cDNA Immunization of Mice with Human Thyroglobulin Generates Both Humoral and T Cell Responses: A Novel Model of Thyroid Autoimmunity

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis

Identification and Comparative Expression Analysis of Interleukin 2/15 Receptor β Chain in Chickens Infected with E. tenella

BACKGROUND: Interleukin (IL) 2 and IL15 receptor β chain (IL2/15Rβ, CD122) play critical roles in signal transduction for the biological activities of IL2 and IL15. Increased knowledge of non-mammalian IL2/15Rβ will enhance the understanding of IL2 and IL15 functions. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] Chicken IL2/15Rβ (chIL2/15Rβ) cDNA was cloned using 5'/3'-RACE. The predicted protein sequence contained 576 amino acids and typical features of the type-I cytokine receptor family. COS-7 cells transfected with chIL2/15Rβ produced proteins of approximately 75 and 62.5 kDa under normal and tunicamycin-treated conditions, respectively. The genomic structure of chIL2/15Rβ was similar to its mammalian counterparts. chIL2/15Rβ transcripts were detected in the lymphoblast cell line CU205 and in normal lymphoid organs and at moderate levels in bursa samples. Expression profiles of chIL2/15Rβ and its related cytokines and receptors were examined in ConA-stimulated splenic lymphocytes and in ceca-tonsils of Eimeria tenella-infected chickens using quantitative real-time PCR. Expression levels of chIL2/15Rβ, chIL2Rα, and chIL15Rα were generally elevated in ceca-tonsils and ConA-activated splenic lymphocytes. However, chIL2 and chIL15 expression levels were differentially regulated between the samples. chIL2 expression was upregulated in ConA-activated splenic lymphocytes, but not in ceca-tonsils. In constrast, chIL15 expression was upregulated in ceca-tonsils, but not in ConA-activated splenic lymphocytes. CONCLUSIONS/SIGNIFICANCE: We identified an avian form of IL2/15Rβ and compared its gene expression pattern with those of chIL2, chIL15, chIL2Rα, and chIL15Rα. Our observations suggest that chIL15 and its receptors, including chIL2/15Rβ, play important roles in mucosal immunity to intestinal intracellular parasites such as Eimeria

Genomic identification of chemokines and cytokines in Opossum

The cytokine repertoire of marsupials is largely unknown. The sequencing of the opossum genome has expedited the identification of many immune genes. However, many genes have not been identified using automated annotation pipelines because of high levels of sequence divergence. To fill gaps in our knowledge of the cytokine gene complement in marsupials, we searched the genome assembly of the gray short-tailed opossum for chemokine, interleukin, colony-stimulating factor, tumor necrosis factor, and transforming growth factor genes. In particular, we focused on genes that were not previously identified through Ensembl's automatic annotations. We report that the vast majority of cytokines are conserved, with direct orthologs between therian species. The major exceptions are chemokine genes, which show lineage-specific duplication/loss. Thirty-six chemokines were identified in opossum, including a lineage-specific expansion of macrophage inflammatory protein family genes. Divergent cytokines IL7, IL9, IL31, IL33, and CSF2 were identified. This is the first time IL31 and IL33 have been described outside of eutherian species. The high levels of similarities between the cytokine gene repertoires of therians suggest that the marsupial immune response is highly similar to eutherians

Effect of iodide on Fas, Fas-ligand and Bcl-w mRNA expression in thyroid of NOD mice pretreated with methimazole

Nonobese diabetic (NOD) mice and a derived strain, NOD.H.2h4, have been used as a model for experimental spontaneous thyroiditis and thyroiditis induced by iodide excess after a goiter-inducing period. Some authors have proposed that iodide, given after methimazole or propylthiouracil, is capable of inducing apoptosis in thyroid cells and that anti-thyroid drugs can modulate the expression of apoptosis components such as Fas and its ligand (Fas-L). Here we evaluated the effect of potassium iodide (20 µg/animal for 4 days, ip) given to NOD mice at the 10th week of life after exposure to methimazole (1 mg/ml) in drinking water from the 4th to the 10th week of life. Fas, Fas-L and Bcl-w expression were analyzed semiquantitatively by RT-PCR immediately after potassium iodide administration (group MI44D) or at week 32 (MI32S). Control groups were added at 10 (C10) and 32 weeks (C32), as well as a group that received only methimazole (CM10). An increase in the expression of Fas-L and Bcl-w (P<0.01, ANOVA) was observed in animals of group MI44D, while Fas was expressed at higher levels (P = 0.02) in group C32 (72.89 ± 47.09 arbitrary units) when compared to group C10 (10.8 ± 8.55 arbitrary units). Thus, the analysis of Fas-L and Bcl-w expression in the MI44D group and Fas in group C32 allowed us to detect two different patterns of expression of these apoptosis components in thyroid tissue of NOD mice