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Departments of
*
Pharmacology 1 and
Internal Medicine 1, Nagasaki University School of Medicine, and
Health Research Center, Nagasaki University, Nagasaki, Japan; and
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactosidase (1 x
1011 particles/mouse, three times at 3-wk intervals) and
followed up to 8 wk after the third immunization. Fifty-five percent of
female and 33% of male BALB/c (H-2d) and 25% of female
C57BL/6 (H-2b) mice developed Graves’-like hyperthyroidism
with elevated serum thyroxine (T4) levels and positive
anti-TSHR autoantibodies with thyroid-stimulating Ig (TSI) and
TSH-binding inhibiting Ig (TBII) activities. In contrast, none of
female CBA/J (H-2k), DBA/1J (H-2q), or SJL/J
(H-2s) mice developed Graves’ hyperthyroidism or
anti-TSHR autoantibodies except SJL/J, which showed strong TBII
activities. There was a significant positive correlation between TSI
values and T4 levels, but the correlations between
T4 and TBII and between TSI and TBII were very weak. TSI
activities in sera from hyperthyroid mice measured with some chimeric
TSH/lutropin receptors suggested that their epitope(s) on TSHR appeared
similar to those in patients with Graves’ disease. The thyroid glands
from hyperthyroid mice displayed diffuse enlargement with hypertrophy
and hypercellularity of follicular epithelia with occasional protrusion
into the follicular lumen, characteristics of Graves’ hyperthyroidism.
Decreased amounts of colloid were also observed. However, there was no
inflammatory cell infiltration. Furthermore, extraocular muscles from
hyperthyroid mice were normal. Thus, the highly efficient means that we
now report to induce Graves’ hyperthyroidism in mice will be very
useful for studying the pathogenesis of autoimmunity in Graves’
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal models are very useful tools to study the pathophysiology of
autoimmune thyroid disease. There are some spontaneous animal models of
autoimmune thyroiditis in which the autoimmune response is directed to
Tg or TPO (4). Immunization of certain animals with Tg or
TPO in combination with classical immunological adjuvants has also been
reported to successfully induce thyroiditis (5, 6). In
contrast, there are no spontaneous animal models of Graves’
hyperthyroidism, and numerous attempts using TSHR protein expressed in
bacteria or insect cells and classical immunization protocols have
failed to establish a disease model (3, 7, 8). However,
some new approaches for generating animal models of Graves’
hyperthyroidism have recently been demonstrated. First, a novel,
pioneering immunization protocol using transfected fibroblasts (a L
cell line) coexpressing TSHR and MHC class II Ag has been described by
Shimojo et al. (9) to induce hyperthyroidism in a small
proportion (
20%) of immunized syngeneic AKR mice. This model was
later confirmed by two other groups (10, 11). Later, B
lymphoblastoid cells (a M12 cell line) expressing TSHR have also been
used in a similar protocol with a high proportion of the syngeneic
BALB/c mice becoming hyperthyroid (12). Furthermore,
genetic immunization with an eukaryotic expression vector containing
TSHR cDNA has proved to be useful in inducing hyperthyroidism in a
small proportion (20%) of outbred NMRI (13), not
inbred BALB/c, mice (14). Although these methods opened
new ways to investigate the autoimmune reaction to TSHR, they have some
drawbacks, such as use of cell lines available only for certain strains
of mice (9, 12) and a low rate of disease induction
(9, 14).
Therefore, the present studies were designed to establish a better means for inducing disease. Our first attempt was the genetic immunization protocol mentioned above modified by combining the TSHR expression plasmid and the cytokine expression plasmids (GM-CSF or M-CSF). However, no significant immune response was observed. We then used recombinant adenovirus to achieve higher transgene expression in the muscle. We show in this study that repeated administration of recombinant adenovirus vector expressing TSHR into the muscle efficiently generates anti-TSHR autoantibodies and induces Graves’-like hyperthyroidism in BALB/c (H-2d), and to a lesser extent in C57BL/6(H-2b), mice. Our data also indicate the similarity of epitope(s) on TSHR ectodomain recognized by TSI from Graves" patients and those from hyperthyroid mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pBluescript-human (h)TSHR (15) was digested
with EcoRI, blunt ended with T4 DNA polymerase, and digested
with XbaI. The TSHR cDNA fragment was then ligated into
pHMCMV6 (15), which had been digested with
NheI, blunt-ended, and digested with XbaI. The
resultant plasmid, pHMCMVTSHR, was then digested with I-Ceu I/PI-Sce I
and ligated into I-Ceu I/PI-Sce I-digested pAdHM4 (16, 17). pAdHM4CMVTSHR was linealized with PacI and
transfected into 293 human embryonal kidney cells with SuperFect
(Qiagen, Tokyo, Japan) according to the manufacturer’s instructions.
Recombinant adenovirus expressing TSHR (designated AdCMVTSHR) was then
plaque-purified. Adenovirus was propagated in 293 human embryonal
kidney cells and purified through two rounds of CsCl density gradient
centrifugation (18). The multiplicity of infection (MOI)
was defined as the ratio of total number of particles used in a
particular infection divided by the number of cells. The viral particle
concentration was determined by measuring the absorbance at 260 nm
following the incubation of the virus solution in 10 mM Tris-HCl, 1 mM
EDTA, and 0.1% SDS at 56°C for 10 min; an absorbance of 1
corresponds to 1.1 x 1012 particles/ml
(19). Adenovirus expressing -galactosidase (AxCALacZ)
(18) was used as a negative control.
Immunization protocols
BALB/c (H-2d), CBA/J (H-2k), C57BL/6 (H-2b), DBA/1J (H-2q), and SJL/J (H-2s) 6-wk-old mice were purchased from Charles River Breeding Laboratories (Tokyo, Japan). All experiments were conducted in accordance with the principles and procedures outlined in the Guideline for the Care and Use of Laboratory Animals in Nagasaki University (Nagasaki, Japan). Mice were kept in a specific pathogen-free condition through the experiments. For DNA vaccination, groups of mice were injected in the leg muscle with 50 µl 25% sucrose in PBS containing 100 µg of pCAGTSHR (20) alone or in combination with the expression plasmid for GM-CSF or M-CSF (RIKEN DNA Bank, Saitama, Japan). Injection was repeated twice at 3-wk intervals. For immunization with adenovirus, mice were i.m. injected with 50 µl PBS containing 1 x 1011 particles of AdCMVTSHR or AxCALacZ. The same immunization schedule was repeated twice at 3-wk intervals.
125I-TSH binding and TSH-induced cAMP synthesis in COS cells or muscle injected with adenovirus or plasmid
A total of 1 x 105 COS cells in a 24-well culture plate were infected with AdCMVTSHR or AxCALacZ at a MOI of 1,000 or 10,000 (particles/cell). Two days later, 125I-TSH binding to intact cells and intracellular cAMP measurements were performed with 125I-bovine TSH (TRAb kit; RSR, Cardiff, U.K.) and with a cAMP radioimmunoassay kit (Yamasa, Tokyo, Japan), respectively, as previously described (20). Unlabeled TSH used in TSH binding study was of bovine origin (Sigma-Aldrich, St. Louis, MO).
125I-TSH binding was also performed with 50 µg crude membranes prepared as previously described (20) from muscles 5 days after injection of either 1 x 1011 particles of AdCMVTSHR or 100 µg of pCAGTSHR.
Thyroid function test
Total thyroxine (T4) in murine sera was measured with a commercially available radioimmunoassay kit (Eiken Chemical, Osaka, Japan). The normal range was defined as the mean ± 3 SD of control mice.
TSI and TBII measurements
TSI activities in murine sera were measured with FRTL5 cells (20) or CHO cells stably expressing wild-type (wt)-hTSHR or chimeric TSH-lutropin receptor (LHR)-6 and TSH-LHR-8 (21). The cDNAs for wt-TSHR and chimeric TSHR/LHRs were ligated into the eukaryotic expression vector pCR3 with the constitutive CMV promoter (Invitrogen, Groningen, The Netherlands). The cells were seeded at 3 x 104 cells/well in a 96-well culture plate and incubated in 50 µl hypotonic HBSS containing 1 mM isobutyl-methylxanthine, 20 mM HEPES, 0.25% BSA, and 5 µl serum for 2 h at 37°C. cAMP >150% of control mice was judged as positive.
TSH-binding inhibiting Ig (TBII) values were determined with a commercially available TRAb kit. Ten microliters of serum was used for each assay. A value >15% for inhibition of control binding was judged as positive.
Thyroid and eye histology
Thyroid tissues and extraocular muscles were removed and fixed with 10% formalin in PBS. Tissues were embedded in paraffin and 5-µm-thick sections were prepared and stained with H&E.
Data analysis
Data were analyzed by unpaired Student’s t test or
by the 
2 test. Correlations among
T4 and autoantibodies were assessed by linear
regression using StatView 4.02 software (Abacus Concepts, Berkeley,
CA). Values of p < 0.05 were considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Therefore, we next challenged the animals with recombinant adenovirus
expressing TSHR (AdCMVTSHR) to increase transgene expression in the
muscle. Integrity of AdCMVTSHR was confirmed by specific
125I-TSH binding and by TSH-induced cAMP
synthesis in COS cells infected with AdCMVTSHR, not with AxCALacZ (a
negative control) (Fig. 1
, A
and C). Furthermore, a higher expression level of TSHR was
observed in muscle with AdCMVTSHR infection as compared
with that withpCAGTSHR (Fig. 1B). In this experiment, TSH
binding induced with pCAGTSHR was negligible.
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A, serum
T4 levels were increased over mean ± 3 SD
in 10 of 19 (53%) female BALB/c, three of nine (33%) male BALB/c, and
two of eight (25%) female C57BL/6 mice, but in none of CBA/J, DBA/1J,
or SJL/J mice. Thus, by including the one female BALB/c mouse, which
died as mentioned above, 11 of 20 (55%) female BALB/c mice appeared to
develop hyperthyroidism. There was no significant difference in the
rate of disease induction between male and female BALB/c mice. No mice
showed any significant decrease in T4. BALB/c
mice immunized with AxCALacZ were all in a euthyroid state
(T4, 4.5 ± 0.57 µg/dL).
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B. Sera
from all but one mice with increased T4 levels
displayed positive TSI. No euthyroid mice of any strain showed TSI
activity. As shown in Fig. 3A,
there was a significant positive correlation between TSI values and
T4 levels (r = 0.89). Thus,
BALB/c and, to a lesser degree, C57B/6 mice are susceptible to TSI
generation and disease induction.
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C, TBII assay revealed that not
only BALB/c and C57B/6 but also SJL/J mice are susceptible to TBII
generation. Besides all the hyperthyroid and some other euthyroid
BALB/c and C57B/6 mice (16 of 19 (84%) female BALB/c, five of nine
(56%) male BALB/c, and six of eight (75%) C57BL/6 mice), six of eight
(75%) SJL/J mice were positive for TBII, the values being the highest
among five groups. There was no significant correlation between
T4 levels and TBII values (r =
0.38) (Fig. 3B), and correlation between TSI and TBII values
was significant but extremely poor (r = 0.40) (Fig. 3C).
To delineate the epitope(s) on TSHR for TSI in the sera of hyperthyroid
mice, TSI activities were also measured with two chimeric TSH/LHRs,
TSHR-LHR-6 and -8, in which the C-terminal and the N-terminal
two-fifths of TSHR ectodomain were, respectively, replaced with the
corresponding region of LHR (Fig. 4A). Although wt-TSHR and
TSH-LHR-6 expressed on CHO cells responded well to stimulation by TSH
and TSI of both human and mouse origin, TSH-LHR-8 did so only to TSH
stimulation, not to TSI (Fig. 4B). These data are
essentially identical to those previously reported with human Graves’
sera (24, 25), suggesting the crucial role of the
N-terminal region of TSHR ectodomain in both human and murine
TSI.
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12 µg/dL), as shown in Fig. 5, C and D (compared with the normal thyroid in Fig. 5, A and B), but are less evident and
heterogeneous in hyperthyroid mice with lower T4;
papillary protrusion of the follicular epithelia was sparsely observed
and the epithelia consisted of either flat cells (inactive) or columnar
cells (hyperactive), as previously reported (14).
Decreased amounts of colloid were also observed. However, no
inflammatory cell infiltration was observed. Furthermore, extraocular
muscles from hyperthyroid mice were normal (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20%). Although Prabhakar
and his colleagues have reported to be able to induce Graves’-like
hyperthyroidism in nearly 100% of BALB/c mice (12),
thyroid histology from affected mice in their study showed
"hypertrophy and enlargement of colloids with thinning of the thyroid
epithelium." This finding contradicts that in Graves’ disease, in
which thyroid epithelial cells are tall and columnar and sometimes
extend as papillary folds into the follicles, indications of
hypertrophy and hypercellularity. Second, in contrast to previously reported methods that are applied only to certain strains of inbred mice or to outbred mice, our method can be used for any mouse strain, which allows analysis of the genetic influence(s) on the immune response to TSHR. Our data clearly show that BALB/c (H-2d) and, to a lesser degree, C57BL/6 (H-2b) mice are susceptible to generation of TSI and induction of Graves’ hyperthyroidism. These data, together with a previous report (26), suggest the genetic backgrounds play a role in susceptibility to an induced form of Graves’ disease in mice. Indeed Graves’ disease is known to be a multigenic disease in humans (27); both HLA and non-HLA genes, such as CTLA-4, are associated with a predisposition to autoimmune thyroid disease (28, 29). In addition, immunized SJL/J mice show negative TSI and strongly positive TBII, the highest among five groups of different strains, suggesting that the genetic factor(s) crucial for Graves’ disease/TSI induction and those for TBII generation may also be different. Our results may explain a lower incidence of disease induction in Shimojo’s model (AKR with H-2k) (9) and Vassart’s model (NMR outbred mice with H-2q) (14) and a high incidence in BALB/c mice (12). It is also suggested that the reason for the failure to produce effective stimulating autoantibodies in BALB/c mice by DNA vaccination may not be related to their genetic background as the authors have speculated (14). It is known that expression level of transgene by adenovirus infection is higher than that with DNA vaccination (30). We showed higher expression level of TSHR in muscle injected with adenovirus than that with plasmid. Although adenovirus-specific neutralizing Abs can be elicited not only following i.v. injection but also following i.m. injection of adenovirus, the concentration of these Abs in the muscle may be lower than that in the serum, allowing effective multiple dosing to the muscle (30). Thus, adenovirus infection seems to elicit superior immune response to DNA vaccination. Indeed, serum T4 levels, which reflect the degree of the immune reaction induced, in our model are higher than those in Vassart’s model (9–20 vs 8–10 µg/dL) (14). Usefulness of adenovirus as a means to induce strong in vivo immune response has also been reported recently by Chen et al. (31).
It is generally believed that autoimmune reaction in human Graves’ disease is Th2 dominant (32). In animal models, previous studies performed by Kita et al. (10) have also demonstrated that immunization of AKR mice with L cells expressing TSHR and MHC class II with the pertussis toxin, a Th2 response-inducing adjuvant, led to Graves’ disease in a higher proportion of mice, and use of CFA, a Th1 adjuvant, delayed the disease onset. Indeed, BALB/c mice are reported to inherently elicit Th2-dominant immune response (33, 34). This could be one of the reasons for high susceptibility of BALB/c mice to Th2-dominant Graves’ hyperthyroidism.
However, recent studies indicate that the immune response induced in
Shimojo’s and Vassart’s models is not simply Th2 dominant. First,
monoclonal anti-TSHR Abs established from BALB/c mice immunized
with DNA vaccination are IgG2a, an isotype predominant in Th1 immune
response (13). Second, splenocytes from immunized mice in
Shimojo’s and Vassart’s models produce IFN-
, a Th1 cytokine,
spontaneously and in response to TSHR Ag, respectively (23, 35). Although Vassart et al. (14) have described
that the immune response occurred in the thyroid gland is Th2 dominant,
DNA vaccination with plasmid generally leads to Th-1-biased immune
response, because plasmids propagated in bacteria contain unmethylated
CpG sequences that induce T cells to a Th1 proinflammatory immune
response (36). Although Th1/Th2 balance in our model is at
present unknown, this finding may be another reason for low prevalence
of Graves’ disease in these models.
In contrast to Vassart’s model (14), female bias, intrathyroidal lymphocyte infiltration, or eye lesion (one of the extrathyroidal manifestations of Graves’ disease) were not observed in hyperthyroid mice in our study as in Shimojo’s models (9, 10, 11). Although the exact reasons for this difference are presently unknown, it is plausible that different experimental protocols may induce the subtle distinct immune responses. It is apparently suggested, as Vassart et al. (14) have mentioned, that a purely humoral immune response is induced in our and Shimojo’s models (9), which, however, does not simply indicate the Th2-dominant immune response as mentioned above. It is at present uncertain whether the adequate murine model of Graves’ disease should include these three findings. Pathophysiology of Graves’ disease in human seems more complex than that in an animal model induced with TSHR as a single immunogen, because not only TSHR but also Tg and TPO are autoantigens in the former (1). Further studies will be required to address these issues.
Our study with chimeric receptors suggests that the epitopes on TSHR recognized by TSI in our model appear very similar to those observed in human disease (24, 25), suggesting the involvement of the N terminus of TSHR ectodomain in both human and murine TSI. This is in agreement with the studies by Kikuoka et al. (37), who have shown the importance of the N terminus of TSHR ectodomain (domains A and B in our chimeras) in induction of Graves’ hyperthyroidism with Shimojo’s model. The N terminus of TSHR ectodomain contains a major portion of the B cell epitope(s) for TSI, although this region does not appear to contain T cell epitopes.
In summary, in this work we report a novel murine model of Graves’ disease in which repeated i.m. injection of adenovirus expressing TSHR efficiently induces anti-TSHR Abs with thyroid-stimulating activity, resembling TSI, and hyperthyroidism. Our results demonstrate that BALB/c (H-2d) and, to a lesser degree, C57BL/6 (H-2b) mice appear susceptible to the disease. This new model will be a useful tool to study the pathogenesis of autoimmunity in Graves’ disease; the high disease penetrance in our model is particularly advantageous over other methods for some studies, such as identification of susceptibility genes and development of new therapeutic approaches.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: TSHR, thyrotropin receptor; T4, thyroxine; Tg, thyroglobulin; TPO, thyroid peroxidase; MOI, multiplicity of infection; TBII, TSH-binding inhibiting Ig; TSI, thyroid-stimulating Ig; LHR, lutropin receptor; wt, wild type.
Received for publication August 31, 2001. Accepted for publication January 16, 2002.
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References |
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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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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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T. Ando, R. Latif, S. Daniel, K. Eguchi, and T. F. Davies Dissecting Linear and Conformational Epitopes on the Native Thyrotropin Receptor Endocrinology, November 1, 2004; 145(11): 5185 - 5193. [Abstract] [Full Text] [PDF]
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Y. Nagayama, S. M. McLachlan, B. Rapoport, and K. Oishi Graves' Hyperthyroidism and the Hygiene Hypothesis in a Mouse Model Endocrinology, November 1, 2004; 145(11): 5075 - 5079. [Abstract] [Full Text] [PDF]
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K. J. Land, J. S. Moll, M. H. Kaplan, and G. S. Seetharamaiah Signal Transducer and Activator of Transcription (Stat)-6-Dependent, But Not Stat4-Dependent, Immunity Is Required for the Development of Autoimmunity in Graves' Hyperthyroidism Endocrinology, August 1, 2004; 145(8): 3724 - 3730. [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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Y. Nagayama, K. Watanabe, M. Niwa, S. M. McLachlan, and B. Rapoport Schistosoma mansoni and {alpha}-Galactosylceramide: Prophylactic Effect of Th1 Immune Suppression in a Mouse Model of Graves' Hyperthyroidism J. Immunol., August 1, 2004; 173(3): 2167 - 2173. [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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L. Schwarz-Lauer, P. N. Pichurin, C.-R. Chen, Y. Nagayama, C. Paras, J. C. Morris, B. Rapoport, and S. M. McLachlan The Cysteine-Rich Amino Terminus of the Thyrotropin Receptor Is the Immunodominant Linear Antibody Epitope in Mice Immunized Using Naked Deoxyribonucleic Acid or Adenovirus Vectors Endocrinology, May 1, 2003; 144(5): 1718 - 1725. [Abstract] [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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T. Ando, M. Imaizumi, P. Graves, P. Unger, and T. F. Davies Induction of Thyroid-Stimulating Hormone Receptor Autoimmunity in Hamsters Endocrinology, February 1, 2003; 144(2): 671 - 680. [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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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Cells infected with AxCALacZ at a MOI of 10,000
particles/cell; •, cells infected with AdCMVTSHR at a MOI of 1,000;
, cells infected with AdCMVTSHR at a MOI of 10,000. Data are
representative of two separate experiments; each point represents the
mean ± SD of triplicate experiments. *, p
< 0.01; **, p < 0.05 vs AxCALacZ
infection.
, LHR segments. LHR is 50-aa shorter
in the D domain than TSHR as shown by a thin horizontal line.
B, TSI activities in CHO cells stably expressing wt-TSHR
or chimeric TSH/LHRs. cAMP synthesis in response to stimulation by TSH
and murine sera was examined in CHO cells stably expressing wt-TSHR,
TSH-LHR-6, or TSH-LHR-8 as described under Materials and
Methods. Two representative sera from hyperthyroid mice and one
human TSI positive serum were used. The data are mean ± SE
(n = 4) of two separate experiments determined in
duplicates.