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Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves' disease.

ABSTRACT: We performed genetic immunization of outbred NMRI mice, using a cDNA encoding the human thyrotropin receptor (TSHr). All mice produced antibodies capable of recognizing the recombinant receptor expressed at the surface of stably transfected Chinese hamster ovary (CHO) cells, and sera from most of the immunized mice blocked TSH-dependent stimulation of cAMP accumulation in cells expressing the TSHr. Five out of 29 female mice showed sign of hyperthyroidism including elevated total T4 and suppressed TSH levels. The serum of these mice contained thyroid-stimulating activity, as measured in a classic assay using CHO cells expressing recombinant TSHr. In contrast, only 1 male out of 30 had moderately elevated serum total T4 with undetectable TSH values. The hyperthyroid animals had goiters with extensive lymphocytic infiltration, characteristic of a Th2 immune response. In addition, these animals displayed ocular signs reminiscent of Graves' ophthalmopathy, including edema, deposit of amorphous material, and cellular infiltration of their extraocular muscles. Our results demonstrate that genetic immunization of outbred NMRI mice with the human TSHr provides the most convincing murine model of Graves' disease available to date.

SUBMITTER: Costagliola S

PROVIDER: S-EPMC377458 | biostudies-literature | 2000 Mar

REPOSITORIES: biostudies-literature

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Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves' disease.

Costagliola S S Many M C MC Denef J F JF Pohlenz J J Refetoff S S Vassart G G

The Journal of clinical investigation 20000301 6

We performed genetic immunization of outbred NMRI mice, using a cDNA encoding the human thyrotropin receptor (TSHr). All mice produced antibodies capable of recognizing the recombinant receptor expressed at the surface of stably transfected Chinese hamster ovary (CHO) cells, and sera from most of the immunized mice blocked TSH-dependent stimulation of cAMP accumulation in cells expressing the TSHr. Five out of 29 female mice showed sign of hyperthyroidism including elevated total T4 and suppress ...[more]

PMID: 10727449

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Project description:Background: Graves' disease, caused by autoantibodies that activate the thyrotropin (TSH) receptor (TSHR), has only been reported in humans. Thyroiditis-prone NOD.H2h4 mice develop autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO) but not to the TSHR. Evidence supports the importance of the shed TSHR A-subunit in the initiation and/or amplification of the autoimmune response to the holoreceptor. Cells expressing the gene for the isolated A-subunit secrete A-subunit protein, a surrogate for holoreceptor A-subunit shedding. NOD.H2h4 mice with the human TSHR A-subunit targeted to the thyroid (a "self" antigen in such transgenic (Tgic) animals), unlike their wild-type (wt) siblings, spontaneously develop pathogenic TSHR antibodies to the human-TSH holoreceptor. These autoantibodies do not recognize the endogenous mouse-TSH holoreceptor and do not cause hyperthyroidism. Methods: We have now generated NOD.H2h4 mice with the mouse-TSHR A-subunit transgene targeted to the thyroid. Tgic mice and wt littermates were compared for intrathyroidal expression of the mouse A-subunit. Sera from six-month-old mice were tested for the presence of autoantibodies to Tg and TPO as well as for pathogenic TSHR antibodies (TSH binding inhibition, bioassay for thyroid stimulating antibodies) and nonpathogenic TSHR antibodies (ELISA). Results: Expression of the mouse TSHR A-subunit transgene in the thyroid was confirmed by real-time polymerase chain reaction in the Tgics and had no effect on the spontaneous development of autoantibodies to Tg or TPO. However, unlike the same NOD.H2h4 strain with the human-TSHR A-subunit target to the thyroid, mice expressing intrathyroidal mouse-TSHR A subunit failed to develop either pathogenic or nonpathogenic TSHR antibodies. The mouse TSHR A-subunit differs from the human TSHR A-subunit in terms of its amino acid sequence and has one less glycosylation site than the human TSHR A-subunit. Conclusions: Multiple genetic and environmental factors contribute to the pathogenesis of Graves' disease. The present study suggests that the TSHR A-subunit structure (possibly including posttranslational modification such as glycosylation) may explain, in part, why Graves' disease only develops in humans.

Project description:Background: The actions of thyrotropin-binding inhibitory immunoglobulins (TBIIs) against thyrotropin receptors in thyroid follicular cells have been studied as important etiological factors in Graves' disease (GD). The purpose of this study was to investigate changes in the TBII levels of patients undergoing total thyroidectomy (TTx) or radioactive iodine (RAI) therapy for GD refractory to antithyroid drugs (ATDs). Methods: We enrolled patients who underwent TTx or RAI for GD with previous ATD use between January 2011 and December 2017 at the Samsung Medical Center in Seoul, Korea. Thorough retrospective reviews of medical records were performed in 130 patients. Results: Patients with goiter, ophthalmopathy, high levels of TBIIs, and high doses of ATDs received TTx. Elderly patients with arrhythmia received RAI. We observed that TBII levels continued to decrease after TTx. On the contrary, TBIIs initially increased for 138 days (estimated median time) and then decreased slowly after RAI. A faster decline in TBII levels was observed in the TTx group than in the RAI group (p < 0.001). The estimated median time for TBIIs to decrease below 4.5 IU (3 × upper normal limit, which is known to be a risk factor for fetal hyperthyroidism) was 318 days in the TTx group and 659 days in the RAI group, respectively. In the RAI group, high levels of TBII (>4.5 IU/L) were present in 70 (82%) at 6 months, 57 (67%) at 1 year, and 3 (3%) at 2 years. In the TTx group, rapid decreases in TBII levels were observed in younger patients and those with lower baseline TBII levels. In the RAI group, smaller thyroid volume was correlated with more rapid decrease in TBII levels. Conclusions: The changes in TBII levels following TTx or RAI were different in patients with refractory GD. When deciding on TTx or RAI, this difference should be considered with patient age, severity of hyperthyroidism, goiter, ophthalmopathy, and future pregnancy plans (for young female patients).

Project description:Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a global threat. The only approved vaccine against TB, Mycobacterium bovis bacillus Calmette-Guérin (BCG), provides insufficient protection and, being a live vaccine, can cause disseminated disease in immunocompromised individuals. Previously, we found that intradermal cDNA tattoo immunization with cDNA of tetanus toxoid fragment C domain 1 fused to cDNA of the fusion protein H56, comprising the Mtb antigens Ag85B, ESAT-6, and Rv2660c, induced antigen-specific CD8+ T cell responses in vivo. As cDNA tattoo immunization would be safer than a live vaccine in immunocompromised patients, we tested the protective efficacy of intradermal tattoo immunization against TB with H56 cDNA, as well as with H56_E, a construct optimized for epitope processing in a mouse model. As Mtb antigens can be used in combination with BCG to boost immune responses, we also tested the protective efficacy of heterologous prime-boost, using dermal tattoo immunization with H56_E cDNA to boost BCG immunization in mice. Dermal H56 and H56_E cDNA immunization induced H56-specific CD4+ and CD8+ T cell responses and Ag85B-specific IgG antibodies, but did not reduce bacterial loads, although immunization with H56_E ameliorated lung pathology. Both subcutaneous and intradermal immunization with BCG resulted in broad cellular immune responses, with increased frequencies of CD4+ T effector memory cells, T follicular helper cells, and germinal center B cells, and resulted in reduced bacterial loads and lung pathology. Heterologous vaccination with BCG/H56_E cDNA induced increased H56-specific CD4+ and CD8+ T cell cytokine responses compared to vaccination with BCG alone, and lung pathology was significantly decreased in BCG/H56_E cDNA immunized mice compared to unvaccinated controls. However, bacterial loads were not decreased after heterologous vaccination compared to BCG alone. CD4+ T cells responding to Ag85B- and ESAT-6-derived epitopes were predominantly IFN-?+TNF-?+ and TNF-?+IL-2+, respectively. In conclusion, despite inducing appreciable immune responses to Ag85B and ESAT-6, intradermal H56 cDNA tattoo immunization did not substantially enhance the protective effect of BCG under the conditions tested.

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Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves' disease.

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Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves' disease.

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