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*
IRIBHN, ULB,
Euroscreen s.a., and
Laboratory of Histology, Louvain Medical School, Brussels, Belgium; and
§
Department of Pathology, University of Wales College of Medicine, Cardiff, Wales, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to its natural ligand, the TSHR is the target of thyroid-stimulating autoantibodies (TSAb) in Graves’ disease (8, 9), or TSH-blocking Abs (TBAb) in some cases of idiopathic myxoedema (10, 11). Up until now, the protein domains and mechanisms implicated in the activation of the receptor by its physiologic agonist TSH or autoantibodies have been approached mainly by site-directed mutagenesis or the study of chimeric receptors (12, 13, 14, 15, 16). This stems from the lack of data or tools allowing direct probing of the three-dimensional structure of the receptor. In particular, most (monoclonal) Abs produced to date recognize preferentially the denatured receptor or sequential epitopes harbored by the native protein (17, 18, 19). The large quantities of the extracellular domain (ECD) of the hTSHR produced in insect cells (20, 21, 22) or in bacteria as recombinant fusion protein (23, 24, 25) were antigenically active (18, 19, 21, 25, 26) and were indeed useful in establishing animal models of thyroiditis (27, 28). However, the immunogens consisted mainly of incorrectly folded proteins that do not bind TSH (22, 23, 24), yielding Abs recognizing preferentially the denatured molecules. These Abs are useful in some analyses, such as Western blotting, but they contribute little to studies of the native functional receptor.
Recently, one group adopted a promising approach consisting of hyperimmunization of mice with fibroblasts coexpressing MHC class II Ag and the hTSHR (29). Twenty percent of the mice presented high T4 level, TBII Abs, and histologic signs of hyperthyroidism, although thyroiditis was not induced. In the present study, we have explored a third approach called genetic immunization. In this case, induction of an immune response is achieved by immunization with an expression DNA vector encoding the protein Ag (30, 31). Mice immunized by intramuscular injection of an expression construct for the hTSHR cDNA construct developed high levels of circulating Abs recognizing the receptor and displaying TBII, TBAb, or TSAb activities; mAbs recognizing the native TSHR were isolated from splenocytes, after fusion with SP2/0 myeloma cells. All immunized mice displayed a severe lymphocytic infiltration of their thyroids, composed mostly of activated B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four groups of five 6-wk female BALB/c mice were used. They were injected in the anterior tibialis muscle on day 0 with 100 µg of pcDNAIII plasmid (control group), pcDNAIII-hTSHR in PBS, pcDNAIII-hTSHR in 25% sucrose, or pcDNAIII-hTSHR in PBS, but 5 days after pretreatment of the muscle with cardiotoxin purified from venom of Naja nigricollis (10 mM, 100 µl/injection; Calbiochem, La Jolla, CA). Injections were repeated 3 and 6 wk thereafter. Blood samples were obtained from retro-ocular capillaries 8 and 11 wk after the initial immunization and at sacrifice, which was after 18 wk, when the spleens and thyroids were also removed. For all determinations, sera obtained at the various time points were tested individually.
FACS analysis
CHO cells expressing the hTSHR (JP09) (32) or CHO cells
expressing the hLHR (FC11) (33) were detached from the plates with PBS
containing EDTA and EGTA (5 mM each) and transferred into Falcon 2052
tubes (200,000 cells/tube). Cells were centrifuged at 500 x
g, at 4°C for 3 min, and the supernatant was removed by
inversion. They were incubated for 30 min at room temperature with 100
µl PBS/0.1% BSA containing 2 µl serum or 10 µl culture
supernatant from hybridoma. The cells were washed with 4 ml PBS/0.1%
BSA and centrifuged as above. They were incubated for 30 min on ice in
the dark with fluorescein-conjugated 
-chain-specific goat
anti-mouse IgG (Sigma Chemical Co., St. Louis, MO) in the same
buffer. Propidium iodide (10 µg/ml) was used for detection of damaged
cells, which were excluded from the analysis. Cells were washed once
again and resuspended in 250 µl PBS/0.1% BSA. The fluorescence of
5000 cells/tube was assayed by a FACScan flow cytofluorometer (Becton
Dickinson, Eerenbodegem, Belgium).
Measurements of TBII activity
TSH-binding inhibiting activity was measured on intact CHO cells expressing the hTSHR, JP09, as previously described, with minor modifications (27). Briefly, 5 x 104 cells/well in 96-well plates were incubated in 0.1 ml of modified Hanks’ buffer without NaCl (isotonicity maintained with 280 mM sucrose), supplemented with 2.5% low fat milk, 125I-labeled TSH (30,000 cpm), and mouse serum, for 4 h at room temperature. At the end of the incubation period, the cells were rapidly rinsed with the same ice-cold buffer and solubilized with 0.2 ml 1 N NaOH, and radioactivity was measured in a gamma counter.
The 125I-labeled TSH tracer (TRAK) was the kind gift of BRAHMS Diagnostica, GmBH (Berlin, Germany). TBII activity was measured using 3 µl of serum/well. All experiments were done in triplicate, and results are expressed as cpm bound.
Measurement of TSAb and TBAb
Thyroid-stimulating and -blocking activities were measured using CHO cells expressing the hTSHR, JP26 (34). Briefly, 5 x 104 cells/well in 96-well plates were incubated in 5 mM KCl, 0.25 mM KH2PO4, 0.5 mM MgSO4, 0.4 mM Na2HPO4, 1 mM CaCl2, 0.1% glucose, 2 mM isobutylmethylxanthine, 20 mM HEPES, and 0.3% BSA containing 3 µl of serum (total volume, 100 µl/well). Incubation was for 4 h at 37°C; cAMP released into the medium was measured using a competitive binding assay (Amersham, Bucks, U.K.). TSAb were measured under the basal conditions described above and TBAb in identical conditions, but with the addition of 200 µIU/ml final concentration bovine TSH (Sigma Chemical Co.). Duplicate samples were assayed in all experiments; results are expressed as pmol cAMP/ml.
Measurement of anti-TSHR Abs by ELISA
Purified ECD of the TSHR, produced as a fusion protein with maltose-binding protein (MBP-ECD) in bacteria, or the fusion partner associated with ß-galactosidase as control (MBP-ßGal) (23) was diluted in 0.1 M of sodium carbonate-bicarbonate buffer, pH 9.6 (5 µg/ml, 100 µl/well) and incubated in microwell plates overnight at 4°C. After washing and saturation with 10 mM sodium phosphate, 150 mM NaCl, pH 7.3 (PBS), containing 0.1% Tween and 5% BSA (blocking buffer), 100 µl of serum samples (1/500 in blocking buffer) from immunized mice were added for 2 h at room temperature. The plates were washed, and alkaline phosphatase-labeled goat anti-mouse Abs were added. After 1-h incubation at room temperature, wells were washed and the substrate was added (1 mg/ml p-nitrophenyl phosphate in 10% diethanolamine buffer, 1 mM MgCl2, pH 9.8). All determinations were done in triplicate, and the absorbance was read at 405 nm in a spectrophotometer.
SDS-PAGE and Western blotting
Purified MBP-ECD or MBP-ßGal as control (approximately 2 µg/lane) were loaded onto 10% SDS polyacrylamide gels (35) after reduction with ß-mercaptoethanol at 100°C for 2 min. After electrophoresis, proteins were stained with Coomassie blue or electrophoretically transferred onto nitrocellulose membranes (36). Blots were incubated for 30 min with a blocking solution (TBSN + milk: 10 mM Tris, pH 8, 150 mM NaCl, 0.05% Nonidet P-40, and 5% milk) at room temperature, and then stained with a pool of sera from mice (1/200) immunized with pcDNAIII-TSHR. Thereafter, the blots were incubated with alkaline phosphatase-labeled anti-mouse IgG and, finally, NBT-BCIP (nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl-phosphate) as substrate (both from Promega Corp., Madison, WI). The serum from a mouse immunized with the recombinant protein MBP-ECD was used as positive control.
Light microscopy and immunohistochemistry
The thyroid glands were removed and carefully dissected in saline from the surrounding connective tissue under a dissecting microscope. One lobe was processed for light microscopy and the other for immunohistochemistry. The lobe designed for light microscopy was immersed for 2 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer postfixed for 1 h in 1% osmium tetroxide and embedded in Lx112 resin. Sections (0.5 µm thick) were stained with toluidine blue.
The lobe for immunohistochemistry was inserted into a liver fragment, embedded in Tissue-Teck, and rapidly frozen in isopentane cooled in liquid nitrogen to generate cryostat sections. The frozen sections were subjected to immunoperoxidase staining using different rat mAbs specific for CD4+ T cells and B cells, as previously described (27, 28, 37, 38).
Biopsies of human thyroid glands were obtained at surgery and also designed for immunohistochemistry to detect the location of the TSHR using the mouse mAb BA8 generated by genetic immunization (see Results). This Ab was used at 1/100 or 1/250 dilutions.
Generation of mAbs
For mAb isolation, mice were boosted by i.v. injection, with K562 cells (2 x 105 cells) expressing high level of TSHR (about 1.106 receptors/cell), recently developed in our laboratory. Three days after this boost, splenocytes or lymphocytes from popliteal or inguinal lymph nodes from BALB/c mouse immunized with pcDNAIII-TSHR were fused with myeloma cells SP2/0 at a 5:1 ratio using 50% polyethylene glycol (39). After fusion, cells were seeded into 96-well plates at 2 x 105 cells/well in selective medium (DMEM containing 10% FBS, 2% Nutridoma (Boehringer Mannheim Corp., Brussels, Belgium), 10 mM sodium pyruvate, 2 mM L-glutamine, 5 mM ß-mercaptoethanol, 2.5 U/ml amphotericin B, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µM hypoxanthine, 400 µM aminopterine, and 16 µM thymidine). Ten days after the fusion, when the cells were actively growing, culture supernatants were tested for anti-hTSHR Abs by FACS analysis on JP09 cells. Selected hybridomas were cloned by limiting dilution. Cloned hybridomas were grown as ascites in pristane-primed BALB/c mice. Sepharose-protein A affinity chromatography was used to purify IgG from mouse ascites fluid. The Ig class of mAb was determined with a mouse mAb isotyping kit (IsoStrip; Boehringer Mannheim Corp.). Ab BA8 was iodinated by the chloramine-T method to a specific radioactivity of 80 µCi/µg.
Binding of I125-labeled Ab to hTSHR
JP09 cells were seeded in 96-well plates (50,000 cells/well) and incubated at room temperature in PBS/2.5% milk with I125-labeled BA8 mAb (100,000 cpm/well) in presence of various concentrations of the same cold Ab. One hour later, the cells were washed twice with the same buffer and solubilized by 1 M of NaOH. Bound radioactivity was determined in a gamma counter. The competition-binding curves have been fitted by nonlinear regression (40), using the fitting module of Sigma Plot for Windows (Jandel Scientific GmbH, Erkrath, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight weeks after the first injection, all mice immunized with
pcDNAIII-hTSHR had high titers of IgG Abs, recognizing the
ECD of hTSHR in an ELISA, based on the bacterially produced fusion
protein MBP-ECD (Fig. 1
). The OD varied
from 0.4 to 1.4 for sera of animals immunized with hTSHR cDNA construct
without or with cardiotoxin pretreatment, respectively. Sera from mice
immunized with the pcDNAIII vector alone did not recognized MBP-ECD (OD
below 0.1). All sera failed to recognize the MBP-ßGal protein used as
control.
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Eight weeks after the first injection, all four sera from mice
immunized with the hTSHR-cDNA construct after cardiotoxin treatment (D1
to D4) recognized the native receptor expressed at the surface of
stably transfected CHO cells (mean fluorescence, 150 arbitrary units);
in comparison, control cells yielded only background fluorescence (25
arbitrary units) (Fig. 2a). Only one serum
from a mouse immunized with hTSHR-cDNA in PBS without cardiotoxin
pretreatment (serum B5) and three sera from mice immunized with the
hTSHR-DNA in sucrose (sera C1, C2, and C3) showed a clear signal on
cells expressing the receptor in comparison with control cells. All
sera from mice immunized with the empty vector failed to recognize the
hTSHR by FACS analysis. Eleven weeks after the first injection, 11 sera
(sera B2, B3, B5, C1 to C4, and D1 to D4) from a total of 14 mice (one
mouse immunized with DNA after cardiotoxin treatment died early after
the beginning of the protocol) immunized with the hTSHR-cDNA recognized
the native hTSHR in FACS analysis (Fig. 2b). All sera
failed to recognize the hLHR when tested under the same conditions.
Sera from animals immunized with the empty vector remained negative on
both hTSHR- and hLHR-expressing cells throughout the immunization
protocol.
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In agreement with the results obtained in ELISA (see above), a
pool of sera from mice immunized with the hTSHR cDNA after cardiotoxin
pretreatment clearly recognized the MBP-ECD fusion protein on Western
blots (Fig. 3a),
without cross-reaction with the fusion partner MBP. This confirms the
presence within the antisera of Abs recognizing (most likely linear)
epitopes present on the amino-terminal portion of the hTSHR produced in
Escherichia coli.
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Eleven weeks after the first injection, sera from all mice were
tested individually for TBII activity on JP09 cells expressing stably
the hTSHR (Table I). No TBII activity was
observed in the sera of mice immunized with the empty vector. In
contrast, the sera from one mouse immunized with hTSHR cDNA in PBS
(serum B5), from four mice immunized with hTSHR cDNA in sucrose (sera
C1, C2, C3, and C4), and from all mice immunized with hTSHR cDNA after
cardiotoxin pretreatment, exhibited TBII activity. Inhibition of
125I-labeled TSH binding ranged from 40 to 80%.
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). No TBAb
activity was measured in sera of mice immunized with the vector alone.
Serum from one mouse immunized with hTSHR cDNA in PBS, which was
positive for TBII activity (serum B5), exhibited an inhibition of
TSH-mediated cAMP production of 50%. With mice immunized with hTSHR
cDNA in sucrose, one serum (serum C2) caused an inhibition of cAMP
production of 60%. Finally, all four sera with TBII activity from mice
immunized with hTSHR cDNA after cardiotoxin pretreatment showed a TBAb
activity; inhibition of cAMP production ranged from 60% (mouse D1) to
85% (mouse D2).
All sera were tested individually for TSAb activity on JP26 cells. Only
one serum, devoid of TBII activity, from a mouse immunized with hTSHR
cDNA in sucrose (mouse C5) exhibited a clear TSAb activity, with a
stimulation of cAMP production about 800%. This stimulating activity
was specific for the hTSHR, as the serum failed to stimulate cAMP
production in JP02 cells, which do not express the hTSHR, or in FC11
cells, which express the hLHR (Fig. 4).
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Two mice immunized with the hTSHR cDNA (mouse C5, TSAb positive
from the protocol with sucrose, and mouse D2, TBAb positive from the
protocol with cardiotoxin) were boosted with K562 cells expressing the
hTSHR and killed 3 days later for mAb production. Three mAbs were
isolated after screening by FACS on JP09 cells (see Materials and
Methods); two were obtained from the spleens (BA8 and
BH10), and one from lymph nodes (AB1). These Abs, of IgG2a isotypes,
clearly recognized the native hTSHR expressed at the surface of CHO
cells (Fig. 5), but failed to recognize
the dog TSHR, rat TSHR, and the hLHR (not illustrated). In contrast
with the 2C11 Ab kindly provided by Dr. A. P. Johnstone (19), they
do not recognize a mutant hTSHR with an amino acid substitution (I167N)
(41), which is expected to destroy the three-dimensional structure of
the ECD (7) and hinders expression of the mutant at the cell surface
(to be published).
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). The binding of labeled BA8 was not displaced by an excess
of cold bovine TSH, nor by autoimmune human sera with a strong TBII
activity (Table II). The labeled BA8 was
displaced by the other two mAbs isolated in this study, but not by the
2C11 Ab (Table II), which recognizes a linear epitope in the
carboxyl-terminal portion of the ECD of the receptor (354-VFFEEQ-359).
Contrary to the antisera from the immunized mice, the three monoclonals
failed to recognize the hTSHR in Western blots containing the MBP-ECD
fusion protein (data not illustrated).
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Human thyroid follicles showed a positive labeling after
immunostaining with the BA8 mAb. The labeling concentrated at the basal
pole of the thyroid cells (Fig. 6).
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Signs of thyroiditis were observed in the 14 immunized BALB/c
mice. Clusters of immune cells were often located at the periphery of
the thyroid (Fig. 7A),
where they comprised CD4+ T cells (Fig. 7B) and were surrounded by B cells (B
220+) (Fig. 7C). In addition to the
periphery, B cells were distributed abundantly all over the thyroid
interstitium (Fig. 7C).
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D). The
follicles located inside the gland and surrounded by immune cells had
greatly variable sizes. There were numerous small follicles, showing an
irregular basal outline, and there were frequent pictures suggestive of
follicular budding, a process implicated in folliculoneogenesis (Fig. 7E). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genetic immunization was also efficient in generating mAbs against the
TSHR. Three mAbs of IgG2a-
isotype were isolated, two from the
spleen and one from lymph nodes. They recognized the native hTSHR at
the surface of CHO cells, but failed to do so with the dog
or the rat TSHR (data not illustrated). They did not recognize the rECD
of the hTSHR in Western blot, suggesting that they are directed against
conformational epitopes. When used as culture supernatant (at about 1
µg/ml IgG), these Abs did not show TBII, TBAb, or TSAb activities.
One of these mAbs, BA8, isolated from a fusion with spleen cells, was
characterized further. It recognized the native hTSHR with high
affinity (apparent Kd about 4.5
10-11 M). Its binding was not displaced by TSH, neither by
autoantibodies from patients, nor by the 2C11 mAb. In contrast, the two
other Abs isolated, BH10 and AB1, competed with the binding of BA8,
suggesting that, despite their coming from two different fusions, the
three monoclonals are probably closely related. The three monoclonals
are of the IgG2a isotype, suggesting presentation of Ag by dendritic
cells rather than macrophages. Dendritic cells are capable of eliciting
both Th1 and Th2 responses (45, 46, 47), and the IgG2a isotype reflects a
switch to Th1, probably as a consequence of IFN- production (45).
The BA8 molecule is able to recognize in FACS a series of 16 TSHR
mutated in the serpentine domain, and available in our laboratory (not
shown) (48, 49). For receptors mutated in the ECD, this Ab recognizes
efficiently the S281T/N (48) and the P162A (41) mutants, as well as the
polymorphic P52T variant (50). In contrast to 2C11, BA8 did not
recognize the I167N (41) mutant receptor, even after permeabilization
of the cells. Together these results indicate that BA8 reacts only with
the native, normally folded TSHR and recognizes conformational epitopes
specific for the human receptor. For this reason, it constitutes an
invaluable tool for the purification of the bioactive receptor. Its
ability to recognize the TSHR on tissue sections will also make it
useful in probing the structure of the receptor in samples from
diseased thyroids.
Genetic immunization was developed primarily to induce protective immunity. In protocols having this goal, autoimmune reactions would be an unwanted side effect, and indeed, when they have been sought they have not generally been found. One exception to this involved the generation of autoantibodies to dsDNA, analogous to those occurring in patients with systemic lupus erythematosus, in mice immunized with the cDNA for the polyoma virus large T Ag. The authors found that a particular cDNA encoding a DNA-binding protein was required and that plasmid DNA in itself was not able to induce DNA Abs. The lack of Abs to other autoantigens suggested that polyclonal activation was not responsible, but rather Ag-cognate T-B cell interaction (51). Furthermore, genetic immunization of the neonate has been shown to induce tolerance, an effect that diminishes and is absent even in mice at 2 wk of age (52). Traditional immunization schedules are often, although not always, associated with tolerance induction in the first weeks of life (53, 54, 55).
To our knowledge, this is the first report of an organ-specific autoimmune response (as evidenced by the thyroiditis present in all mice treated with the hTSHR cDNA construct) being obtained via genetic immunization and in the absence of conventional adjuvants. The human and murine TSHR display sequence similarity, both at the nucleotide and amino acid levels (86.7% identity in amino acids). In these experiments, in which tolerance has been broken, one must assume that differences between the two receptors are sufficient to trigger T cell responsiveness. After intramuscular injection, the hTSHR gene is probably expressed by the myocyte and presented at its surface in the context of MHC class I. Expression of genes in myocytes from injected cDNA constructs has been well documented (56, 57, 58). However, these cells do not have the costimulatory signal necessary to elicit an immune response and are more likely to induce tolerance or cytotoxic reactions. The myocyte is thus unlikely to be the APC responsible for the humoral response and thyroiditis, which suggests presentation by a professional APC such as a dendritic cell. If the protein is overproduced, or if a cytotoxic response is elicited first, lysis of myocytes could lead to the release of hTSHR peptides that could be recovered by APC present around the injection site and subsequently presented within the context of MHC class II. Peptides differing from the mouse TSHR would be seen as nonself, but in the course of the ensuing proliferation and hypermutation, T cells having specificities for the murine TSHR could be produced, resulting in homing to the thyroid gland and thyroiditis. Alternatively or in addition, cDNA at the injection site could possibly be endocytosed and expressed by APC. Treatment with cardiotoxin could recruit large numbers of APC, particularly dendritic cells that would explain the acute humoral response observed. The receptor Abs induced by the protocol appear to be specific for the hTSHR. They exhibit bioactivity in in vitro assays using the hTSHR, but this is not mirrored by changes in circulating T4 levels. This indicates that, despite the infiltration of activated B cells into the mouse thyroids, the receptor Abs exhibit little or no bioactivity on the endogenous host TSHR. How does this equate with human disease? It has been suggested that Graves’ disease could result from immunization against cross-reacting Ag(s) of Yersinia enterocolitica (59, 60, 61), a phenomenon known as molecular mimicry (62, 63). In our model, there is obvious close mimicry between the human Ag encoded by our immunogen and mouse TSHR. This is a likely explanation for the breakage of tolerance in our model, while it has been reported that defects in tolerance mechanisms exist in autoimmune disease (64, 65).
Future refinement of our model might build on the recent demonstration that immunization with fibroblast coexpressing the TSHR and class II Ags leads to thyroiditis with TSAb (29). Expression of the TSHR cDNA in isolated dendritic cells, followed by their infusion in mice, or direct intradermal injection of TSH cDNA, aiming at Langerhans cells, might lead to an even stronger immune stimulation, due to the simultaneous presence at the cell surface of the native TSHR, class II Ag loaded with TSHR peptide and costimulatory molecules.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. S. Costagliola, IRIBHN, ULB, Campus Hôpital Erasme, bâtiment C, 808 route de Lennik, B-1070 Bruxelles, Belgique. E-mail address:
3 Abbreviations used in this paper: TSH, thyrotropin; ßGal, ß-galactosidase; CHO, Chinese hamster ovary; ECD, extracellular domain; h, human; LH, luteinizing hormone; LHR, luteinizing hormone receptor; MBP, maltose-binding protein; TSH-binding inhibiting Ig; TBAb, thyrotropin-blocking Ab; TSAb, thyroid-stimulating autoantibody; TSHR, thyrotropin receptor.
Received for publication June 2, 1997. Accepted for publication October 9, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E. Urizar, S. Claeysen, X. Deupi, C. Govaerts, S. Costagliola, G. Vassart, and L. Pardo An Activation Switch in the Rhodopsin Family of G Protein-coupled Receptors: THE THYROTROPIN RECEPTOR J. Biol. Chem., April 29, 2005; 280(17): 17135 - 17141. [Abstract] [Full Text] [PDF]
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G. Baker, G. Mazziotti, C. von Ruhland, and M. Ludgate Reevaluating Thyrotropin Receptor-Induced Mouse Models of Graves' Disease and Ophthalmopathy Endocrinology, February 1, 2005; 146(2): 835 - 844. [Abstract] [Full Text] [PDF]
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 I. Migeotte, E. Riboldi, J.-D. Franssen, F. Gregoire, C. Loison, V. Wittamer, M. Detheux, P. Robberecht, S. Costagliola, G. Vassart, et al. Identification and characterization of an endogenous chemotactic ligand specific for FPRL2 J. Exp. Med., January 3, 2005; 201(1): 83 - 93. [Abstract] [Full Text] [PDF]
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S. Costagliola, M. Bonomi, N. G. Morgenthaler, J. Van Durme, V. Panneels, S. Refetoff, and G. Vassart Delineation of the Discontinuous-Conformational Epitope of a Monoclonal Antibody Displaying Full in Vitro and in Vivo Thyrotropin Activity Mol. Endocrinol., December 1, 2004; 18(12): 3020 - 3034. [Abstract] [Full Text] [PDF]
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P. N. Pichurin, G. D. Chazenbalk, H. Aliesky, O. Pichurina, B. Rapoport, and S. M. McLachlan "Hijacking" the Thyrotropin Receptor: A Chimeric Receptor-Lysosome Associated Membrane Protein Enhances Deoxyribonucleic Acid Vaccination and Induces Graves' Hyperthyroidism Endocrinology, December 1, 2004; 145(12): 5504 - 5514. [Abstract] [Full Text] [PDF]
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 M. Tonacchera, A. Perri, G. De Marco, P. Agretti, M. E. Banco, C. Di Cosmo, L. Grasso, P. Vitti, L. Chiovato, and A. Pinchera Low Prevalence of Thyrotropin Receptor Mutations in a Large Series of Subjects with Sporadic and Familial Nonautoimmune Subclinical Hypothyroidism J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5787 - 5793. [Abstract] [Full Text] [PDF]
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C.-R. Chen, H. Aliesky, P. N. Pichurin, Y. Nagayama, S. M. McLachlan, and B. Rapoport Susceptibility Rather than Resistance to Hyperthyroidism Is Dominant in a Thyrotropin Receptor Adenovirus-Induced Animal Model of Graves' Disease as Revealed by BALB/c-C57BL/6 Hybrid Mice Endocrinology, November 1, 2004; 145(11): 4927 - 4933. [Abstract] [Full Text] [PDF]
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J. Guo, P. N. Pichurin, J. C. Morris, B. Rapoport, and S. M. McLachlan "Naked" Deoxyribonucleic Acid Vaccination Induces Recognition of Diverse Thyroid Peroxidase T Cell Epitopes Endocrinology, August 1, 2004; 145(8): 3671 - 3678. [Abstract] [Full Text] [PDF]
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T. Muehlberg, J. A. Gilbert, P. V. Rao, A. M. McGregor, and J. P. Banga Dynamics of Thyroid-Stimulating and -Blocking Antibodies to the Thyrotropin Receptor in a Murine Model of Graves' Disease Endocrinology, April 1, 2004; 145(4): 1539 - 1545. [Abstract] [Full Text] [PDF]
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P. N. Pichurin, O. Pichurina, R. C. Marians, C.-R. Chen, Terry. F. Davies, B. Rapoport, and S. M. McLachlan Thyrotropin Receptor Knockout Mice: Studies on Immunological Tolerance to a Major Thyroid Autoantigen Endocrinology, March 1, 2004; 145(3): 1294 - 1301. [Abstract] [Full Text] [PDF]
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C.-R. Chen, P. Pichurin, G. D. Chazenbalk, H. Aliesky, Y. Nagayama, S. M. McLachlan, and B. Rapoport Low-Dose Immunization with Adenovirus Expressing the Thyroid-Stimulating Hormone Receptor A-Subunit Deviates the Antibody Response toward That of Autoantibodies in Human Graves' Disease Endocrinology, January 1, 2004; 145(1): 228 - 233. [Abstract] [Full Text] [PDF]
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B. S. Prabhakar, R. S. Bahn, and T. J. Smith Current Perspective on the Pathogenesis of Graves' Disease and Ophthalmopathy Endocr. Rev., December 1, 2003; 24(6): 802 - 835. [Abstract] [Full Text] [PDF]
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V. Wittamer, J.-D. Franssen, M. Vulcano, J.-F. Mirjolet, E. Le Poul, I. Migeotte, S. Brezillon, R. Tyldesley, C. Blanpain, M. Detheux, et al. Specific Recruitment of Antigen-presenting Cells by Chemerin, a Novel Processed Ligand from Human Inflammatory Fluids J. Exp. Med., October 6, 2003; 198(7): 977 - 985. [Abstract] [Full Text] [PDF]
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 G. Smits, O. Olatunbosun, A. Delbaere, R. Pierson, G. Vassart, and S. Costagliola Ovarian Hyperstimulation Syndrome Due to a Mutation in the Follicle-Stimulating Hormone Receptor N. Engl. J. Med., August 21, 2003; 349(8): 760 - 766. [Full Text] [PDF]
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Y. Nagayama, H. Mizuguchi, T. Hayakawa, M. Niwa, S. M. McLachlan, and B. Rapoport Prevention of Autoantibody-Mediated Graves'-Like Hyperthyroidism in Mice with IL-4, a Th2 Cytokine J. Immunol., April 1, 2003; 170(7): 3522 - 3527. [Abstract] [Full Text] [PDF]
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S. Brezillon, V. Lannoy, J.-D. Franssen, E. Le Poul, V. Dupriez, J. Lucchetti, M. Detheux, and M. Parmentier Identification of Natural Ligands for the Orphan G Protein-coupled Receptors GPR7 and GPR8 J. Biol. Chem., January 3, 2003; 278(2): 776 - 783. [Abstract] [Full Text] [PDF]
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P. V. Rao, P. F. Watson, A. P. Weetman, G. Carayanniotis, and J. P. Banga Contrasting Activities of Thyrotropin Receptor Antibodies in Experimental Models of Graves' Disease Induced by Injection of Transfected Fibroblasts or Deoxyribonucleic Acid Vaccination Endocrinology, January 1, 2003; 144(1): 260 - 266. [Abstract] [Full Text] [PDF]
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V. Vlaeminck-Guillem, S.-C. Ho, P. Rodien, G. Vassart, and S. Costagliola Activation of the cAMP Pathway by the TSH Receptor Involves Switching of the Ectodomain from a Tethered Inverse Agonist to an Agonist Mol. Endocrinol., April 1, 2002; 16(4): 736 - 746. [Abstract] [Full Text] [PDF]
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R. Metcalfe, N. Jordan, P. Watson, S. Gullu, M. Wiltshire, M. Crisp, C. Evans, A. Weetman, and M. Ludgate Demonstration of Immunoglobulin G, A, and E Autoantibodies to the Human Thyrotropin Receptor Using Flow Cytometry J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1754 - 1761. [Abstract] [Full Text] [PDF]
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P. Pichurin, O. Pichurina, G. D. Chazenbalk, C. Paras, C.-R. Chen, B. Rapoport, and S. M. McLachlan Immune Deviation Away from Th1 in Interferon-{gamma} Knockout Mice Does Not Enhance TSH Receptor Antibody Production after Naked DNA Vaccination Endocrinology, April 1, 2002; 143(4): 1182 - 1189. [Abstract] [Full Text] [PDF]
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Y. Nagayama, M. Kita-Furuyama, T. Ando, K. Nakao, H. Mizuguchi, T. Hayakawa, K. Eguchi, and M. Niwa A Novel Murine Model of Graves' Hyperthyroidism with Intramuscular Injection of Adenovirus Expressing the Thyrotropin Receptor J. Immunol., March 15, 2002; 168(6): 2789 - 2794. [Abstract] [Full Text] [PDF]
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P. Pichurin, X.-M. Yan, L. Farilla, J. Guo, G. D. Chazenbalk, B. Rapoport, and S. M. McLachlan Naked TSH Receptor DNA Vaccination: A TH1 T Cell Response in Which Interferon-{gamma} Production, Rather than Antibody, Dominates the Immune Response in Mice Endocrinology, August 1, 2001; 142(8): 3530 - 3536. [Abstract] [Full Text] [PDF]
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 M. Crisp, K. J. Starkey, C. Lane, J. Ham, and M. Ludgate Adipogenesis in Thyroid Eye Disease Invest. Ophthalmol. Vis. Sci., October 1, 2000; 41(11): 3249 - 3255. [Abstract] [Full Text]
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J. Pohlenz, L. Duprez, R. E. Weiss, G. Vassart, S. Refetoff, and S. Costagliola Failure of Membrane Targeting Causes the Functional Defect of Two Mutant Sodium Iodide Symporters J. Clin. Endocrinol. Metab., July 1, 2000; 85(7): 2366 - 2369. [Abstract] [Full Text]
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L. Bartalena, A. Pinchera, and C. Marcocci Management of Graves' Ophthalmopathy: Reality and Perspectives Endocr. Rev., April 1, 2000; 21(2): 168 - 199. [Abstract] [Full Text]
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M. Tonacchera, P. Agretti, A. Pinchera, V. Rosellini, A. Perri, P. Collecchi, P. Vitti, and L. Chiovato Congenital Hypothyroidism with Impaired Thyroid Response to Thyrotropin (TSH) and Absent Circulating Thyroglobulin: Evidence for a New Inactivating Mutation of the TSH Receptor Gene J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1001 - 1008. [Abstract] [Full Text]
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S. Kaithamana, J. Fan, Y. Osuga, S.-G. Liang, and B. S. Prabhakar Induction of Experimental Autoimmune Graves' Disease in BALB/c Mice J. Immunol., November 1, 1999; 163(9): 5157 - 5164. [Abstract] [Full Text] [PDF]
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J. C. Jaume, J. Guo, Y. Wang, B. Rapoport, and S. M. McLachlan Cellular Thyroid Peroxidase (TPO), Unlike Purified TPO and Adjuvant, Induces Antibodies in Mice That Resemble Autoantibodies in Human Autoimmune Thyroid Disease J. Clin. Endocrinol. Metab., May 1, 1999; 84(5): 1651 - 1657. [Abstract] [Full Text]
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M.-C. Many, S. Costagliola, M. Detrait, J.-F. Denef, G. Vassart, and M. Ludgate Development of an Animal Model of Autoimmune Thyroid Eye Disease J. Immunol., April 15, 1999; 162(8): 4966 - 4974. [Abstract] [Full Text] [PDF]
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M. Kita, L. Ahmad, R. C. Marians, H. Vlase, P. Unger, P. N. Graves, and T. F. Davies Regulation and Transfer of a Murine Model of Thyrotropin Receptor Antibody Mediated Graves' Disease Endocrinology, March 1, 1999; 140(3): 1392 - 1398. [Abstract] [Full Text]
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S. Costagliola, N. G. Morgenthaler, R. Hoermann, K. Badenhoop, J. Struck, D. Freitag, S. Poertl, W. Weglöhner, J. M. Hollidt, B. Quadbeck, et al. Second Generation Assay for Thyrotropin Receptor Antibodies Has Superior Diagnostic Sensitivity for Graves' Disease J. Clin. Endocrinol. Metab., January 1, 1999; 84(1): 90 - 97. [Abstract] [Full Text]
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P. Rodien, C. Bremont, M.-L. R. Sanson, J. Parma, J. Van Sande, S. Costagliola, J.-P. Luton, G. Vassart, and L. Duprez Familial Gestational Hyperthyroidism Caused by a Mutant Thyrotropin Receptor Hypersensitive to Human Chorionic Gonadotropin N. Engl. J. Med., December 17, 1998; 339(25): 1823 - 1826. [Full Text] [PDF]
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B. Rapoport, G. D. Chazenbalk, J. C. Jaume, and S. M. McLachlan The Thyrotropin (TSH)-Releasing Hormone Receptor: Interaction with TSH and Autoantibodies Endocr. Rev., December 1, 1998; 19(6): 673 - 716. [Abstract] [Full Text]
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P. Rodien, F. Cetani, S. Costagliola, M. Tonacchera, L. Duprez, T. Minegishi, C. Govaerts, and G. Vassart Evidences for an Allelic Variant of the Human LC/CG Receptor rather than a Gene Duplication: Functional Comparison of Wild-Type and Variant Receptors J. Clin. Endocrinol. Metab., December 1, 1998; 83(12): 4431 - 4434. [Abstract] [Full Text]
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C. Govaerts, A. Lefort, S. Costagliola, S. J. Wodak, J. A. Ballesteros, J. Van Sande, L. Pardo, and G. Vassart A Conserved Asn in Transmembrane Helix 7 Is an On/Off Switch in the Activation of the Thyrotropin Receptor J. Biol. Chem., June 15, 2001; 276(25): 22991 - 22999. [Abstract] [Full Text] [PDF]
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