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Endocrinology Vol. 145, No. 2 809-816
Copyright © 2004 by The Endocrine Society

Development of a Murine Model of Autoimmune Thyroiditis Induced with Homologous Mouse Thyroid Peroxidase

H. P. Ng, J. Paul Banga and Annie W. C. Kung

Department of Medicine (H.P.N., A.W.C.K.), The University of Hong Kong, Queen Mary Hospital, Hong Kong, People’s Republic of China; and Division of Medicine (J.P.B.), Guy’s, King’s and St. Thomas’ School of Medicine, London SE11 6SP, United Kingdom

Address all correspondence and requests for reprints to: Annie W. C. Kung, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, People’s Republic of China. E-mail: awckung{at}hkucc.hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Autoimmune thyroid disease (AITD) is a common autoimmune disease. Thyroid peroxidase (TPO) is a well characterized autoantigen in AITD. Autoantibodies and autoreactive T lymphocytes to TPO are believed to play a major role in the pathogenesis of lymphocytic thyroiditis. To understand the pathogenic mechanisms of AITD and the role of TPO, we have established a mouse model of lymphocytic thyroiditis by immunizing C57Bl/6 (H-2b), CBA (H-2k), and C57Bl/6 x CBA F1 mice with recombinant murine TPO (rmTPO) ectodomain comprising amino acid residue 1–837 produced in Escherichia coli. Mice were immunized with 30 µg purified ectodomain in complete Freund’s adjuvant. Antibodies against rmTPO were detected in the serum of all mice from day 21 onward. Draining lymph node cells from rmTPO-immunized animals showed dose-dependent proliferation to TPO stimulation. Mice killed at d 50 and 90 revealed variable degrees of thyroiditis with infiltration of mononuclear cells and destruction of thyroid follicles. C57Bl/6 and the F1 mice, in comparison with CBA mice, showed a greater degree of thyroiditis. There was a lack of correction between the intensity of thyroiditis and the anti-TPO response. Immunotyping of the thyroid cellular infiltrates showed predominantly CD4+ T cells and B220+ B cells but scanty CD8+ T cells. None of the control mice injected with the purified fusion partner developed anti-TPO antibodies and thyroiditis. In conclusion, a genuine autoimmune mouse model of lymphocytic thyroiditis was established using autologous mouse TPO. This new model induced with autologous TPO will lead to a better understanding of the mechanisms in destructive thyroiditis and will assist in the development of new strategies for modulating the pathogenic immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AUTOIMMUNE THYROID DISEASES (AITD) are among the most common autoimmune conditions affecting especially adult female populations (1, 2). The thyroid-specific antigens include thyroperoxidase (TPO), thyroglobulin (Tg), and the TSH receptor (3). Autoantibodies and autoreactive T lymphocytes to these autoantigens are believed to play a major role in the pathogenesis of AITD (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Autoantibody to TPO in patients with Hashimoto’s thyroiditis leads to destruction of the thyroid gland and hypothyroidism (4, 5, 6, 7, 8, 9, 10), whereas antibodies to TSH receptor cause hyperthyroidism in Graves’ disease (3, 13). Antithyroperoxidase antibody is cytotoxic to the thyroid cells by their ability to fix complement or by antibody-dependent cell cytotoxicity, suggesting that TPO may be a major contributor to thyroid cell injury in autoimmune disease (4, 5, 6, 7, 8, 9, 10, 14).

Experimental autoimmune thyroiditis (EAT) has been used as a model for studying the pathological mechanisms of Hashimoto’s thyroiditis. Immunization of C57Bl/6 (H-2b) mice with purified porcine TPO (pTPO) led to a dose- dependent thyroiditis in the majority of the animals (15, 16). In a different study, immunization of other strains such as H-2k or H-2d with pTPO failed to give any pathogenicity (17). Importantly, in the pathogenic H-2b strain, the induced antibodies to TPO were specific for the pTPO and did not cross-react with mouse TPO (mTPO) (15, 16). In addition, the animals remained euthyroid (15). It is well recognized that injection with heterologous antigen in adjuvant preferentially leads to the induction of species cross-reactive epitopes (18), which may explain in part the specificity of the anti-TPO response restricted solely to the pTPO in the study reported by Kotani et al. (15). Indeed, it has recently been shown that to obtain an induced autoantibody response to human TPO resembling that present in patients with thyroid autoimmunity, it is necessary to use cellular immunization or naked plasmid DNA vaccination rather than purified TPO for injection (19, 20). In this study, we examined the pathogenicity of recombinant murine TPO (rmTPO) to induce lymphocytic infiltration in H-2b, H-2k, and H-2b x H-2k F1 mice. We show that using autologous mouse thyroid peroxidase, it is possible to break tolerance with adjuvant to allow a bona fide model of lymphocytic thyroiditis to be established, which was dependent on the major histocompatibility complex genetic background of the animal.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Production of mTPO ectodomain
The 2.5-kb cDNA fragment with EcoRI cohesive site coding for amino acids 1–837 of the ectodomain of the mTPO was amplified from the full-length cDNA of mTPO (21) using a pair of primers (5'-GCGGAATTCATGAGAACACTTGGAGCTATC-3' and 5'-GCGGAATTCGGATGCCCGAGGTAGCCTGCC-3') and Expand High Fidelity PCR system (Roche Diagnostics Corp., Mannheim, Germany), which provide an error rate of 8.5 x 10–8. mTPO ectodomain cDNA was introduced into the polylinker of vector pGEX-4T-1, downstream from the glutathione-S-transferase (GST) gene (22). Expression of a GST-mTPO fusion protein and GST were induced by 0.1 mM isopropylthiogalactoside (IPTG). Cells were harvested by centrifugation, and pellets were resuspended in 20 ml PBS containing 1% Sarkosyl, 1 mM phenylmethylsulfonylfluoride, 1% aprotinin, and 50 mg DNase I and sonicated twice for 1 min. The GST-mTPO fusion protein was purified with a glutathione-agarose beads column. rmTPO was separated from the fusion protein by digestion with thrombin. Purified rmTPO ectodomain was analyzed by Western blotting using rabbit antihuman TPO polyclonal antibody Rb6 (23).

Immunization
Eight-week-old female mice of C57Bl/6 (H-2b), CBA (H-2k), and C57Bl/6 (H-2b) x CBA (H-2k) F1 strains were used for the experiments in groups of 10 animals. rmTPO ectodomain was emulsified at a ratio of 1:1 in complete Freund’s adjuvant (CFA) (Sigma, St. Louis, MO), and 30 µg rmTPO ectodomain in 200 µl emulsion was injected sc in the hind footpads and at the back. The boosting injection was carried out 7 d later with the same dose of rmTPO ectodomain emulsified in incomplete Freund’s adjuvant (Sigma). Control mice were immunized with 30 µg GST protein plus adjuvant. Sera for determination of anti-TPO antibody were collected at d 21, 35, 50, and 90 through a retro-orbital route under anesthesia. Sera for total T4 measurement were collected at d 50 and 90. All animals were housed in the Laboratory Animal Unit of the Faculty of Medicine, University of Hong Kong, under sterile barrier-free controlled conditions, and the study was approved by the Ethics Committee of Hong Kong and performed according to the standards laid out by the University of Hong Kong.

Measurement of mTPO antibody by ELISA
For ELISA, rmTPO was diluted in 0.06 M sodium bicarbonate coating buffer (pH 9.6) at 3 µg/ml. As control, 3% BSA was used for coating, and 100 µl of each diluted antigen was added to the wells of a microwell plate (Costar, Cambridge, MA) and incubated overnight at 4 C. After washing three times with PBS containing 0.1% Tween 20, all wells were saturated with blocking solution (5% skimmed milk powder in PBS). Then, 100 µl of 50x diluted sera from immunized mice was added and incubated for 1.5 h at 37 C. The plate was again washed and incubated with alkaline phosphatase-conjugated antimouse IgG (Sigma) for 1 h at 37 C. The plate was washed and developed with p-nitrophenol phosphate (Sigma Fast p-nitrophenol phosphate tablet set) for 30 min at room temperature. All determinations were done in duplicate, and the absorbance was read at 405 nm.

Cross-reactivity of mTPO autoantibody
Cross-reactivity of anti-rmTPO antibody was tested against native mTPO and human TPO (hTPO). Crude extract of native mTPO was extracted from the thyroid of 20 hypothyroid mice fed with 0.1% propylthiouracil for 10 d. The thyroid microsomes were prepared as described previously (24). Briefly, murine thyroid glands were homogenized in lysis buffer (5 M KCl containing 1 mM phenylmethylsulfonylfluoride and 10% aprotinin). The lysate was centrifuged at 100,000 x g for 1 h, and the microsomes were resuspended in lysis buffers. The denatured rmTPO was prepared as follows: 1 mg/ml rmTPO in PBS was heated for 15 min at 95 C (heat-denatured rmTPO) or exposed to 6 M urea (Amersham, Little Chalfont, UK) for 12 h at 37 C and dialyzed against PBS for 48 h (urea-denatured rmTPO). Purified hTPO was prepared as previously described (25). Briefly, highly purified hTPO was obtained from pooled Graves’ disease thyroid glands by the combination of detergent solubilization, monoclonal antibody affinity, and ion-exchange chromatography. After determination of anti-rmTPO activity, five sera with high activity from each strain of mice were used to ascertain reactivity by ELISA with either crude native mTPO, heat- or urea-denatured rmTPO, or purified hTPO.

Histology
Five mice of each strain of the animals were killed at d 50 and the rest at d 90 to examine for evidence of thyroiditis. The thyroid glands were removed and carefully embedded in OCT compound (Sakura Finetechnical Co., Tokyo, Japan) and rapidly frozen in isopentane cooled in liquid nitrogen to generate cryostat sections. The frozen section was subjected to hematoxylin and eosin staining and immunostaining using different rat antimouse monoclonal antibodies specific for CD4+ and CD8+ T cells and B220+ B cells (PharMingen, San Diego, CA). The purified rat IgG2a and IgG2b (PharMingen) were used as isotype controls. The severity of thyroiditis was graded on a scale of 0–4 as follows: grade 0, normal histology; grade 1, interstitial accumulation of inflammatory cells distributed around one or two follicles; grade 2, one or more foci of inflammatory cells reaching at least the size of one follicle; grade 3, 10–40% of thyroid replaced by inflammatory cells; and grade 4, more than 40% of thyroid replaced by inflammatory cells. Scoring was performed blind to the animal treatment groups. For immunostaining, after fixing the section with acetone for 10 min, rat antimouse monoclonal antibody diluted 1:100 in 3% BSA/PBS was applied onto the section for 1 h. The sections were rinsed gently with PBS, and the peroxidase-conjugated polyclonal antirat Ig antibody (PharMingen) was applied for 1 h. The diaminobenzidine substrate (Sigma 3,3'-diaminobenzidine tablet set) was given after washing with PBS.

Proliferation assay of T lymphocytes
Draining lymph nodes of immunized F1 mice were excised 8–10 d after the second immunization, and a single-cell suspension was prepared. Cell number was adjusted to 1 x 107 cells/ml in RPMI 1640 (Life Technologies, Inc., Invitrogen Corp., Carlsbad, CA) supplemented with L-glutamine, 100 U/ml antibiotic-antimycotic, and 10% fetal calf serum, and 100 µl of cell suspension was added to each well of a flat-bottom 96-well plate (Costar). rmTPO ectodomain was diluted with RPMI and was added to the cell suspension at different concentration. All experiments were performed in triplicate. After 4 d of incubation in humidified air containing 5% CO2, each well was pulsed with [methyl-3H]thymidine (Amersham). The cultured cells were then harvested on glass fiber filters, and thymidine incorporation was measured by a liquid scintillation counter.

Total T4 measurement
Serum total T4 was measured by fluorescent polarization immunoassay using a commercial kit (Abbott Laboratories, Abbott Park, IL). The mean T4 levels of different strains of mice were determined in 10 8-wk-old adult female mice. The normal range was defined as mean ± 3 SD with C57Bl/6 being 50–91 nmol/liter; CBA, 39–86 nmol/liter, and C57Bl/6 x CBA F1, 46–130 nmol/liter. Comparisons before and after treatment as well as between groups were by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production of mTPO ectodomain
Upon IPTG induction, GST-mTPO ectodomain fusion protein was induced to express at approximately 116 kDa in Escherichia coli transformed with pGEX-4T-1 (Fig. 1AGo). After purification by GST-glutathione agarose bead column and digestion with thrombin, the recombinant mTPO ectodomain (rmTPO) comigrated to the correct molecular size of approximately 89 kDa on Western blot (Fig. 1BGo). The yield of rmTPO from l liter of bacterial culture after IPTG induction was 0.5 mg/liter bacterial culture.



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  		FIG. 1. Production of rmTPO ectodomain from E. coli cDNA fragment of mTPO ectodomain was inserted into pGEX-4T-1 vector polylinker downstream from the GST gene. A, Upon IPTG induction, GST mTPO ectodomain fusion protein was induced to express at 116 kDa; lane a, marker; lane b, E. coli lysate without IPTG induction; lane c, E. coli induced with 0.1 mM IPTG to produce mTPO ectodomain. B, Purified mTPO ectodomain was recognized by polyclonal antihuman antibody Rb6 in Western blotting after purification with GST-glutathione agarose bead column and thrombin digestion; lane a, marker; lane b, purified mTPO recognized by rabbit anti-TPO polyclonal antibody.

 
Production of mTPO ectodomain-specific antibody
Immunization of the animals resulted in the production of antibodies against mTPO ectodomain in all strains of mice (Fig. 2Go). When sera were diluted at 1:50, anti-mTPO ectodomain antibodies could be detected at d 21 and the concentration of antibody increased gradually after immunization. None of the control animals had detectable anti-TPO antibodies. Because the animals were immunized with recombinant TPO produced in E. coli, we also determined the cross-reactivity of the induced anti-TPO antibodies with native mTPO extracted from thyroid glands of hypothyroid mice as well as the cross-reactivity with hTPO. As shown in Fig. 3Go, different strains of mice displayed differences in the cross-reactivity of the induced anti-TPO antibodies. Thus, anti-TPO serum from C57/BL6 mice show statistically significant binding to native mTPO compared with control (GST-immunized) sera. More interestingly, the CBA and F1 mice induced anti-rmTPO antibodies do not appear to significantly recognize the native TPO (Fig. 3Go). Moreover, denaturation of rmTPO by heat or with urea did not alter significantly the reactivity of anti-TPO antibodies in all three strains of mice compared with control GST-injected sera; however, C57Bl/6 anti-TPO responses to the denatured TPO were weaker than those in CBA mice. Finally, the induced anti-rmTPO antibodies in all three strains of mice show weak cross-reactivity with hTPO (Fig. 3Go).



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  		FIG. 2. Detection of anti-mTPO antibodies after immunization with rmTPO. Sera were collected from 10 mTPO-immunized mice and 10 control mice immunized with GST plus adjuvant at d 21, 35, 50, and 90 and assayed at 1:50 dilution. The data of 10 mice in each group are shown as mean ± 1 SD: C57Bl/6 mice immunized with CFA+GST ({triangleup}) or with CFA+rmTPO ({blacktriangleup}); CBA mice immunized with CFA+GST ({circ}) or with CFA+rmTPO ({bullet}); and F1 mice immunized with CFA+GST ({square}) or with CFA+rmTPO ({blacksquare}). Antibody against mTPO ectodomain was detected in mTPO-immunized mice at d 21 and after.

 


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  		FIG. 3. Reactivity of anti-rmTPO antibodies. Five sera from each strain collected at d 50 with high anti-rmTPO activity, and five sera from control mice were reacted against either native mTPO, heat-denatured rmTPO, urea-denatured rmTPO, or purified hTPO. The dilution of anti-rmTPO antibodies were at 1:50 except for native mTPO, which was 1:25. ELISA data of five mice in each group are shown as mean ± 1 SD: sera from rmTPO-immunized mice ({blacksquare}) and control mice ({square}); positive control rabbit anti-hTPO antibody Rb6 ( ); and negative control normal rabbit serum ( ). The dilution of Rb6 and normal rabbit serum was 1:1000.

 
Histology
At d 50, all three strains of mice showed evidence of different degrees of infiltration by mononuclear cells on thyroid section (Table 1Go). C57Bl/6 (H-2b) and F1 mice developed more severe thyroiditis compared with CBA (H-2k) animals at d 90 (ANOVA, P < 0.02). Furthermore, destruction of follicles and reduction in the follicular size were also seen in some sections (Fig. 4Go). Interestingly, the degree of thyroiditis by histology showed no correlation with the antibody response in individual animals, as CBA (H-2k) did not develop a significant degree of thyroiditis, although this strain produced strong anti-mTPO antibody response. Characterization of the phenotype of the infiltrates by immunostaining showed that the majority of the mononuclear cells found in between the follicles were CD4+ T cells and CD 220+ B cells, and only scanty CD8+ T cells were seen (Fig. 5Go).


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  		TABLE 1. Severity of thyroiditis induced by immunization with rmTPO

 


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  		FIG. 4. Hematoxylin-eosin staining of frozen sections of the thyroid glands obtained from immunized mice. A and E, At d 90, control C57Bl/6 [A (x100)] and F1 [E (x100)] mice immunized with GST showed normal histology; B, C, F, and G, mononuclear cell infiltrates in between thyroid follicles in C57Bl/6 [B (x100) and C (x200)] and F1 [F (x100) and G (x200)] immunized with rmTPO; D, destruction of thyroid follicles in rmTPO-immunized mice [D (x100) and H (x200)].

 


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  		FIG. 5. Immunoperoxidase staining of frozen sections of the thyroid gland from F1 mice at d 50 after immunization. A–C, CD4+ T cells infiltrating in between the follicles [A (x400)], B220+ B cells [B (x400)], and CD8+ cells [C (x400)]; D, control mice with no infiltrating mononuclear cells [D (x200)]; E and F, negative controls rat IgG2b [E (x200)] and IgG2a [F (x200)].

 
Proliferation assay of T lymphocytes of mTPO- immunized mice
T lymphocytes obtained from animals immunized with mTPO ectodomain when cultured in the presence of rmTPO ectodomain showed a dose-dependent proliferative response with the stimulation index being greater than 30 (Fig. 6Go). In contrast, lymph node cells obtained from control animals immunized with GST protein did not show any proliferative response to rmTPO ectodomain.



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  		FIG. 6. Proliferation assay (mean ± 1 SD of cpm) of mTPO ectodomain-primed lymph node T lymphocytes to rmTPO ectodomain. Lymph node cells (LNCs) were pooled from three rmTPO- immunized F1 mice. LNC suspension cultured in the presence of mTPO ectodomain showed a dose-dependent proliferative response when compared with control LNCs primed with GST plus adjuvant. The rmTPO concentrations for proliferation assays were 0 ({square}), 1 ( ), 5 (), and 25 µg/ml ({blacksquare}).

 
Serum total T4 level
In the rmTPO-immunized animals, none of the animals in three different strains showed subnormal T4 levels at d 50. By d 90 only one C57Bl/6 had subnormal T4 level of 48 nmol/liter. None of the control animals had abnormal T4 levels throughout the study.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We describe the first successful attempt of establishing a bona fide autoimmune thyroiditis model by inducing an inflammatory cell infiltrate into the thyroid glands of mice with syngeneic mTPO ectodomain. These animals developed autoantibodies against the autoantigen mTPO as well as sensitized lymphocytes against mTPO. There were histological features of thyroiditis with infiltration of T and B lymphocytes. Despite the fact that anti-TPO antibodies were readily induced in both C57Bl/6 and CBA mice with insignificant difference, the incidence and degree of thyroiditis in CBA is relatively lower, suggesting that the H-2b haplotype is more susceptible to induction of thyroiditis by rmTPO in adjuvant. However, the H-2k haplotype in F1 mice does not contribute either susceptibility or resistance to the thyroiditis, implicating that the H-2b haplotype contributes as a genetic susceptible element for pathogenicity. Furthermore, even in the inbred F1 strain of H-2b x H-2k mice, there was variation in disease susceptibility, because not all animals demonstrated thyroiditis despite all developing antibodies to TPO. In addition, there was no correlation of anti-TPO response to the severity of thyroiditis. These findings agree with the results found in the study using pTPO to induce thyroiditis (15), in which the degree of thyroiditis was more severe in H-2b than H-2K mice. Similar to our present study, specific pTPO autoantibodies did not correlate with the severity of thyroiditis in any strain of the mice studied (15). This observation is in contrast to those seen in the Tg-induced EAT mouse model, where there was a strong correlation of the anti-Tg antibody titer with the degree of thyroiditis (26, 27).

We found that rmTPO antibody reacted to both native mTPO and hTPO. The weak reactivity to mTPO in ELISA may be due to several reasons. First, an E. coli system was used to produce the rmTPO in this study. It is known that recombinant protein generated from a prokaryotic expression system may have improper folding, and it is likely that the anti-rmTPO autoantibodies induced in our animals may not be able to recognize conformational epitopes of native TPO. Second, the amount of mTPO in the crude thyroid microsome extract may be insufficient for strong binding in ELISA. Third, to increase the yield of TPO, we induced hypothyroidism in the mice before protein extraction. It has been demonstrated that increased TSH stimulation may alter the expression of autoantibody binding domains of TPO in cultured human thyroid cells in vitro (28). Denaturation of rmTPO by heat or urea reduced the recognition of anti-rmTPO antibody, suggesting that the secondary structure of the epitopes is important for antibody recognition. When compared with mTPO, hTPO shares 73% homology in the protein sequence (21, 29). The binding of anti-rmTPO antibody to hTPO may similarly be directed against linear hTPO epitopes that share high homology to mTPO. From human data we know that anti-TPO antibodies against conformational epitopes play a more important role in the development of AITD than antibodies against linear epitopes (3, 4, 30). In a study comparing the quality of the antibodies raised from immunization with either cellular hTPO or recombinant hTPO, it was found that anti-hTPO antibody raised from cellular hTPO resembled closely patients’ autoantibodies in terms of their high affinities, restricted epitopes, and TPO epitopic fingerprints (19, 31).

Similar to the pTPO-induced EAT model (15), immunizing mice with mTPO in CFA induced a lymphocytic form of EAT in which the thyroid was infiltrated primarily by T lymphocytes and other mononuclear cells. Immunophenotyping of the intrathyroid infiltrates of our animals showed mainly CD4+ T cells and B220+ B cells. In patients with autoimmune thyroid disease, it has been demonstrated that intrathyroid B cells are important for the production of anti-thyroid antibody (32, 33). A high proportion of intrathyroid B cells corresponded to a high titer of thyroid antibodies in patients with both Graves’ disease and Hashimoto’s thyroiditis (34). B cell accumulation in the thyroid may be mediated by T cells because an increase in intrathyroid CD4+ T cells was also seen. In our present study, only scanty CD8+ cells were detected among the intrathyroid infiltrates. Our immunization regimen with syngeneic mTPO and adjuvant might disturb the balance between the self-reactive lymphocytes and regulatory cells in the mice, but failed to elicit strong autoreactive cytotoxic T cell response during the observation period of our experiment. Previous studies have shown that CD4+ T cells are both sufficient and necessary in EAT induction because the disease can be transferred into naive recipients by Tg-specific CD4+ T cell lines or clones (35, 36), and it does not develop in nude mice (37). Initiation of a CD8+ T cell response to injected protein is thought to be indirect through cross-priming by dendritic cells (38, 39), and the kinetics of the cross-priming is late. The mechanism of cross-priming for inducing cytotoxic T cells remains unclear, but it is known that apoptotic cells in the inflammatory tissue are potent inducers for priming a cytotoxic T cell response. Recent studies in apoptosis provide evidence for involvement of death receptors and cytokine-regulated apoptotic pathways rather than antibody- and T cell-mediated death mechanisms as being responsible for autoimmune thyrocyte depletion in the pathogenesis of thyroid autoimmunity (14).

None of the animals, except one C57Bl/6, had subnormal T4 levels during the observation period. In mice, measurement of the total T4 level alone is not a good indicator for documentation of hypothyroidism. Mice are not very susceptible to the disturbance of thyroid function, and their T4 levels fluctuate widely even within inbred strains (40). Unfortunately, we were unable to measure serum TSH levels in these animals. Another reason for not developing hypothyroidism in our animals may be related to the mild immunization protocol used in our experiments, or perhaps we had not observed our animals for a sufficient period to detect the evolution of the autoimmune process. In humans, total thyroid failure is apparent only after a long period of subclinical mild hypothyroidism (41), and smoldering thyroiditis can exist for decades before glandular failure and the onset of clinical symptoms. In conclusion, we were able to establish a bona fide model of autoimmune thyroiditis and document the role of TPO as an autoantigen in autoimmune thyroiditis.


   Footnotes
 
This work was supported by the Hong Kong Research Grant Council.

Abbreviations: AITD, Autoimmune thyroid disease; EAT, experimental autoimmune thyroiditis; GST, glutathione-S-transferase; hTPO, human TPO; IPTG, isopropylthiogalactoside; rmTPO, recombinant murine TPO.

Received May 28, 2003.

Accepted for publication October 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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