Interleukin 13 is a B cell stimulating factor.

The recently cloned human interleukin 13 (IL-13) is a novel cytokine expressed in activated T cells that has been shown to inhibit inflammatory cytokine production by lipopolysaccharide-activated monocytes. The protein encoded by the IL-13 cDNA is the human homologue of a mouse Th2-product called P600. Here, we show that IL-13 acts at different stages of the B cell maturation pathway: (a) it enhances the expression of CD23/Fc epsilon RII and class II MHC antigens on resting B cells; (b) it stimulates B cell proliferation in combination with anti-Ig and anti-CD40 antibodies; and (c) it induces IgE synthesis. Thus, the spectrum of the biological activities of IL-13 on B cells largely overlaps that previously ascribed to IL-4. The present observations suggest that IL-13 may be an important factor, in addition to IL-4, in the development of allergic diseases.

Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy.

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.

The kinin B1 receptor antagonist SSR240612 reverses tactile and cold allodynia in an experimental rat model of insulin resistance.

BACKGROUND AND PURPOSE: Diabetes causes sensory polyneuropathy with associated pain in the form of tactile allodynia and thermal hyperalgesia which are often intractable and resistant to current therapy. This study tested the beneficial effects of the non-peptide and orally active kinin B(1) receptor antagonist SSR240612 against tactile and cold allodynia in a rat model of insulin resistance. EXPERIMENTAL APPROACH: Rats were fed with 10% D-glucose for 12 weeks and effects of orally administered SSR240612 (0.3-30 mg kg(-1)) were determined on the development of tactile and cold allodynia. Possible interference of SSR240612 with vascular oxidative stress and pancreatic function was also addressed. KEY RESULTS: Glucose-fed rats exhibited tactile and cold allodynia, increases in systolic blood pressure and higher plasma levels of insulin and glucose, at 12 weeks. SSR240612 blocked tactile and cold allodynia at 3 h (ID(50)=5.5 and 7.1 mg kg(-1), respectively) in glucose-fed rats but had no effect in control rats. The antagonist (10 mg kg(-1)) had no effect on plasma glucose and insulin, insulin resistance (HOMA index) and aortic superoxide anion production in glucose-fed rats. CONCLUSIONS AND IMPLICATIONS: We provide the first evidence that the B(1) receptors are involved in allodynia in an experimental rat model of insulin resistance. Allodynia was alleviated by SSR240612 most likely through a direct inhibition of B(1) receptors affecting spinal cord and/or sensory nerve excitation. Thus, orally active non-peptide B(1) receptor antagonists should have clinical therapeutic potential in the treatment of sensory polyneuropathy.

Expression of tpo mRNA in thyroid tumors: quantitative PCR analysis and correlation with alterations of ret, Braf , ras and pax8 genes.

Immunocytochemistry (ICC) of thyroid peroxidase (TPO) using the monoclonal antibody MoAb47 has been used as malignancy marker on thyroid fine needle aspiration. However, little is known about the fate of TPO in thyroid carcinoma. We performed a qualitative PCR (Q-PCR) analysis to measure the expression of variants of tpo mRNA in 13 normal tissue samples, 30 benign tumors (BT), 21 follicular carcinomas (FC), 20 classical papillary carcinomas (PCc), 12 follicular variants of papillary carcinomas (PCfv) and nine oncocytic carcinomas (OC). We also studied mutations involving the ras, Braf, ret or pax8 genes. Results of Q-PCR were closely correlated with those of ICC (P < 0.0001; R = 0.59) and showed that overall tpo expression was lower in all carcinomas than in normal and BT (P < 0.05). The ratio tpo2 or tpo3 to tpo1 was inversed in follicular tumors. Genetic mutations were observed in 90% of PCc, 61.9% of FC, 41.7% of PCfv, 0% of OC and 10% in BT. pax8-ppar gamma1 rearrangement was correlated with qualitative changes in tpo mRNA (P < 0.01). These results confirmed the decrease of TPO expression in 97% of thyroid carcinomas regardless of histological type and the overexpression of shorter splice variants in follicular tumors. Both reduction in quantity of TPO and impairment of its maturation process could account for the atypical immunohistochemical reaction of MoAb47 with TPO.

Determination of sensitivity of fresh leukemia cells to immunotoxins.

In clinical practice, sensitivity of malignant cells to a given immunotoxin remains hypothetical, since standard test systems such as the protein synthesis inhibition assay or the cloning assay are not appropriate. This study evaluated the feasibility of a semi-routine procedure based on dye exclusion assay enumerating the percentage of living cells after fluorescein diacetate-propidium iodide staining. The validity of the method was evaluated using five different subclones derived from the CEM cell line, which expressed a wide range of sensitivity to T101 A-chain immunotoxin. The comparison between dye exclusion assay and standard test systems suggested that this method might allow an easy and reproducible semi-quantitative evaluation of the sensitivity of leukemia cells. In a series of 21 patients suffering from various blood diseases in which the malignant cells expressed the T65 antigen, dye exclusion assay could detect clear T101 immunotoxin cell sensitivity in about 50% of the cases. The mean density of T65 antigen on malignant cells was found to influence dramatically the sensitivity of target cells to T101 immunotoxin.

ret/PTC1 and ret/PTC3 in thyroid tumors from Chernobyl liquidators: comparison with sporadic tumors from Ukrainian and French patients.

Like children exposed to Chernobyl fallout, the workers who cleaned up after the accident, also known as liquidators, have exhibited an increased incidence of thyroid cancer. A high prevalence of ret/PTC3 rearrangement has been found in pediatric post-Chernobyl thyroid tumors, but this feature has not been investigated in liquidator thyroid tumors. In this study we analyzed the prevalence of ret/PTC1 and ret/PTC3 in thyroid tumors from 21 liquidators, 31 nonirradiated adult Ukrainian patients, and 34 nonirradiated adult French patients. ret rearrangements in carcinomas were found in 83.3% of liquidators, 64.7% of Ukrainian patients, and 42.9% of French patients. The prevalence of ret/PTC1 was statistically similar in the three groups. The prevalence of ret/PTC3 was significantly higher in liquidators than in French patients (P = 0.03) but it was also high in nonirradiated Ukrainian patients who exhibited values intermediate between liquidators and French patients. In adenomas the prevalence of rearrangement was significantly higher in all Ukrainians than in French patients (P = 0.004). Like children exposed to Chernobyl fallout, liquidators showed a high prevalence of ret/PTC3. This finding suggests that irradiation had the same effect regardless of age. However, given the high rate of ret/PTC3 in nonirradiated adult Ukrainians, the possibility of genetic susceptibility or low-level exposure to radiation in that group cannot be excluded.

Transient protection by peripheral benzodiazepine receptors during the early events of ultraviolet light-induced apoptosis.

The peripheral benzodiazepine receptor (PBR) is a mitochondrial protein involved in the formation of mitochondrial permeability transition (PT) pores which play a critical role during the early events of apoptosis. PBRs are located in many tissues and are strongly expressed in the superficial layers of human epidermis. PBRs play a protective role against free radical damage and PBR ligands modulate apoptosis. To investigate the role of PBR during the early events of ultraviolet (UV)-mediated apoptosis we compared the effects of UVB on PBR-transfected Jurkat cells and their wild type counterparts devoid of any PBR expression. Results indicate that early after UVB exposure (up to 4 h), PBR-transfected cells were more resistant to apoptosis and exhibited a delayed mitochondrial transmembrane potential drop, a diminished superoxide anions production, and a reduced caspase-3 activation. Taken together these findings suggest that PBR may regulate early death signals leading to UV induced apoptosis.

Monoclonal antibody approach to the relationship between immunological structure and biological activity of thyrotropin.

In order to locate the domains involved in the biological activity of TSH and to get some insight in the relationship between immunological and biological properties of TSH, 24 monoclonal antibodies (mAb) to 11 different antigenic regions of hTSH were tested for both binding to hTSH and inhibition of hTSH stimulation of adenylate cyclase in human thyroid membranes. These mAb were also investigated for binding to bovine TSH (bTSH), and interference with bTSH binding to the receptor and stimulation of adenylate cyclase. Radioiodinated human TSH (hTSH) was incubated with increasing concentrations of mAb. Maximum hTSH binding by the various mAb ranged from 15-75% and was not related to the apparent affinity of the mAb for hTSH. Maximum inhibition by the mAb of hTSH stimulation of adenylate cyclase ranged from 3-92%. As compared to the antigenic map of hTSH, it was observed that mAb reacting with the same antigenic regions might display varying inhibition of hTSH. Nevertheless, it was clearly shown that the most potent inhibitors of hTSH stimulatory activity interacted with epitopes located on the alpha- and beta-subunits or expressed only by holo hTSH. Only 11 of the 24 mAb cross-reacted significantly with bTSH. Seven exhibited the same inhibition of hTSH and bTSH stimulatory activity; the four remaining mAb rather than to inhibit adenylate cyclase stimulation as observed with hTSH, did not interfere or even increased adenylate cyclase stimulation by bTSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Different concentrations of thyroid peroxidase and thyroglobulin in the nuclear envelope and the endoplasmic reticulum throughout the cytoplasm.

Thyroid peroxidase (TPO) and thyroglobulin (TG) represent two major glycoproteins of thyroid follicular cells performing biological functions such as iodination, transcytosis of thyroglobulin, and formation of thyroid hormones. They are involved in thyroid autoimmunity and thyroid inborn metabolic disorders. Studying these processes at a molecular level includes the determination of their precise intracellular distribution. An evaluation of the relative concentrations of TG and TPO in different subcellular compartments was carried out in stimulated human follicular cells using thin-frozen sections and the immunogold technique. It is documented that TG is transported from the endoplasmic reticulum and the Golgi apparatus to the follicular lumen by transport vesicles; most of it being present in the expanded endoplasmic reticulum throughout the cytoplasm. On the other hand, gold particles indicating TPO are adjacent to the membranes of the exocytotic pathway. They do not label the basolateral membrane but show the strongest density in the nuclear envelope and the apical membrane. The labeling density of TPO is about four times higher in the nuclear envelope than in the endoplasmic reticulum throughout the cytoplasm. In contrast, TG is concentrated three times higher in the rough endoplasmic reticulum throughout the cytoplasm than in the nuclear cisternae. Our results give the first quantitative evidence that TPO and TG are concentrated in different subcompartments of the endoplasmic reticulum. Because previous studies demonstrated the nuclear envelope as the site where the synthesis of endogenous peroxidase (Brökelmann, J., D. W. Fawcett, Biol. Reprod. 1, 59-71 (1969)) begins, we suggest that synthesis of these functionally related proteins happens in specialized parts of the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of human chorionic gonadotropin and human luteinizing hormone with human thyroid membranes.

In an attempt to identify a possible pathogenetic role for the hCG molecule in the mechanism of the hyperthyroidism which occurs in choriocarcinoma, we have looked for evidence that the hCG molecule has a thyrotropic action on the human thyroid. The thyrotropic activity of various hCG preparations on the human thyroid was assessed by measuring the stimulation of adenylate cyclase activity in human thyroid plasma membranes purified by sucrose density gradient centrifugation. The highly purified hCG CR119 preparation stimulated human thyroid adenylate cyclase activity. Its activity was more than 654 times greater than could be accounted for by human TSH (hTSH) contamination of the preparation, as determined by RIA. The thyrotropic activity intrinsic to 1.0 IU hCG was equivalent to roughly 0.27 microU hTSH. Significant saturable binding of the 125I-labeled highly purified hCG preparation to human thyroid membranes was demonstrated, and the bound component was characterized. Its apparent molecular size, subunit composition, and testis receptor-binding characteristics were those of the hCG molecule. Examination of a crude urinary hCG preparation in adenylate cyclase and TSH radioligand assays using human thyroid membranes showed no evidence of any molecule other than hCG with a thyrotropic action on the human thyroid. Given that hCG binds to and stimulates adenylate cyclase activity in human thyroid tissue, as the above data indicate, then human LH (hLH) would be expected to do the same, since hLH and hCG have such strong structural and functional similarities. As anticipated, a highly purified hLH preparation exhibited TSH binding inhibition and adenylate cyclase stimulation. Its activity was more than 1030 times greater than could be accounted for by hTSH contamination of the preparation. The thyrotropic activity intrinsic to 1.0 IU hLH was equivalent to roughly 44 microU hTSH. Thus, in addition to other shared properties, the hLH molecule and the hCG molecule share the ability to interact with human thyroid tissue. These results strongly indicate that the hCG molecule has a thyrotropic action on the human thyroid and support the hypothesis that hCG is the thyrotropic factor that mediates the hyperthyroidism which occurs in patients with hCG-secreting neoplasms.

Effect of carboxypeptidase digestion of the human choriogonadotropin molecule on its thyrotropic activity.

Digestion of hCG by a mixture of carboxypeptidases B and Y results in an enzyme dose- and incubation time-dependent increase in its ability to stimulate the adenylate cyclase (AC) of human thyroid membranes. Treated under the same conditions [3 h at 37 C; enzyme to hCG ratio, 0.04 (wt/wt)], partially and highly purified hCG preparations display an increase of about 300% in thyroid AC-stimulating activity, while TSH displays a 30% decrease. In contrast, carboxypeptidase digestion of hCG under these conditions has no significant effect on its activity in the rat testis AC assay. The carboxypeptidase digestion results in cleavage of carboxyl-terminal amino acid residues 142--145 from the hCG beta-subunit; digestion of the hCG alpha-subunit is much less effective, as the carboxy-terminal amino acid residue 92 is removed from only about 13% of the hCG molecules. In accord with the results of amino acid analysis, a slight, if any, decrease in apparent molecular weight is found by gel filtration and polyacrylamide gel electrophoresis. In addition, carboxypeptidase digestion results in antigenic alterations of the molecule, as shown by a flatter slope of the dose-response curve in a hCG RIA and a 70% decrease in potency in a RIA that uses an antiserum to the hCG beta carboxy-terminal peptide. These data demonstrate that partial digestion of the hCG molecule with carboxypeptidase results in an increase in human thyroid AC-stimulating activity, with retention of the rat testis AC-stimulating activity.

Monoclonal antiidiotypic antibodies interact with the 93 kilodalton thyrotropin receptor and exhibit heterogeneous biological activities.

Ten antiidiotypic monoclonal antibodies (AI mAb) reacting with the TSH receptor were produced by immunization of mice with a mouse mAb directed to a TSH epitope involved in the binding of the hormone to the receptor. The AI mAb were tested for their effects on the TSH receptor-adenylate cyclase system. Four AI mAb behave as agonists of TSH as they compete with TSH for binding to the receptor and stimulate the adenylate cyclase activity. Five AI mAb inhibited both TSH binding to the receptor and adenylate cyclase activity in the presence and absence of TSH; they were thus considered as antagonists of the hormone. One mAb inhibited only slightly the binding of TSH to the receptor and did not interfere significantly with the adenylate cyclase activity. The 10 AI mAb bound with various apparent affinities to solubilized thyroid membrane proteins but not to kidney, spleen, and liver membranes. Membrane desialylation did not significantly alter the binding of the AI mAb whereas deglycosylation modified more or less strongly the binding of mAb to thyroid membranes. It was also shown that the binding to solubilized thyroid membranes of the 10 mAb was inhibited by anti-TSH receptor autoantibodies present in patients with autoimmune disease. The mAb were further analyzed by Western blot analysis. One mAb did not react with the antigen, which suggested that it was directed to a conformational epitope. The other 9 mAb reacted with a protein of 70 kilodaltons and 5 revealed another band of 93 kilodaltons in accordance with the TSH receptor size deduced from the recently molecular cloning.

Relevance of the low and high affinity thyrotropin-binding sites of human thyroid membranes to the stimulation of adenylate cyclase.

TSH is known to interact on thyroid membranes with two classes of binding sites that differ in affinity and capacity. To assess the relevance of the class of TSH-binding sites characterized by low affinity and high capacity to the stimulation of adenylate cyclase, we studied the interactions of desialylated hCG (as-hCG) and its beta-subunit (as-hCG beta) with human thyroid membranes. In low ionic strength buffer, pH 7.8, where both classes of sites are operant, as-hCG fully inhibited and as-hCG beta partially inhibited [125I] bovine (b) TSH binding. Scatchard analysis of the [125I]bTSH binding inhibition curve in the presence of 1.0 X 10(-5) M as-hCG beta clearly indicated that as-hCG beta interacted only with the low affinity class of binding sites, leaving the high affinity class unaffected. In the presence of 140 mM NaCl, [125I]bTSH interacted predominantly with the high affinity class of binding sites; as-hCG fully inhibited [125I]bTSH binding to this class of sites, whereas as-hCG beta displayed essentially no interaction. Scatchard analysis of [125I]as-hCG beta binding to human thyroid membranes in low ionic strength buffer revealed a single apparent class of sites with low affinity (Kd = approximately 1.0 X 10(-6) M) and high capacity (Q = approximately 300 pmol/mg membrane protein). The bTSH preparation (Thytropar) showed a 10-fold greater binding inhibition potency at these sites than either the as-hCG or the as-hCG beta preparation, in keeping with the inference that as-hCG beta interacts with the low affinity class of TSH-binding sites. At a concentration more than 3 times that necessary to inhibit TSH binding to the low affinity class of sites, the as-hCG beta molecule neither stimulated adenylate cyclase nor inhibited the ability of TSH to do so. In contrast, the as-hCG molecule, which interacts with both classes of TSH-binding sites, fully inhibited TSH stimulation of adenylate cyclase. We conclude that the low affinity class of TSH-binding sites is not the class of sites through which TSH stimulates adenylate cyclase, and that this role is best ascribed to the high affinity class of TSH-binding sites.

Relationship between immunological structure and biochemical properties of human thyroid peroxidase.

Although the primary structure of human thyroid peroxidase (hTPO) has been recently deciphered, little is known about its spatial conformation. Such information is of crucial importance in any attempt to relate the structure with the function of hTPO. To probe the antigenic surface of hTPO and to correlate its immunological structure to its biochemical properties, we used 13 monoclonal antibodies (mAb) displaying various affinity for hTPO. Criss-cross experiments showed 7 clusters of reactivity which were interpreted as reflecting 7 epitopes on the surface of the hTPO molecule. Extending our analysis to partial and nonsymmetrical cross-reactivities, these epitopes were shown to be localized in 4 antigenic domains of the hTPO. We further investigated the nature of these 7 hTPO epitopes by testing mAb binding to peroxidases from various origins and chemically modified hTPO; 3 epitopes were shown to be evolutionary conserved, and 5 resistant to reduction and denaturation. We also analyzed the role of the hTPO epitopes in the enzymatic activity and autoimmune targeting of the molecule. Nine epitopes were shown to be localized at the vicinity of both catalytic sites as the binding of their respective mAb modulated the enzyme activity. Autoantibodies from patients presenting with autoimmune thyroid disorders were essentially directed to epitopes similar or adjacent to those recognized by 8 of the 13 mAb and present on only 2 antigenic domains of hTPO. Taken together these data allowed us to propose a tentative map of the surface of the hTPO molecule which associates its epitopic structure with its biochemical functions.

Studies on the mechanism responsible for thyrotropin-induced expression of microsomal/peroxidase antigen in FRTL-5 cells.

The expression of the microsomal (M) antigen on the surface and in the cytoplasm of a strain of rat thyroid cells (FRTL-5) is under the regulation of TSH. In the present report the mechanism by which TSH induces such expression was investigated with the use of human microsomal antibody-positive serum and an indirect immunofluorescence technique. Studies were also performed to ascertain whether the M antigen of FRTL-5 cells could be identified with thyroid peroxidase (TPO), as suggested by recent data obtained in human thyroid tissue. Preabsorption experiments showed that, like solubilized human thyroid microsomes, purified human TPO completely abolished the binding of microsomal antibody to FRTL-5 cells. No inhibition was obtained by preabsorption with control human tissues (placenta, liver, and spleen) or human thyroglobulin, indicating that the antigen recognized by microsomal antibody in FRTL-5 cells was TPO. After 72 h of TSH withdrawal from the culture medium the M/TPO antigen disappeared from the surface and the cytoplasm of FRTL-5 cells. Readdition of TSH (250 microU/ml) to the culture medium of cells lacking the M/TPO antigen elicited its reappearance within 24-48 h. This effect of TSH was prevented by 10 microM cycloheximide or 0.5-5 micrograms/ml actinomycin D. Two well known stimulators of the adenylate cyclase-cAMP system, cholera toxin and forskolin, mimicked TSH in inducing the reappearance of the M/TPO antigen. A similar effect was observed with use of the phosphodiesterase inhibitor isobutylmethylxanthine. Reappearance of M/TPO antigen was also produced by the cAMP analog 8-bromo-cAMP. The tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate, which stimulates thyroid cell growth through a cAMP-independent pathway, was ineffective in inducing the M/TPO antigen in FRTL-5 cells. The present data indicate that 1) thyroid peroxidase accounts for most, if not all, of the microsomal antigen of FRTL-5 cells; and 2) TSH modulates the expression of the M/TPO antigen in FRTL-5 cells by a mechanism that involves cAMP production and requires mRNA formation and subsequent protein synthesis.

Monoclonal antibody mapping of the antigenic surface of human thyrotropin and its subunits.

Although the amino acid sequence of the alpha- and beta-subunits of glycoprotein hormones in various species has been deciphered, data on their tertiary structure are not abundant. This impedes correlation between structure and function. The availability of monoclonal antibodies to human TSH (hTSH) offers the opportunity to enumerate the antigenic determinants present on the surface of hTSH and its subunits and to examine their spatial relationships. Twenty-eight monoclonal antibodies to hTSH were obtained from several fusions, and screens carried out separately in the laboratories involved in this study. Affinities for hTSH ranged from 10(8)-10(11) M-1. Cross-reactivity with bovine TSH (bTSH), human gonadotropins (hLH, hFSH, and hCG), and the alpha- and beta-subunits of hTSH distinguished 10 groups of monoclonal antibodies (mAb) according to their main cross-reactions: 1) hTSH alpha, hLH, hFSH, and hCG; 2) hTSH alpha, bTSH, hLH, hFSH, and hCG; 3) hFSH; 4) bTSH and hFSH; 5) bTSH, hLH, and hFSH; 6) bTSH, hLH, hFSH, and hCG; 7) hTSH beta; 8) hTSH beta and bTSH; 9) hTSH beta and hFSH; and 10) hTSH beta, hLH, hFSH, and hCG. mAb were incorporated into 2-site binding assays to probe hTSH by a 28 X 28 matrix, the free alpha-subunit by a 4 X 4 matrix, and the free beta-subunit by a 18 X 18 matrix. Regarding intact hTSH, 12 different clusters of mAb were distinguished and interpreted as reflecting 12 distinct antigenic regions on the surface of the hTSH molecule. Two of them were localized on the alpha-subunit, and 6 on the beta-subunit; 4 were only expressed by the holo-hormone and, thus were designated conformational antigenic regions (alpha beta). Surface mapping of the free alpha- and beta-subunits was virtually identical to that observed with the holo-hormone. Modification of the operative conditions of mAb reacting only with holo-hTSH shows that they recognize the alpha-subunit, but not the beta-subunit of hTSH. These results indicate that 1) hTSH beta presents epitopes that are evolutionary conserved; 2) hTSH alpha presents several epitopes that are species specific and 2 that are not hormone specific; 3) dissociation of hTSH does not modify the antigenic surface expressed by both subunits when they are associated; and 4) some of the conformational determinants expressed only by holo-hTSH are more likely derived from the alpha-subunit than from the beta-subunit.

Production of immunoreactive thyroglobulin C-terminal fragments during thyroid hormone synthesis.

Here, we studied the fragmentation of the prothyroid hormone, thyroglobulin (Tg), which occurs during thyroid hormone synthesis, a process which involves iodide, thyroperoxidase, and the H2O2-generating system, consisting of glucose and glucose oxidase. Various peptides were found to be immunoreactive to autoantibodies to Tg from patients and monoclonal antibodies directed against the immunodominant region of Tg. The smallest peptide (40 kDa) bore thyroid hormones and was identified at the C-terminal end of the Tg molecule, which shows homologies with acetylcholinesterase. Similar peptides were obtained by performing metal-mediated oxidation of Tg via a Fenton reaction. It was concluded that the oxidative stress induced during hormone synthesis generates free radicals, which, in turn, cleave Tg into immunoreactive peptides.

Heterogeneity of the Graves' immunoglobulins directed toward the thyrotropin receptor-adenylate cyclase system.

The TSH-displacing and the thyroid-stimulating activities of Graves' immunoglobulins G (GIgG) have been accounted for by either homogeneous TSH receptor antibodies or heterogeneous antibodies directed toward the TSH receptor-adenylate cyclase system. To clarify this matter, the study of the interactions of GIgG preparations from 14 untreated Graves' patients with human thyroid membranes was undertaken. Dose-response curves of GIgG, diluted in IgG from normal subjects, were carried out in the [125I]TSH radioreceptor assay and the adenylate cyclase assay in the presence or absence of TSH. In the radioreceptor assay, GIgG were constantly negative in 3 cases (21%) and positive, depending of the dose, in 11 cases. In the adenylate cyclase assay, the dose-activity profiles in the absence of TSH were bell-shaped curves in 3 cases and sigmoid curves in 9 cases; in 2 cases (14%), GIgG preparations were devoid of any effect. Binding-isotherms and dose-activity profiles did not appear to share simple relationships. In the presence of TSH, GIgG preparations elicited a decrease in 6 cases, an increase in 3 cases, and no effect in 5 cases (36%) in the adenylate cyclase activity. The data obtained by radioreceptor assay and adenylate cyclase assay in the presence or absence of TSH were found statistically correlated (P less than 0.05 to P less than 0.001) but not linearly related, the points being scattered on specific parts of the diagrams. These observations could not be accounted for by TSH receptor antibodies in GIgG being an entity of constant properties, albeit varying in titer among patients. Rather, GIgG effects fit well the patterns of action of a heterogeneous ligand, as shown by computing a theoretical model for ligand heterogeneity with respect to binding equilibrium constant and intrinsic biological activity. Accordingly, GIgG activity in the TSH receptor-adenylate cyclase system could be attributed to heterogeneous antibodies varying with regard to binding constant and acting as stimulating or blocking antibodies of the adenylate cyclase.

Meta-analysis evaluation of the impact of thyrotropin receptor antibodies on long term remission after medical therapy of Graves' disease.

Patients with the hyperthyroidism of Graves' disease (GDH) have a higher risk of relapse after antithyroid drug therapy (ATD) therapy when TSH receptor antibodies (TRAb) are positive, but the practical clinical implication of TRAb as a predictor for relapse is still much debated. This study was undertaken to investigate by meta-analysis the results from the literature on the use of TRAb as predictor of long term (i.e. at least 1 yr) relapse after ATD. Eighteen publications from 1975-1991 fulfilled the criteria of 1) availability of TRAb at the end of ATD treatment, 2) at least 1 yr of follow-up after ATD, 3) data presentation in a form suitable for meta-analysis, and 4) no other thyroid-related therapy during the follow-up period. The 10 prospective studies, 5 of which measured TSH binding inhibiting immunoglobulins (total n = 597) and 5 of which measured thyroid-stimulating antibodies (n = 340), were computed together because no significant differences were found. In contrast, retrospective and prospective studies differed. In the prospective studies, the odds reduction of relapse showed 65% less risk of relapse when TRAb were absent compared to that in TRAb-positive patients (P < 0.00001). The present meta-analysis has, thus, confirmed in a large number of patients (n = 1524) that absence of TRAb is significantly protective against relapse of GDH after ATD treatment. However, 25% of the patients are "misclassified," and the main questions arising from the study are, therefore, the following. 1) Is it worthwhile to use TRAb as predictor of relapse? 2) Should patients with GDH continue ATD until TRAb becomes negative, rather than for a fixed period? The available methods for TRAb do not allow sufficiently high prediction of relapse or remission after ATD for the individual patient.