Genetic variability in CRTH2 polymorphism increases eotaxin-2 levels in patients with aspirin exacerbated respiratory disease.

INTRODUCTION: CRTH2 is expressed on the surface of eosinophils and has been shown to mediate PGD2-induced eosinophil migration in vitro. Eosinophilic infiltration in the upper and lower airways is the key feature of asthma. Considering the fact that eosinophil infiltration is prominent in the upper and lower airways of aspirin exacerbated respiratory disease (AERD) compared to aspirin-tolerant asthma (ATA) patients, we hypothesized that activation of eosinophils via dysregulation of the CRTH2 gene may play an important role and be an important marker for AERD. METHODS: The three study groups - 107 with AERD, 115 with ATA and 133 normal healthy controls (NC) - were recruited from Ajou University Hospital, South Korea. Two polymorphisms of the CRTH2 gene at -466T>C and -129C>A were genotyped using primer extension methods. RESULTS: AERD patients had significantly higher serum eotaxin-2 levels than did those with ATA (P = 0.034). A significant difference in the genotype frequencies of CRTH2 -466T>C was detected between AERD and ATA patients (P < 0.05). The serum eotaxin-2 level was significantly higher in AERD patients carrying the TT genotype of CRTH2 -466T>C than those with the CT and CC (P < 0.05). In vitro functional study demonstrated that the -466T allele had lower luciferase activity (P < 0.001) and lower mRNA expression with higher production of eotaxin-2 (P = 0.003) in human lung epithelial cells. EMSA showed that CRTH2 -466T produced a specific band with a higher affinity than CRTH2 -466C had. CONCLUSION: The CRTH2 -466T>C polymorphism increases serum and cellular eotaxin-2 production through lowered CRTH2 expression, leading to eosinophilic infiltration in AERD patients.

Alpha-T-catenin (CTNNA3) gene was identified as a risk variant for toluene diisocyanate-induced asthma by genome-wide association analysis.

BACKGROUND: Toluene diisocyanate (TDI) is the most important cause of occupational asthma, but the genetic mechanism of TDI-induced asthma is still unknown. OBJECTIVE: The objective of the study was to identify susceptibility alleles associated with the TDI-induced asthma phenotype. METHODS: We conducted a genome-wide association study in 84 patients with TDI-induced asthma and 263 unexposed healthy normal controls using Affymetrix 500K SNPchip. We also investigated the relationships between genetic polymorphisms and transcript levels in Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with TDI-induced asthma enrolled in this study. RESULTS: Genetic polymorphisms of CTNNA3 (catenin alpha 3, alpha-T catenin) were significantly associated with the TDI-induced asthma phenotype (5.84 x 10(-6) for rs10762058, 1.41 x 10(-5) for rs7088181, 2.03 x 10(-5) for rs4378283). Carriers with the minor haplotype, HT2 [GG], of two genetic polymorphisms (rs10762058 and rs7088181) showed significantly lower PC(20) methacholine level (P=0.041) and lower mRNA expression of CTNNA3 than non-carriers (P=0.040). A genetic polymorphism in the 3' downstream region of CTNNA3 (rs1786929), as identified by DNA direct sequencing, was significantly associated with the TDI-induced asthma phenotype (P=0.015 in recessive analysis model) and the prevalence of serum-specific IgG to cytokeratin 19 (P=0.031). CONCLUSION: These findings suggested that multiple genetic polymorphisms of CTNNA3 may be determinants of susceptibility to TDI-induced asthma.

Combined effect of IL-10 and TGF-beta1 promoter polymorphisms as a risk factor for aspirin-intolerant asthma and rhinosinusitis.

BACKGROUND: It has been known that interleukin (IL)-10 promoter polymorphisms at -1082A/G, -819T/C and -592A/C, may influence IL-10 expression and associate with asthma. Interleukin-10 facilitates the regulatory function of transforming growth factor (TGF)-beta. The goal of this study was to investigate a gene-gene interaction between IL-10 and TGF-beta1 polymorphisms in Korean asthmatics with aspirin hypersensitivity. METHODS: Single-nucleotide polymorphism genotyping of IL-10 and TGF-beta1 genes was performed and the functional effect of the IL-10 polymorphisms was analysed applying a luciferase reporter assay and an electrophoretic mobility shift assay. RESULTS: Among the patients with asthma, polymorphism at -1082A/G was significantly associated with the phenotype of aspirin-intolerant asthma, AIA (P = 0.007, P(c) = 0.021). Moreover, a synergistic effect between the TGF-beta1-509C/T and IL-10-1082A/G polymorphisms on the phenotype of AIA was noted; when stratified by the presence of rhinosinusitis, the frequency of rare alleles (the CT or TT genotype of TGF-beta1-509C/T and AG or GG genotype of IL-10-1082A/G) was significantly higher in the patients with AIA (15.2%) when compared with those with ATA (6.3%, P = 0.031; odds ratio 4.111; 95% confidence interval 1.504-11.235). In an in vitro functional assay, the -1082G reporter plasmid exhibited significantly greater promoter activity when compared with the -1082A construct in Jurkat T cells (P = 0.011). Moreover, we found that the transcription factor Myc-associated zinc-finger protein preferentially bound the -1082G allele. CONCLUSION: Our results suggest that IL-10 promoter polymorphisms contribute to the development of AIA and that rhinosinusitis may interact genetically with TGF-beta1.

Histamine N-methyltransferase 939A>G polymorphism affects mRNA stability in patients with acetylsalicylic acid-intolerant chronic urticaria.

BACKGROUND: Histamine plays an important role in allergic inflammation. Histamine levels are regulated by histamine N-methyltransferase (HNMT). OBJECTIVE: To investigate the functional variability of HNMT gene in relation to genetic polymorphisms in patients with aspirin intolerant chronic urticaria (AICU). METHODS: Two single-nucleotide polymorphisms of the HNMT gene (314C>T, 939A>G) were genotyped in chronic urticaria patients. The functional variability of 3'-untranslated region polymorphism (3'-UTR) was assessed using the pEGFP-HNMT 3'-UTR reporter construct to examine mRNA stability and fluorescence-tagged protein expression. The HNMT enzymatic activities related to the 939A>G polymorphism were examined both in the human mast cells (HMC-1) transfected with the pHNMT CDS-3'-UTR construct and in the patients' red blood cells (RBCs). Histamine release from the basophils of AICU patients was examined. RESULTS: The 939A>G polymorphism was significantly associated with the AICU phenotype, while no association was found with the 314C>T polymorphism. An in vitro functional study using HMC-1 cells demonstrated that the 939A allele gave lower levels of HNMT mRNA stability, HNMT protein expression, and HNMT enzymatic activity and higher histamine release than the 939G allele. The in vivo functional study demonstrated that the AICU patients with the 939A allele had lower HNMT activity in RBC lysates and higher histamine release from their basophils. CONCLUSION: The HNMT 939A>G polymorphism lowers HNMT enzymatic activity by decreasing HNMT mRNA stability, which leads to an increase in the histamine level and contributes to the development of AICU.

Association of TNF-alpha promoter polymorphisms with aspirin-induced urticaria.

OBJECTIVE: Although the pathogenesis of aspirin-induced urticaria (AIU) is not fully understood, mast cell activation has been noted in patients with AIU. Tumour necrosis factor (TNF)-alpha, a potent pro-inflammatory cytokine, is released by human skin mast cells and other inflammatory cells in patients with urticaria. To investigate the role of TNF-alpha promoter polymorphisms in the development of AIU, we performed an association study of TNF-alpha promoter polymorphisms with AIU phenotype. METHODS: Two hundred thirty-nine patients with AIU consisting of 120 patients with aspirin intolerant chronic urticaria (AICU) and 119 with aspirin-intolerant acute urticaria (AIAU), and 524 normal controls were enrolled. AIU was confirmed by oral aspirin challenge test. Five SNPs in the TNF-alpha gene (-1031T>C, -863C>A, -857C>T, -308G>A, -238G>A) were genotyped by a single-base extension method. Haplotype analyses were done. RESULTS: The genotype frequencies of TNF-1031T>C and TNF-863C>A were significantly higher in the AIU patients than in the normal controls in both co-dominant (P = 0.014, P = 0.007) and dominant (P = 0.007, P = 0.004) models. The frequency of TNF-ht2[CACGG] containing a genotype in the AIU group was significantly higher in the normal controls with both co-dominant (P = 0.004, Pc = 0.02) and dominant models (P = 0.002, Pc = 0.01). CONCLUSIONS: These findings suggest that the two promoter polymorphisms of TNF-alpha at -1031T>C and -863C>A may contribute to the development of AIU.

Biometric and refractive changes after orbital decompression in Korean patients with thyroid-associated orbitopathy.

PURPOSE: To determine the biometric and refractive changes after orbital decompression in Korean patients with thyroid-associated orbitopathy (TAO). METHODS: Retrospective, observational study (between October 2012 and September 2014) was performed. Patients with TAO undergoing orbital decompression for stable proptosis received ophthalmic examinations, including Hertel exophthalmometry, A-scan biometry, autorefraction measures, corneal topography, and wavefront aberration measures, before orbital decompression and again 2 months after surgery. RESULTS: Included in the study were 43 eyes from 23 patients. The mean exophthalmometric value decreased by 4.1 mm 2 months after orbital decompression (P<0.001). On average, axial length (AL) increased significantly by 0.08 mm (P<0.001); specifically, 37 (86%) of the 43 eyes had increased AL. Whereas anterior chamber depth and lens thickness showed no significant changes (P=0.086 and P=0.905, respectively), the mean spherical refraction and spherical equivalent (SE) decreased by 0.35 and 0.48 D, respectively (P=0.008 and P<0.001, respectively). However, cylindrical refraction and axis showed no significant changes (P=0.057 and P=0.218, respectively). The changes in AL and SE were significantly correlated (R=-0.411, P=0.009). Notably, there were no changes in corneal topography or wavefront aberration after orbital decompression. CONCLUSIONS: TAO patients who underwent orbital decompression showed myopic refractive change via increase in AL. Possible refractive changes should be considered in cases of TAO complaining of decreased visual acuity after orbital decompression.

p66Shc expression in proliferating thyroid cells is regulated by thyrotropin receptor signaling.

It is almost unanimously accepted that thyrocyte proliferation is synergistically activated by TSH and insulin/IGF-I. Moreover, it was recently suggested that p66Shc, which is an adaptor molecule of the IGF-I receptor, might play a critical role in this synergistic effect. In this study, we undertook to confirm the role and the mechanism underlying the regulation of p66Shc expression via TSH receptor in thyrocytes. We have found that p66Shc expression is elevated in proliferating human thyroid tissues, including adenomatous goiter, adenoma, Graves' disease, and thyroid cancer, but not in normal thyroid. Among growth factors, TSH increased p66Shc expression both in vivo and in vitro; however, IGF-I, epidermal growth factor, or insulin did not. TSH and Graves' Ig increased the p66Shc expression via the TSH receptor-G(s)-cAMP pathway. However, interestingly, IGF-I or epidermal growth factor increased the tyrosine phosphorylations of p66Shc, and this was enhanced by TSH pretreatment. A similar synergism was observed during the DNA synthesis. When we measured the p66Shc levels induced by individual Igs from 130 patients with Graves' disease, TSH receptor stimulating activity and goiter size showed a weak correlation. We conclude that the expression of p66Shc is regulated by signaling through the TSH receptor in proliferating thyroid cells and that p66Shc appears to be an important mediator of the synergistic effect between TSH and IGF-I with respect to thyrocyte proliferation. Moreover, we suggest that TSH potentiates the regulatory effect of IGF-I on thyrocyte growth, at least in part, by increasing the expression of p66Shc.

Thyrotropin induces SOCS-1 (suppressor of cytokine signaling-1) and SOCS-3 in FRTL-5 thyroid cells.

TSH has multiple physiological roles: it is required for growth, differentiation, and function of the thyroid gland, and it regulates transcription of interferon-gamma (IFN-gamma)-responsive genes in thyrocytes, including genes for the major histocompatibility complex and intercellular adhesion molecule-1. This report demonstrates that TSH induces the expression of suppressor of cytokine signaling (SOCS)-1 and -3 proteins and alters the phosphorylation state of signal transducer and activator of transcription (STAT) proteins STAT1 and STAT3. The expression of SOCS-1 and SOCS-3 and the phosphorylation state of STAT1 and STAT3 were examined after treatment with TSH or IFN-gamma in either TSH-sensitive FRTL-5 thyroid cells or TSH-insensitive FRT and buffalo rat liver (BRL) cells, which lack functional TSH receptors. SOCS-1 and SOCS-3 are constitutively expressed in FRTL-5 cells, but not in FRT and BRL cells. IFN-gamma up-regulated SOCS-1 and SOCS-3 RNA and protein in FRTL-5 cells, as reported previously for nonthyroid cells. Interestingly, TSH also significantly induced SOCS-1 and SOCS-3 in FRTL-5 cells, but not in FRT and BRL cells. When SOCS-1 or SOCS-3 was overexpressed in FRTL-5 cells, STAT1 phosphorylation at Y701 and STAT1/DNA complex formation in response to IFN-gamma were reduced. Furthermore, overexpression of either SOCS-1 or SOCS-3 significantly inhibited the IFN-gamma-mediated transactivation of the rat ICAM-1 (intercellular adhesion molecule-1) promoter. TSH and IFN-gamma had different effects on STAT1 and STAT3 phosphorylation. The phosphorylation of Y701 in STAT1, which is responsible for homodimer formation, nuclear translocation, and DNA binding, was specifically stimulated by IFN-gamma, but not by TSH or forskolin. However, the phosphorylation of S727 in STAT1 was induced by IFN-gamma, TSH, and forskolin. TSH induced phosphorylation of both Y705 and S727 in STAT3, while IFN-gamma phosphorylated only the Y705. In addition, we found that SOCS-3 was associated with JAK1 and JAK2 and that these associations were stimulated by TSH. These findings demonstrate that TSH induces SOCS in thyroid cells and provides the evidence of signal cross-talk between TSH and cytokines in thyroid cells.

Involvement of JAK/STAT (Janus kinase/signal transducer and activator of transcription) in the thyrotropin signaling pathway.

TSH is an important physiological regulator of growth and function in thyroid gland. The mechanism of action of TSH depends on interaction with its receptor coupled to heterotrimeric G proteins. We show here that TSH induces the phosphorylation of tyrosine in the intracellular kinases Janus kinase 1 (JAK1) and -2 (JAK2) in rat thyroid cells and in Chinese hamster ovary (CHO) cells transfected with human TSH receptor (TSHR). The JAK family substrates STAT3 (signal transducers and activators of transcription) are rapidly tyrosine phosphorylated in response to TSH. We also find that JAK1, JAK2, and STAT3 coprecipitate with the TSHR, indicating that the TSHR may be able to signal through the intracellular phosphorylation pathway used by the JAK-STAT cascade. TSH increases STAT3-mediated promoter activity and also induces endogenous SOCS-1 (suppressor of cytokine signaling-1) gene expression, a known target gene of STAT3. The expression of a dominant negative form of STAT3 completely inhibited TSH-mediated SOCS-1 expression. These findings suggest that the TSHR is able to signal through JAK/STAT3 pathways.

Cycloheximide inhibits interferon-gamma-induced class II major histocompatibility complex antigen expression in cultured rat thyroid cells.

The effects of the agents that are related to intracellular events on interferon-gamma-induced class II major histocompatibility complex antigen expression were studied using the technique of immunocytochemistry. Rat class II major histocompatibility complex antigen (RT1.B) was expressed in 88.3 +/- 3.3% (n = 3) of the functioning rat thyroid cells (FRTL-5) cultured in a medium containing 100 U/ml recombinant rat interferon-gamma (IFN gamma). Deprivation of bovine TSH had no effect on the expression of RT1.B antigen by IFN gamma. A23187 (1 nM to 2 microM) and/or 10 nM to 10 microM phorbol 12-myristate 13-acetate did not induce the expression of RT1.B antigen. IFN gamma-induced RT1.B expression was not inhibited by either 10 nM to 100 microM 1-(5-isoquinolysulfonyl)-2-methylpiperazine or 200 nM to 200 microM 8-(N,N-dimethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride. It was also not inhibited by either 5-200 microM verapamil or 500 nM to 20 microM trifluoperazine. However, 0.01-10 micrograms/ml cycloheximide inhibited IFN gamma-induced RT1.B antigen expression in a dose-dependent manner. These results suggest that IFN gamma induces RT1.B antigen expression in FRTL-5 cells via de novo protein synthesis independent of the cAMP system, phosphatidylinositide system, and voltage-dependent calcium channel.

Identification of the peptides that inhibit the stimulation of thyrotropin receptor by Graves' immunoglobulin G from peptide libraries.

Graves' disease is characterized by the overproduction of thyroid hormones due to the persistent stimulation of TSH receptor by autoantibodies. To determine the epitopes recognized by the autoantibodies, an enzyme-linked immunosorbent assay was developed that uses the human TSH receptor extracellular domain attached to plastic wells. The total IgG from some of the Graves' patients interacted with the bound TSH receptor (TSHR) at a significantly higher level than that in normal individuals. The IgG preparation that showed the highest binding activity was used for the identification of peptide sequences that prevent binding of Graves' IgG to TSHR from positional scanning synthetic peptide combinatorial libraries. A hexapeptide mixture, X1X2FDDA (X1 is a mixture of E, M, and Y; X2 is a mixture of E, H, and T), was found to be effective for inhibiting the binding of Graves' IgG to the TSHR. Further fractionation of X1X2FDDA showed that the following three sequences were highly effective: EEFDDA, ETFDDA, and EHFDDA. The second position of the three peptides did not appear to be important. The peptides also inhibited the cAMP synthesis induced by IgG of four of eight patients with Graves' disease tested. The synthesis of cAMP by TSH was also inhibited by the peptides to some extent. The peptide sequences most likely mimic a part of the conformational epitopes recognized by at least one class of Graves' IgG.

Expression of the functional extracellular domain of human thyrotropin receptor using a vaccinia virus system: its purification and analysis of autoantibody binding.

We produced substantial amount of the extracellular domain of the human TSH receptor (TSHRE) that has a tag of six histidines at C-terminus as a soluble form in the human cell line HeLa using a vaccinia virus system. By sequential nickel-chelating and lentil lectin column chromatography, TSHRE was purified to about 70% purity, with the recovery of around 0.1-0.2 mg TSHRE/L culture (5 x 10(8) cells/liter culture). The purified TSHRE interacted with TSH as well as Graves' autoantibodies to TSHR. However, the affinity of TSHRE for TSH was much lower than that of intact TSHR. The IC50 value for inhibition of TSH-dependent cAMP synthesis by TSHRE was about 10(-8) mol/L. Most importantly, the purified TSHRE inhibited the binding of the IgG of Graves' patients to thyroid membrane. About 1 microg/mL (2 x 10(-8) mol/L) TSHRE neutralized most of the autoantibody activity of patients' sera tested in the TSH binding inhibitory immunoglobulin (TBII) assay. Moreover, this protein neutralized thyroid stimulatory antibody-induced cAMP synthesis with an IC50 of 1 x 10(-9) mol/L and completely at 0.5-1 microg/mL (1-2 x 10(-8) mol/L). In the simple enzyme-linked immunosorbent assay, the TSHRE immobilized on the wells coated with nickel showed significantly higher binding with the IgGs from Graves' patients than in those from normal individuals. This autoantibody-reactive TSHRE will be useful for further studies on the diagnosis, pathogenesis, and the development of therapy of Graves' disease.

Epitopes for thyroid-stimulating antibodies in Graves' sera: a possible link of heterogeneity to differences in response to antithyroid drug treatment.

To evaluate the extent and clinical relevance of epitope heterogeneity for stimulating TSH receptor antibodies (TSHRAbs), we measured the activity of IgG preparations from 66 untreated patients with Graves' disease using Chinese hamster ovary (CHO) cells transfected with wild-type human TSHR and two TSHR chimeras with residues 9-165 (Mc1 + 2) or 90-165 (Mc2) substituted by equivalent residues of the LH/CG receptor. IgG from 68% of patients lose all of the stimulating TSHRAb activity with the chimeras; IgG from 27% lose most of the activity. Thus, we show that 95% of patients have stimulating TSHRAbs that require epitopes on the N-terminal portion of the extracellular domain of the TSHR and demonstrate the importance of epitopes within residues 90-165 for the first time. Heterogeneous epitope distribution, residual activity with one or both chimeras, i.e. with epitopes other than on the N-terminus of the TSHR, occurred in 21 patients (group A). Forty-five patients with homogeneous epitope distribution (group B) had stimulating TSHRAbs that depended only on epitopes on the N-terminus of the TSHR. Patients in group A were more likely to become euthyroid during antithyroid drug therapy and to do so more quickly than group B patients. The CHO-human TSHR cell system described herein appears to be as effective as the FRTL-5 rat thyroid system in stimulating TSHRAb detection; however, the two systems appear to measure different antibody populations in about 30% of cases. Further, stimulating TSHRAb activities measured in the FRTL-5 system tend to correlate better with goiter size and 99mTc pertechnetate uptake, whereas stimulating activities measured in the CHO-human TSHR/chimera system correlate better with free T4 and T3 levels.

A strong association between thyrotropin receptor-blocking antibody-positive atrophic autoimmune thyroiditis and HLA-DR8 and HLA-DQB1*0302 in Koreans.

We investigated whether the associations between HLA alleles of patients with autoimmune hypothyroidism varied according to the presence or absence of TSH receptor-blocking antibody (TRBab). We analyzed the HLA-A, -B, -C, and -DR antigens by serotyping and the DQA1 and DQB1 genes using both enzymatic DNA amplification and sequence-specific oligonucleotide hybridizations. The patient population consisted of 47 Korean patients with atrophic autoimmune thyroiditis and 62 patients with goitrous autoimmune thyroiditis. The antigen frequency of HLA-DR8 was significantly increased in 23 atrophic autoimmune thyroiditis patients that were positive for TSH binding inhibitor immunoglobulin (TBII) compared to 136 controls [52% vs. 16%; chi 2 = 13.1; Pc (corrected P value) = 0.003]. This relative risk was 5.7; the etiological fraction was 0.43. HLA-DQB1*0302 was also increased in patients with TBII-positive atrophic autoimmune thyroiditis (24% vs. 7%; chi 2 = 11.2; Pc = 0.012; relative risk = 4.4; etiological fraction = 0.19). No specific DR antigens or DQB1 alleles were increased in either TBII-negative atrophic autoimmune thyroiditis or goitrous autoimmune thyroiditis. A significant decrease in the frequency of HLA-DR6 antigen was observed in both TBII-positive atrophic autoimmune thyroiditis (0% vs. 32%; chi 2 = 8.4; Pc = 0.03) and goitrous autoimmune thyroiditis (0% vs. 32%; chi 2 = 23.2; Pc < 0.001) patients. The frequency of the HLA-Cw1 antigen was significantly increased in all patient groups. We conclude that TRBab-positive atrophic autoimmune thyroiditis is immunogenetically different from both goitrous autoimmune thyroiditis and TRBab-negative atrophic autoimmune thyroiditis. It is possible that HLA-DR8 and/or DQB1*0302 may be related to the susceptibility genes involved in the production of TRBab in Koreans.

Changes in epitopes for thyroid-stimulating antibodies in Graves' disease sera during treatment of hyperthyroidism: therapeutic implications.

To determine whether there are changes in epitope recognition by stimulating TSH receptor antibodies (TSHRAbs) during treatment of hyperthyroidism and to evaluate the clinical relevance of such changes, we serially measured the activity of IgG preparations from 39 patients with Graves' disease over an 8-month period. To measure epitope changes of the stimulating TSHRAbs, we used Chinese hamster ovary (CHO) cells transfected with wild-type human TSHR (hTSHR) or TSHR chimeras with residues 90-165 (Mc2) substituted by equivalent residues of the rat LH/CG receptor. When initially examined, 37 of the 39 patients had significant stimulating TSHRAb activity measured with wild-type CHO-hTSHR cells. Serial measurements of stimulating TSHRAb activity in Mc2 chimera-transfected cells divided the 39 patients into three distinct groups. Thus, 10 patients (heterogeneous epitope group) exhibited low but significant activity in Mc2 chimera assays at the start of the study; 10 patients who were initially negative in Mc2 chimera assays remained negative (persistently homogeneous epitope group); and 19 patients who were initially negative in Mc2 chimera assays became transiently or persistently positive during treatment, despite a simultaneous decrease in TSHRAb activity measured with wild-type TSHR (changing epitope group). The functional stimulating TSHRAb epitope thus changed from residues 90-165 to residues outside this region in the last group, which comprises nearly two-thirds of the initially Mc2-negative patients (19 of 29) and one-half of all patients (19 of 39). Patients in the changing epitope group responded more quickly and to lower doses of methimazole than patients in the persistently homogeneous epitope group, behaving in this respect exactly as the patients in the heterogeneous epitope group. Additionally, although the decrease in stimulating TSHRAb activities during the 8-month treatment period was similar in the two groups, the thyrotropin binding inhibitor immunoglobulin (TBII) activities decreased more rapidly in patients in the persistently homogeneous epitope group than in patients in the changing epitope group (P < 0.05). There were no differences in initial stimulating TSHRAb or TBII activities, degree of hyperthyroidism, goiter size, or prior duration of symptoms between the persistently homogeneous epitope group and changing epitope group. In summation, we show that the epitopes of stimulating TSHRAbs in Graves' disease patients may change during their clinical course or treatment period, and that the change is from antibodies recognizing N-terminal TSHR residues 90-165 to antibodies recognizing other regions of the TSHR. We also show that the development of stimulating TSHRAbs with this heterogeneous epitope or their presence at the initial screening for disease activity seems to be associated with increased responsiveness to antithyroid drug therapy. We suggest, therefore, that Mc2 chimera assays may be useful to predict the response of patients to antithyroid drug therapy.

Negative correlation between the change in bone mineral density and serum osteocalcin in patients with hyperthyroidism.

To assess the changes in bone mineral density and osteoblastic activity in patients with hyperthyroidism and to investigate the relationship between those changes, we measured bone mineral density and serum osteocalcin in 109 patients with Graves' disease, comprising 75 untreated patients and 34 patients under treatment, and 200 normal controls. The degree of change in bone mineral density was quantified with Z transformation, in which we used means and SDs of bone mineral densities obtained in the process of age- and sex-matched 1:1 pairing developed by ourselves. Bone mineral density was low in female patients with hyperthyroidism in the spine, femur neck, trochanter, and Ward's triangle, but was not low in male patients. Serum osteocalcin was elevated in patients with untreated Graves' disease and correlated negatively with the Z values of bone mineral densities of the spine, femur neck, and trochanter. In conclusion, indices of osteoblastic activity were elevated in patients with hyperthyroidism, probably secondary to a thyroid hormone-induced increase in bone resorption which resulted in reduced bone mineral density. Quantification of the change in bone mineral density by using the parameters derived in the process of age- and sex-matched pairing seems to be an efficient method for statistical analyses.

Characterization of monoclonal thyroid-stimulating and thyrotropin binding-inhibiting autoantibodies from a Hashimoto's patient whose children had intrauterine and neonatal thyroid disease.

A multiplicity of TSH receptor autoantibodies (TSHRAbs) have been characterized after subcloning heterohybridomas produced from the lymphocytes of a patient who has Hashimoto's thyroiditis and had three children with intrauterine or neonatal hyperthyroidism. Twelve clones produced stimulating TSHRAbs that increased cAMP levels and iodide uptake in rat FRTL-5 thyroid cells and increased cAMP levels in Chinese hamster ovary (CHO) cells transfected with the human TSHR; like 95% of Graves' stimulating TSHRAbs, all 12 have their functional epitope on the N-terminus of the TSHR extracellular domain, requiring residues 90-165 for activity. All 12 bind to human thyroid membranes in the absence, but not the presence, of TSH, but are only weak inhibitors of TSH binding in assays measuring TSH binding-inhibiting Igs (TBIIs). In contrast, 8 different clones produced TSHRAbs that did not increase cAMP levels, but, instead, exhibited significant TBII activity. Four inhibited the ability of TSH or a stimulating TSHRAb to increase cAMP levels and had their functional epitope on the C-terminal portion of the TSHR external domain, residues 261-370, mimicking the properties of blocking TSHRAbs that cause hypothyroidism in patients with idiopathic myxedema. The 4 other TBIIs inhibited the ability of TSH, but not that of a stimulating TSHRAb, to increase cAMP levels, like TBIIs in Graves' patients. The functional epitope for 3 of these Graves'-like TBIIs was residues 90-165; the functional epitope for the fourth was residues 24-89. The fourth also increased arachidonic acid release and inositol phosphate levels in FRTL-5 thyroid cells and exhibited conversion activity, i.e. the ability to increase cAMP levels in the presence of an anti-human IgG. Thus, this TBII exhibited signal transduction activity, unlike the other 3 Graves'-like TBIIs. The patient, therefore, has stimulating TSHRAbs and 3 different types of TBIIs, each with different functional properties and different epitopes on the TSHR.

Clinical significance of hepatic visualization on iodine-131 whole-body scan in patients with thyroid carcinoma.

UNLABELLED: The purpose of this study was to evaluate the frequency and clinical significance of diffuse hepatic uptake on 131I whole-body scan in 399 patients (53 males, 348 females) with well-differentiated adenocarcinomas of the thyroid. METHODS: Two hundred and ninety-one diagnostic scans were performed 2 days after the administration of 74-370 MBq (2-10 mCI) 131I, and 824 post-therapy scans were done 3-5 days after the administration of 1.11-7.4 GBq (30-200 mCI) 131I. There was no evidence of liver metastasis in these patients. Liver and thyroid visualization on each 131I scan were graded from 0-4. To evaluate the incorporation of radioiodine to thyroglobulin and thyroid hormones, a patient's serum was extracted by 80% ethanol/20% trichloroacetic acid solution and analyzed by silica gel thin-layer chromatography. RESULTS: Diffuse hepatic uptake (> Grade 2) was definitely seen in 239 of 399 (59.9%) of the patients and 397 of 1115 (35.6%) of the studies. In the diagnostic scans, 36 (12.0%) showed uptake in the liver. In post-therapy scans, however, the incidence of liver uptake increased according to increased doses of 131I (39.1% with 1.11 GBq, 61.5% with 2.775-3.7 GBq and 71.3% with 5.55-7.4 GBq). The more that uptake appeared in the residual thyroid, the more it appeared in the liver. There were 13 patients whose scans showed metastatic and liver uptake without any thyroid uptake. Fifteen patients showed diffuse liver uptake without uptake by the thyroid or metastasis. Follow-up studies of seven of these patients revealed metastatic lesions. Liver uptake on scan related to the fraction of 131I-labeled thyroglobulin in the serum. CONCLUSION: Diffuse liver uptake indicated functioning thyroid remnant or metastasis. In a few cases, liver uptake without uptake by the thyroid or metastasis on whole-body scans suggests hidden metastases.

Value of FDG PET in papillary thyroid carcinoma with negative 131I whole-body scan.

UNLABELLED: The management of metastatic thyroid carcinoma patients with a negative 131I scan presents considerable problems. Fifty-four athyrotic papillary thyroid carcinoma patients whose 1311 whole-body scans were negative underwent 18F-fluorodeoxyglucose (FDG) PET; the purpose was to determine whether this procedure could localize metastatic sites. We also assessed its usefulness in the management of these patients. METHODS: Whole-body emission scan was performed 60 min after the injection of 370-555 MBq 18F-FDG, and additional regional attenuation-corrected scans were obtained. Metastasis was pathologically confirmed in 12 patients and was confirmed in other patients by overall clinical evaluation of the findings of other imaging studies and of the subsequent clinical course. RESULTS: In 33 patients, tumor had metastasized, whereas 21 patients were in remission. FDG PET revealed metastases in 31 patients (sensitivity 93.9%), whereas thyroglobulin levels were elevated in 18 patients (sensitivity 54.5%). FDG PET was positive in 14 of 15 metastatic cancer patients with normal thyroglobulin levels. In 20 of 21 patients in remission, FDG PET was negative (specificity 95.2%), whereas thyroglobulin levels were normal in 16 patients (specificity 76.1%). The sensitivity and specificity of FDG PET were significantly higher than those of serum thyroglobulin. In patients with negative 1311 scans, FDG PET detected cervical lymph node metastasis in 87.9%, lung metastasis in 27.3%, mediastinal metastasis in 33.3% and bone metastasis in 9.1%. In contrast, among 117 patients with 131I scan-positive functional metastases, 131I scan detected cervical lymph node metastasis in 61.5%, lung metastasis in 56.4%, mediastinal metastasis in 22.2% and bone metastasis in 16.2%. In all 5 patients in whom thyroglobulin was false-negative with negative antithyroglobulin antibody, PET showed increased 18F-FDG uptake in cervical lymph nodes, mediastinal lymph nodes, or both. Among patients with increased 18F-FDG uptake only in the cervical lymph nodes, the nodes were dissected in 11. Metastasis was confirmed in all, even in normal-sized lymph nodes. CONCLUSION: FDG PET scan localized metastatic sites in 131I scan-negative thyroid carcinoma patients with high accuracy. In particular, it was superior to 131I whole-body scan and serum thyroglobulin measurement for detecting metastases to cervical lymph nodes. FDG PET was helpful for determining the surgical management of these patients.

Solid-phase immunoassay using a flow cytometer: quantitative and qualitative determination of protein antigens and a hapten.

A fluoroimmunoassay employing a flow cytometer as the fluorescence-detecting device is described. Three kinds of antigens, murine immunoglobulin (Ig), human chorionic gonadotropin (hCG) and progesterone were chosen as examples of the assay using fluorescein-labeled antibodies. Cyanogen bromide-activated agarose beads were used as solid-phase supporters. The flow cytometric immunoassay was applied to both qualitative and quantitative analyses; determination of murine Ig isotypes, quantitative determination of Ig, hCG and a hapten, progesterone. This assay produced very reproducible and less-fluctuating data since thousands of particles in the assay were collected and processed to produce a single value for fluorescence intensities. Furthermore, the working range of the assay in terms of antigen concentration was much broader than that of enzyme immunoassay. Therefore, we believe that microparticles like agarose beads could be useful solid-phase supporters in immunoassay, and the flow cytometer could provide a reliable alternative to the fluorescence-detecting device in immunoassay.