Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens.
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AKR/N mice injected with fibroblasts expressing MHC class II (RT4.15HP cells) and the TSH receptor (TSHR) develop antibodies similar to those in Graves' disease. We were unable to analyse the subclass of these antibodies because of unexpectedly high non-specific binding by ELISA or flow cytometry. The non-specific binding reflected generalized immune activation which occurred even when the fibroblasts did not express the TSHR. However, the IgG subclasses were determined for thyroid peroxidase (TPO) antibodies induced using TPO-expressing RT4.14HP cells and found to be IgG2a > IgG1. This Thl pattern is consistent with spontaneous secretion of interferon-gamma (but not IL-4 or IL-10) by splenocytes from injected mice. The Th1 bias was related to fibroblast injection because conventional immunization of the same mouse strain with purified TPO and adjuvant induced a Th2 response (IgG1 >> IgG2a). Further, untransfected fibroblasts themselves induced powerful, non-specific proliferative responses when used as antigen-presenting cells (APC) in vitro. Flow cytometry revealed that the RT4.15HP fibroblasts (and TSHR- and TPO-transfected derivatives) expressed B7-1. Unexpected constitutive expression of this key molecule may bypass the requirement for up-regulation of other costimulatory molecules involved in T cell stimulation. Our data support the concept that RT4.15HP fibroblasts present the TSHR (or TPO), at least for initiating the immune response. However, the accompanying generalized immune stimulation creates difficulties for analysis of TSHR-specific T and B lymphocytes. On the other hand, extension of the model to TPO, an easier antigen to study, will facilitate analysis of murine T cell responses likely to resemble those in human thyroid autoimmunity. (+info)
Effects of proinflammatory cytokines on anterior pituitary 5'-deiodinase type I and type II.
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Cytokines are locally produced in the anterior pituitary and act through para-/autocrine mechanisms to modulate cell growth and hormone production. 5'-Deiodinases type I (D1) and type II (D2) are also expressed in the anterior pituitary and play an integrative role in the regulation of hormone production and pituitary feedback. D1 activity is known to be regulated by proinflammatory cytokines in liver and thyroid. Therefore, we examined the effects of IL-1 beta, IL-6 and TNF alpha on 5'-deiodinase activities in reaggregates of rat anterior pituitaries and rat somatomammotroph GH3 cells cultured alone, or in a bicameral culture system together with the murine folliculo-stellate (FS) cell line TtT/Gf. In reaggregate cultures of rat anterior pituitaries IL-1 beta stimulated D1 and D2 dose-dependently and D2 activity was increased by TNF alpha. When GH3 cells were cocultured with TtT/Gf cells, D2 activities were 2.3-fold lower than in GH3 cells cultured alone. TNF alpha (50 ng/ml) and IL-6 (100 U/ml) stimulated D2 in GH3 cells when the cells were cultured alone and treated with these cytokines for 24 h. When TtT/Gf cells in the coculture model were treated with IL-1 beta, TNF alpha and IL-6, no effect on D1 or D2 activities in GH3 cells was observed. In male, adult rats a single LPS injection (i.p.) stimulated D2 and D1 activities in the anterior pituitary, and decreased liver D1 activities and serum TSH levels. In vitro, LPS stimulation of the coculture model of GH3 and FS cells also increased D1 activity. Electrophoretic mobility shift assays (EMSAs) revealed that IL-1 beta and TNF alpha activate the transcription factor NF kappa B in reaggregates of rat anterior pituitaries and in TtT/Gf cells cultured alone or cocultured with GH3 cells. Taken together, these findings imply that in anterior pituitary cells 5'-deiodinase activities are stimulated by locally produced cytokines in a para-/autocrine manner but cell types other than FS cells seem to mediate some of the effects. (+info)
SECIS-SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy.
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Selenocysteine incorporation at UGA codons requires cis-acting mRNA secondary structures and several specialized trans-acting factors. The latter include a selenocysteine-specific tRNA, an elongation factor specific for this tRNA and a SECIS-binding protein, SBP2, which recruits the elongation factor to the selenoprotein mRNA. Overexpression of selenoprotein mRNAs in transfected cells results in inefficient selenocysteine incorporation due to limitation of one or more of these factors. Using a transfection-based competition assay employing overexpression of selenoprotein mRNAs to compete for selenoprotein synthesis, we investigated the ability of the trans-acting factors to overcome competition and restore selenocysteine incorporation. We report that co-expression of SBP2 overcomes the limitation produced by selenoprotein mRNA overexpression, whereas selenocysteyl-tRNA and the selenocysteine-specific elongation factor do not. Competition studies indicate that once bound to SECIS elements, SBP2 does not readily exchange between them. Finally, we show that SBP2 preferentially stimulates incorporation directed by the seleno protein P and phospholipid hydroperoxide glutathione peroxidase SECIS elements over those of other selenoproteins. The mechanistic implications of these findings for the hierarchy of selenoprotein synthesis and nonsense-mediated decay are discussed. (+info)
Search for the autoantibody immunodominant region on thyroid peroxidase: epitopic footprinting with a human monoclonal autoantibody locates a facet on the native antigen containing a highly conformational epitope.
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Autoantibodies to thyroid peroxidase (TPO) are the hallmark of the humoral autoimmune response in human autoimmune thyroiditis (Hashimoto's thyroiditis). The majority of TPO autoantibodies in individual patients' sera interact with a restricted immunodominant region on TPO. Although this region can be mapped, previous studies have failed to localize its position on the TPO molecule. We, therefore, used a footprinting approach that can localize a highly conformational, discontinuous epitope on a very large molecule. Extensive biotinylation ( approximately 15 biotins/molecule protein) of lysine residues on the surface of purified, native TPO resulted in loss of multiple tryptic cleavage sites, as determined by analysis of tryptic polypeptide fragments on reverse-phase HPLC. TPO was then complexed with a monoclonal human autoantibody Fab (TR1.9) before biotinylation. After dissociation from TR1.9, TPO was recovered by gel filtration. A trypsin site, previously observed to be lost after TPO biotinylation, was restored when biotinylation was performed on the TPO-TR1.9 complex. The epitope-protected lysine (K) was present in a 30-aa TPO fragment that, by N-terminal sequencing, was found to be K713. Altered recognition by TR1.9 of a TPO-myeloperoxidase chimeric molecule involving this region supported the epitope protection data. In conclusion, we provide the first identification of an amino acid residue (K713) comprising part of an epitope within the TPO immunodominant region. This focal residue localizes the facet on the large, highly complex TPO molecule that contains the immunodominant region and provides the basis for rational guided mutagenesis studies to more fully characterize this region. (+info)
Changes of peripheral tissue thyroid hormone metabolism in rats fed with selenium- and vitamin E-deficient artificial semisynthetic diet.
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OBJECTIVE: To examine the relationship between selenium (Se)- and vitamin E(VE)-deficiency and thyroid hormone (TH) metabolic disturbance, especially type I iodothyronine 5'-deiodinase (ID-I) activity. METHODS: We observed the metabolic changes of TH-3, 3',5-triidothyronine (T3) and thyroxin (T4) and their free radicals-glutathione peroxidase (GSH-Px) and lipid peroxides (LPO) in the peripheral tissues (liver, kidney and blood) of Wistar rats maintained on Se- and VE-deficient artificial semi-synthetic diet for 8 weeks. RESULTS: In the Se- and VE-deficient rats (compared with Se- and VE-supplement rats): 1). Hepatic and renal ID-I activities decreased by 60% and 50% respectively, serum levels of T3 reduced 36%, and T4 increased by 32%. Hepatic ID-I activity and serum T3 concentration in Se- and VE-supplement rats were significantly higher than those in the Se-supplement and VE-deficient rats. 2). GSH-Px activities in the whole blood and liver decreased by 61% and 82% respectively. LPO concentrations in serum and liver increased by 53% and 40% respectively. When Se content remained the same, changes of VE content did not significantly affect GSH-Px activity, but the combined supplement of Se and VE decreased significantly the LPO concentration. CONCLUSIONS: The data suggests that GSH-Px activity is influenced mainly by Se level, but ID-I activity is influenced by VE concentration in addition to Se level. VE seemed to play a protective role on ID-I and its mechanism might be related to the fact that VE protects the stability of microsomal membrane in which ID-I exists, avoiding from free radical damage as well as the coordinated action and mutual sparing effect of Se and VE. (+info)
Thyroid hormone activation in human vascular smooth muscle cells: expression of type II iodothyronine deiodinase.
(62/847)
Thyroid hormone has been reported to have significant effects on the peripheral vascular system, including relaxation of vascular smooth muscle cells and antiatherosclerotic effects. To exert its biological activity, thyroxine, which is a major secretory product of thyroid gland, needs to be converted to 3,5,3'-triiodothyronine (T(3)) by iodothyronine deiodinase. Type I iodothyronine deiodinase (DI) is widely distributed and maintains circulating T(3) level, whereas type II iodothyronine deiodinase (DII) is present in a limited number of tissues to provide local intracellular T(3). In the present study, we have identified iodothyronine deiodinase in cultured human coronary artery smooth muscle cells (hCASMCs) and human aortic smooth muscle cells (hASMCs). All of the characteristics of the deiodinating activity in hCASMCs and hASMCs were compatible with DII. Northern analysis demonstrated that DII mRNA was expressed in both hCASMCs and hASMCs, and DII mRNA levels as well as DII activities were rapidly increased by dibutyryl-cAMP or forskolin. These data demonstrate, for the first time, the expression of DII in human vascular smooth muscle cells, which is regulated by a cAMP-mediated mechanism. The present results suggest a previously unrecognized role of local T(3) production by DII in the pathophysiology of human vascular smooth muscle cells. (+info)
Differences between the effects of thyroxine and tetraiodothyroacetic acid on TSH suppression and cardiac hypertrophy.
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OBJECTIVE: We earlier reported marked qualitative differences between the effect of 3,5,3'-tri-iodothyroacetic acid (Triac) and tri-iodothyronine (T3) on cardiac hypertrophy at equivalent thyroid-stimulating hormone (TSH)-suppressive doses. We have now extended these studies to specific cardiac parameters. Due to its rapid metabolic clearance rate, Triac is not suitable for TSH suppression and therefore the slowly metabolized 3,5,3',5'-tetraiodothyroacetic acid (Tetrac), the precursor of Triac, was studied. METHODS: Hypothyroid rats were infused over 13 days with 1.5-40.5 nmol Tetrac/day per 100 g body weight (BW) or with 0.5-13.5 nmol thyroxine ((4)T4)/day per 100 g BW. RESULTS: The responses of serum TSH and of hepatic monodeiodinase type 1 were parallel for both hormones, their potency ratios could therefore be compared. Tetrac was revealed as being only half as active on hepatic monoiodinase type 1 despite a similar serum TSH levels. Tetrac can therefore be considered to have a preferential action on serum TSH suppression. The cardiac effects on Ca2+-ATPase (SERCA 2a) and monodeiodinase type 1 activity were qualitatively different and therefore one cannot give an overall quantitative estimate of these differences. The results showed clearly, however, that Tetrac is less efficient for all parameters studied, namely induction of cardiac hypertrophy, alpha-myosin heavy chain mRNA, monodeiodinase type 1 activity and mRNA levels of the sarcoplasmic SERCA 2a. CONCLUSION: We postulate therefore that, in the rat and possibly in man, Tetrac could represent a favorable alternative for suppression of serum TSH levels. (+info)
Measurement of anti-thyroglobulin and anti-thyroid peroxidase antibodies using highly sensitive radioimmunoassay: an effective method for detecting asymptomatic focal lymphocytic thyroiditis in the elderly.
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Subclinical thyroiditis or thyroid dysfunction is relatively common in the elderly. To estimate the effectiveness of measurement of serum levels of anti-thyroglobulin and anti-microsomal or thyroid peroxidase antibodies for detecting focal lymphocytic thyroiditis (FLT) in the elderly, we examined the relationships between antibody titer and postmortem histological finding of the thyroid gland in 180 consecutive autopsies (69 women and 111 men) over 60 years of age without any overt clinical thyroid or collagen diseases. FLT was found in 25 cases (13.9%) with female predominance (21.7% in female vs. 9.0% in male). Measurements of serum levels of anti-thyroglobulin and anti-thyroid peroxidase antibodies by radioimmunoassay (TgAb and TPOAb, respectively) were compared with the measurements of anti-thyroglobulin and anti-microsomal antibodies by a hemagglutination technique (TGHA and MCHA, respectively), using sera from 25 patients with FLT and age- and sex-matched 51 patients without FLT. Among 25 cases with FLT, TgAb and TPOAb were positive in 17 (68%) and 12 (48%), respectively. There was a close relationship between degree of FLT and serum level of TgAb or TPOAb (P<0.0001). On the other hand, TGHA and MCHA were positive only in 8 (32%) and 10 (40%), respectively. TgAb and TPOAb were more sensitive than TGHA (68% vs. 32%, P<0.05) and MCHA (48% vs. 40%) to detect FLT. Positive findings in either TgAb or TPOAb significantly improved sensitivity (76%) compared with that of TGHA or MCHA (44%) (P<0.05). Specificities of combined measurements of TgAb and TPOAb (90%) were not significantly different from those of TGHA and MCHA (100%). These findings indicate that TgAb is a more sensitive method for detecting FLT and that its diagnostic sensitivity for FLT increases by using it in combination with TPOAb. Therefore, in the elderly without clinically or biochemically overt thyroid dysfunction, positive TgAb and/or TPOAb could imply presence of FLT, and their titers might reflect degree of inflammation. (+info)