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S Costagliola

Publications and source records attributed to S Costagliola.

At least 55 records · Page 3Linked to original sources
PubMed1997

Diversity and prevalence of somatic mutations in the thyrotropin receptor and Gs alpha genes as a cause of toxic thyroid adenomas.

A total of 33 different autonomous hot nodules from 31 patients, originating mainly from Belgium, were investigated for the presence of somatic mutations in the TSH receptor and Gs alpha genes. This constitutes an extension of our previous study, including the first 11 nodules of the series. The complete coding sequence of the TSH receptor gene and the segments of Gs alpha known to harbor mutations impairing guanosinetriphosphotase activity were studied by direct sequencing of genomic DNA extracted from the nodules. DNA from the juxtanodular tissue or peripheral white blood cells was analyzed in all patients to confirm that the mutations identified were somatic. Twenty-seven mutations (82%) were found in the TSH receptor gene, affecting a total of 12 different residues or locations. All these mutations but 2 (see below) have been identified previously as activating mutations. Only 2 mutations were found in Gs alpha (6%). In 4 nodules, no mutation was detected. Five residues (Ser281, Ile486, Ile568, Phe631, and Asp633) were found mutated in 3 or 4 different nodules, making them hot spots for activating mutations. Phe631 and Asp633 belong to a cluster of 5 consecutive residues (629-633) in the N-terminal half of transmembrane segment VI; which harbor together 44% of the mutations identified in this cohort. Two novel mutations were identified: a point mutation causing substitution of Phe for Leu at position 629 (L629F); and a deletion of 12 bases removing residues 658-661 at the C-terminal portion of exoloop 3 (del658-661). When tested by transfection in COS-7 cells, both mutant receptors display increase in constitutive stimulation of basal cAMP accumulation. Although it is still capable of binding TSH, the del658-661 mutant has completely lost the ability to respond to the stimulation by the hormone. Our results demonstrate that, in a cohort of patients from a moderately iodine deficient area, somatic mutations increasing the constitutive activity of the TSH receptor are the major cause of autonomous hot nodules.

Adenoma↗
PubMed1996

Functional characteristics of a variant thyrotropin receptor.

A claim has been made that a variant of the human thyrotropin receptor in which Pro52 is replaced by Thr ([Thr52]thyrotropin receptor) is associated with autoimmune thyroid diseases and displays increased responsiveness to thyrotropin. We have analysed the functional characteristics of this variant receptor. Equivalent numbers of of the wild type and of the variant thyrotropin receptor, measured both by 125I-thyrotropin binding and by flow cytofluorimetry, were transiently expressed in COS-7 cells. Under these conditions, the two receptors showed the same degree of constitutive activity for the cAMP pathway, the same affinity for bovine thyrotropin, and a virtually identical responsiveness to bovine thyrotropin for activation of both the cAMP and inositol-phosphate regulatory pathways. Our results show that the [Thr52]thyrotropin receptor variant of the human thyrotropin receptor, which is present in the 12% of the population, does not affect receptor function and represents most likely a simple polymorphism.

Alleles↗
PubMed1996

Specific activation of the thyrotropin receptor by trypsin.

The identification of 16 different activating mutations in the TSH receptor, found in patients suffering from toxic autonomous adenomas or congenital hyperthyroidism, leads to the concept that this receptor is in a constrained conformation in its wild-type form. We used mild trypsin treatment of CHO-K1 cells or COS-7 cells, stably or transiently transfected with the human TSH receptor, respectively, and measured its consequences on the TSH receptor coupled cascades, i.e. cyclic AMP and inositol-phosphates accumulation. A 2-min, 0.01% trypsin treatment increased stably cyclic AMP but not inositol-phosphates formation. This was not observed after chymotrypsin, thrombin and endoproteinase glu C treatment. The TSH action on cyclic AMP was decreased by only 25%. The effect was also observed in cells expressing the dog TSH receptor. It was not observed in MSH receptor, LH receptor expressing or mock transfected cells (vector alone). It is therefore specific for the TSH receptor, for its action on the Gs/adenylate cyclase cascade, and for the proteolytic cleavage caused by trypsin. Using monoclonal (A. Johnstone and P. Shepherd, personal communication) and polyclonal antibodies directed against the extracellular domain of the TSH receptor, it was shown that treatment by trypsin removes or destroys a VFFEEQ epitope (residues 354-359) from the receptor. The effect mimics the action of TSH as it activates Gs alpha and enhances the action of forskolin. It is not reversible in 1 h. The results support the concept that activation of the receptor (by hormone, autoantibodies, mutations or mild proteolysis) might involve the relief of a built-in negative constrain. They suggest that the C-terminal portion of the large extracellular domain plays a role in the maintenance of this constrain.

Adenylyl Cyclases↗
PubMed1996

[Basedow disease following metastatic thyroid cancer].

OBJECTIVES: Metastatic thyroid carcinoma rarely provokes hyperthyroidism. We describe here the main characteristics of this association observed in a 48-year-old woman. CASE REPORT: The patient had presented signs of hyperthyroidism for one year. Clinical examination revealed severe Graves' disease with nodular goiter. Presurgery investigations demonstrated thyroid carcinoma and lung metastasis. Thyroid stimulating immunoglobulins were elevated. After total thyroidectomy, hyperthyroidism persisted, indicating the functional nature of the lung metastases. DISCUSSION: The effect of thyroid stimulating immunoglobulins on the clinical course of thyroid carcinoma and distant metastases remains unknown.

Female↗
PubMed1996

Self-reactive cytotoxic gamma delta T lymphocytes in Graves' disease specifically recognize thyroid epithelial cells.

In this paper we report the isolation of a self-reactive cytotoxic gamma delta T cell line, 158RE.2, that originates from the T lymphocyte population infiltrating the thyroid gland of a patient with Graves' disease. Functional data using this cell line demonstrate that gamma delta T cells expanded in the thyroid tissue specifically recognize a ligand expressed by thyroid epithelial cells and cell lines of endocrine epithelial origin. The TCR expressed by these gamma delta T cells--V gamma I/V delta 5--is unusual in peripheral blood lymphocytes, and its specificity is clearly different from that observed in a high percentage of gamma delta T cells from PBL, which express the common TCR V gamma 9/V delta 2. The V gamma I/V delta 5 receptor is involved in the recognition of the ligand expressed by the thyroid cells, but not in the NK-like activity also displayed by 158RE.2. These cells express CD8 alpha alpha dimers, which participate in the thyroid ligand recognition but not in the NK-like activity. The epithelial cell recognition is not restricted by classical MHC class I or class II molecules, although the CD8 alpha alpha participation in the recognition suggests the involvement of nonclassical MHC molecules. These are the first data to be presented on self-reacting gamma delta T cells in human epithelium.

Animals↗
PubMed1996

Cloning of candidate autoantigen carboxypeptidase H from a human islet library: sequence identity with human brain CPH.

A number of proteins, many of them enzymes, i.e. glutamic acid decarboxylase (GAD), carboxypeptidase H, 37-40 K tyrosine phosphatase (ICA512, IA2/IA2 beta), have been proposed as islet autoantigens involved in the pathogenesis of IDDM. Until recently, progress in their characterization has been impeded by the inaccessibility of the human pancreas, resulting in many of them being cloned from animal or non-islet sources. Carboxypeptidase H, one of these enzymes, has been cloned and sequenced from human brain and from rat islets but not from human islets. In this study, we describe the production of a human islet cDNA library and the cloning of islet CPH from it. Since CPH clones were also detected in a human thyroid library, we have sequenced CPH from these two endocrine tissue libraries and compared them to the known brain sequence. The sequences from islets and thyroid were identical and differed from brain only in the absence of a second ATG in the predicted 5'non-coding region. Northern blot analysis revealed the presence of an identical 2.5 kb transcript in human islets, thyroid and brain. The confirmation of the existence of a single isoform of CPH expressed in brain and endocrine tissues simplifies future experiments to elucidate the role of CPH as autoantigen.

Animals↗
PubMed1996

European collaborative study of LH assay: 3. relationship of immunological reactivity, biological activity and charge of human luteinizing hormone.

This report describes the results of the third part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The aim of the present work was to establish the link between immunoreactivity and biological activity of human LH, thus allowing to determine the antigenic domains of the molecule involved in the induction of the biological effect. The relationship between immunoreactivity and electric charge of hLH was also studied. This work allowed to further apprehend hLH isomorphism and its role in discrepancies observed among hLH assays and clinical status. It also made the feasibility of measuring biologically active isoforms by an immunological method to be assessed. The effect of 36 mAb with known epitopic specificity, was evaluated on both hLH binding to rat membrane receptor and hLH induced production of testosterone by porcine Leydig cells. All the epitopes located on the beta subunit were found to be strongly involved in the biological activity whereas 4/9 and 10/18 epitopes present on the alpha subunit or specific for the holomolecule respectively appeared weakly involved. Assaying biological hLH using immunological method would require that mAb specific for all the epitopes involved in the receptor activation be tested, and thus appears presently unsuitable for routine clinical evaluation. In the previous work some LH immunoassays were found to underestimate LH concentrations (J. Endocrinol. Invest 1994, 17: 397-406 and 407-416). The mAb used in liquid phase in these kits were found in the present work to be directed against the domains of LH weakly involved in the activation of the receptor and would suggest that bioactive LH would be misevaluated by these kits. The immunoreactivity of hLH isoforms separated by isoelectric focusing (IEF) in liquid phase was also determined. IEF allowed to separate three groups of hLH isoforms but none of them exhibited a specific discriminating pattern of immunoreactivity when they were tested against a panel of mAb. It suggests that, in our experimental conditions, the electric charge and the immunoreactivity of hLH were not closely linked.

Animals↗
PubMed1996

Transfer of thyroiditis, with syngeneic spleen cells sensitized with the human thyrotropin receptor, to naive BALB/c and NOD mice.

In previous studies we have induced TSH binding-inhibiting Igs and thyroiditis in BALB/c mice and thyroiditis alone in NOD mice immunized with the extracellular domain of the human TSH receptor produced as a maltose-binding protein fusion in bacteria (MBP-ECD). In this study, our aim was to determine whether thyroiditis can be transferred to syngeneic naive recipients with in vivo and in vitro primed spleen cells. Groups of 6-week-old female BALB/c and NOD mice were immunized ip with MBP-ECD in an adjuvant of alum plus attenuated Bordetella pertussis toxin, on days 0 (100 micrograms), 14, 28, and 35 (50 micrograms). These mice (in vivo primed) and nontreated age- and sex-matched controls were killed on day 43, and their spleens and thyroids were removed, the latter to verify the induction of thyroiditis in the antigen-treated mice. Splenocytes were disrupted mechanically and cultured at 3 x 10(6)/ml in RPMI supplemented with 20 micrograms/ml MBP-ECD for 48-64 h. After this in vitro priming, some of the splenocytes received no further treatment, but a portion was fractionated into a CD4+-enriched population. Groups of 6-week-old female BALB/c and NOD mice were immunized into the tail vein with 100-200 microliters PBS containing approximately 10(5)-10(7) unfractionated T cells (both in vivo primed and not) and CD4+-enriched (in vivo primed) splenocyte populations. The animals were killed 16 days later, and their thyroids were examined histologically and by immunohistochemistry. In addition, levels of antibody to the MBP-ECD priming antigen were assessed by enzyme-linked immunosorbent assay in the antigen- and spleen-treated mice. In the donor animals, in vivo priming resulted in an extensive lymphocytic infiltration of the thyroids in both BALB/c and NOD mice and follicular destruction in the latter. There was no evidence of thyroiditis in all 9 BALB/c mice and all 4 NOD mice who received unfractionated T cells from mice that had not been primed in vivo. In contrast, transfer of MBP-ECD in vivo primed unfractionated T cells resulted in thyroiditis in 9 of 13 BALB/c mice and 5 of 6 NOD mice; similarly, the equivalent CD4+-enriched population produced extensive thyroiditis in 2 of 3 BALB/c mice and all three NOD mice. The most striking difference between the antigen- and spleen-treated mice was in the quantity of the infiltrate, which was much greater in the latter and extended throughout the thyroid glands of these animals. In common with mice treated directly with the MBP-ECD antigen, the infiltrates of both BALB/c and NOD recipient mice contained large numbers of activated T cells expressing the receptor for interleukin-2, and macrophages and dendritic cells were plentiful, particularly in the BALB/c mice, in which B cells and interleukin-10-positive T cells were also present. The most abundant infiltrates, containing numerous CD8+ T cells and follicular destruction, were observed in NOD mice receiving primed unfractionated T cells or CD4+-enriched T cells. In contrast to the donors, none of the recipient animals had circulating antibodies to the MBP-ECD antigen. In conclusion, we have shown that it is possible to transfer thyroiditis with spleen cells from mice primed in vivo with a human TSH receptor preparation. Furthermore, the thyroiditogenic activity appears to reside in the CD4+ population.

Animals↗
PubMed1996

Patient with monoclonal gammopathy, thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy; demonstration of direct binding of autoantibodies to the thyrotropin receptor.

We describe a patient with monoclonal gammopathy who subsequently developed thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy. The pathogenesis of thyrotoxicosis in Graves' disease is due to the presence of autoantibodies that mimic the action of thyrotropin (TSH), called thyroid-stimulating antibodies (TS-ab); these antibodies may or may not inhibit the binding of TSH to the receptor (thyroid-binding inhibiting immunoglobulin, TBII). The patient's immunoglobulins were TS-ab positive and TBII negative when measured on CHO cells expressing the human TSH receptor. The pathogenetic link between the thyroid, orbit and skin is yet to be established but several candidate shared antigens have been proposed, including the TSH receptor itself. The monoclonal immunoglobulins were in evidence before the symptoms of pretibial myxedema, thyrotoxicosis and ophthalmopathy. In addition, the patient had no autoantibodies to thyroglobulin or thyroperoxidase, which are classic markers of thyroid autoimmunity. This combination led us to postulate that the monoclonal gammopathy could be the cause of all the observed pathology. One method to test this hypothesis would be to show that the monoclonal immunoglobulin is a TS-ab. Various methods were used to separate the monoclonal from the polyclonal components of the patient's serum. Preparative isoelectric focusing enabled us to obtain fractions containing only the monoclonal (as revealed by polyacrylamide gel electrophoresis), which were devoid of TS-ab activity (measured as cAMP accumulation in CHO cells expressing the human TSH receptor in a hypotonic bioassay). Subsequently, different oligoclonal fractions were shown to have varying degrees of TS-ab activity, with one fraction having faint biological activity and able to recognize a recombinant TSH receptor preparation in a Western blot. In conclusion, the monoclonal antibody does not seem to be responsible for the thyrotoxicosis, pretibial myxedema and ophthalmopathy. We confirm previous data showing that TSH receptor antibodies in patients with Graves' disease are heterogeneous in nature and we present the first demonstration of autoantibodies capable of binding the TSH receptor but devoid of TBII activity.

Adult↗
PubMed1995

The thyroperoxidase doublet is not produced by alternative splicing.

Thyroperoxidase is a membrane-bound, heme-containing enzyme which catalyses iodination of thyroglobulin and coupling of resulting iodotyrosines to produce thyroid hormone. In addition to the full length molecule of 933 amino acids (TPO1), Northern blotting and sequencing have revealed several shorter transcripts. The most abundant is a species lacking 171 nucleotides in which the alternative splicing results in the deletion of codons 533-590 in exon 10 (TPO2). Evidence for TPO2 transcripts being translated into a protein is lacking, but in Western blots TPO invariably appears as a doublet of 110 and 105 kDa. In the present study we have produced two recombinant fusion proteins for: (i) the 57 amino acids which are spliced out in TPO2 and (ii) for the 20 amino acids which bridge the splice site (10 amino acids on both sides). Both recombinant fragments have been produced in the pMAL-cRI vector as a maltose-binding protein (MBP) fusion, permitting their purification from a bacterial lysate on an amylose column. Rabbits have been immunized by intradermal injection of 500 micrograms of fusion protein, initially in complete Freund's adjuvant followed by two boosts, at 2-week intervals, in incomplete Freund's adjuvant. The resulting high titre immune sera (IS) were reactive with the relevant immunising antigens, when tested by ELISA. Depletion of each serum by passage through an MBP-CNBr Sepharose column allowed purification of antibodies against the relevant peptides, as demonstrated by ELISA with the appropriate fusion protein and MBP. This demonstrates that we have produced specific polyclonal antibodies for the 57 amino acids unique to TPO1 and for the amino acid segment bridging the splice site, found in TPO2. These polyclonal antibodies were used in Western blotting experiments with normal and Graves' thyroid membranes, in reducing and non-reducing conditions. Monoclonal 47/C21 which recognises a linear epitope (amino acids residues 710-722) common to TPO1 and TPO2 was used as a control. In non-reducing conditions, we observed a broad signal at 105-110 kDa, which appeared to comprise two bands, with both polyclonal antibodies and the monoclonal. There was no difference in the image between the normal and the Graves' thyroid. In reducing conditions, the broad signal resolved clearly into two distinct bands, one at 105 and the other at 110 kDa. Once again we observed exactly the same pattern of reactivity with all three antibodies both in normal and Graves' glands. We conclude that the TPO doublet is not the consequence of translation of TPO2.

Alternative Splicing↗
PubMed1995

The autoimmune response induced by immunising female mice with recombinant human thyrotropin receptor varies with the genetic background.

In a previous study, we have described the induction of thyroid blocking (TBAB) and thyrotropin binding inhibiting antibodies accompanied by thyroiditis in female BALBc mice (H2d) immunised with the extra-cellular domain (ECD) of the human thyrotropin receptor (TSHR) expressed as a maltose binding protein (MBP) fusion. In the present study we have investigated the response induced in mice of varying MHC haplotype. Two groups of female NOD (H2g), CBA (H2k) and C57 (H2b) mice were immunised intra-peritoneally with MBP-ECD or MBP on days 0 (100 micrograms), 15, 30 and 43 (50 micrograms). Blood samples from individual mice were obtained on days 0, 22, 36 and 50 and assessed for thyroid binding inhibiting immunoglobulins (TBII), thyroid stimulating (TSAB) and TBAB. On day 50 the treated mice and five age/sex matched NOD mice were sacrificed, their thyroids removed, examined histologically and any infiltrate characterised. Induction of antibodies to the ECD was tested by ELISA in which plates had been coated with either MBP-ECD or an ECD-protein A fusion. All of the mice developed a strong antibody response to the relevant immunogen but none of them contained TBII, TSAB or TBAB activities. No lymphocytic infiltration of the thyroid glands of the CBA or C57 mice was observed. In contrast, all of the NOD mice displayed severe thyroiditis, whilst one of seven MBP-treated mice had moderate infiltration and none of five untreated controls. Immunohistochemical analysis revealed that the infiltrate was predominantly activated T helper cells with little evidence of B cells or the cytokines IL-10 or IL-4, indicating that a Th1 response had been induced, contrary to our findings in BALBc mice which mount a Th2 response. In conclusion we have shown that the type and extent of response induced by immunising with the TSHR varies in mice of differing genetic background. H2d mice develop thyroiditis and TBAB/TBII, H2g mice develop thyroiditis in the absence of functional TSHR antibodies, whilst H2b and H2k mice are resistant.

Animals↗
PubMed1995

Mapping thyroid peroxidase epitopes using recombinant protein fragments.

The identification of autoantibody epitopes is important to the understanding of autoimmune thyroid diseases. In the case of thyroid peroxidase antibodies (TPO-ab), recent reports have disagreed about the number and type of autoantibody epitopes found in human TPO. In order to clarify the nature of these epitopes, we used an approach that provides recombinant human TPO produced by bacterial cells. The cDNA of four slightly overlapping fragments of human TPO-TPO 1(Glu 17-Ser 227), TPO 2(Tyr 226-Thr 476), TPO 3(Glu 471-Ser 720) and TPO 4(Phe 709-Leu 993)--were amplified by polymerase chain reaction and subcloned into the expression vector pMAL. In addition, a TPO 3 species for an alternatively spliced form of TPO of 876 amino acids was constructed (TPO 3M). Each of these constructs encodes a fusion protein, in which the amino terminal portion is maltose-binding protein, followed by the sequence of the fragment of human TPO. The plasmid constructs were transformed in Escherichia coli and, after growth, bacterial cells were harvested, lysed and the lysate was passed over an amylose affinity column and eluted with maltose. Western blots were performed using 33 sera from patients with autoimmune thyroid disease (group 1) and 17 sera from patients with nodular goiter and focal thyroiditis (group 2), all positive for TPO-ab measured by radio-immunoassay; sera from 10 healthy people with no clinical evidence of thyroiditis and positive for TPO-ab measured by radioimmunoassay (group 3) and sera from 30 patients with antigastric parietal cell antibodies without signs or symptoms of thyroiditis, 16 negative for TPO-ab (group 4a) and 14 positive for TPO-ab (group 4b), were included in the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Identification of the thyroid Na+/I- cotransporter as a potential autoantigen in thyroid autoimmune disease.

The thyroid gland is the target of several autoimmune diseases. Specific thyroid proteins have been identified as autoantigens associated with these diseases (e.g. thyroperoxidase, thyroglobulin and the thyrotrophin (TSH) receptor). In this paper, we report that the serum of a patient suffering from Hashimoto's thyroiditis, autoimmune gastritis and rheumatoid arthritis was able to inhibit the chronic TSH-induced I- uptake of dog thyrocytes in culture, even at a 1:1000-fold dilution, without affecting their 86Rb+ uptake. This blocking activity is rare as 147 sera (from patients positive for antibodies to the thyroid microsomes and the gastric parietal cell antigen, patients with Sjögren's syndrome, patients with a high titre of microsomal antibodies and low or negative for antibodies to thyroperoxidase, and patients with a high titre of microsomal antibodies and frank hypothyroidism) were negative when tested for their ability to inhibit I- uptake. Subsequently we tested 20 murine monoclonal antibodies previously obtained by immunizing mice with a crude human thyroid membrane preparation, which were all negative when tested against thyroglobulin and thyroperoxidase. One of the monoclonal antibodies displayed a 50% inhibition of the chronic TSH-induced 125I- uptake of dog thyrocytes without affecting the 86Rb+ uptake of the cells. Immunoglobulins purified from the ascite fluid by affinity chromatography on a protein A cellulose column had the same characteristics. Taken together, the data suggest that thyroidal 125I- uptake can be inhibited by antibodies, that autoantibodies in the patient's serum are most probably responsible for the observed inhibition and therefore that the Na+/I- cotransporter is probably an autoantigen.

Animals↗
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

European collaborative study of luteinizing hormone assay: 1. Epitope specificity of luteinizing hormone monoclonal antibodies and surface mapping of pituitary and urinary luteinizing hormone.

This report describes the results of the first part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The whole study was designed to understand the causes of discrepancy among LH immunoassay methods. In the parent work, we studied the characteristics of 55 monoclonal antibodies to LH which allowed us to establish a detailed map of the antigenic surface of the hormone. In the present report we used this information to interpret the discrepancy in LH concentrations assayed with 12 different methods in 300 sera from subjects with various clinical conditions. The 55 monoclonal antibodies provided by 11 commercial companies were tested in various experiments: the apparent affinity of the antibodies was, generally but not always, higher for LH presented by a second antibody coated to plastic than for LH directly coated to plastic; 26% of the antibodies recognized the alpha subunit, 26% the beta subunit and 48% reacted only with the holomolecule (anti-alpha beta); only 28% of the antibodies were strictly specific for LH. Criss-cross experiments allowed us to distinguish 13 antigenic regions on the surface of LH: 6 were located on the alpha subunit, 3 on the beta subunit and 4 on the holomolecule. The monoclonal antibodies to the alpha beta regions further separated into 12 clusters of reactivity. Accordingly, LH appeared to exhibit at least 21 epitopes. Comparison of the immunoreactivity of various LH preparations indicated that highly purified pituitary LH and immunoaffinity purified urinary LH reacted similarly with the monoclonal antibodies and strongly differed from crude urinary LH. These data indicated that the immunoreactivity of an LH preparation depends mainly upon the degree of purification and not that much upon the origin of the preparation. The epitope specificity of the monoclonal antibodies used in 11 commercially available LH assay kits was also determined: 10 kits used at least one anti-alpha beta monoclonal antibody associated with an anti-beta monoclonal antibody in 7 cases or another anti-alpha beta monoclonal antibody in 3 cases; one kit used an anti-alpha monoclonal antibody associated with an anti-beta monoclonal antibody. None of the kits were strictly identical with regards to the epitope specificity of the monoclonal antibodies used.

Antibodies, Monoclonal↗
PubMed1994

European collaborative study on luteinizing hormone assay: 2. Discrepancy among assay kits is related to variation both in standard curve calibration and epitope specificity of kit monoclonal antibodies.

This report describes the results of the second part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The whole study was designed to understand the causes of discrepancy among LH immunoassay methods. In the parent report we described the characteristics of LH monoclonal antibodies. In the present work we focused on the comparison of 11 commercially available monoclonal antibody based kits and one polyclonal antibody based RIA. Recovery experiments of the second IS 80/552 and of a related LH preparation showed that curve calibration differed among kits. The ratio between the LH recovery of the two most different kits was 2.30. LH concentrations were determined, using the 12 assay methods in the sera of prepubertal children (n = 46), normal women (n = 26) and men (n = 39) before and after LHRH stimulation, post-menopausal women (n = 29) and patients with renal failure (n = 71) or polycystic ovaries (n = 28). In children LH was detected in 0 to 80% of the sera depending on the kit. In adults, the mean LH concentration provided by the 12 kits varied from 6.0 to 13.55 IU/L showing a ratio of 2.26 between the two most different kits. A thorough statistical analysis allowed to distinguish two groups of kits. The first groups consisting of 6 kits provided results close to those obtained by the RIA. The 5 other kits misrecognized circulating LH in subjects with various clinical status. These 5 kits were the only ones to use, as labelled probes, monoclonal antibodies specific for the holohormone (anti-alpha beta) whereas the 6 other kits used, as labelled probes, monoclonal antibodies directed to the alpha (1 kit) or the beta-subunit (5 kits). In both groups, the coated monoclonal antibodies are directed to either the beta-subunit (1/6 and 2/5 kits) or the holohormone (5/6 and 3/5 kits). Taken together these data suggest that discrepancy among LH assay kits is related to variation in standard curve calibration and in epitope specificity of monoclonal antibodies used in the kits. These findings may prove to be instrumental to alleviate differences among LH assay methods.

Adult↗