Association of NFKB1 -94ins/del ATTG promoter polymorphism with susceptibility to and phenotype of Graves' disease.

Recently, a functional polymorphism in the NFKB1 gene promoter (-94ins/del ATTG) has been identified and associated with chronic inflammatory diseases. The aim of this study was to analyze the association of NFKB1 polymorphism with susceptibility to and phenotype of Graves' disease (GD). The initial case-control association study, performed in a Polish-Warsaw cohort (388 GD patients and 688 controls), was followed by the two replication studies performed in Polish-Gliwice and Japanese-Kurume cohorts (198 GD patients and 194 controls, and 424 GD patients and 222 controls, respectively). The frequency of the -94del ATTG (D) allele was increased in GD compared to controls in Warsaw cohort. This finding was replicated in Gliwice cohort. Combining both Polish-Caucasian cohorts showed that the NFKB1 polymorphism was significantly associated with susceptibility to GD with a codominant mode of inheritance (P=0.00005; OR=1.37 (1.18-1.60)). No association with GD was found in Japanese cohort. However, subgroup analysis in Japanese GD patients revealed a correlation between the NFKB1genotype and the development of ophthalmopathy (P=0.009; OR=1.49 (1.10-2.01)), and the age of disease onset (P=0.009; OR=1.45 (1.09-1.91)). Our results suggest that NFKB1 -94ins/del ATTG polymorphism may be associated with susceptibility to and/or phenotype of GD.

IL-18 gene polymorphism confers susceptibility to the development of anti-GAD65 antibody in Graves' disease.

AIMS: This study aimed to investigate whether interleukin-18 (IL-18) gene polymorphisms are associated with the development of antibody against the 65-kDa isoform of recombinant human glutamic acid decarboxylase (GAD65Ab) in patients with Graves' disease. METHODS: A total of 398 unrelated Japanese patients with Graves' disease, with and without GAD65Ab, were recruited. Three single nucleotide polymorphisms in the IL-18 gene were examined and the polymorphic allele and the genotype and haplotype frequencies calculated. RESULTS: The frequency of the GG genotype at position -4675 of the IL-18 gene was significantly lower in Graves' disease patients with GAD65Ab than those without (4% vs. 24%, P = 0.0126). The -4675C allele frequency was significantly greater in patients with GAD65Ab than those without (69% vs. 53%, P = 0.0168). The homozygous -4675G/-607A/-137G haplotype was less common in Graves' disease patients with GAD65Ab than those without (4% vs. 23%, P = 0.0144). CONCLUSIONS: These findings in a Japanese population indicate that Graves' disease patients carrying the GG genotype at position -4657 of the promoter of the IL-18 gene or a gene in linkage disequilibrium with the -4675G/-607A/-137G haplotype have a low risk for the development of GAD65Ab in Graves' disease.

Growth factor-expressing mast cells accumulate at the thyroid tissue-regenerative site of subacute thyroiditis.

The localization and biological roles of the multifunctional cell type mast cells remain unclear in subacute thyroiditis that is characterized by both epithelioid granuloma formation and thyroid tissue repair. We examined their immunolocalization with tryptase of a mast cell marker, using the biopsy specimens from 12 cases. In the epithelioid granuloma, no mast cells were detected in any of the cases, although a small number of them (4.6 +/- 2.4) were seen at the fibrous stroma around the granuloma in all cases. By contrast, in all cases, increased mast cells (28 +/- 7.2) localized at the thyroid tissue-regenerative site where both thyroid folliculogenesis and angiogenesis take place. To elucidate possible roles of mast cells in the disease, we also examined their immunoexpressions of vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-BB (PDGF), transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF), which affect thyroid folliculogenesis and angiogenesis. In all 12 cases, mast cells displayed all of these growth factors in a manner not specific to the infiltrating site. The data suggest that growth factor-expressing mast cells may play crucial roles in the thyroid tissue repair of subacute thyroiditis, modulating thyroid folliculogenesis and angiogenesis; and that the multifunctionality of the cells may be partly dependent on their expressions of various growth factors.

A polymorphism of the 5' flanking region of tumour necrosis factor alpha gene is associated with thyroid-associated ophthalmopathy in Japanese.

OBJECTIVE: We have studied the polymorphism of the 5' flanking region of the tumour necrosis factor (TNF)-alpha gene in order to better understand the genetic background of autoimmune thyroid disorders and thyroid-associated ophthalmopathy. PATIENTS AND METHODS: We studied the polymorphism of the 5' flanking region of the TNF-alpha gene at positions - 1031 (T to C change, termed as - 1031C), - 863 (C to A, - 863 A), - 857 (C to T, - 857T), - 308 (G to A, - 308 A) and - 238 (G to A, - 238 A) in Japanese patients with Graves' disease [n = 173, 62 of whom had associated ophthalmopathy (American Thyroid Association (ATA) class III or greater)] and healthy control subjects (n = 575), using a polymerase chain reaction sequence-specific oligonucleotide probe method. RESULTS: The allele frequency of - 857T in the Graves' disease patients (22. 5% vs. 17.7%, OR = 1.35, P = 0.045, corrected P = 0.23) was slightly greater than in the Japanese healthy subjects, respectively. However, the difference was not statistically significant. In Graves' disease patients with evident ophthalmopathy (ATA class III or greater), the allele frequencies of - 1031C and - 863 A were significantly greater than those with no or mild ophthalmopathy (ATA class 0-II) (31.5% vs. 13.5%, OR =2.94, P < 0.0001, corrected P < 0. 0005; 23.4% vs. 11.7%, OR =2.30, P = 0.0044, corrected P = 0.022, respectively) and in control subjects. The strength of the association of the polymorphism - 1031C increased with the severity of ophthalmopathy, with odds ratios of 2.36 for ATA class III, and 5. 43 for ATA class IV-VI, respectively, compared with Graves' disease with no or mild ophthalmopathy (ATA class 0-II). Although the phenotype frequency of DRB1*0901 was not different among Graves' disease patients with or without ophthalmopathy and control subjects, the phenotype frequency of DRB1*0901(-)/-1031C(+) was significantly increased in Graves' disease patients with ophthalmopathy compared to those with no or mild ophthalmopathy (OR = 4.91, P = 0.0005) or control subjects (OR = 4.59, P < 0.0001). CONCLUSIONS: These results suggest that the - 1031C or - 863 A alleles, or a gene in linkage disequilibrium with the TNF-alpha gene, predispose to the development of ophthalmopathy in Japanese patients with Graves' disease.

Technetium-99m tetrofosmin parathyroid imaging in patients with primary hyperparathyroidism.

OBJECTIVE: To confirm the clinical significance of 99mTc-tetrofosmin imaging for the localization of hyperfunctioning parathyroid glands in patients with primary hyperparathyroidism. METHODS: All patients were imaged with 99mTc-tetrofosmin at 10 minutes and 2 hours after radiotracer injection, and with ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI). The parathyroid/thyroid uptake ratio of 99mTc-tetrofosmin (P/T uptake ratio) was calculated. PATIENTS: Twenty patients with primary hyperparathyroidism were referred to our clinic, underwent surgical neck exploration or mediastinotomy and were diagnosed as having parathyroid adenoma. These patients were investigated for the preoperative localization by 99mTc-tetrofosmin scintigraphy. RESULTS: 99mTc-tetrofosmin imaging demonstrated focal uptake in 19 out of 20 patients with parathyroid adenoma. Two of the lesions were ectopic. US identified 17 parathyroid glands. CT and MRI initially detected 17 parathyroid glands. However, two additional parathyroid glands were localized on repeated CT and MRI in tandem with the results of the 99mTc-tetrofosmin imaging. Thus, the sensitivity and specificity of tetrofosmin imaging were 95% (19/20) and 95% (19/20); US, 85% (17/20) and 94% (16/17); initial CT, 85% (17/20) and 94% (16/17); and initial MRI, 88% (17/20) and 94% (16/17), respectively. The P/T uptake ratio at 2 hours after tetrofosmin injection was correlated with the serum concentration of intact PTH (rs=0.47, p<0.05) and the resected tumor weight (rs=0.53, p<0.05). CONCLUSION: 99mTc-tetrofosmin scintigraphy is useful for localization of parathyroid adenoma. Tetrofosmin uptake depends on the tumor weight and serum intact PTH levels.

Cytokine profiles in eye muscle tissue and orbital fat tissue from patients with thyroid-associated ophthalmopathy.

Eye muscle (EM) and retroorbital fat tissue are two major sites of involvement in thyroid-associated ophthalmopathy (TAO). Lymphocytic infiltration in these tissues is a prominent histological feature of TAO. We have investigated the cytokine gene profiles in EM and orbital fat (OF) tissues from patients with TAO. Total RNA was isolated from EM tissue of 14 patients and from OF tissues of 29 patients with TAO. Cytokine gene expression was assessed by RT-PCR using paired primers for interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, CD4, CD8, and glyceraldehyde-3-phosphate dehydrogenase. IFNgamma, TNFalpha, IL-1beta, and IL-6 messenger RNA (mRNA) were mainly detected in EM tissue, whereas IL-4 and IL-10 mRNA were detected in only one patient. On the other hand, in OF tissue, IL-4 and IL-10 mRNA were detected in 24% and 38% of the patients, respectively, and IFNgamma, IL-1beta, and IL-6 mRNA were less often detected compared with EM tissue. The enlargement of EM tissue as assessed by computed tomography correlated significantly with TNFalpha mRNA expression in EM tissue. The orbital volume was positively correlated with IL-6 mRNA expression and negatively correlated with IL-4 mRNA and IL-10 mRNA expression in OF tissue. These results suggest that T helper (Th) 1-like cytokines predominate in EM tissue in most patients and that the predominant cytokine profile in OF tissue varies from patient to patient. Both Th1-like and Th2-like immune responses may play roles in the development of two components of ophthalmopathy.

Functional Fas ligand expression in thyrocytes from patients with Graves' disease.

Fas/Fas ligand (FasL) interaction has been suggested to play a role in the pathogenesis of Hashimoto's thyroiditis. This manuscript addressed a role for Fas/FasL interaction in the pathogenesis of Graves' disease (GD). Apoptosis was detected in 0.5-5.0% of GD thyrocytes, but not in normal thyrocytes from patients with adenoma (N). Fas was constitutively expressed on the basement membrane of both GD and N thyrocytes. Thyrocytes expressed Bcl-2 constitutively in both GD and N thyrocytes. FasL was detected at the messenger ribonucleic acid level in thyroid tissue and cultured thyroid cells by Northern blotting and RT-PCR. FasL protein was detected in the cytoplasm and basolateral surface of thyrocytes from GD, but not in N. Cell surface expression of FasL on cultured thyrocytes disappeared within 48 h after their isolation. However, it was retained by culturing the cells with a matrix metalloproteinase inhibitor. Coculture with thyrocytes induced apoptosis of Fas transfectants, which was blocked by an anti-FasL antibody. Although cultured thyrocytes expressed Fas on the surface, they were not killed by an agonistic anti-Fas antibody. Interferon-gamma-induced Fas up-regulation was suppressed by TSH. These results suggest that the increased expression of FasL in GD thyrocytes, the down-regulation of Fas expression by TSH or possibly by TSH receptor autoantibody, and the overexpression of Bcl-2, which could render thyrocytes resistant to FasL-mediated elimination, may thus be involved in the pathogenesis of GD.

Pre-surgical localization of ectopic parathyroid glands using three-dimensional CT imaging, 99Tcm sestamibi, and 99Tcm tetrofosmin imaging.

We describe two patients with ectopic parathyroid glands evaluated with 99Tcm sestamibi, 99Tcm tetrofosmin, and three-dimensional computed tomography (3D-CT). Radionuclide images of the neck were acquired at 10 min, and at 2-3 h after radiopharmaceutical injection, and showed intense uptake in the ectopic parathyroid tissue. These patients also underwent contrast enhanced CT imaging with 3D reconstructions which were evaluated for ability to visualize critical anatomical structures, e.g. blood vessels and parathyroid glands. Based on 3D-CT images, surgical planning was altered in one of the two patients studied. In conclusion, reconstructed 3D-CT images provided useful anatomical localization of ectopic parathyroid glands identified on 99Tcm sestamibi and 99Tcm tetrofosmin imaging. This anatomical information aided surgical planning of gland resection.

Immunohistochemical analysis of Bcl-2, Bax, and Bak expression in thyroid glands from patients with subacute thyroiditis.

Bcl-2 family proteins are important regulators of apoptosis. To clarify a role of apoptosis and the expression of Bcl-2 family proteins in the pathogenesis of subacute thyroiditis (SAT), we evaluated the expression of Bcl-2, Bax, and Bak by immunohistochemistry and apoptosis by in situ end labeling of fragmented DNA in thyroid tissues from 11 patients with SAT. Apoptotic nuclei were found in granulomas, especially in macrophages/histiocytes and lymphocytes, and in the regenerating follicular cells, but were rarely found in the area of fibrosis. The mean (+/-SD) percentage of apoptotic follicular cells was significantly greater in SAT than that in controls (1.4 +/- 0.8% vs. 0.4 +/- 0.6%). Bcl-2, Bak, and Bax were strongly expressed in the granulomas and regenerating thyroid follicular cells from patients with SAT. Bcl-2 and Bak, but not Bax, were expressed in follicular cells from normal controls. The percentage of apoptotic cells and the expression of Bax in follicular cells did not correlate with age or serum levels of thyroid hormones, C-reactive protein, or thyroglobulin. These data suggest that apoptosis may be involved in the development of SAT and that Bax expression in regenerating thyrocytes may be important for the recovery of SAT.

Increased serum soluble Fas in patients with Graves' disease.

We addressed the role of soluble Fas (sFas), which suppresses Fas-mediated apoptosis, in the pathogenesis of Graves' disease (GD). The serum concentration of sFas was measured by enzyme-linked immunosorbent assay and the expression of sFas mRNA in thyroid tissues by reverse transcriptase-polymerase chain reaction. The serum concentration of sFas was significantly increased in untreated GD (mean+/-SD: 1.57+/-0.48 ng/mL) compared to age-matched control subjects (0.77+/-0.46 ng/mL). The serum sFas level tended to decrease after the medication of antithyroid drugs for 6 to 8 weeks and was significantly decreased in patients who were euthyroid for more than 3 years (0.98+/-0.23 ng/mL), compared to that in untreated GD. The concentration of serum sFas was significantly correlated with anti-thyrotropin (TSH) receptor antibody titers, but not with the other clinical parameters (free triiodothyronine [FT3], free thyroxine [FT4], TSH, antithyroglobulin antibody titer, antimicrosomal antibody titer, or 123I uptake). The sFas mRNA was detected in thyroid tissue, cultured thyrocytes, and intrathyroidal lymphocytes. sFas was detected in supernatant of cultured thyrocytes from patients with GD. Its production by thyrocytes was induced by culture with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). The present study confirms serum sFas increases in GD and provides evidence of local production of sFas by thyrocytes and its regulation by cytokines. These data suggest that sFas may play a role in the pathogenesis of GD.

Autoantibodies reactive with extracellular matrix proteins in patients with thyroid-associated ophthalmopathy.

Autoantibodies reacting with extracellular matrix proteins have been extensively studied in various autoimmune connective tissue diseases. Because of the possibility that such antibodies may play a role in orbital connective tissue inflammation in thyroid-associated ophthalmopathy (TAO), we studied the humoral immune response against specific extracellular matrix (ECM) proteins, namely: collagen types I, III, IV, V (CI, CIII, CIV, CV), fibronectin (FN), and laminin (LM). Anti-ECM antibodies of immunoglobulin G (IgG), IgA, and IgM classes were determined by enzyme linked immunosorbent assay (ELISA). Overall, sera from 50% of patients with TAO contained antibodies reactive against one or more ECM proteins, compared to 27% with Graves' disease (GD) without evident eye involvement, 28% with Hashimoto's thyroiditis (HT), and 9% of normal subjects. Serum anti-CI, anti-CIII, anti-CV and anti-LM levels were significantly (p<0.05) higher in patients with TAO than in normals. Anti-CI, anti-CV and anti-LM reactivity was antigen-specific in most TAO sera, while anti-CIII antibodies cross-reacted with other antigens. Anti-collagen antibodies were mainly of the IgG class. To determine the structural epitopes of these proteins, we performed immunoblotting studies on cyanogenbromide (CNBr)-derived peptides of CI and CV. While sera from 9 of 10 patients with TAO reacted with CI peptides, the response was polyclonal and uniform in all patients. However, only 2 of 10 TAO sera reacted with CV peptides. In conclusion, our study suggests that a variety of ECM proteins (CI, CV, LM) may be secondary autoantigens that are recognized by antibodies in TAO. While these antibodies appear to react with epitopes expressed on both native and denatured proteins, and may therefore have the potential to bind to ECM in vivo, their pathogenic role in TAO remains unknown.

Dominant infiltration of T(H)1-type CD4+ T cells at the retrobulbar space of patients with thyroid-associated ophthalmopathy.

Lymphocyte infiltration in the retrobulbar space is a prominent histological feature of thyroid-associated ophthalmopathy (TAO). We have characterized phenotypic and functional features of T cells derived from retrobulbar infiltrates of 3 TAO patients to better understand their roles in the disease. One hundred four T-cell clones (TCC) were directly established from cells of retrobulbar tissues using a highly efficient cloning procedure. Phenotypic analysis of TCC showed approximately 70% to 80% were CD3+ CD4+ CD8- T cells, and approximately 20% to 30% were CD3+ CD8+ CD4- T cells. None of the TCC were CD3+ CD4- CD8- T cells. Analysis of the cytokine profile of TCC, as documented by the ability to express interferon-gamma, interleukin (IL)-2, IL-4, and IL-10 demonstrated that the majority of TCC expressed T helper (T(H))1-like profile in both the mRNA and protein levels. A few TCC showed T(H)0-like profile, but no TCC showed T(H)2-like profile. These results suggest that T(H)1-type CD4+ T cells play important roles in the pathogenesis of TAO.

Color Doppler ultrasonography in patients with subacute thyroiditis.

We studied the utility of color Doppler ultrasonography in patients with subacute thyroiditis. Eighteen patients with subacute thyroiditis (SAT) with painful goiter and thyrotoxicosis underwent color Doppler ultrasonography during the acute and recovery stages of the disease. Thyroid vascularization in these patients was compared with that of 15 untreated patients with Graves' disease and 17 control subjects. During the acute stage of subacute thyroiditis, color Doppler ultrasonography showed low echogenicity without increased tissue vascularity in the affected swollen thyroid. In the recovery stage, color Doppler ultrasonography showed isoechogenicity with slightly increased vascularization. Vascularization became normal at 1 year follow-up time. In contrast, marked by increased vascularization was observed in patients with untreated Graves' disease. Color Doppler ultrasonography showed clear differences between SAT and Graves' disease patients. Vascularity was significantly correlated with serum free thyroxine (FT4) and thyrotropin (TSH) concentrations in the recovery stage (3 months after the initial ultrasonography). Color Doppler ultrasonography accurately visualized lesions without increased vascularity in the acute stage of SAT and lesions of slightly increased vascularity in the recovery stage. Color Doppler ultrasonography may be a useful, noninvasive, and rapid method for differentiating SAT from Graves' disease and for evaluating and monitoring the location and activity of lesions in SAT.

Technetium-99m tetrofosmin imaging in patients with subacute thyroiditis.

We studied the significance of technetium-99m tetrofosmin scintigraphy in patients with subacute thyroiditis (SAT). Six patients with SAT, who had painful goitre with thyrotoxicosis, underwent 99mTc-pertechnetate scintigraphy and 99mTc-tetrofosmin imaging during the acute and recovery stages of SAT. The thyroid uptake ratio of tetrofosmin was compared with the clinical parameters associated with SAT. 99mTc-pertechnetate scintigraphy showed markedly reduced uptake in the thyroid during the acute stage of SAT, suggesting that the appropriate metabolic pathway is not functioning. Conversely, 99mTc-tetrofosmin images showed diffuse increased uptake in the thyroid region on early and delayed imaging. Tetrofosmin images in the acute stage and in the recovery stage of SAT showed different clearance curves for tetrofosmin uptake. The uptake ratio assessed as thyroid uptake/background (T/B) correlated with the serum C-reactive protein concentration. In conclusion, 99mTc-tetrofosmin uptake may reflect the inflammatory process associated with SAT, and thus this tracer may have potential as a marker of disease activity and severity.

Possible involvement of Fas-mediated apoptosis in eye muscle tissue from patients with thyroid-associated ophthalmopathy.

In order to investigate the possible involvement of apoptosis in the pathogenesis of thyroid-associated ophthalmopathy (TAO), we studied the expression of Fas, Fas ligand (FasL), and Bcl-2 in extraocular muscle tissues from patients with TAO by immunohistochemistry using monoclonal antibodies against Fas, FasL, and Bcl-2. Apoptosis was detected by in situ end-labeling of fragmented DNA. Apoptosis was detected in extraocular muscle tissues from 17 of 19 patients with TAO and, to a smaller degree in 4 of 7 normal control subjects. The mean percentages of apoptotic nuclei were 12.1% in TAO eye muscle fibers and 16.4% in TAO interstitial cells, compared with 2.2% and 0.4% in normal eye muscle fibers and interstitial cells, respectively. The percentage of apoptotic nuclei in eye muscle fibers correlated with the enlargement of eye muscle tissue (r = 0.47, p < 0.05). Fas was demonstrated on the surfaces of extraocular muscle fibers in 11 of 18 patients with TAO, but not in normal extraocular muscle tissues. Neither TAO nor normal extraocular muscle tissues expressed Bcl-2. FasL was detected in infiltrating mononuclear cells in extraocular muscle tissue from a patient with TAO. These data suggest that Fas-mediated apoptosis may occur in extraocular muscle tissue from patients with TAO and may be involved in the late stage of TAO.

The 64-kilodalton eye muscle protein is the flavoprotein subunit of mitochondrial succinate dehydrogenase: the corresponding serum antibodies are good markers of an immune-mediated damage to the eye muscle in patients with Graves' hyperthyroidism.

Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder associated with thyroid autoimmunity, particularly Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye muscle membrane proteins reportedly of 55 and 64 kDa are the best markers of the ophthalmopathy. The main focus of our recent studies has been to purify the pertinent proteins from porcine eye muscle membranes and characterize them. The 64-kDa protein is now shown from a partial sequence and by Western blotting using specific antibody probes to be the flavoprotein (Fp) subunit of succinate dehydrogenase and to have a correct molecular mass of 67 kDa. The protein was purified and cleaved with cyanogen bromide, and the N-terminal region of an immunoreactive partial peptide was determined. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively. Succinate dehydrogenase is both a citric acid cycle enzyme and a component (complex II) of the mitochondrial respiratory chain. It is thus essential for aerobic energy production and is highly conserved. The mature human and bovine Fp subunits are 92% homologous and have a molecular mass of approximately 67 kDa, the same as our redetermined value for the 64-kDa marker protein. Sera from patients with TAO and from those with Graves' hyperthyroidism without evident ophthalmopathy highlighted the 64-kDa marker protein in crude porcine eye muscle membranes and the Fp subunit of highly purified bovine succinate dehydrogenase at the identical position on Western blots. Anti-beef Fp antibodies were detected in sera from 67% of patients with active TAO of more than 1-yr duration, in 30% with stable TAO of more than 3-yr duration, and in 30% of patients with Graves' hyperthyroidism without ophthalmopathy, but in only 7% of age- and sex-matched normal subjects. As succinate dehydrogenase is bound to the matrix (inside) surface of the mitochondrial inner membrane, it is unlikely to be accessible to circulating autoantibodies. We would postulate that eye muscle damage in ophthalmopathy is probably caused by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and eye muscle shared protein G2s, and that presentation of succinate dehydrogenase is secondary. On the other hand, an autoantibody response to succinate dehydrogenase may be a good marker of immune-mediated damage to the eye muscle fiber and may support the idea that the extraocular muscles are targets of the autoimmune reactions of TAO.