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Biomedical subjects

M Ludgate

Publications and source records attributed to M Ludgate.

At least 55 records · Page 3Linked to original sources
PubMed1994

Western blot analysis of thyrotropin receptor expression in human thyroid tumours and correlation with TSH-binding.

Expression of thyrotropin receptor (TSHr) was analyzed in normal human thyroids and differentiated thyroid tumours by Northern blotting of total RNA and by Western blotting of detergent-solubilized membrane proteins with an antibody specific for the extracellular domain of TSHr. Under nonreducing conditions TSHr stained as a 90-kDa protein in normal thyroid tissue. Expression varied in differentiated carcinomas from normal to not detectable in parallel to steady state TSHr mRNA levels and TSH binding in a TSH binding assay. The negative expression of TSHr in some patients with differentiated carcinomas may have consequences for the TSH suppressive treatments of these patients.

Binding Sites↗
PubMed1994

Induction of thyrotropin receptor (TSH-R) autoantibodies and thyroiditis in mice immunised with the recombinant TSH-R.

Two groups of 10 Balbc by Jico male mice were immunised on days 0, 15 and 35, with the extra cellular domain (ECD) of the human thyrotropin receptor (TSH-R) expressed as a fusion protein in bacteria (group 1) or with the maltose binding protein (MBP) fusion partner alone (group 2). Blood was obtained on days 0, 22, 32, 42 and 49 and samples from the individual animals pooled for each group. Serum and immunoglobulin (IgG) preparations were tested, using CHO cells expressing the human TSH-R (JP26 and JP09) for thyroid stimulating (TSAb); thyroid blocking (TBAb) and thyrotropin binding inhibiting (TBII) activities. Neither serum nor IgGs were found to contain TSAb at any time point. TBII activity was present in the serum of both groups on day 32 and in group 1 only on day 49; when the test was performed on IgGs, only the MBP-ECD day 49 preparation remained significantly positive for TBII (p < 0.005). Significant TBAb activity was present in both the serum and IgG of group 1 day 49 (p < 0.005) and to a lesser extent on 42 (p < 0.02). Following the second immunisation (day 15) both groups and had decreased circulating T4 levels (p < 0.05) when compared with day 0 in each case. Group 2 were unaffected by the third immunisation on day 35 but the MBP-ECD group again had significantly decreased T4 levels (p < 0.02) compared with MBP day 49 and (p < 0.03) when compared with MBP-ECD day 0. Histological examination of thyroids from group 1 animals revealed extensive vascularisation and an atypical lymphoblastoid infiltration which was not observed in control mice. These preliminary results indicate that care is required in interpreting data since a non-receptor antigen was shown to decrease circulating thyroxine and serum from these animals had apparent TBII like activity. However, the results obtained with the IgGs suggest that receptor autoantibodies can be induced by immunising with the human TSH-R, in addition, the immunised mice show histological evidence for the development of thyroiditis.

Animals↗
PubMed1994

Antibodies against 1D, a recombinant 64-kDa membrane protein, are associated with ophthalmopathy in patients with thyroid autoimmunity.

We have tested for serum antibodies reactive with 1D, a recombinant 65-kDa human thyroid protein which is also expressed in eye muscle, in patients with thyroid autoimmunity and ophthalmopathy by immunofluorescence and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. We also measured antibodies to a 64-kDa pig eye muscle membrane protein which is identified by SDS-PAGE and Western blotting, correlating the two reactivities. While antibodies to 1D, expressed in Chinese hamster ovary (CHO) cell membrane, were detected in approximately 40% of patients with ophthalmopathy, in both tests the greatest prevalence, by immunofluorescence, 73%, was demonstrated in patients with Graves' hyperthyroidism without clinically evident eye disease, although only 50% of these patients were positive in immunoblotting. When the two tests for anti-1D antibodies were compared, immunofluorescence appeared to be the more specific and immunoblotting appeared to be the more sensitive. The greatest prevalence of antibodies reactive with a 64-kDa pig eye muscle protein, 71%, was in patients with TAO of less than 1 year duration; tests were positive in 49% of patients with more chronic ophthalmopathy and in 50% of patients with Graves' hyperthyroidism without evident eye disease. Antibodies reactive with 1D were detected in 17% of normals by immunofluorescence and 24% by immunoblots, while antibodies reactive with the 64-kDa pig eye muscle protein were detected in only 10% of the normal subjects tested. Lesser prevalences of antibodies to the two 64-kDa proteins in patients with established eye disease suggest that such antibodies may be an early abnormality in patients with Graves' hyperthyroidism who are predisposed to develop ophthalmopathy. Although the association was not close, reactivity against 1D by immunoblotting, but not immunofluorescence, was significantly correlated with reactivity to a 64-kDa eye muscle membrane protein by immunoblotting. On the other hand, when sera containing antibodies reactive with both 1D and the 64-kDa eye muscle protein were incubated with CHO (1D) cell membrane, reactivity against 1D was absorbed while that against the eye muscle protein was not. The precise relationship between the two 64-kDa proteins can only be clarified by cloning the 64-kDa protein from an eye muscle expression library and comparing the sequences with those of 1D.

Adolescent↗
PubMed1994

Detection of antibodies blocking thyrotropin effect using Chinese hamster ovary cells transfected with the cloned human TSH receptor.

Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) were used to develop an assay to detect thyroid autoantibodies blocking the TSH-dependent cAMP production (TSHBAb). The study group included 38 patients with goitrous Hashimoto's thyroiditis (HT) and 47 subjects with atrophic thyroiditis (AT). In the HT group, 8 patients had subclinical hypothyroidism (HT-SH) and 30 had overt hypothyroidism (HT-H). Thirty normal subjects served as controls. Immunoglobulin G (IgG) was prepared from serum by double chromatography on DEAE-Sephadex. CHO-R cells were seeded in 96-well plates and were cultured for 48 h before the assay in RPMI-1640 medium plus 1 mmol/L glutamine, 10% fetal calf serum, and 0.4 g/L geneticin. In the assay for TSHBAb, CHO-R cells were incubated with IgG alone (0.5-2 mg/ml), TSH alone (0.2-625 mU/L), or IgG plus TSH; all samples were diluted in hypotonic medium containing 0.5 mmol/L isobutylmethylxanthine (IBMX). After 2 h of incubation at 37 degrees C in 5% CO2-95% air atmosphere, TSH-stimulation was quantified by measuring extracellular cAMP by a RIA. IgGs from normal subjects did not significantly modify the stimulation of adenylate cyclase produced by TSH, the results obtained ranging between -30% and +18% (mean +/- SD = -3 +/- 14%). All IgGs producing an inhibition greater than 2SD from the mean of controls (> 25%) were considered positive for blocking antibodies. TSHABAb were detected in 1/8 (12.5%) patients with HT-SH, in 7/30 (23.3%) with HT-H and 16/47 (34.0%) patients with AT.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Identification of high molecular weight forms of inositol 1,4,5-trisphosphate 3-kinase in rat thymus and human lymphocytes.

A soluble inositol 1,4,5-trisphosphate 3-kinase (InsP3 3-kinase) has been characterized from extracts of rat thymus. The enzyme was shown to have a molecular weight within the range 98,000-114,000 M(r) as determined by regeneration of enzyme activity from sodium dodecyl sulphate polyacrylamide gels. The enzyme phosphorylates inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5-tetrakisphosphate (InsP4) with an apparent Km of 3.1 +/- 0.4 microM. The enzyme is stimulated 4-6-fold by Ca2+/calmodulin and is not recognised by polyclonal antisera raised against rat brain InsP3 3-kinase A. High levels of InsP3 3-kinase activity were also detected in soluble extracts of human lymphocyte preparations. The human lymphocyte enzyme was shown to have a molecular weight between 61,000 and 70,000 M(r) as judged by SDS-PAGE, and was stimulated approximately 10-fold in the presence of Ca2+/calmodulin. These results establish that InsP3 3-kinase from rat thymus and human lymphocyte preparations represent high molecular weight isoenzymes of the InsP3 3-kinase family.

Animals↗
PubMed1994

T cell responses to orbital antigens in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is most likely to be a T cell-mediated disease, in which cytokines released in the extraocular muscles activate fibroblasts, increasing glycosaminoglycan production. The nature of the orbital antigen recognized by the infiltrating T cells is unclear, although it is possible that there is cross-reactivity between this and a thyroid autoantigen to explain the close association with thyroid autoimmunity. We have tested the ability of human and porcine eye muscle antigen preparations to stimulate proliferation of circulating T cells from healthy subjects and patients with TAO or Graves' disease without clinical TAO. Occasional responses were seen, particularly after depletion of CD8+ T cells, and two out of 10 TAO patients responded to eye muscle proteins of 25-50 kD after fractionation of antigens on gels and subsequent elution. There was no disease-specific response of T cells to R1, R14, D1 and 1D3, recombinant proteins identified from screening an eye muscle cDNA library with sera from patients with autoimmune thyroid disease. We have also found that interferon-gamma (IFN-gamma) production by T cells from TAO patients was not stimulated by eye muscle membrane antigens or by 1D3. These results suggest that the frequency of circulating T cells responding to eye muscle antigens in TAO is low, and that several candidate orbital antigens, including the 64-kD protein 1D3, are unlikely to be important T cell autoantigens in this condition.

Animals↗
PubMed1994

Recombinant thyrotropin receptor and the induction of autoimmune thyroid disease in BALB/c mice: a new animal model.

In a preliminary study, we observed the production of TSH binding-inhibiting (TBII) and thyroid-blocking (TBAb) antibodies accompanied by lymphocytic infiltration of the thyroid in a pool of male BALB/c mice immunized with the extracellular domain (ECD) of the human TSH receptor (TSHR) expressed as a maltose-binding protein (MBP) fusion in bacteria. In the present study we evaluated the humoral response to the same antigenic preparation in a new series of individual male and female BALB/c mice immunized ip on day 0 with 100 micrograms MBP-ECD and days 25, 39, and 53 with 50 micrograms MBP-ECD in an adjuvant composed of aluminum oxide, magnesium hydroxide, and Bordetella pertussis vaccine. Mice immunized with MBP served as control. Individual sera and immunoglobulins were tested for TBII, TBAb, and thyroid-stimulating antibodies (TSAb) on days 0, 32, 46, and 60, and total circulating T4 levels were measured by RIA. Animals were killed on day 120, their thyroids were examined histologically, the infiltrates were characterized using monoclonal antibodies specific for T-cells (total, activated, helper, and suppressor), B-cells, and macrophages. Sera and immunoglobulins G of the MBP-treated control group were all negative for TSAb, TBAb, and TBII activity. The receptor-immunized mice, despite having high titers of antibodies to the immunogen in an enzyme-linked immunosorbent assay, displayed a heterogeneous response in terms of biological activity, with 3 of 7 female and 4 of 8 male mice having TBAb/TBII activities that persisted and whose activity increased throughout the experiment. No significant TSAb antibody activity was observed. Total T4 levels were also heterogeneous even before immunization, but 9 of 15 MBP-ECD-treated mice had levels below the normal range after immunization, and 7 of these also had TBII/TBAb activities. At the end of the experiment, only 4 of the MBP-ECD-treated female mice survived, but all of them had a severe lymphocytic infiltration of their thyroid, composed mostly of activated T-cells, although B-cells and macrophages were also present. A similar infiltrate was seen in 4 of 8 male MBP-ECD-treated mice. No infiltrate was observed in male or female MBP-treated mice. The model described demonstrates the feasibility of using the TSHR as an immunogen to overcome tolerance and mimics some characteristics of human autoimmune disease of the thyroid.

Animals↗
PubMed1994

Presence of nonfunctional thyrotropin receptor variant transcripts in retroocular and other tissues.

The TSH receptor (TSHR) has been proposed as an antigenic link between the thyroid and the orbit; TSHR transcripts have been demonstrated by other groups, one in orbital tissue and the other in orbital and dermal fibroblasts. In a previous study we were unable to demonstrate transcripts for the complete TSHR in retroocular muscle containing also fibroblasts. We now confirm this finding. A 1.3-kilobase variant of the TSHR messenger ribonucleic acid (mRNA) has been described in normal and Graves' thyroids; it contains exons 1-8 of the major mRNA species and a unique 3'-sequence predicted to encode further amino acids and a polyadenylated tail. Lacking the membrane-spanning region, the corresponding variant protein, if expressed, is not expected to couple to G-proteins. Using primers specific for this variant in reverse polymerase chain reaction experiments, Southern blotting and frequencies, we demonstrate the presence of this transcript in normal and Graves' thyroid, extraocular muscle, peripheral blood mononuclear cells, and, to a lesser extent, in fat and fibroblasts. TSH-mediated protein synthesis, cAMP, and glycosaminoglycan production have been measured in cultured fibroblasts. At 5 mU/mL, bovine TSH stimulated glycosaminoglycan production, but recombinant TSH did not, even at higher concentrations, suggesting that contaminating factors are responsible. Together the data do not support the presence of a functional complete TSHR in orbital tissue. However, they are compatible with a role for the extracellular portion of the receptor as a nonfunctional autoantigen and provide some explanation for the conflicting results with regard to the relevance of the TSHR in the pathophysiology of thyroid-associated ophthalmopathy.

Actins↗
PubMed1994

Overexpression of the extracellular domain of the thyrotrophin receptor in bacteria; production of thyrotrophin-binding inhibiting immunoglobulins.

The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21-414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting. To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1993

Lack of evidence supporting the presence of mRNA for the thyrotropin receptor in extra-ocular muscle.

The search for autoantigenic targets in thyroid associated ophthalmopathy (TAO) and a possible link with the thyroid continues. Several authors have presented data suggesting that extrathyroidal tissues, e.g. extraocular muscle (EOM), may contain a functional thyrotropin receptor (TSH-R). We have applied Northern blotting and PCR amplification in an attempt to demonstrate the mRNA for the TSH-R in EOM, which although predominantly composed of muscle, may contain a significant proportion of fibroblasts. In Northern blots of poly(A) mRNA of normal human thyroid, EOM and skeletal muscle (SM) probed with a p32 labeled hTSH-R cDNA, transcripts of 4.6 and 4.4 kb were observed only in the thyroid. PCR amplification was performed on thyroid and EOM cDNAs using pairs of primers designed to amplify exons 1-->9, the extracellular domain (ECD), and exon 10, the intracellular domain (ICD). Both the ECD and ICD were amplified from a thyroidal cDNA library but only the ICD in the EOM cDNA library, this probably being due to the inevitable contamination with genomic DNA. These results do not support the hypothesis that the TSH receptor, as such, would be an autoantigen of EOM involved in TAO.

Adipose Tissue↗
PubMed1993

Antibodies reactive with an intracellular epitope of a recombinant 64 kDa thyroid and eye muscle protein in patients with thyroid autoimmunity and ophthalmopathy.

We have developed an enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies reactive with a 98 amino acid fragment, called D1, of a recombinant thyroid and eye muscle membrane protein corresponding to a MW of 64 kDa (called 1D) in the serum of patients with thyroid autoimmunity with and without ophthalmopathy. Antibodies against the D1 fragment expressed as a fusion protein with beta galactosidase, were detected in 29% of patients with thyroid-associated ophthalmopathy (TAO) of < 1 yr duration, in 33% of those with disease of > 3 yr duration, in 40% of patients with Graves' hyperthyroidism (GH) without evident eye disease, in 31% of patients with lid lag and retraction but no other signs of progressive ophthalmopathy, in 25% of patients with euthyroid Graves' disease and in 43% of patients with untreated Hashimoto's thyroiditis (HT), but in none of 14 patients with other (non-immunological) thyroid disorders. Although tests were positive in 6 out of the 15 patients with ophthalmopathy and no overt thyroid autoimmunity overall there was no close association of the antibodies with clinical features of the eye disease or its course. In those sera in which Western blotting for antibodies reactive with a 64 kDa eye muscle membrane protein and ELISA were both carried out there was no close correlation between the two tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Detection of thyroid-stimulating antibody using Chinese hamster ovary cells transfected with cloned human thyrotropin receptor.

A clone of Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) was used to optimize an assay for thyroid-stimulating antibody (TSAb), measuring adenylate cyclase stimulation by purified immunoglobulin G from patients with Graves' disease. Optimal sensitivity to bovine TSH (1 mU/L) and TSAb was obtained using hypotonic buffer and measuring extracellular cAMP. In time-response experiments, TSAb stimulation was maximal after 2 h of incubation in hypotonic buffer. Under these conditions, a significant stimulation by Graves' immunoglobulin G was obtained with 33 of 35 (94%) samples from patients with untreated Graves' disease and with 21 of 23 (91%) from patients who relapsed after a course of antithyroid drugs. On the other hand, TSAb was detected in only 12 of 20 (60%) patients who were euthyroid during methimazole treatment and in 4 of 11 (36%) who were euthyroid after a course of antithyroid drugs. All samples from Graves' patients were also tested for TSAb activity on FRTL-5 cells. The results of cAMP stimulation in FRTL-5 and CHO-R showed a fairly good correlation (r = 0.60; P < 0.0001). In particular, of the 58 patients with active Graves' disease (35 with untreated hyperthyroidism and 23 relapsed after methimazole), 43 (74%) were positive in both assays, 3 (5%) were negative in both, 11 (19%) were negative in FRTL-5 and positive in CHO-R, and 1 (1.7%) was negative in CHO-R and positive in FRTL-5. In conclusion, CHO cells transfected with the cloned human TSH receptor are suitable for the clinical assay of TSAb. The sensitivity of this assay is higher than that obtained using FRTL-5 cells, having the additional advantages of expressing the human TSH receptor and requiring less cumbersome procedures for cell culture.

Adenylyl Cyclases↗
PubMed1993

Tissue specificity and serologic reactivity of an autoantigen associated with autoimmune thyroid disease.

A recent report has identified a new autoantigen called D1 that appears to be associated with Graves' ophthalmopathy and is expressed in the thyroid and eye muscle. To better characterize the tissue specificity and disease relevance of this antigen, we evaluated the expression of D1 RNA in various human tissues using a reverse transcriptase polymerase chain reaction assay. These studies indicate a wide tissue distribution of the messenger RNA for this antigen, including the thyroid, eye muscle, parathyroid, spleen, skeletal muscle, and uterus. There were variations in the relative amounts of specific message for D1 in the different tissues, with the uterus, thyroid, and eye muscle having the greatest amount of product per microgram of total RNA. A maltose binding protein-D1 fusion protein was expressed in Escherichia coli, purified, and used to assess serologic reactivity to D1 by Western blot. Autoantibodies to this antigen were noted in 19 of 24 (78%) of Hashimoto's disease patients, 26 of 41 (63%) of Graves' disease patients, and in 9 of 17 (53%) of normal controls. Sixty percent of Graves' disease patients with clinical ophthalmopathy had antibodies to D1, as did 63% of Graves' patients without signs or symptoms of clinical ophthalmopathy. There was no correlation between reactivity to D1 and either clinical measures of hyperthyroidism or antibody titers to thyroid peroxidase or thyroglobulin. The presence of autoantibodies to this antigen in patients with Hashimoto's disease, in Graves' disease patients without ophthalmopathy and in normal controls indicate that serologic recognition of this antigen is not restricted to patients with ophthalmopathy. In addition, the expression of messenger RNA for this antigen in multiple types of cells questions the tissue specificity of this autoantigen.

Adult↗
PubMed1992

Thyroid stimulating immunoglobulins, like thyrotropin activate both the cyclic AMP and the PIP2 cascades in CHO cells expressing the TSH receptor.

In human thyrocytes and in a permanent CHO cell line expressing the human thyroid stimulating hormone (TSH) receptor cDNA (JP09 cells), TSH activates both the cyclic AMP and the phosphatidylinositol 4,5-bisphosphate (PIP2) cascade, although the latter effect requires higher TSH concentrations. Thyroid stimulating autoantibodies (TSAb) activate also the human thyroid leading to the hyperthyroidism of Graves' disease. They bind to the TSH receptor and mimic the TSH stimulation of the gland by increasing intracellular cyclic AMP, but they do not enhance PIP2 hydrolysis in human thyroid slices. We show in this study that TSAb are able to activate the PIP2 cascade in JP09 cells, a cell line expressing high levels of TSH receptor. This suggests that the mechanism of action of TSAb on the TSH receptor is qualitatively similar to that of TSH.

Animals↗
PubMed1992

Recombinant TSH-receptor for determination of TSH-receptor-antibodies.

We have expressed the complete coding sequence of the human TSH-R in eucaryotic cells. Limiting dilution enabled us to select two clones, JP09 and JP26, which have formed the basis of binding and bioassays respectively, for TSH-R antibodies. Results obtained in the binding assay correlated well with those obtained in the TRAK (Henning Berlin) assay, while the bioassay was at least as sensitive as measurements made with FRTL5 or human thyroid cells. These cell lines provide a reliable source of human TSH-R which will be useful in routine diagnosis.

Animals↗
PubMed1992

Characterization of monoclonal antibodies to the human thyrotropin receptor.

We have produced four monoclonal antibodies (mAbs), 34A, 49G, 11E7, and 12E3, which bind the human TSH receptor (hTSH-R) when expressed on a human thyroid cell line (GEJ), freshly dissociated human and murine thyroid cells, or Chinese hamster ovary cells stably transfected with the hTSH-R gene. These mAbs were obtained after immunization of DBA/1 mice with affinity-purified TSH-binding sites from GEJ cells. Biochemical studies, including sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, Western blot, and immunoprecipitation of solubilized GEJ cell membranes or human thyroid cells showed that most of the mAbs recognized two bands: one located at 46-48 kilodaltons and the other at 86-88 kilodaltons. Inhibition of [125I]hTSH binding to solubilized porcine membranes (TSH-receptor auto-antikörper assay) or Chinese hamster ovary cell membranes previously transfected with hTSH-R gene showed that mAb 34A recognizes the hTSH-binding site of both receptors. In contrast, mAbs 49G, 11E7, and 12E3 recognize a structure located near the hTSH-binding site. Lastly, the ability of these mAbs to stimulate murine thyroid function was investigated by measuring cAMP production and iodide accumulation. The 34A mAb, which fully competes with [125I]TSH for binding to hTSH-R, was able to induce both functions. Conversely, the 12E3 mAb, which was the least potent inhibitor of [125I]TSH binding to hTSH-R-transfected cells had no effect. A relationship was, therefore, established between the capacity of mAb to hTSH-R to inhibit [125I]hTSH binding and their ability to induce thyroid functions.

Animals↗