Clinical and Investigative Medicine

Thyroid xenografts from patients with Graves' disease in severe combined immunodeficient mice and NIH-beige-nude-xid mice

Toshio Mukuta, MD
Guillermo Arreaza, MD
Mitsushige Nishikawa, MD, PhD
Erika Resetkova, MD, PhD
Christopher Jamieson, MD
Hajime Tamai, MD
Robert Volpé, MD

Clin Invest Med 1997; 20 (1): 5-15.

[résumé]

Drs. Mukuta and Arreaza were Fellows of the Angus Foundation, Wellesley Hospital; Dr. Nishikawa was a Fellow of the Thyroid Foundation of Canada; Dr. Resetkova was a Fellow of Medical Research Council of Canada/ICI Pharma; Dr. Jamieson was with the Department of Surgery, Wellesley Hospital, University of Toronto, Toronto, Ont.; Dr. Tamai is with the Department of Psychosomatic Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; and Dr. Volpé is with the Division of Endocrinology and the Endocrinology Research Laboratory, Wellesley Hospital, Toronto, Ont.

(Original manuscript submitted Feb. 6, 1996; received in revised form July 8, 1996; accepted July 23, 1996)

Reprint requests to: Dr. Robert Volpé, Endocrinology Research Laboratory, Wellesley Hospital, University of Toronto, 160 Wellesley St. E, 112D Jones Bldg., Toronto ON M4Y 1J3; fax 416 966-5046

Contents

Abstract

 Objective: To compare human thyroid xenografts from patients with Graves' disease in severe combined immunodeficient (SCID) mice and triple immunodeficient NIH-beige-nude-xid (NIH-3) mice to obtain an improved animal model for studying these xenografts.

Design: Animal study.

Participants and animals: Patients with Graves' disease; SCID and NIH-3 mice.

Interventions: Thyroid tissue from six patients with Graves' disease was xenografted to SCID and NIH-3 mice; in addition, peripheral blood mononuclear cells (PBMC) from 12 patients with Graves' disease were grafted intraperitoneally to separate SCID and NIH-3 mice.

Outcome measures: Levels of human immunoglobulin (IgG), thyroperoxidase antibodies (TPO-Ab), thyroglobulin (Tg-Ab), and expression of thyrocyte intercellular adhesion molecule-1 (ICAM-1) and histocompatibility leukocyte antigen (HLA-DR) in mice after xenografting.

Results: IgG was detected in all mice grafted with Graves' thyroid tissue and some mice grafted with PBMC; levels of human IgG peaked 6 to 10 weeks after xenografting. Human IgG levels reached a mean of 500 mg/L (standard error of the mean [SEM] 150 mg/L) in the NIH-3 mice with thyroid xenografts. This was similar to results in SCID mice with thyroid xenografts, which had a mean level of human IgG of 640 mg/L (SEM 230 mg/L). PBMC xenografting resulted in a mean IgG level of 1200 mg/L (SEM 250 mg/L) in NIH-3 mice, which was similar to the mean level of 1000 mg/L (SEM 280 mg/L) in SCID mice. The rate of rise in human IgG in the sera of the NIH-3 mice with thyroid xenografts was similar to that in the SCID mice. TPO-Ab were also detected in some mice with Graves' thyroid grafts and in a few mice injected with PBMC, with levels peaking 4 to 6 weeks after xenografting. TPO-Ab levels reached a mean 109.3 U/mL (SEM 57.2 U/mL) in the NIH mice with thyroid xenografts, which were similar to the mean level of 91.7 U/mL (SEM 34.2 U/mL) in the SCID mice. There were no significant differences in the Tg-Ab levels in each type of mice (13.9 [SEM 12.1] U/mL v. 17.9 [SEM 7.9] U/mL). Eight weeks after xenografting into mice, the expression of xenograft thyrocyte ICAM-1 decreased significantly in both the SCID and NIH-3 mice (from 43.4%, SEM 4.9%, to 35.9%, SEM 4.6%, in the NIH-3 mice, p<0.05, and from 43.4%, SEM 4.9%, to 32.5%, SEM 5.2%, in the the SCID mice, p<0.05). However, the expression of thyrocyte HLA-DR did not change significantly in the NIH-3 mice (from 11.5%, SEM 3.3%, to 10.8%, SEM 3.3%), whereas it decreased significantly in the SCID mice (from 11.5%, SEM 3.3%, to 4.2%, SEM 2.0%, p<0.02).

Conclusions: Not only SCID mice but also NIH-3 mice may be useful as animal models for xenografted thyroid tissue, which will help us elucidate the pathogenesis of autoimmune thyroid disease. NIH-3 mice are superior to SCID mice in maintaining the expression of thyrocyte HLA-DR in Graves' thyroid xenografts at levels as high as those before xenografting; this maintenance of expression may be due to the lack of natural killer cells in NIH-3 mice.

Résumé

 Objectif : Obtenir un modèle animal amélioré pour l'étude des xénogreffes de tissus thyroïdiens humains provenant de sujets avec maladie de Graves, en comparant les résultats de telles xénogreffes chez des souris avec déficit immunitaire combiné sévère (SCID) et chez des souris avec déficit immunitaire triple (NIH-3).

Devis : Étude chez l'animal.

Sujets : Patients avec maladie de Graves; souris SCID et NIH-3.

Interventions : Une xénogreffe a été pratiquée chez des souris SCID et NIH-3 avec le tissu thyroïdien provenant de six sujets avec maladie de Graves. Une greffe intra-péritonéale de cellules mononuclées du sant périquérique (CMSP) provenant de 12 sujets avec maladie de Graves a été pratiquée chez d'autres souris SCID et NIH-3. Variables mesurées : Niveaux d'IgG humaines, d'anticorps anti-thyroperoxydase (AcTPO) et d'anticorps anti-thyroglobuline ainsi que l'expression de la molécule d'adhésion inter-cellulaire de type 1 (ICAM-1) et des antigènes leucocytaires d'histocompatibilité (HLA-DR) chez les souris greffés.

Résultats : Des IgG humaines ont été décelées chez toutes les souris greffées avec le tissu thyroïdien et chez certaines souris greffées avec les CMSP. Le niveau maximal d'IgG humaines fut atteint 6 à 10 semaines après la greffe. Le niveau moyen d'IgG humaines était de 500 mg/L (erreur standard de la moyenne [ESM] 150 mg/L) chez les souris NIH-3 avec xénogreffes thyroïdiennes. Ce résultat était similaire aux souris SCID avec xénogreffes thyroïdiennes dont le niveau moyen d'IgG humaines était de 640 mg/L (ESM 230 mg/L). La xénogreffe de CMSP résulta en un niveau moyen d'IgG de 1200 mg/L (ESM 250 mg/L) chez les souris NIH-3, et ceci était similaire au niveau atteint chez les souris SCID (1000 mg/L, ESM 280 mg/L). Le taux d'augmentation d'IgG humaines était similaire chez les deux types de souris. Des AcTPO furent décelés chez certaines souris greffées avec tissus thyroïdiens et chez quelques souris receveuses de CMSP, le niveau maximal étant atteint 4 à 6 semaines après la greffe. Les niveaux moyens d'AcTPO étaient similaires chez les souris NIH-3 et SCID greffées avec tissu thyroïdien (109.3 U/mL [ESM 57.2 U/mL] versus 91.7 U/mL [ESM 34.2 U/mL]). Il n'y avait pas de différence significative quant au niveau moyen d'anticorps anti-thyroglobuline entre les deux types de souris (13.9 [ESM 12.1] U/mL versus 17.9 [ESM 7.9] U/mL). Une diminution significative dans l'expression de la ICAM-1 par les thyrocytes greffés survint dans les deux types de souris (de 43.4% [ESM 4.9%] à 35.9% [ESM 4.6%]) dans le groupe NIH-3, p<0.05). Par contre, l'expression des antigènes leucocytaires d'histocompatibilité (HLA-DR) par les thyrocytes n'était pas modifiée de manière significative chez la souris NIH-3 (de 11.5% [ESM 3.3%] à 10.8% [ESM 3.3%]), tandis qu'une diminution significative était observée chez la souris SCID (11.5% [ESM 3.3%] à 4.2% [ESM 2.0%], p<0.02).

Conclusions : En plus des souris SCID, les souris NIH-3 sont un modèle animal utile de xénogreffes de tissu thyroïdien et ce modèle pourrait aider à élucider la pathogénèse des maladies thyroïdienne auto-immunes. Le modèle NIH-3 est supérieur au modèle SCID, puisque l'expression des marqueurs HLA-DR dans les thyrocytes xénogreffés s'est maintenue à un niveau aussi élevé qu'avant la greffe. Ce maintien de l'expression des marqueurs HLA-DR pourrait être dû à l'absence de cellules tueuses naturelles chez les souris NIH-3.

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Introduction

In vivo studies of human autoimmune thyroid disease (AITD) have been difficult, primarily because of ethical considerations. Furthermore, although nude mice accept human thyroid xenografts, they lyze passenger human lymphocytes because of the presence of functional murine natural killer (NK) and B cells.[1­3] Severe combined immunodeficient (SCID) mice have a defect in the recombinase system for antigen receptor genes that results in a lack of mature T and B cells,[4­6] but they have NK cells;[7] therefore, both a thyroid xenograft and its lymphocytes survive in SCID mice.[8­10] In the last few years, we have been using an SCID-mouse model to study AITD.[11­16] We have demonstrated successful reconstitution of SCID mice with intrathyroidal lymphocytes of thyroid xenografts and detection of autoantibodies in sera of SCID mice xenografted with peripheral blood mononuclear cells (PBMC) from patients with Graves' disease.[13] Our results are similar to other reported results from studies involving patients with autoimmune disease, including AITD.[17­19] However, Martin and associates[20] have reported that, in this model, the lymphocyte infiltrates remained unchanged or diminished in size, compared with those of the original donor tissue.[20]

The NIH-3 mice (nu-nu, bg/bg, xid/xid), first reported by Andriole and associates,[21] are immunodeficient mice with three separate mutations that affect regulation of the immune system. By combining these mutations, experimenters produced mice with deficiencies in not only T and B but also NK cells. In 1992, Defosse, Duray and Johnson[22] reported that NIH-3 mice were useful as a model for infectious disease. Thyroid xenografts in these mice have not been reported to date. We hoped that an animal model that simulates human AITD could be found. We also wished to investigate whether NIH-3 mice are superior to SCID mice as an animal model for the study of human AITD. We therefore compared the effects of human thyroid xenografts from patients with Graves' disease in these two different kinds of immunodeficient mice; we studied the nature of the histologic changes and the immune responses to the xenografts in these two mouse strains.

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Material and methods

Mice

SCID male mice (C.B-17, scid/scid) were obtained from the Ontario Cancer Institute­Princess Margaret Hospital, Toronto. NIH triple immunodeficient male mice (NIH-3, nu/nu, bg/bg, xid/xid) were obtained from Taconic Farms, Germantown, NY. Mice were 8 to 12 weeks old at the time of xenografting and were not treated with antibiotics. During the experiments, the mice did not show any overt signs of secondary infections, i.e., lethargy, ruffled or lost fur, or laboured breathing. The standards of animal care guidelines were observed.

Xenotransplantation of human thyroid tissue and human peripheral blood mononuclear cells

Xenotransplantation of human thyroid tissue was performed, as described elsewhere.[23,24] Briefly, 18.9 to 36.8 g of human thyroid tissue from six patients with hyperthyroid Graves' disease (patients 1 to 6 in Table 1) were obtained at surgery after the patients had given informed consent. These tissues were cut into small pieces and xenografted subcutaneously into three SCID and three NIH-3 mice (each mouse weighing 0.8 g) within 24 hours after surgery. No animal received more than one xenograft. The tissues were reasonably homogeneous in each case, so that individual mice receiving grafts from one donor received analogous tissue. The number of SCID and NIH-3 mice receiving thyroid from a given patient was the same.

Human PBMC from 12 other patients with hyperthyroid Graves' disease (patients 1 to 12 in Table 2) were separated by Ficoll­Hypaque density gradient centrifugation (Pharmacia, Piscataway, NJ).[25] Twelve other SCID mice and 12 other NIH-3 mice were injected intraperitoneally with 20 ×10[6] cells each of PBMC.

Detection of human immunoglobulin, thyroperoxidase antibody and thyroglobulin in sera of mice

Human immunoglobulin (IgG) was measured by the single radial immunodiffusion method using immunodiffusion plates (NOR-Partigen IgG MC and LC-Partigen IgG, Behringwerke AG, Marburg, Germany) as previously described.[13,14] There was no crossreactivity with mouse IgG in this system. The quantitative determination of thyroperoxidase antibody (TPO-Ab) and thyroglobulin (Tg-Ab) were assayed by radioimmunosorbent assay (RIA) kits (Kronus, Dana Point, Calif.).

Detection of thyroid-stimulating antibody

The cloned rat thyroid cell line FRTL-5[26] was cultured, as described elsewhere.[27] Thyroid-stimulating antibody (TSAb) was assayed by measuring adenosine 3 prime:5 prime-cyclic phosphate (cAMP) production in FRTL-5 cells, as described,[13,14] with the use of an RIA kit (Amersham, Arlington Height, Ill.). The normal range (within two standard deviations of the mean values in normal sera) is 56% to 144%. Any level above 144% was considered TSAb-positive. All TSAb assay measurements were run simultaneously in a single assay.

Thyrocyte histocompatibility leukocyte antigen and intercellular adhesion molecule-1 expression

Measurements of thyrocyte histocompatibility leukocyte antigen-DR (HLA-DR) or intercellular adhesion molecule-1 (ICAM-1) expression were carried out by flow cytometric analysis, as described elsewhere,[28] with minor modifications. Briefly, thyrocytes digested with 2 mg/mL collagenase (type II, Sigma, St. Louis, Mo.) were cultured for 2 days. Cultured cells (1 ×times 10 to the sixth) were allowed to incubate for 45 minutes at 4 degrees C with 10 microlitres phycoerythrin (PE)-conjugated anti-HLA-DR mAb or anti-ICAM-1 mAb. PE-mouse IgG was used as a nonspecific antibody control. After incubation, cells were washed with cold phosphate-buffered saline solution containing 0.1% sodium azide, and fixed with 1% paraformaldehyde. After fixation, labeled cells were analysed with a flow cytometer (FACScan, Becton Dickinson, Mountain View, Calif.) and a computer system (Lysis II, Becton Dickinson). The percentage of HLA-DR positive or ICAM-1 positive thyrocytes was determined.

Light microscopic studies

Before and after xenografting, the thyroid tissues were fixed in neutral formalin, embedded in paraffin and submitted to the Department of Pathology, Wellesley Hospital, Toronto, for routine light microscopic studies.

Experimental protocols

Human thyroid tissues from six patients with Graves' disease (patients 1 to 6 in Table 1) were xenografted into three SCID (SCID-TH) and three NIH-3 (NIH-3-TH) mice. Eight weeks after xenografting, the SCID-TH and NIH-3-TH mice were killed. HLA-DR and ICAM-1 expression were determined, and histologic assessment of the thyroid xenografts was undertaken. Human PBMC from 12 patients with Graves' disease (patients 1 to 12 in Table 2) were grafted intraperitoneally in 12 SCID (SCID-PB) and 12 NIH-3 (NIH-3-PB) mice. During 8 consecutive weeks, blood samples were obtained from the lateral tail vein of the SCID and NIH-3 mice every 2 weeks for the measurement of human IgG, TPO-Ab, Tg-Ab and TSAb.

Statistical analysis

Student's t-test and multiple comparison tests (analysis of variance and Sheffé's method) were employed. A p value of less than 0.05 was considered significant.

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Results

Human IgG production in SCID mice and NIH-3 mice xenografted with human thyroid or human PBMC

Human IgG was detected in all of the mice xenografted with thyroid tissues from patients with Graves' disease and in some of the mice grafted with PBMC. Levels peaked 6 to 10 weeks after xenografting. The rate of increase in human IgG in sera of the NIH-TH mice was similar to that in the SCID-TH mice (data not shown). The maximum human IgG levels obtained were a mean of 500 mg/L (standard error of the mean [SEM] 150 mg/L) in the NIH-3-TH mice, levels similar to those obtained in the SCID-TH mice (mean 640 mg/L, SEM 230 mg/L). The levels obtained in the NIH-3-PB mice (mean 1200 mg/L, SEM 250 mg/L) were also similar to those in the SCID-PB mice (mean 1000 mg/L, SEM 280 mg/L) (Fig. 1). There were no significant differences between the maximum IgG levels in the two types of mouse.

TPO-Ab and Tg-Ab in SCID mice and NIH-3 mice xenografted with human thyroid or human PBMC

TPO-Ab and Tg-Ab were detected in some of the mice with Graves' thyroid grafts and in a few of the mice injected with PBMC. Levels peaked 4 to 6 weeks after xenografting. The maximum TPO-Ab levels were a mean of 109.3 U/mL (SEM 57.2 U/mL) in the NIH-TH mice, and similar (mean 91.7 U/mL, SEM 34.2 U/mL) in the SCID-TH mice. There were no significant differences between the maximum TPO-Ab levels in the two types of mouse. Moreover, there were no significant differences in the maximum Tg-Ab levels in the two types of mouse (mean 13.9, SEM 12.1 U/mL v. mean 17.9, SEM 7.9 U/mL) (Fig. 2).

TSAb in SCID mice and NIH-3 mice xenografted with human thyroid tissues

TSAb was detected in most of NIH-3 mice with thyroid grafts, and levels peaked 4 to 6 weeks after xenografting. The maximum TSAb level was a mean of 550% (SEM 91%) in the NIH-3-TH mice, significantly higher than the mean level of 137% (SEM 23%) in the SCID-TH mice (p<0.02) (Fig. 3).

Thyrocyte ICAM-1 and HLA-DR expression after thyroid xenografting

Eight weeks after xenografting of thyroid tissues into mice, the expression of ICAM-1 on thyrocytes of xenografted thyroid tissues decreased significantly in both the NIH-3 and the SCID mice, compared with the expression in the tissue after human thyroid surgery. Expression decreased from a mean of 43.4% (SEM 4.9%) to 35.9% (SEM 4.6%) in the NIH-3 mice and to 32.5% (SEM 5.2%) in the SCID mice, p<0.05) (Fig. 4). There were no significant differences between thyrocyte ICAM-1 expression in the NIH-3 mice and the SCID mice. However, the expression of HLA-DR on thyrocytes did not change significantly in the NIH-3 mice (from a mean of 11.5%, SEM 3.3%, to a mean of 10.8%, SEM 3.3%), but decreased significantly in the SCID mice (from a mean of 11.5%, SEM 3.3%, to 4.2%, SEM 2.0%, p<0.02) (Fig. 5). Moreover, thyrocyte HLA-DR expression in the NIH-3 mice was significantly higher than that in the SCID mice (p<0.05).

Histologic findings from xenografted thyroid tissue

Histologic studies showed that human lymphocyte infiltration in grafts was maintained 8 weeks after xenografting into the NIH-3 mice, and that infiltration was similarly maintained in the xenografts in the SCID mice, with the exception of the tissue from one patient. In the tissue from this patient, the lymphocytic infiltration was more prominent in tissue grafted into the NIH-3 mouse than in that grafted into the SCID mouse (Figs. 6 to 8).

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Discussion

We have previously reported thyroid antibody production in SCID mice containing xenografts of human thyroid tissue from patients with Hashimoto's thyroiditis and Graves' disease.[12,13] These antibodies (TPO-Ab, Tg-Ab and TSH receptor antibodies [TRAb]) persisted in the SCID mice for at least 8 weeks after Graves' thyroid xenografting and gradually decreased with time; only transient hyperthyroxinemia was observed. The histologic picture appeared similar to destructive thyroiditis. Martin and associates[20] reported that many of the grafts in their SCID mice manifested a diminution in the infiltrates over time, with an absence of HLA-DR antigen expression by both T cells and thyrocytes. They concluded that a factor in the patients with AITD that activates their thyroid-specific T cells could be absent from this mouse model. One possible reason for difficulty in simulating hyperthyroidism in SCID mice with thyroid xenografts could be the presence of NK cells, which remain despite the lack of mature T and B cells. In investigating another potential mouse model for Graves' xenografts, we have compared the immune response in NIH-3 mice with that in SCID mice.

Byers and associates[29] investigated organ-specific metastases in nude, NIH-3 and SCID mice with human melanoma cells. Although the NIH-3 mice had more pancreatic and brown fat lesions than nude or SCID mice, the overall pattern of organ metastases was similar among the strains. Fishwild and colleagues[30] established a model for studying the efficacy of immunoconjugates and successfully depleted human T leukemic cells from lymphoid tissue in NIH-3 mice by treating the mice with an anti-CD7 immunoconjugate. Moreover, NIH-3 mice were susceptible to progressive infection with Borrella burgdorferi, which resulted in pancarditis, synovitis and myositis.[22] On the other hand, Mead and colleagues31 compared the susceptibility of SCID and NIH-3 mice to Cryptosporidium parvum infections. Colonization of the gallbladder and hepatobiliary duct epithelium occurred in most of the NIH-3 mice but a smaller proportion of the SCID mice. Garofalo and associates[32] also reported that spontaneous metastases were observed only in mice with advanced subcutaneous tumours and more frequently in NIH-3 mice and SCID mice than in nude mice. A 375M melanoma formed more lung colonies in nude and NIH-3 mice than in SCID mice. Thus, many investigators reported that NIH-3 mice may offer some advantages for studying some viral infections and the malignant behaviour of human melanoma, T-cell leukemia and human solid tumours. These reports support our finding that NIH-3 mice showed some advantages in producing TSAb and maintaining the expression of thyrocyte HLA-DR, compared with SCID mice.

SCID mice can be made more receptive to xenograft survival by ablating NK activity. Cavacini and coworkers[33]reported that a single dose (500 g) of anti-asialo GM1 (AsGM1) reduced NK activity in SCID mice by 60%. In contrast, gamma irradiation suppressed NK cell activity by more than 80% of baseline levels. Shpitz and associates34 reported that pretreatment of SCID mice with radiation plus anti-AsGM1 significantly improved short-term human PBMC grafting and provided a potentially useful model for the study of cancer immunotherapy. Barry and colleagues[35] reported successful grafting of human postnatal thymus in SCID mice after irradiation or treatment with anti-AsGM1. These investigators showed that depletion of NK cells in SCID mice permitted more reconstitution with T cells and a higher percentage of graft survival than in untreated SCID mice. In this study, we demonstrated that NIH-3 mice with xenografts of thyroid tissue from patients with Graves' disease produced higher values of TSAb and manifested a greater expression of thyrocyte HLA-DR than untreated SCID mice. These findings may be due to the lack of NK cells in NIH-3 mice. On the other hand, there were no significant differences between maximum TPO-Ab and Tg-Ab levels between the two strains of mouse, but this finding may be due to a wide variability of response in these antibodies. When we grafted PBMC alone from 12 patients with Graves' disease in SCID mice or NIH-3 mice, only 5 of the 12 SCID mice (42%) and 7 of the 12 NIH-3 mice (58%) were found to have detectable human IgG, whereas all of the SCID and NIH-3 mice (100%) with grafted thyroid tissue from patients with Graves' disease showed detectable levels of human IgG. PBMC alone were more easily rejected in both types of mouse than were intrathyroidal lymphocytes contained in thyroid xenografts. This finding may be due to a greater number of lymphocytes in thyroid xenografts or to thyroid tissues' effect of protecting intrathyroidal lymphocytes from the immune system.

Three of the NIH-3 mice died and one NIH-3 mouse suffered from lymphoma. The most likely cause of all of their deaths is lymphoma: these mice had extremely large spleens (more than 1.0 g), some areas of necrosis in the liver and several intraperitoneal lymph nodes. Flow cytometric analysis showed massive increases in human T and B cells in the spleen. NIH-3 mice may thus be more susceptible to cancer or infectious disease and may be more likely to die because they are more severely immunodeficient. Their care may require even more attention to detail than the care of SCID mice.

We conclude that SCID mice and NIH-3 mice may be useful animal models for xenografted thyroid tissue. NIH-3 mice are superior to SCID mice in maintaining expression of thyrocyte HLA-DR, in producing higher TSAb levels and in maintaining the severity of human lymphocytic infiltration in Graves' disease thyroid xenografts; these effects may be due to the lack of NK cells in NIH-3 mice. Studies in NIH-3 mice may lead to better simulations of Graves' disease than have been possible with SCID mice, with consequent greater facility for studying pathogenic and immunotherapeutic elements.

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Acknowledgements

We would like to thank Drs. Kanji Kuma, Fumio Matsuzuka and Edward Young for supplying thyroid tissues. We also gratefully acknowledge the staff of the animal colony in Princess Margaret Hospital for their care of the mice. We are further grateful to Mrs. Arjumand Hasan and Mrs. Colleen Ash for their technical assistance. We thank Jo Geary for her secretarial assistance.

This work was supported by a grant (MT 859) from the Medical Research Council of Canada.

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| CIM: February 1997 / MCE : février 1997 |
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