent manner in the rat thyroid cell line, FRTL-5 cells, which had been prelabelled with [3H]inositol. The threshold concentration of TSH required to stimulate inositol phosphate formation was more than 20 munits/ml, which is approx. 10(3)-fold greater than that required for cyclic AMP accumulation and growth in these cells. We also demonstrate that membranes prepared from FRTL-5 cells possess a guanine nucleotide-activatable polyphosphoinositide phosphodiesterase, which suggests that activation of inositide metabolism in these cells may be coupled to receptors by the G-protein, Gp. Our findings suggest that two second-messenger systems exist to mediate the action of TSH in the thyroid.

Animals↗

PubMed1987

Characterization of monoclonal antibodies directed against the TSH receptor, as revealed with the cytochemical bioassay.

The monoclonal antibody 11E8 (Kohn et al. 1984) is a novel proof of the actions of thyroid stimulator, since it is not only a potent stimulator of thyroid cells but also an inhibitor for TSH-binding. This might reflect an interaction of 11E8 with a discrete domaine of the TSH receptor, which is an essential component for TSH but not for TSab actions. These unique properties of 11E8 can be applied to the characterization of other thyroid stimulators which may interact with different domaines of the receptor. Moreover, 'TSab-like' stimulators, such as the sheep anti-TSH antiserum, could potentially be identified by 11E8.

Animals↗

PubMed1987

Forskolin and thyrotrophin stimulation of rat FRTL-5 thyroid cell growth: the role of cyclic AMP.

The adenylate cyclase stimulator forskolin increases intracellular cyclic AMP (cAMP) in rat FRTL-5 cells within minutes and, after a lag phase of 20-24 h, an increase of cells in metaphase is seen. The dose-response relationships were similar in both systems, with significant increases in the number of metaphases observed at approximately 0.1 mumol/l and a doubling of cAMP levels at 1 mumol/l, whilst doses of 0.1 mmol/l and above proved cytotoxic. An involvement of intracellular cAMP as a positive intermediate in cell division was further suggested by the finding that a low dose of forskolin (0.1 mumol/l) potentiated TSH stimulation of mitosis. Isobutyl methyl xanthine (IBMX), a phosphodiesterase inhibitor, also acted as a mitogen and potentiated TSH action. Moreover, the simultaneous inclusion of low doses of IBMX and forskolin additionally potentiated TSH stimulation of mitosis. An analogue of cAMP, dibutyryl cAMP, also stimulated mitosis and acted over a restricted dose range, with maximal stimulation at 1 mmol/l. We conclude that cAMP may act as a positive signal for FRTL-5 thyroid cell proliferation.

1-Methyl-3-isobutylxanthine↗

PubMed1985

Characterization of monoclonal antibodies raised against solubilized thyrotropin receptors in a cytochemical bioassay for thyroid stimulators.

Selected clones of monoclonal antibodies from mice immunized with solubilized preparations of bovine TSH receptors have been characterized in a cytochemical bioassay (CBA) for thyroid stimulators. This assay is based upon quantification of changes in naphthylamidase activity of sections of guinea pig thyroids with use of a chromogenic substrate. Monoclonal 22A6 is a thyroid-stimulating antibody directed at a site within the TSH receptor. Thus, although it is a weak inhibitor of 125I-TSH binding to thyroid membranes, 22A6 is inhibited from binding to membranes by TSH, exhibits a more than additive agonist effect on adenylate cyclase activity when tested at low TSH concentrations in thyroid cells, and is a competitive antagonist of TSH enhancement of adenylate cyclase activity at high TSH concentrations. In the CBA, 22A6 is a stimulator whose maximal activity is obtained with 77 pg/ml (3-min exposure). Dose-response curves of a long acting thyroid stimulator (LATS)-B standard and 22A6 have slopes which are not significantly different; as anticipated, the response to LATS-B is inhibited by antihuman immunoglobulin G (IgG) and that due to 22A6 by antimouse IgG. In contrast to 22A6, monoclonal 11E8 is a relatively potent inhibitor of 125I-TSH binding as well as TSH stimulation of adenylate cyclase activity, while failing to act as a stimulator itself. 11E8 is itself inactive as a stimulator in the CBA over a wide dose range; it does, however, inhibit TSH stimulation in the CBA. This inhibition is abolished by antimouse IgG. The transient peak of response observed in time courses to TSH occurs later in the presence of 11E8. Unlike its effect on TSH 11E8 shows relatively low potency (greater than 10,000-fold lower) when inhibiting stimulation by the thyroid stimulating antibodies, 22A6 or LATS-B. Since this difference cannot be explained by quantitative differences in the ability of 22A6 or 11E8 to bind to thyroid membranes, the CBA data suggest that the stimulating antibodies, 22A6 and LATS-B, may interact with different determinants on TSH receptors then either TSH or the blocking antibody, 11E8. This also implies that in Graves' disease blocking antibodies may be incompletely expressed in the presence of stimulating antibodies, although they may be potent inhibitors of TSH binding, as measured in receptor assays.

Adenylyl Cyclases↗

PubMed1985

Forskolin stimulation of naphthylamidase in guinea pig thyroid sections detected with a cytochemical bioassay.

Forskolin, from the roots of the Indian medicinal plant Coleus forskohlii, has recently been shown to be a potent stimulator of adenylate cyclase in many systems, including endocrine tissues such as the thyroid gland. We describe forskolin activation of beta-naphthylamidase activity in guinea pig thyroid tissue using the cytochemical bioassay (CBA) for thyroid stimulators. This CBA is the most sensitive bioassay for TSH and LATS-B currently available, being able to detect stimulation by doses as low as 10(-5) mU TSH/l and 10(-9) mU LATS-B/l. The dose-response curve to forskolin was bell-shaped (as is seen with TSH and LATS-B) with the ascending limb of the curve produced by 10(-13) M to 10(-12) M forskolin after a 3 min exposure time. Maximal stimulation was observed with 10(-12) M forskolin. However, the dose-response curve to forskolin was not parallel to that given by TSH, the slope of the ascending limb being much greater. It has been suggested that stimulation of beta-naphthylamidase activity in the CBA is via cAMP. We report that dibutyryl cAMP at doses from 10(-16) M to 10(-11) M produces a bell-shaped dose-response curve with a very broad peak response, again not parallel to that produced by TSH. Forskolin activation of beta-naphthylamidase in the CBA is unaffected by a 1:10(6) dilution of 11E8, a monoclonal antibody raised against solubilised TSH receptors, which binds to the TSH receptor and inhibits TSH stimulation. Although the precise location of forskolin action is not known, this is further evidence that forskolin acts at a post-surface receptor site.

Aminopeptidases↗

PubMed1985

VIP stimulation of beta-naphthylamidase activity in guinea-pig thyroid sections.

Subsequent to the discovery of vasoactive intestinal peptide (VIP) in the thyroid gland, VIP has been shown to stimulate various thyroid functions. The site of interaction of VIP with the thyroid follicular cell is at present not known, and this study has used the ultrasensitive cytochemical bioassay (CBA) for thyroid stimulators to investigate this further. Exposure of thyroid sections for 3 min to VIP resulted in increased naphthylamidase activity, with half-maximal response observed at 3 X 10(-13) M VIP. This response to such low doses of VIP is consistent with the CBA being ultrasensitive to other thyroid stimulators e.g. TSH, thyroid stimulating antibodies and forskolin. The response to VIP was abolished by rabbit anti-VIP antiserum. The dose-response curve to VIP was bell-shaped (as with the other stimulators), maximal stimulation occurring at 10(-12) M VIP. In contrast, however, to other thyroid stimulators, namely TSH, LATS-B and 3 monoclonal stimulating antibodies, whose ascending limbs of the dose-response curves extended over 3-4 orders of magnitude, the VIP curve rose rapidly from basal to maximal tissue stimulation from 10(-13) to 10(-12) M VIP, i.e. one order of magnitude. This unusual dose-response curve to VIP was parallel to that produced by forskolin. 11E8, a monoclonal 'blocking' antibody which is a potent inhibitor of TSH stimulation, did not 'block' forskolin stimulation, consistent with the belief that forskolin acts at a post-receptor site.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗

PubMed1985

Thyrotrophin stimulation of mitogenesis of the rat thyroid cell strain FRTL-5: a metaphase index assay for the detection of thyroid growth stimulators.

Thyroid-stimulating hormone (TSH) has been shown to stimulate mitosis in cultures of the continuous thyroid cell strain FRTL-5, and this system may be used to quantify the growth-promoting effects of thyroid stimulators. Removal of TSH from the culture medium led to a progressive decline in the metaphase index (MI) to zero, after 7 days. Thus the cell culture conditions may be manipulated so that metaphases are absent in control cultures, i.e. in the absence of TSH. Restimulation with TSH caused an increase in mitosis only after a lag-phase of 20-24 h. A maximum MI was observed between 40 and 50 h, with a secondary peak between 70 and 75 h. An immunoglobulin G (IgG) preparation from a thyrotoxic patient with a small goitre which was a potent stimulator of adenylate cyclase in these cells produced a similar time-course. A dose-response relationship to TSH was obtained 47 h after addition of the hormone. Significant stimulation was observed with 10 mu. TSH/l, and maximal stimulation with 1 unit TSH/l; the highest dose tested (10 units TSH/1) slightly decreased the MI below the maximum. Stimulation of these cells appeared to be TSH specific, since FSH, human chorionic gonadotrophin, LH and isoproterenol did not induce mitosis. Epidermal growth factor under the experimental conditions employed was unable to induce mitosis. However, an increase in mitosis was observed with the adenylate cyclase stimulator forskolin. These experiments confirm the mitogenic properties of TSH and we describe a metaphase index assay for the detection of thyroid growth promotors.

Animals↗

PubMed1984

A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland.

A technique for the cytochemical demonstration of peroxidase activity in unfixed guinea-pig thyroid tissue is described in this paper. The substrate 3,3'-diaminobenzidine tetrahydrochloride (DAB) is oxidized by the peroxidase to form an insoluble reaction product. Optimal results were obtained after 20 min incubation at 37 degrees C in reaction medium containing 1.4 mM DAB (in 0.1 M Tris-HCl) and 0.15 mM hydrogen peroxide at pH 8.0. Peroxidase activity was seen in the thyroid follicle cells as a diffuse brown reaction product (which was more dense and granular in erythrocytes). The enzyme activity was quantified using a scanning-integrating microdensitometer, and the effects of two specific peroxidase inhibitors were evaluated. Both 3-amino-1,2,4-triazole and methimazole inhibited peroxidase activity in the follicle cells (enzyme activity was still seen in the erythrocytes), maximal inhibition occurring at 10 mM. Stimulation of peroxidase in the thyroid was observed in vivo (1 I.U. TSH administered every 8 h for two days), with the maximal stimulation occurring after 1 day.

3,3'-Diaminobenzidine↗

PubMed1984

Further studies on the response of a cytochemical bioassay to thyroid stimulators, using reference preparations of thyrotropin and long acting thyroid stimulator.

The fundamental response of this assay is shown to be a shortening of a latency period prior to a peak of staining after tissue sections have been exposed to either TSH or LATS. The magnitude of the peak for a given tissue is both stimulator and dose independent. Simultaneous exposure to a combined dose of the two stimulator resulted in a further shortening of the latency period, and no evidence of two peaks was observed in the presence of both stimulators. Dose-response curves after exposure to reference preparations of the two stimulators for the same time period (180 s.) were parallel: this indicates that given incremental doses of either stimulator result in indistinguishable increases in the fundamental response of this assay.

Animals↗

PubMed1984

The application of a cytochemical bioassay to measure thyroid stimulating antibodies; a study of patients with euthyroid Graves' ophthalmopathy.

Using a cytochemical bioassay for thyroid stimulators, we have studied in detail the responses to IgG preparations from sera of 11 patients with euthyroid Graves' ophthalmopathy. This assay is based upon changes in naphthylamidase activity in response to the addition of thyroid stimulators. A wide range of dilutions of each IgG preparation was tested, and bell-shaped dose response curves were obtained where thyroid stimulating antibody (TSAb) was present. Such dose-response curves, giving a maximum response at one dilution (higher or lower concentrations giving a decreased or nil response) are characteristic of this assay. Results were expressed as a titer which is defined as the reciprocal of the dilution giving the maximum response for a given sample. Our routine strategy for titer determination for a given IgG preparation, including the use of "negative" and "positive" controls is described. Retesting of IgG's in separate bioassays showed that the titers were reproducible. Serum from one of the 11 patients was TSAb negative, i.e. did not provoke a response at any dilution tested in this ultrasensitive bioassay. The others were TSAb positive with a wide range of titers (1:10(2) to 1:10(8)). In addition one patient (initial titer 1:10(5)) was plasmapheresed, first in the absence of immunosuppression, when rebound resulted in a titer of 1:5 X 10(7), and secondly in combination with immunosuppression when the titer was reduced to less than 1:10(2). These large changes in titer for samples from one patient were consistent with the strikingly wide ranges of titers found for the 10 other patients.

Adult↗

PubMed1984

Characterization of monoclonal antibodies derived from lymphocytes from Graves' disease patients in a cytochemical bioassay for thyroid stimulators.

Monoclonal antibodies 208F7 and 307H6, derived from Graves' lymphocytes, were previously shown to stimulate thyroid function. We characterized these antibodies in the ultrasensitive cytochemical bioassay for thyroid stimulators. Bell-shaped dose-response curves were obtained for both antibodies, confirming their actions as thyroid stimulators; 307H6 was 10(7) times more potent than 208F7, and the ascending limb of the response curve to 307H6 was not significantly different from that of a reference preparation of thyroid-stimulating antibodies, namely LATS-B. Stimulation by both 208F7 and 307H6 was inhibited by antihuman, but not antimouse, immunoglobulin. Stimulation by 208F7, but not 307H6, was inhibited by 11E8 (a monoclonal antibody raised against the TSH receptor), which is a relatively potent inhibitor of TSH, but not thyroid-stimulating antibodies. These findings together with previous observations on the interactions of 208F7 and 307H6 with thyroid cells in both the presence and absence of TSH and of 208F7 and 307H6 with solubilized thyroid membrane components are summarized in a model relating the appropriate epitopic regions of the TSH receptor.

Animals↗