Bridge Technology with TSH Receptor Chimera for Sensitive Direct Detection of TSH Receptor Antibodies Causing Graves' Disease: Analytical and Clinical Evaluation.

Graves' disease is caused by stimulating autoantibodies against the thyrotropin receptor inducing uncontrolled overproduction of thyroid hormones. A Bridge Assay is presented for direct detection of these thyroid-stimulating immunoglobulins using thyrotropin receptor chimeras. A capture receptor, formed by replacing aa residues 261-370 of the human thyrotropin receptor with residues 261-329 from rat lutropin/choriogonadotropin receptor and fixed to microtiter plates, binds one arm of the autoantibody. The second arm bridges to the signal receptor constructed from thyrotropin receptor (aa 21-261) and secretory alkaline phosphatase (aa 1-519) inducing chemiluminescence. The working range of the assay is from 0.3 IU/l to 50 IU/l with a cutoff of 0.54 IU/l and functional sensitivity of 0.3 IU/l. Sensitivity and specificity are 99.8 and 99.1%, respectively, with a diagnostic accuracy of 0.998. The low grey zone is from 0.3-0.54 IU/l. The stimulatory character of the assayed antibodies is shown through a good correlation (r=0.7079, p<10(-7)) to serum T4 levels of untreated patients. In Graves' disease, titers are increased in associated eye disease. In 3 hypothyroid patients with sera positive in the thyrotropin receptor competition assay and in the blocking bioassay, antibodies are not detected by the Bridge Assay, while the monoclonal blocking antibody K1-70 was detected. In Hashimoto disease thyrotropin receptor autoantibodies are detected in some patients, but not in goiter. This Bridge Assay delivers good diagnostic accuracy for identification of Graves' disease patients. Its high sensitivity may facilitate early detection of onset, remission, or recurrence of Graves' disease enabling timely adaption of the treatment.Human genes: TSHR, Homo sapiens, acc. no. M31774.1.

Modulation of iodide uptake by dialkyl phthalate plasticisers in FRTL-5 rat thyroid follicular cells.

Plasticisers imparting flexibility to plastics are man-made chemicals abundantly present in the environment. Effects of six different dialkyl phthalates were studied in vitro in the rat thyroid cell line FRTL-5 on their ability to modulate basal iodide uptake mediated by the sodium/iodide symporter (NIS). The present study shows that diisodecyl phthalate (DIDP), dioctyl phthalate (DOP), diisononyl phthalate (DINP) and bis (2-ethylhexyl) phthalate (DEHP) significantly enhance iodide uptake when concentrations in the magnitude between 10(-4) M and 10(-3) M were applied. In this range, these phthalates do not assess toxicity on the cells. Specific inhibiton of NIS demonstrated that enhancement of iodide uptake is due to NIS. In contrast, benzyl butyl phthalate (BBP) also augments iodide uptake at 1mM but this concentration has just exceeded the toxicity threshold and dibutyl phthalate (DBP), the most toxic compound did not modulate iodide uptake at any concentration applied. As we can deduce from our results, plasticisers are capable of significantly modulating NIS mediated iodide uptake activity.

Once-daily oral gatifloxacin vs three-times-daily co-amoxiclav in the treatment of patients with community-acquired pneumonia.

A double-blind, double-dummy, multicentre, multinational, parallel-group study was designed to establish proof of equivalence between oral gatifloxacin and oral co-amoxiclav in the treatment of 462 patients with mild-to-moderate community-acquired pneumonia. Eligible patients were randomised equally to either gatifloxacin 400 mg once-daily plus matching placebo for 5-10 days, or amoxycillin 500 mg + clavulanic acid 125 mg three-times-daily for 5-10 days. The primary efficacy endpoint was clinical response (clinical cure plus improvement) at the end of treatment. Overall, a successful clinical response was achieved in 86.8% of gatifloxacin-treated patients, compared with 81.6% of those receiving co-amoxiclav, while corresponding rates of bacteriological efficacy (eradication plus presumed eradication) were 83.1% and 78.7%, respectively. The safety and tolerability profile of gatifloxacin was comparable to that of co-amoxiclav, with adverse gastrointestinal events, e.g., diarrhoea and nausea, being the most common treatment-related adverse events in both groups. The study showed no evidence of gatifloxacin-induced phototoxicity, musculoskeletal disorders, or hepatic and renal problems. Overall, this study showed that gatifloxacin was equivalent clinically to a standard course of co-amoxiclav in patients with community-acquired pneumonia, and that gatifloxacin was safe and well-tolerated.

Improved reperfusion and clinical outcome with enoxaparin as an adjunct to streptokinase thrombolysis in acute myocardial infarction. The AMI-SK study.

AIMS: To establish whether the addition of enoxaparin (a low-molecular-weight heparin) to streptokinase therapy improves early and sustained coronary patency and clinical outcome in patients with evolving myocardial infarction. METHODS AND RESULTS: A total of 496 patients with acute myocardial infarction treated with streptokinase were randomized to an intravenous bolus (30 mg) and subcutaneous injections (1mg x kg(-1), twice daily) of enoxaparin (n=253), or placebo (n=243) for 3-8 days. The median duration of treatment in both groups was 5 days. ST-segment resolution at 90 min and 180 min measured by electrocardiogram was improved in patients receiving enoxaparin. Complete, partial and no ST-segment resolution at 180 min was observed in 36%, 44% and 19% in the enoxaparin group vs 25%, 44% and 31% in the placebo group, respectively (P=0.004). Assessment of the primary end-point revealed improved TIMI-3 flow with enoxaparin vs placebo (70% vs 58%, P=0.01). Combined TIMI-2 and -3 flow was also improved (88% vs 72%, P=0.001), as was TIMI frame count (P=0.003). The triple clinical end-point of death, reinfarction and recurrent angina at 30 days was reduced with enoxaparin (13% vs 21%, P=0.03). CONCLUSION: Streptokinase in combination with enoxaparin is associated with better ST-segment resolution and better angiographic patency at days 5-10, suggesting more effective reperfusion. This was associated with a significant reduction in clinical events, indicating less reocclusion.

The promoter of the human sodium/iodide-symporter gene responds to retinoic acid.

It was shown previously that hNIS mRNA expression is stimulated by retinoic acid (RA) in human follicular thyroid carcinoma cell lines FTC-133 and FTC-238, and patients with thyroid carcinomas lacking iodide uptake respond to RA treatment with increased radioiodide transport. Here, in transient transfection experiments using FTC-238 cells, hNIS promoter/luciferase reporter constructs showed an up to 2.5-fold increase in transcriptional activity after incubation with 1 microM RA. Stimulation by 10 nM T3 was up to 2.4-fold. Deletion or block mutation of a putative nuclear receptor recognition site, 'DR10', abolished RA and T3 responses. Four copies of the DR10 cloned 5' to the thymidine kinase promoter gave a 2.6-fold and a 1.4-fold increase in transcriptional activity after RA and T3 stimulation, respectively. In electrophoretic mobility shifts, a wildtype DR10 oligonucleotide, but not block mutants of either DR10 halfsite, interacted with nuclear receptors. Thus, RA redifferentiation of advanced thyroid carcinomas may reinduce iodide uptake by stimulating hNIS expression and thereby make tumours accessible for radioiodide therapy again.

Transcriptional regulation of the human sodium/iodide symporter gene by Pax8 and TTF-1.

The regulation of the human Na(+)/I(-) symporter (NIS) gene is of considerable interest for both the diagnosis and therapy of thyroid pathologies. We investigated the influence of the thyroid-specific transcription factors TTF-1 and Pax8 on the NIS promoter and its 5'-flanking sequence. Reporter genes containing the corresponding genomic fragments coupled to a luciferase reporter gene were cotransfected with expression vectors carrying the the cDNA's for TTF-1 and Pax8. Transient transfection assays were performed in HeLa and COS-7 cells, which do not express endogenously these transcription factors. The experiments showed, that TTF-1 had no influence on the NIS promoter. Pax8, on the other hand, had a moderate stimulating effect (threefold) on the proximal NIS promoter. ABCD assays indicated an interaction of in vitro-translated Pax8 with the NIS promoter. However, DNase I footprinting experiments with bacterially expressed Pax8 were negative, suggesting an indirect mode of action with the participation of other proteins. A putative NIS upstream enhancer (NUE) 9000 bp upstream of the NIS gene, which was cloned based on its sequence homology to the rat NUE, was not transactivated by either Pax8 or TTF-1. The present data demonstrate, that the combination of Pax8 and TTF-1 is less important for NIS gene transcription than for other thyroid-specific genes. This is presumably related to the fact, that NIS is expressed also in other tissues such as mammary and salivary gland.

Thyroid transcription factor 1 and Pax8 synergistically activate the promoter of the human thyroglobulin gene.

Thyroglobulin (Tg) is an essential thyroid-specific protein, which serves as the matrix for thyroid hormone biosynthesis. To obtain new insights in the regulation of Tg gene expression, we investigated the interaction of the human Tg promoter with the thyroid-specific transcription factors TTF-1 and Pax8. A reporter gene, containing a 202 bp fragment from the human Tg 5'-flanking region including the promoter sequence and the transcriptional start site, and expression vectors containing the cDNAs for human TTF-1 and Pax8 were used in cotransfection experiments, in the non-thyroidal cell lines COS-7 and HeLa. Pax8 increased the specific transcriptional activity of the Tg promoter about threefold, whereas cotransfection with the homeodomain-containing protein TTF-1 stimulated promoter activity from six- to tenfold. The simultaneous expression of both factors stimulated the Tg promoter activity in a multiplicative manner up to 25-fold. TTF-1 binding sites could be localized precisely by lectron mobility shift assay. The two binding elements corresponded to sites A and C in the rat Tg promoter. Site-directed mutagenesis of three nucleotides in each binding element inhibited binding of TTF-1 to the two oligonucleotides. In cotransfection experiments, the mutant site C decreased TTF-1 transactivation to 26% of the wild-type, whereas an additional mutation in the site A reduced this value to almost zero, thus proving the physiological relevance of these sites. The present results demonstrate that the activity of the human Tg promoter is closely dependent on the function of TTF-1 and Pax8, opening the field for further investigations of pathological alterations of Tg gene expression.

Detection of functionally different types of pathological autoantibodies against thyrotropin receptor in Graves' patients sera by luminescent immunoprecipitation analysis.

We describe a new method for the detection of different types of pathological autoantibodies against TSH receptor (TSHR) in Graves' patients sera by luminescent immunoprecipitation analysis. For this purpose three different chimeras composed of human TSHR and rat luteotropin/choriogonadotropin receptor (LH-CGR) were constructed, as was described previously (Tahara K, Ishikawa N, Yamamoto K, Hirai A, Ito K, Tamura Y, Yoshida S, Saito Y, Kohn LD. 1997 Thyroid 7:867-877). They were used in the immunoprecipitation reactions: (i) the wild type TSHR (for the detection of total TSHR autoantibodies), (ii) TSHR/LH-CGR chimera wherein TSHR amino acid residues 8-165 (epitopes for thyroid stimulating antibodies) are replaced by comparable LH-CGR residues, (iii) TSHR/LH-CGR chimera wherein TSHR amino acids 261-370 (epitopes for thyroid blocking antibodies) are replaced by comparable LH-CGR residues, and (iv) TSHR/LH-CGR chimera wherein TSHR amino acids 8-165 and 261-370 are replaced by comparable LH-CGR residues (for the detection of neutral TSHR autoantibodies). DNA encoding the N-terminal 725 (of 764) amino acids of wild type TSHR (or TSHR/LH-CGR chimera) was fused to the cDNA for the 550-amino acid firefly luciferase. The hybrid proteins were produced in HeLa cells using recombinant vaccinia viruses. All fusion proteins retained the enzymatic activity of firefly luciferase and TSHR-LUC interacted with TSH with the same affinity as wild type receptor. The luciferase tagged TSHR and TSHR/LH-CGR chimeras were used for the detection of different types of TSHR autoantibodies (i.e. total, neutral, thyroid stimulating and thyroid blocking) in 63 Graves' disease and 62 normal sera by immunoprecipitation analysis. The data demonstrated positive correlation between results of immunoprecipitation assay and results obtained using cAMP bioassay or assay for TSH binding inhibitory immunoglobulins in test sera.

Cloning and characterization of repressory and stimulatory DNA sequences upstream the Na/I-symporter gene promoter.

To investigate the existence of potential enhancer or silencer elements in the 5'-flanking region of the human Na+/I-symporter (NIS) gene, we cloned 2,512 bp of genomic DNA further upstream of the previously defined proximal promoter. When tested in reporter gene assays, this sequence had no transcriptional activity per se, but was able to repress the activity of the heterologous SV40 promoter. Conversely, when fused to the homologous NIS gene promoter and thus comprising 3,800 bp 5'-flanking region, the transcription of the proximal NIS promoter was stimulated in the human cell lines FTC-133 (from thyroid) and HeLa, but inhibited in the rat thyroid cell line FRTL-5. This might be due to differences between the upstream regions of the rat and human NIS gene. Comparative analysis with standard promoters (SV40) led to the conclusion that the 5'-flanking region of the human NIS gene also exhibited transcriptional activity in non-thyroid cells. Thyroid-stimulating hormone (TSH) had a moderately stimulating effect on the full length NIS reporter gene construct in FRTL-5 cells. This stimulation is presumably mediated by a putative cAMP responsive element found in the first half of the cloned sequence.

Immunoprecipitation analysis of pathological autoantibodies in Graves' patients' sera using biotinylated human thyrotropin receptor labeled with 125I-neutravidiny.

In the present article we describe a method for the direct immunoprecipitation analysis of pathological autoantibodies against TSH receptor (TSHR) in sera of patients with Graves' disease. For this purpose the fusion TSH receptor (TSHR-BIO-6HIS) was constructed. This fusion consists of the N-terminal 725 amino acids of the human TSHR linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of E. coli acetyl-CoA carboxylase (this domain directs the efficient posttranslational biotinylation of the protein) followed by 6 histidine sequence. TSHR-BIO-6HIS was produced in HeLa cells using recombinant vaccinia virus. The expressed receptor was complete active and was biotinylated with a high efficiency (about 90%). Biotinylated TSHR-BIO-6HIS was immobilized on Ni-NTA agarose and selectively labeled with a biotin binding protein-- 125I-neutravidin. The 125I-neutravidin labeled TSHR-BIO-6HIS, freed of the excess of nonbound radioactivity, was eluted from Ni-NTA agarose and used for the detection of pathological autoantibodies in 50 Graves' disease, 10 Hashimoto's disease, 10 insulin-dependent diabetes mellitus and 50 normal sera. 46 of 50 (92%) Graves' disease sera were positive in immunoprecipitation assay, as they have bound 125I-TSHR more effectively than the normal sera. There was a clear positive correlation between the immunoprecipitating activity and TSH-binding inhibiting activity of different Graves' sera (r = 0.69, P < 0.001). These findings pave the way for the development of a new practical assay, capable of detecting all pathological autoantibodies to the TSHR, particularly those which bind but do not affect the hormone-receptor interaction.

Isolation of radiochemically pure 125I-labeled human thyrotropin receptor and its use for the detection of pathological autoantibodies in sera from Graves' patients.

We report a method for the purification and radioactive labeling of human TSH receptor (TSHR). The method is based on the construction of a fusion TSHR (TSHR-Xa-BIO) which consists of the N-terminal 725 amino acids of human TSHR linked to the 4-amino acid Xa protease cleavage site and the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase (the C-terminal domain directs the efficient posttranslational biotinylation of the protein). TSHR-Xa-BIO was produced in HeLa cells using recombinant vaccinia virus. The expressed protein was fully functional and was biotinylated with an efficiency of about 90%. Streptavidin-agarose-immobilized TSHR-Xa-BIO was labeled with 125I using the chloramine T oxidation procedure and specifically eluted from the solid phase after cleavage with protease Xa. Isolated native radiochemically pure 125I-labeled TSHR specifically interacted with pathological autoantibodies in the sera of patients with Graves' disease, and thus could be useful for the detection of these autoantibodies by immunoprecipitation analysis.

Pharmacokinetics of melphalan in isolated limb perfusion.

The pharmacokinetics of melphalan was studied by sampling of tissue and plasma in 72 rats that underwent isolated hyperthermic limb perfusion under different conditions. A miniaturized extracorporeal circulation system for small animals was used for perfusion of the rat hindlimb. Melphalan levels (L-phenylalanine mustard, L-PAM) were determined by high-performance liquid chromatography (HPLC). The temperature of the perfusate plasma and tissue, pH, administration method, and flow rate were modified and compared with regard to their influence on pharmacokinetic parameters. The highest tissue penetration of melphalan was observed under the following conditions: (a) pH range of the perfusate plasma between 7.3 and 7.7 (physiological environment), (b) temperature range of the perfusate from 40 degrees to 41.5 degrees C (destruction of cellular carrier systems at higher temperatures and increased inactivation by hydrolysis of melphalan above 41.5 degrees C), (c) application of melphalan as a single dose into the reservoir of the extracorporeal circuit (optimal tissue penetration), and (d) reduced perfusate flow (prolonged contact time between perfusate and tissue).

Thyroid autoimmunity in children and adolescents with type 1 diabetes mellitus. Effect of age, gender and HLA type.

Type 1 diabetes is often associated with additional autoimmune phenomena. However, data reported on the frequency of thyroid autoimmunity differ vastly. Therefore, the prevalence of thyroid autoantibodies was evaluated at a large pediatric diabetes center in Southern Germany. 2,305 determinations (TPO and TG, ELISA) were performed in 495 patients with type 1 diabetes (234 boys, 261 girls; age at last measurement: 15.4 +/- 0.3 years, duration of diabetes 7. 5 +/- 0.2 years). The prevalence of elevated thyroid antibodies increased dramatically with age: from 3.7% in patients less than 5 years of age up to 25.3% in the age group 15-20 years (p < 0.0001). For children older than 10 years, girls were significantly more affected than boys (p < 0.0001). Thyroid autoimmunity tended to be more prevalent in the subgroup of patients with the HLA type DR3/DR4 compared to patients with other HLA types (p = 0.08). In children older than 10 years, basal TSH concentrations were significantly elevated in antibody-positive patients (p < 0.05). In conclusion, thyroid autoimmunity is prevalent in children and adolescents with type 1 diabetes. Adolescent girls and young women are especially affected. Yearly routine determinations of thyroid antibodies are therefore recommended.

Cloning of a functional promoter of the human sodium/iodide-symporter gene.

We have cloned and sequenced genomic DNA from a human library extending 1300 bp upstream the 5'-untranslated sequence of the cDNA coding for the sodium/iodide symporter. In transient transfection assays this sequence exhibited promoter activity, which could be confined to nucleotides -443 to -395 relative to the ATG start codon. This minimal promoter, including a putative GC- and TATA- box, was preferentially activated in the rat thyroid cell line FRTL-5, but was also active in non-thyroidal cells, such as COS-7 and Chinese-hamster ovary, albeit to a markedly lower extent.

Expression of a biotinylated human thyrotropin receptor in HeLa cells using recombinant vaccinia virus and its application for the detection of Graves' autoantibodies.

We have prepared a biotinylated thyrotropin receptor (TSHR-BIO), and characterized its activity in cells and when bound to solid phase (streptavidin agarose). TSHR-BIO consists of the N-terminal 725 amino acids of the human thyrotropin (TSH) receptor linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotinylation of the protein. TSHR-BIO was expressed using a vaccinia virus expression system. HeLa cells infected with recombinant virus produced large amounts of TSH receptor of approximately 120,000 molecules per cell. Vaccinia virus produced TSHR-BIO was fully functional interacting with TSH (Kd of 2.3+/-0.1 x 10(-10) M) and coupling to cyclic adenosine monophosphate (cAMP) second messenger system. The expressed protein was biotinylated with high efficiency; more than 90% of TSHR-BIO was bound to streptavidin. We have shown the application of streptavidin agarose immobilized TSHR-BIO for the detection of thyroid-binding inhibiting immunoglobulines in unfractionated sera. There was a good positive correlation between the results obtained in this assay and the commercially available TRAK assay performed with solubilized porcine TSH receptor (r = 0.71; p < 0.001, in 45 sera of patients with Graves' disease and 17 normal sera).

Deoxyribonucleic acid binding and transcriptional silencing by a truncated c-erbA beta 1 thyroid hormone receptor identified in a severely retarded patient with resistance to thyroid hormone.

We describe the analysis of a thyroid hormone receptor (TR) beta causing resistance to thyroid hormone, the patient exhibiting hypothyroid symptoms (severe mental retardation, hypoactivity, obesity) and hyperthyroid symptoms (tachycardia, low serum cholesterol) and, additionally, relative early puberty, advanced bone age, and short stature. The patient was heterozygous, with a point mutation producing a premature stop-codon in TR beta-gene exon 10, resulting in a 28-amino acid carboxy-terminal deletion in the cognate TR beta (TR beta-EZ). T3 binding was abolished. Homodimer binding of TR beta-EZ to DR4- and F2-T3 response elements (TREs) was weaker, and to a palindromic TRE (PAL) was stronger than that of wild-type TR beta (TR beta-WT) in the absence of T3. T3 dissociated TR beta-WT, but not TR beta-EZ homodimer, from DR4, F2, and Pal. Heterodimerization of TR beta-EZ with retinoid x receptor beta was seen. TR beta-EZ repressed basal thymidine kinase-promotor activity, coupled to DR4, F2, or PAL. Silencing of basal gene transcription via PAL was weaker, and via DR4 and F2 was more pronounced, compared with TR beta-WT. TR beta-EZ had a strong dominant negative effect on TR beta-WT, attenuated in a TRE- and cell-specific manner by high T3 concentrations. Finally, the degree of TR beta-EZ homodimer-binding affinity to DNA did not correlate with the degree of transcriptional dominant negative activity.

Expression of a functional tagged human thyrotropin receptor in HeLa cells using recombinant vaccinia virus.

We report a new effective system for expression of FLAG and six histidine tagged TSH receptor in mammalian cells using recombinant vaccinia virus. HeLa cells infected with recombinant virus produced large amounts of human TSH receptor of approximately 150,000 functional molecules per cell. This value is one to two orders of magnitude higher than those in thyroid cells and is comparable with receptor number of the best stably transfected mammalian cell clones previously described. Vaccinia virus produced TSH receptor was able to bind TSH (Kd of 2.1 +/- 0.1 x 10(-10) M) and Graves'disease autoantibodies. TSH caused an increase of the intracellular cAMP level in infected HeLa cells in a concentration-dependent manner, demonstrating the coupling of expressed recombinant TSH receptor to the cAMP second messenger system of the cells. 6His-tagged recombinant TSH receptor was immobilized on Ni2+ nitrilotriacetic acid-agarose. Bound receptor was fully functional, interacting with both TSH and Graves' disease autoantibodies in patient sera. The solid phase bound TSH receptor technique provides a new and simple method for the diagnosis of autoimmune diseases.