Biopsy proven and biopsy negative temporal arteritis: differences in clinical spectrum at the onset of the disease. Groupe de Recherche sur l'Artérite à Cellules Géantes.

OBJECTIVES: To assess the clinical features of biopsy proven and negative biopsy temporal arteritis at the time of diagnosis and during a three year follow up. METHODS: Newly diagnosed cases of giant cell arteritis were included in a prospective, multicentre study. Initial clinical and biological features, season of diagnosis, and cardiovascular events occurring during the follow up were recorded. Biopsy proven and negative biopsy cases were compared. RESULTS: Two hundred and seven biopsy proven, and 85 negative biopsy cases were included from 1991 to 1997. Fifty eight per cent of the biopsy proven cases, compared with 39.29% of the negative biopsy cases, were diagnosed during the autumn or winter (p = 0.003). Visual problems (31.5%, v 19.1%, p = 0.031), blindness (9.7% v 2.38%, p = 0.033), jaw claudication (40.8%, v 28.243%, p = 0.044), and temporal artery palpation abnormalities (61.3% v 29.5%, p = 7.10(-7)) were more frequent in the biopsy proven than in the negative biopsy group. Less specific symptoms, such as headache (82.5% v 92. 9%, p = 0.021), or associated polymyalgia rheumatica (40.1% v 65.9%, p = 9 x 10(-5)) were more prevalent in the negative biopsy cases. Biological markers of inflammation were significantly more increased in the biopsy proven group. All cases of blindness occurring after treatment belonged to the biopsy proven group. CONCLUSION: Biopsy proven cases seem to be more severe than biopsy negative cases at the time of diagnosis and during follow up. Seasonal difference at diagnosis may suggest a different aetiological pattern.

Identification of thyroid hormone residues on serum thyroglobulin: a clue to the source of circulating thyroglobulin in thyroid diseases.

Thyroglobulin (Tg) present in the serum of normal individuals and patients with thyroid disorders could be partly newly synthesized non-iodinated Tg and partly Tg containing iodine and hormone residues originating from the lumen of thyroid follicles. With the aim of examining the contribution of the latter source of Tg to the elevation of serum Tg concentration in thyroid pathophysiological situations, we devised a procedure to identify thyroxine (T4) and tri-iodothyronine (T3) residues on Tg from unfractionated serum. A two-step method, basedon (i)adsorption of Tg on an immobilized anti-human Tg (hTg) monoclonal antibody (mAb) and (ii)recognition of hormone residues on adsorbed Tg by binding of radioiodinated anti-T4 mAb and anti-T3 mAb, was used to analyze serum Tg from patients with either Graves' disease (GD), subacute thyroiditis (ST) or metastatic differentiated thyroid cancer (DTC). Purified hTg preparations with different iodine and hormone contents were used as reference. Adsorption of purified Tg and serum Tg on immobilized anti-hTg mAb ranged between 85 and 90% over a wide concentration range. Labeled anti-T4 and anti-T3 mAbs bound to adsorbed purified Tg in amounts related to its iodine content. Tg adsorbed from six out of six sera from ST exhibited anti-T4 and anti-T3 mAb binding activities. In contrast, significant mAb binding was only observed in one out of eight sera from untreated GD patients and in 1 out of 13 sera from patients with DTC. The patient with DTC, whose serum Tg contained T4 and T3, represented a case of hyperthyroidism caused by a metastatic follicular carcinoma. In conclusion, we have identified, for the first time, T4 and T3 residues on circulating Tg. The presence of Tg with hormone residues in serum is occasional in GD and DTC but is a common and probably distinctive feature of ST.

Analyses of the molecular forms of serum thyroglobulin from patients with Graves' disease, subacute thyroiditis or differentiated thyroid cancer by velocity sedimentation on sucrose gradient and Western blot.

Serum thyroglobulin (Tg) concentration increases in diverse thyroid pathophysiological situations. We examined whether Tg molecules appearing in the serum of patients with Graves' disease (GD), subacute thyroiditis (ST) or differentiated thyroid cancer (DTC) have distinctive biochemical properties. We used ultracentrifugation on sucrose gradient and Western blot to analyze structural parameters of immunoreactive Tg in complete serum from 40 patients with GD, ST or DTC. Purified human Tg was used as reference. Immunoreactive Tg from ST or DTC sera sedimented in a single, rather symmetrical peak as purified 19S Tg. In GD sera without detectable anti-Tg autoantibodies (TgaAb), about 80% of immunoreactive Tg was recovered in a Tg dimer peak that often split into two components; the remaining Tg immunoreactivity (10-30%) sedimented faster and was polydispersed. In GD sera containing TgaAb, immunoreactive Tg was recovered in a peak sedimenting faster than 19S Tg corresponding to immune complexes identified by protein A adsorption. Using a Western blot procedure, optimized to detect 0.1 ng Tg in serum. a single band of Tg, migrating as the intact Tg subunit, was always found in non-reducing conditions; the intensity of the band correlated with the immunoassayable Tg concentration. In reducing conditions, the Tg band obtained with GD or ST sera was decreased by up to 70% compared with that of purified Tg or serum Tg from patients with DTC. In conclusion, serum Tg from DTC is remarkably homogeneous and in the form of dimers dissociable into uncleaved monomers. In contrast, serum Tg from GD or ST is heterogeneous with respect to its sedimentation properties and/or the structural integrity of its polypeptide chains. These data provide information on the processes whereby Tg is released into the circulation.

Antibody-dependent cell-mediated cytotoxicity in autoimmune thyroid disease: relationship to antithyroperoxidase antibodies.

Thyroid antibody-dependent cell-mediated cytotoxicity (ADCC) has been reported in autoimmune thyroid disease, and its relationship with antithyroperoxidase antibodies (TPOAb) questioned. We studied the effect of highly purified human thyroperoxidase (TPO) on thyroid ADCC activity elicited by serum from patients with autoimmune thyroid disease. ADCC promoted by a pool of Graves' disease sera could be inhibited by the addition of TPO in a dose-dependent manner. TPO at 40 micrograms/mL decreased the ADCC observed in the presence of this serum pool by 50%. In the presence of 40 micrograms/mL TPO, ADCC was significantly reduced (P < 0.0005) from 39.6 +/- 10.6% (mean +/- SD) to 14.0 +/- 12.9% for the 18 Graves' disease sera tested and from 39.1 +/- 10.5% to 6.1 +/- 1.7% for the 16 thyroiditis sera tested. Purified thyroglobulin had no effect. Immunoaffinity-purified TPOAb could mediate ADCC in a dose-dependent manner, whereas purified antithyroglobulin antibodies could not. Three TPOAb-positive, but ADCC-negative, sera appear to contain an activity able to protect thyroid cells from ADCC. This protective effect is also observed on human fibroblasts. In conclusion, TPO is the major antigen involved in thyroid ADCC.

Human thyroglobulin reference material (CRM 457). 1st Part: Assessment of homogeneity, stability and immunoreactivity.

This paper describes the assessment of the homogeneity and stability of a purified and lyophilized human thyroglobulin (Tg), and characterizes its immunoreactivity. The purified and lyophilized Tg is intended to be used as a primary reference material to establish calibration of working serum based reference material. The programme involved the participation of 15 European laboratories and one laboratory from the United States. The homogeneity of the content of the ampoules was considered acceptable (< 9%). The stability was tested by accelerated temperature degradation showing predicted annual relative losses of 0.01% at -70 degrees C and 1.04% at -20 degrees C. The immunoreactivity of the Tg material as measured in different laboratories varied mostly according to the method used rather than the laboratory. The interlaboratory variability showed that the two commercial methods used in several laboratories (kit 1 and 2) had an interlaboratory variation (CV) of 15.9% (N = 5) and 7.1% (N = 3), respectively, whereas the total interlaboratory CV was 64.3% (N = 18). The immunoreactive Tg had dilution curves parallel with other Tg calibrators (those of the methods). Dilution curves of the Tg material after storage at various temperatures and time were parallel in both RIA and IRMA. In conclusion, we have prepared a Tg reference material which in extensive studies in several participating laboratories has demonstrated a sufficient homogeneity and stability as well as dilution curves parallel to the calibrators of all the immunoassays tested in the study. This reference material is considered the first step towards decreasing the interlaboratory variability between Tg immunoassays.

Human thyroglobulin reference material (CRM 457). 2nd Part: Physicochemical characterization and certification.

This report describes the characterization of a purified human thyroglobulin (Tg) reference material, and details the procedures used in its certification. The purified Tg is intended to be used as a primary reference material to establish calibration of working serum based reference material for immunoassay procedures. The programme involved the participation of 15 European laboratories and one laboratory from the United States. The physicochemical characterization showed by polyacrylamide gel electrophoresis and immunoblotting that the purified Tg had for the major part the expected molecular size of 660 kDa with traces of lower molecular forms. The amino acid composition was close to that demonstrated for the cDNA and the content of iodine was in keeping with a moderately to highly iodinated Tg. The mass concentration in reference material RM 457 is certified to be (0.324 +/- 0.018) g/L on the basis of protein determined by the Lowry method and supported by nitrogen determination, absorbance measurement, and amino acid analysis. This reference material is considered the first step towards decreasing the interlaboratory variability between Tg methods of measurement.

[Description of cell line established from human thyroid papillary cancer and secreting human chorionic gonadotropin hormone]. / Description d'une lignée-cellulaire établie à partir d'un cancer papillaire thyroidien humain et sécrétant l'hormone gonadotrophine chorionique humaine.

One of the difficulties in characterization of the oncogenes involved in thyroid carcinogenesis is the production of cell lines. Arising from a poorly differentiated thyroid papillary carcinoma we have established a cell line synthesizing the thyroglobulin and human chorionic gonadotropin (alpha and beta subunits) (HCG) hormones. These cells will allow research of the oncogenes involved or potentially involved in thyroid papillary carcinomas and evaluation of the role of the autocrine secretion of HCG.

Assessment of antibody dependent cell cytotoxicity in autoimmune thyroid disease using porcine thyroid cells.

Antibody Dependent Cell Cytotoxicity (ADCC) appears to be involved in Autoimmune Thyroid Disease (AITD). Homologous system may trigger non-specific reactions which might obscure specific ADCC. Heterologous target cells may be useful for studying ADCC, provided relevant antigen(s) are expressed. We therefore tested the capacity of porcine thyroid cells to elicit ADCC reaction in the presence of sera from various patients with AITD. Porcine thyroid cells were used in a 4-hr chromium release assay in the presence of 1/10 heat inactivated human sera and human peripheral blood lymphocytes at a 30:1 effector-target ratio. There was a significant correlation (r = 0.64; P < 0.01) between ADCC activities tested on human or porcine thyroid cells. Serum or IgG effects on porcine thyroid ADCC were dose-dependent between 1/10 to 1/10,000 dilutions. Non-thyroid cell systems were unaffected by thyroid cytotoxic sera. Porcine thyrocyte susceptibility to ADCC peaked at the fourth day of culture and was enhanced by addition of TSH or TSH and methimazole in the culture medium. Using this heterologous system, we demonstrated ADCC activity in a significant proportion of patients with thyroiditis (14/19), Graves' opthalmopathy (19/44) or of mothers of children with congenital hypothyroidism (14/39) and in the children themselves (15/39). Discrepancies observed in some sera between ADCC activity and antithyroperoxidase antibody suggest that thyroperoxidase is not the only antigen involved in ADCC. These results indicate that porcine thyroid cells appear suitable for ADCC assay in patients with AITD. Also this system should be helpful to characterize the antigen-antibody involved.

Anti TSH-receptor antibodies in pregnant patients with autoimmune thyroid disorder.

The study was designed to test further the usefulness of the radioreceptor assay of thyroid stimulating hormone (TSH) binding inhibitory immunoglobulins (TBII) and the bioassay of thyroid stimulating antibodies (TSAb) or TSH stimulated cAMP response inhibitory antibodies (TBkAb) in the prediction of neonatal thyroid dysfunction. Of 63 pregnant women with a current or past history of autoimmune thyroid disorder, 11 (one with active and six with a past history of Graves' disease and four with autoimmune thyroiditis) gave birth to a baby with transient hyper or hypo-thyroidism. Only high maternal titres (which could persist after partial thyroidectomy) of anti TSH-receptor antibodies (TRAb) led to neonatal hyperthyroidism. Both types of assay were able to detect the antibodies responsible for transitory neonatal autoimmune thyroid disease. TBII values reflected TSAb titres so that there was a significant correlation between the results of both assays in women with Graves' disease and in neonatal sera. Positive TBII and TBkAb activities were present in 5 of the 28 women with autoimmune thyroiditis. Therefore, when TBII is positive, the functional characterization of the antibodies warrants the use of the bioassay.

[Serum thyroglobulin assay after total unilateral thyroid lobectomy in differentiated thyroid cancer]. / Dosage de la thyroglobuline sérique après lobectomie thyroïdienne totale unilatérale pour cancer thyroïdien différencié.

Thyroglobulin (Tg) levels (ng/ml) were measured in the sera of 51 healthy volunteers (range: 0 to 20), 116 unselected blood donors (0 to 50) and 431 patients under thyrotropin suppression therapy for differentiated thyroid carcinoma. Among these 431 patients, 133 had undergone total thyroidectomy: 107 were in remission (mean +/- standard error of the mean = 0.84 +/- 2.29) and 26 had a recurrence (1974 +/- 586); 298 had undergone unilateral thyroid lobectomy: 292 were in remission (4.65 +/- 12.79) and 6 had a recurrence (1003 +/- 673 range: 18 to 12,000). The degree of thyrotropin suppression had no effect on serum Tg levels, but these were significantly increased by the presence of anti-Tg antibodies. Moderately elevated but stable Tg levels were observed without detectable recurrence during a follow-up period of at least 5 years. Tg was frankly elevated in 4 patients with overt post-lobectomy recurrence (range: 77 to 5,200). A rise, even moderate, of Tg levels after lobectomy is suggestive of a recurrence if it progresses regularly as time goes on (n = 2). It is concluded that in the absence of anti-Tg antibodies Tg assays should be included in the follow-up of patients with differentiated thyroid carcinoma under thyrotropin suppression therapy after unilateral thyroidectomy.

Thyroid hormone residues are released from thyroglobulin with only limited alteration of the thyroglobulin structure.

We have tried to characterize thyroglobulin (Tg) degradation products in purified pig thyroid lysosomes to determine whether the release of thyroid hormone residues from Tg involves a random proteolytic attack or discrete and selective cleavage reactions. The intralysosomal soluble protein fraction was prepared by osmotic pressure-dependent lysis of lysosomes purified by isopycnic centrifugation on Percoll gradients. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a fraction of Tg (5-10% of total lysosomal protein) with the same molecular weight as that of the intact Tg subunit. This high molecular weight Tg was the only intralysosomal species detected by Western blot using antipig Tg antibodies. In nondenaturing conditions, lysosomal Tg (LTg) identified by radioimmunoassay was in the form of a dimer with a sedimentation coefficient lower than that of either iodinated Tg (colloid Tg) or noniodinated Tg (microsomal Tg). LTg had a lower iodine content than colloid Tg:9-12 versus 39-42 iodine atoms/molecule. Pronase hydrolysates of LTg did not contain any 3,5,3',5'-tetraiodo-L-thyronine or 3,3',5-triiodo-L-thyronine residues detectable by reverse-phase high pressure liquid chromatography; iodine present in LTg was in the form of iodotyrosines. Under reducing conditions, LTg almost completely disappeared and gave rise to various polypeptides of smaller size. These results suggest that Tg transferred to lysosomes is subjected to selective proteolytic cleavage reaction(s) that release thyroid hormone residues. This early step would lead to the formation of hormone-depleted Tg molecules that are cleaved at discrete sites, the resulting polypeptides remaining bound through disulfide bonds to yield Tg molecules with an apparently normal size and a slightly altered structure.

Rapid effectiveness of prednisone and thionamides combined therapy in severe amiodarone iodine-induced thyrotoxicosis. Comparison of two groups of patients with apparently normal thyroid glands.

Treatment of amiodarone iodine-induced thyrotoxicosis is often unsuccessful. Nevertheless, severe forms require a rapidly efficient therapy. Twelve patients with severe amiodarone iodine-induced thyrotoxicosis, as demonstrated on clinical and biological findings, were studied. After amiodarone withdrawal, 6 patients (group A) were treated with thionamides alone (carbimazole 60 mg daily and benzylthiouracile 1.5 g daily), and 6 patients (group B) received in addition to the same antithyroid drugs prednisone, 0.50 to 1.25 mg/kg/day for 40 days; in group A, T4 levels did not change over the study period of 40 days; T3 levels decreased only after 30 days; clinical status did not improve. In group B: T3 and T4 levels decreased dramatically at 10 days of treatment, to values significantly lower than in group A; clinical improvement occurred mainly in patients treated with high doses of prednisone; elevated thyroglobulin levels diminished rapidly. Improvement was maintained after cessation of prednisone. The rapid effect of prednisone suggests an impairement of proteolysis of thyroglobulin possibly due to a lysosomal action.

Selective enrichment of phytosphingosine in glycosphingolipids of isolated human thyrocytes as compared to the whole thyroid.

The glycosphingolipids of isolated human thyrocytes have been analyzed. As compared to the total thyroid gland, the pattern of gangliosides was found to be similar, whereas the neutral glycolipid profile was quite different, with glucosylceramide as the major glycosphingolipid of thyrocytes. Moreover, this glucosylceramide contains almost exclusively phytosphingosine (4-D-hydroxy-sphinganine) which is only a minor component in the long-chain bases of the glycosphingolipids extracted from the whole thyroid gland.

[Immunohistochemistry of thyroglobulin in the diagnosis of clear cell carcinomas of the cervical region]. / Immunohistochimie de la thyroglobuline dans le diagnostic des cancers à cellules claires de la région cervicale.

6 thyroid gland and 3 cervical lymph node clear-cells carcinomas are investigated by means of indirect immunofluorescence detection of thyroglobulin. Immunofluorescence was positive only in 1 case, which was a primitive thyroid carcinoma. In 7 other cases, its negativity pointed at the metastatic nature of the lesion, from a clear cell carcinoma of kidney (in 4 cases, nephrectomy was very remote, and in 3 cases, the renal lesion was discovered after our examination of the thyroid tumour). One observation showed negative staining reaction, and was a propagation of a parathyroid carcinoma to the thyroid gland. It is concluded that thyroglobulin can be used as an immunohistochemical marker to establish whether a clear cell cervical tumour originates from thyroid gland, or is a metastatic neoplasm.

Alterations of the cerebroside fraction of human thyroid glycosphingolipids in Graves' disease.

The influence of Graves' disease in human thyroid neutral glycosphingolipids was investigated. The major alteration was in the cerebroside fraction. Although the total amount of cerebroside was not different, the relative proportion of the bands of glucosylceramide separated on borated thin-layer plates was greatly modified in the disease. The band of glucosylceramide containing phytosphingosine and hydroxylated fatty acids decreased strongly, whereas the band with C18 sphingosine and normal fatty acids increased simultaneously. No change was observed in the content of galabiosylceramide. A slight elevation was seen in the amount of globoside at the expense of globotriaosylceramide.

Sex-specific difference of the galabiosylceramide level in the glycosphingolipids of human thyroid.

The glycosphingolipids of human thyroid were isolated and characterized by gas-liquid chromatography and sequential enzymic hydrolysis. The major purified components were identified as glucosyl- and galactosyl-ceramides, lactosyl- and galabiosylceramides, globotriaosyl- and globotetraosylceramides. The long-chain base analyses showed a high proportion of phytosphingosine in glycosylceramide and galabiosylceramide. Fatty acids in 22:0, 24:0, 24:1 prevailed, especially in the cerebroside fraction, with a significant content of alpha-hydroxylated species in galactosylceramide. Female thyroid had a very low content of galabiosylceramide and a higher content of glucosylceramide, as compared to male. No significant difference was found in the other neutral glycosphingolipids and gangliosides.

Major gangliosides in normal and pathological human thyroids.

The major gangliosides of human thyroids were extracted, purified and then analyzed by gas-liquid chromatography. In normal thyroid, GM3 and GD3 represented about 80% of lipid-bound sialic acid. GM3 contained more than 50% of long-chain fatty acids, whereas GD3 contained mostly short ones. 4D hydroxy sphinganine represented 20% of long-chain base content in both cases. In pathological thyroids (Graves' disease, cancer, toxic adenoma), GM3 represented about 60% of lipid-bound sialic acid; its fatty acid content was mostly short chain fatty acids, as in normal GD3. 4D hydroxy sphinganine proportion was decreased.