Porcine Tg Research Articles

Experimental rat models for Hashimoto's thyroiditis.

Hashimoto's thyroiditis (HT) is an autoimmune thyroid disease characterized by T lymphocyte-mediated destruction of thyroid follicles. To study the pathogenesis of HT and the efficacy of new substances for its treatment, an easily obtained and adequate to the human disease experimental model is needed. The aim of our study was to find out whether it is possible to induce experimental autoimmune thyroiditis (EAT) similar to Hashimoto's thyroiditis by injecting with thyroglobulin (Tg) without using agents that enhance its thyroiditogenicity and without taking into account the genetic sensitivity of animals. Wistar rats were immunized with freshly isolated rat Tg or porcine Tg. In 8weeks, histological studies of the thyroid and parathyroid glands were performed. Thyroid function and total serum calcium level were also evaluated. Immunization with both rat and porcine freshly isolated Tg caused T lymphocytic infiltration of the thyroid gland, thyroid follicle atrophy and degradation in Wistar rats. EAT caused by porcine Tg was characterized by greater severity than EAT induced with rat Tg. In 55% of rats with porcine Tg-induced EAT, oxyphilic metaplasia was detected in the parathyroid glands. In addition, low total serum calcium was observed in these rats. Two rat models of autoimmune thyroiditis were obtained. EAT caused in Wistar rats by immunization with rat Tg is similar to Hashimoto's thyroiditis. EAT induced with porcine Tg was accompanied by oxyphil cell metaplasia in the parathyroids and hypocalcemia.

Induction of experimental autoimmune thyroiditis (EAT) by heat-denaturated thyroglobulin (Tg).

Experimental Autoimmune Thyroiditis (EAT) is characterized by autoreactive T and B cell responses, assessed by a marked lymphocytic infiltration of the thyroid gland by T cells and the occurrence of circulating autoantibodies (AAb) to thyroglobulin (Tg). It was recently reported that administration of denaturated exogenous antigens primes class I-restricted cytotoxic T cells in vivo. Since cytotoxic T cells are involved in EAT development, porcine Tg (pTg) was heat-denaturated, i.v. injected into CBA/J mice and features of EAT evaluated. Simultaneously, mice were immunized with pTg and adjuvants and evaluation of EAT performed. We found that heat-denaturated pTg (hdpTg) induced EAT in recipient mice similar to native pTg/adjuvants. Surprisingly, whereas Tg-specific cytotoxic T cells were regularly found in lymph node cells from hdpTg or native pTg immunized mice, proliferative responses were only detected using T cells from native pTg immunized mice. Autoantibodies to pTg were decreased by a factor 30 in sera from mice immunized with hdpTg. These data further emphasized the role of Tg-specific cytotoxic T cells in EAT.

Evolutionary Conservative and Species-Specific Antigenic Determinants of Mammalian Thyroglobulins

Evolutionary conservative and species-specific epitopes of thyroglobulins (Tg) from different mammalian species were studied using panel of 20 monoclonal antibodies (mAb) raised in our laboratory and recognizing 16 antigen determinants on human Tg molecule. Cross-reactivity of mAb with preparations of purified mammalian Tg from other species was studied by the method of indirect enzyme-linked immunosorbent assay (ELISA). Interaction of mAb with Tg in histological sections of thyroid gland tissue was studied by direct immunofluorescence assay using mAb conjugated with fluorescein isothiocyanate (FITC). Since certain Tg epitopes lose their ability to bind mAb after fixation and paraffin embedding, only 12 out of 20 mAb turned out to be suitable for detection of Tg in the tissue sections. The obtained data have allowed establishing that four human Tg epitopes are species-specific. Meanwhile, six human Tg epitopes are shared with bovine Tg epitopes, seven epitopes with porcine Tg, four with rat, and two with mouse Tg. Each Tg of two species (cat and dog) has three common epitopes with human Tg.

KClO 4 inhibits thyroidal activity in the larval lamprey endostyle in vitro

An in vitro experimental system was devised to assess the direct effects of the goitrogen, potassium perchlorate (KClO 4), on radioiodide uptake and organification by the larval lamprey endostyle. Organification refers to the incorporation of iodide into lamprey thyroglobulin (Tg). Histological and biochemical evidence indicated that endostyles were viable at the termination of a 4 h in vitro incubation. A single iodoprotein, designated as lamprey Tg, was identified in the endostylar homogenates by polyacrylamide gel electrophoresis and Western blotting. Lamprey Tg was immunoreactive with rabbit anti-human Tg serum and had an electrophoretic mobility similar to that of reduced porcine Tg. When KClO 4 was added to the incubation medium, both iodide uptake and organification by the endostyle were significantly reduced relative to controls as determined by gamma counting, and gel-autoradiography and densitometry, respectively. Western blotting showed that KClO 4 significantly lowered the total amount of lamprey Tg in the endostyle. Based on the results of this in vitro investigation, we conclude that KClO 4 acts directly on the larval lamprey endostyle to inhibit thyroidal activity. These data support a previous supposition from in vivo experimentation that KClO 4 acts directly on the endostyle to suppress the synthesis of thyroxine and triiodothyronine, resulting in a decrease in the serum levels of these two hormones.

H2-E transgenic class II-negative mice can distinguish self from nonself in susceptibility to heterologous thyroglobulins in autoimmune thyroiditis.

Susceptibility to experimental autoimmune thyroiditis (EAT) is linked to H2-A class II genes; k and s haplotypes are susceptible, while b and f are resistant. EAT is inducible with thyroglobulins (Tgs) from several mammalian species which share portions of identical sequences. But cross-activation and cross-tolerance studies with mouse (m), human (h), and porcine (p) Tg have indicated mTg-unique T-cell epitope(s), in addition to conserved, in EAT induction. The recent introduction of the HLA-DRB1*0301 (DR3) transgene rendered major histocompatibility complex (MHC) class II-negative (Ab(0)) mice susceptible to EAT induction by both hTg and mTg, suggesting usage of conserved epitopes. Here, we introduced the H2-Ea(k) transgene into resistant B10 (H2(b)) or Ab(0) mice with a defective Ea gene to provide functional surface H2E (b haplotype) expression. Surprisingly, both transgenic strains showed severe inflammation only after hTg, but not mTg, immunization, although the moderating influence of the A(b) gene in B10 was evident. In proliferative assays, hTg-primed cells did not respond to mTg, nor to conserved 12mer peptides from three primary hormonogenic sites, two of which can activate T cells for thyroiditis transfer and cytotoxicity. The vigorous response to hTg stimulation was reduced only by Ebeta(b)-specific monoclonal antibody. EAT induction with bovine and pTg showed responses similar to hTg, suggesting thyroiditogenic epitopes shared with hTg, but not mTg. This is the first demonstration of: (1) nonpermissiveness for EAT induction with mTg, normally the most thyroiditogenic Tg and the one with unique epitopes for susceptible mice, and (2) the separation of hTg from mTg in EAT induction in H2-E-transgenic mice.

Experimental Autoimmune Thyroiditis Induced by Thyroglobulin-Pulsed Dendritic Cells

Dendritic cells (DCs), which are the most effective professional antigen-presenting cells (APCs), initiate and regulate immune responses. In this report, we examine the role of DCs in the induction of autoimmune thyroiditis. Experimental autoimmune thyroiditis (EAT) induced by immunization with thyroglobulin (Tg) plus adjuvant is considered to be an animal model of autoimmune thyroiditis, and is categorized as a T cell-mediated autoimmune disease. To examine the contribution of DCs to EAT, naive DCs were purified from high responder B10BR mice and pulsed with various concentrations of porcine Tg (pTg). These pTg-pulsed DCs were transferred without adjuvant to syngenic BIOBR mice to induce EAT. Mice that had received pTg-pulsed DCs showed thyroiditis, and the degree of thyroiditis induced was positively correlated to the amounts of pTg used for the incubation (pulsing) of DCs. The severity of thyroiditis was also correlated to the amounts of anti-pTg IgG2a antibodies and IFN-γ in the recipient sera, but not to IL-4 or IL-10, indicating that Th1 cells are mainly activated by pTg-pulsed DCs and attributable to the pathogenesis of EAT.

Carbohydrate analysis of porcine thyroglobulin isoforms with different iodine contents

To further validate the relationship between thyroid hormone formation and the carbohydrate structure of thyroglobulin (Tg), we reinvestigated the relationship between the iodine content and the asparagine-linked oligosaccharide structures of porcine Tg. Purified porcine Tg was further separated into isoforms (Tg-F1, -F2 and -F3) with a DEAE–cellulose ion-exchange chromatography column. The iodine residues, neutral sugar and sialic acid were analyzed for the separated Tg isoforms and their asparagine-linked oligosaccharide structures were analyzed. The asparagine-linked oligosaccharides were released from Tg-F1, -F2 and -F3 by hydrazinolysis and each oligosaccharide was labeled with p-aminobenzoic acid octyl ester (ABOE). The ABOE-labeled oligosaccharides from Tg-F1, -F2 and -F3 were analyzed for their relative content in oligosaccharides of each structure type by chemical methods and DEAE- and ConA high-performance liquid chromatography (HPLC) columns. As a result, it was revealed that the Tg fraction eluted at higher ionic strength from a DEAE–cellulose column is apt to contain more of each iodoamino acid, as well as total content of iodine, larger negative zeta-potential, conforming to sialic acid content in the Tg molecule and to a higher content of di-sialo-bi-antennary complex and to high mannose-type oligosaccharides. These results support the conclusion that iodine organification of the Tg molecule is correlated with asparagine-linked oligosaccharide completion.

Carbohydrate and protein determinants are involved in thyroglobulin recognition by FRTL 5 cells.

To avoid premature lysosomal degradation, thyrocytes have a system able to recycle internalized immature thyroglobulin molecules (Tg) to the follicular lumen via the Golgi apparatus. It has been shown that this quality control system depends on recognition of exposed N-acetylglucosamine (GlcNAc) determinants (Miquelis et al., J Cell Biol, 1993, 123, 1695) present on immature Tg (Bastiani et al., 1995, Endocrinology, 1995, 136, 4204). However, the same in vitro kinetics studies also showed that GlcNAc residues alone induce only weak recycling. The latter finding led us to investigate the possibility that protein determinants might also be involved in binding. For this purpose, we studied binding of Tg to FRTL 5 cells, a widely available TSH-dependent cell line and found that binding to plasma membranes occurred at acidic pH in the presence of calcium, i.e. under conditions previously reported for binding of GlcNAc-BSA to porcine thyroid cell membranes. As expected, binding was GlcNAc- and oligosaccharide-dependent because Bandeiraea Simplificiola II affinity column analysis indicated that GlcNAc-bearing Tg were preferentially bound and N-glycanase treatment of Tg inhibited interaction. Ovomucoid, GlcNAc-BSA, and porcine Tg oligosaccharides did not inhibit binding, indicating that carbohydrates were not the sole determinants for binding. The fact that pronase digestion of Tg totally abolished binding implied that peptide determinants were involved in the interaction. This involvement is supported by the observation that porcine, rat, bovine, and human Tg bound FRTL 5 cell membranes and that monoclonal antibodies raised against human Tg interfered with the binding of both human and porcine Tg. Based on these findings we conclude that, besides the involvement of GlcNAc-bearing oligosaccharides, Tg receptors form a stable bond with peptide determinants.

On the relationship between completion of N-acetyllactosamine oligosaccharide units and iodine content of thyroglobulin: a reinvestigation.

Lack of completion of N-acetyllactosamine-type glycosylation on thyroglobulin (Tg) has been implicitly considered as an etiological factor of some thyroid disorders, i.e. goiter and hypothyroidism. However, there is some evidence that Tg with incompletely processed N-acetyllactosamine glycans occurs in the normal gland. Recent findings demonstrated that exposed N-acetylglucosamine (GlcNAc) residues present on internalized glycoprotein in the thyrocyte may act as a retention signal that prevents lysosomal homing and triggers recycling of GlcNAc-bearing molecules through galactosyltransferase- and thyroperoxidase-containing compartments of the Golgi apparatus. This finding raises the possibilities 1) that exposed GlcNAc residues are not randomly distributed, but are mainly present on immature Tg; and 2) that this process promotes elongation of complex glycans, thereby eliminating the retention signal. To further validate this hypothesis, we reinvestigated the relationship between the iodine content and the glycan completion of porcine Tg of luminal origin. Tg subpopulations were separated according to their iodine content on rubidium chloride centrifugation gradients, and their interactions with various plant and animal lectins were analyzed in solid phase assays. Iodine content used as an index of age ranged from 0.6-1.2%. There was no significant correlation between iodine content and either neutral sugar or oligosaccharide content, as judged by chemical methods or interaction with [125I]Solanum tuberosum and [125I]Pisum sativum agglutinins. In contrast, the number of GlcNAc-accessible residues (as judged by interaction with [125I]Bandeiraea simplificolia II) decreased as iodine content increased. These changes were concomitant with an increase in galactose (measured by interaction with [125I]R-icinus communis and [125I]galactosidase (Gal)/GalNAc rat hepatic lectin) and sialic acid content. Related experiments using a Tg subpopulation depleted in GlcNAc-exposed residues by passage through a B. simplificolia II affinity column showed that the capacity of this subpopulation to bind to membranes was lowered compared to that of the total Tg. These results support the following conclusions: 1) in normal glands, all or part of the Tg molecules are secreted in an incompletely glycosylated form; and 2) iodine organification is correlated with glycan completion. Therefore, asialoagalactothyroglobulin appears to be a physiological precursor for an efficient recycling mediated by the GlcNAc receptor to the iodination site. New insights in thyroid disorders are discussed.

The number of oligosaccharides borne by porcine thyroglobulin is variable.

Purified porcine thyroglobulin (Tg) was fractionated on a concanavalin A-Sepharose 4B column by a step-wise elution with increasing concentrations of methyl alpha-mannoside (fraction A, 50 mM; B, 100 mM; C, 200 mM; D, 500 mM, and E, 1 M), and its fractional ratio was 12.8:28.6:26.4:19.7:12.4. These five fractions showed the same profile in polyacrylamide gel electrophoresis. The subfractions were analyzed for their relative contents in oligosaccharides of each structure type and for their monosaccharide contents. In fractions B, C, D, and E the former varied between 15-22% for triantennary complex-type, 47-60% for biantennary complex-type, and 22-30% for high mannose-type oligosaccharide. Fraction A showed a higher percentage of triantennary complex-type structures (36%) and a lower percentage of biantennary complex-type structures (17%). The monosaccharide numbers increased from fraction A to E: 85 to 135 mannose residues, 60 to 82 galactose residues, 84 to 115 N-acetyl glucosamine residues, and 22 to 28 sialic acid residues. After analysis of the number of mannose residues contained in the high mannose-type structures, it was possible to calculate the number of oligosaccharides borne by each Tg subfraction. This number was approximately the same for fractions A and B (22.4 and 21.7), then it increased from B to E (21.8 to 32.9). These results account for the separation obtained on the concanavalin A-Sepharose 4B column. Separation of the two first subfractions bearing the same number of oligosaccharides is certainly due to the higher number of high mannose-type structures in B. In conclusion, the studies reported here show that porcine Tg is heterogeneous, and mainly so in terms of total number of N-glycan structures.

Identification of a thyroiditogenic sequence within the thyroglobulin molecule.

Thyroglobulin (Tg)-specific T cells are important in the induction of experimental autoimmune thyroiditis (EAT), but the nature and the number of the Tg T cell epitopes involved in the disease process are unknown. Through the use of computerized algorithms that search for putative T cell epitopes, a 17-mer peptide (TgP1) was identified within the known portion of the rat Tg sequence (corresponding to amino acids 2495 to 2511 of the human Tg sequence) that induced strong mononuclear cell infiltration of the thyroid in classic EAT-susceptible murine strains such as SJL, C3H, and B10.BR and low or undetectable infiltration in EAT-resistant strains such as BALB/c and B10. TgP1 appears to be phylogenetically conserved since it is completely homologous to its bovine counterpart and differs at a single amino acid position from its human analogue. After priming with TgP1 in vivo, significant proliferative T cell responses to TgP1 in vitro were observed only with lymphocytes from susceptible (high responder) strains, thus correlating proliferative capacity with EAT induction. TgP1-primed T cells did not respond to intact mouse Tg (MTg) or rat Tg in vitro and, conversely, T cells primed in vivo with MTg or rat Tg did not respond to TgP1 in culture, suggesting that TgP1 is comprised of non-immunodominant T cell determinants. TgP1 was defined as a serologically nonimmunodominant epitope as well, since in vivo priming of all strains with MTg led to strong MTg-specific IgG responses but no TgP1-specific responses in ELISA assays. This was not due to lack of immunogenic B cell determinants on TgP1, however, because peptide challenge of EAT-susceptible strains elicited TgP1-specific IgG that also cross-reacted with MTg and rat, human, bovine, and porcine Tg. The data demonstrate that TgP1 delineates nonimmunodominant but highly immunogenic determinants at both the T and B cell level, which may play an important role in the development of autoimmune thyroiditis.

Experimental autoimmune thyroiditis induced by a 5-10-kDa tryptic fragment from porcine thyroglobulin.

Monoclonal autoantibodies obtained using the native porcine thyroglobulin (Tg) molecule for immunization were shown to react against a defined epitope of the Tg molecule. Its biochemical characterization was undertaken, using trypsin treatment of Tg, followed by gel filtration, affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography analysis. Immunoreactivity tested by immunoblotting with appropriate monoclonal anti-Tg antibody allowed the detection of one peptide fragment of 5-10 kDa, after limited proteolysis with trypsin. We demonstrated that this peptide reacts exclusively with monoclonal anti-Tg antibodies which block in vitro proliferative T cell response; moreover, the 5-10-kDa tryptic fragments from porcine Tg were able to induce thyroiditis when injected into normal CBA mice, while porcine Tg depleted from these fragments was not able to do so.

Monoclonal antithyroglobulin antibodies derived from immunizations of mice with human eye muscle and thyroid membranes.

A mouse hybridoma clone secreting an immunoglobulin M monoclonal antibody (K-5-4), which reacted with human thyroglobulin (Tg), was obtained from spleen cells of mice immunized with crude membranes of human eye muscle tissues (Em). Its binding to Tg could be inhibited by another monoclonal anti-Tg (F1-11-1) derived from spleen cells of mice immunized with human thyroid cell membranes, but K-5-4 did not inhibit the binding of F1-11-1 to Tg. This finding suggests that K-5-4 may react with a site on the Tg molecule which is susceptible to conformational changes, such as that induced by binding of another anti-Tg antibody at another site on Tg. K-5-4 reacted with human, mouse, rat, guinea pig, bovine, and porcine Tg. Binding and immunohistological staining experiments failed to detect binding of K-5-4 to Em tissue. The very low frequency of one Tg-reacting hybridoma from 6 X 10(8) spleen cells fused after Em immunization contrasts with the relative ease with which monoclonal anti-Tgs were generated from spleen cells of mice immunized with crude human thyroid membranes. In the latter case, 1 anti-Tg hybridoma was generated for every 100,000 spleen cells fused, and an extensive library of monoclonal anti-Tgs was collected. Some of these antibodies were specific for human Tg only, while others cross-reacted with Tg of other animal species. None had the species reativity pattern of K-5-4. The anti-Tgs were used to affinity purify human Tg directly from supernatant of thyroid homogenate; the purified Tg was, in turn, used to affinity purify human polyclonal but monospecific anti-Tg directly from serum of patients in a simple and rapid procedure. We conclude that the monoclonal anti-Tgs are useful reagents in isolating and purifying Tg and anti-Tg.

Production of anti-thyroglobulin antibody by blood lymphocytes from patients with autoimmune thyroiditis, induced by the insolubilized autoantigen.

Abstract Mononuclear cells (MNC) from the blood of patients with autoimmune thyroiditis cannot be stimulated in vitro with the autoantigen thyroglobulin (Tg). However, when Tg was coupled with a solid matrix, MNC from 13 of 19 patients tested responded to this insolubilized Tg (i-Tg) by generating cells which produced IgG anti-Tg antibodies, as demonstrated in a "spot ELISA". This response was specific not only in the assay phase but also in the induction phase, because 1) cultures with insolubilized ovalbumin, bovine Tg, or porcine Tg were negative, and 2) i-Tg did not induce polyclonal IgG production. Generation of anti-Tg-producing cells was completely abrogated when free Tg was added to i-Tg cultures. Stimulation with i-Tg after separation of T and non-T MNC revealed that: 1) the response was independent of T cells; 2) in three of the six patients whose MNC did not respond, removal of T cells did result in anti-Tg production; 3) re-addition of the T cells--but not of irradiated T cells--abrogated the response in two of these three patients; and 4) preincubation of monocytes with i-Tg resulted in suboptimal anti-Tg production. These findings indicate that B cells from patients with autoimmune thyroiditis can be specifically stimulated with Tg coupled with a solid matrix, resulting in generation of anti-Tg-producing cells. The essence of this mode of presenting the antigen would appear to be the cross-linkage of surface immunoglobulin. The response was T cell-independent; in some patients, however, the presence of a radiosensitive suppressor T cell population can be demonstrated.