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Power converters are considered the major source of electromagnetic interference (EMI) in power conversion devices in electric vehicles and in more electrical aircraft. Several works study the impact of power converters on conducted emissions (CE), due mainly to common-mode (CM) currents, most of them with the traditional two-level inverter in power drive systems. An inverter is commonly modeled as a voltage source with input and output impedances. This paper presents an equivalent circuit of parallel and series multilevel inverters that can be used to analytically calculate CM currents. The method developed here, precisely estimates CM currents which is very important to prevent CM chokes to saturate and to fail on respecting standard limits.

International audience; Key points in the development of More Electrical Aircraft (MEA) are currently DC power distribution in higher voltage levels (540 V) and the use of disruptive technology such as Wide BandGap (WBG) semiconductors in power inverters. Using WBG components (SiC and GaN) increases the power converter mass density. However, fast switching of WBG components (tens of kV/s) induces voltage transient overshoots due to parasitic elements within the inverter. In addition, propagation and reflection phenomena along the harness connected to this inverter, even for small lengths, cause a significant voltage overshoot across the loads. Such overvoltage in Adjustable Speed Drives (ASD: association of inverter, harness and motor) supplied by the new HVDC 540 V aeronautical network could be fatal for the Electrical Insulation System (EIS). This paper proposes a fast and accurate modeling methodology to predict transient overvoltage; it allows us to analyze the impact of SiC inverter technology on overvoltage at motor terminals.

International audience; The use of fast semiconductor components such as IGBT and SiC in Adjustable Speed Drives (ASD) increases power density and provides better controllability. Nevertheless, secondary effects such as overvoltage at the motor terminals appear and can affect the reliability of the Electrical Insulation System (EIS). The overvoltage is due to the propagation and reflection phenomena along the harness. In this paper, we propose a fast frequency model to simulate motor overvoltage. Their maximum level is studied according to the following parameters: inverter dv/dt, harness length and motor impedance.

Key areas in the development of More Electrical Aircraft (MEA) are, currently, DC power distribution in higher voltage levels and the use of disruptive technology such as Wide BandGap (WBG) semiconductor. Using WBG components (SiC and GaN) increases the power converter mass density. However, fast switching of WBG components (tens of kV/µs) induces voltage transient overshoots due to parasitic coupling within the inverter. In addition, propagation and reflection phenomena along the harness, even for small lengths, cause voltage overshoots across the loads. Such overvoltage in an electromechanical chain (association of inverter, harness and motor) supplied by the new HVDC 540V aeronautical network could be fatal for the Electrical Insulation System (EIS). This paper proposes an accurate and fast model to predict overvoltage along a harness; it allows to analyze the impact of SiC inverter output voltage waveforms.

International audience; In power electronics applications, high frequency models for cables are necessary to understand EMI issues in pulsewidth modulation drives. This paper shows the approach developed at the French Institute of Technology (IRT) Saint-Exupery, in order to take account of the frequency dependency of unshielded power cables per-unit-length parameters for EMC simulations. Fast, predictive models are compared to different shapes numerical models. The method was applied to unshielded two and three wires cables. Finally, common mode (CM) emissions modeling is proposed to predict the CM noise currents, which are the most disturbing in any variable-speed drive systems. The modeling principle is to consider the complete CM circuit as a chain of quadripolar matrices.

abc.2013.0885 Auteur(s) : Pierre Carayon pierre.carayon@univ-uma.fr Professeur de biochimie et biologie moleculaire, Faculte de medecine, Universite d’Aix-Marseille, France Le 18 juillet dernier, la Cour des comptes a rendu public un rapport sur la biologie medicale. Ce document appelle a une economie de 200 a 300 millions d’euros, une reduction de 7,5 % de la tarification des actes de biologie et la denonciation de la convention liant biologistes et assurance-maladie. [...]

Les 26 et 27 novembre 2015 ont eu lieu « Les Rencontres maladies rares 2015 » organisees par Eurobiomed a Montpellier. Ces deux journees ont rassemble, autour du theme maladies rares, des personnels de sante tels que cliniciens, biologistes, chercheurs, auxiliaires de vie, administratifs, et des structures telles qu’associations de malades et entreprises impliquees dans le diagnostic ou le medicament, ayant tous en commun la volonte d’intervenir dans le domaine des maladies rares.Du [...]

PurposeThe frequency simulation and optimisation of electromagnetic compatibility (EMC) filter is often computation time consuming. The purpose of this paper is to propose an approach for easy and fast modelling and optimization of power electronics structures.Design/methodology/approachThe paper proposes an approach for easy and fast modelling and optimization of power electronics structures. It focuses on the EMC filter design. To achieve this task time simulation, FFT and automatic frequency modelling are combined.FindingsAn automatic frequency modelling is proposed and also gives automatically the model gradients. Therefore, the model can be used to optimize the EMC filter, but also can help in choosing its topology. Several optimization algorithms are used and compared.Research limitations/implicationsThe power electronics load is supposed to be a set of predefined harmonic sources, obtained by time simulation + FFT before the optimisation process.Practical implicationsThe frequency model allows for the rapid designing and comparing of several structures or modelling hypothesis with regard to the parasitic elements and circuit imperfections.Originality/valueThe frequency model is automatically generated, and sizing criteria on the component (e.g. inductors, capacitor) can be added in an analytical form, for example, to deal with volume or mass criteria.

Dososahexaenoate (DHA) is highly concentrated in mammalian nervous and visual systems and its deficiency during gestation, lactation and early life, could have dramatic impacts on brain functions and mental health. Achieving an appropriate DHA status in the neonatal brain is an important goal of neonatal nutrition. We evaluated how a-linolenic acid (ALA) provided by different dietary fat matrices improved DHA content in the brains of both young male and female rats. Young rats born from dams fed during gestation and lactation with a low ALA diet (0.4% of fatty acids) were subjected for 6 weeks after weaning to an anhydrous dairy fat blend-based diet that provided 1.5% ALA or to a palm oil blend-based diet that provided the same ALA level: either 1.5% ALA or 1.5% ALA and 0.12% DHA with 0.4% arachidonic acid (ARA). With each diet the n-6/ n-3 ratio was similar (10) to follow the values generally recommended for infant formula. Fatty acids analysis in whole brain showed that 1.5% ALA dairy fat blend was superior to both 1.5% ALA palm-oil blends, supplemented or not with dietary DHA, for increasing brain DHA. Females compared to males had significantly higher brain DHA with the 1.5% ALA palm-blend diet, but the dietary supplementation with DHA smoothed the differences by a specific increase of males DHA brain. In conclusion, dairy fat blend enriched with ALA appear to be an interesting strategy for achieving optimal DHA levels in the brain of post-weaning rats. Inclusion of dairy fat in infant formulas should be reconsidered.

Background: Mast cells participate in immune defense and allergic disease. At baseline, serum tryptase levels primarily reflect mast cell burden, while mast cell degranulation leads to granule tryptase release, which may be detectable as a transitory elevation of serum tryptase levels. Thus, mast cell burden and mast cell activity are reflected by serum tryptase levels, but reports are scarce in infants under 1 yr. We aimed at defining levels of total serum tryptase levels in this population. Methods: Total serum tryptase levels (ImmunoCAP; Phadia) were measured in 372 sera from infants younger than 1 yr. Two hundred and forty-two sera came from non-atopic, non-allergic infants in good condition, who had blood drawn for routine follow-up or diagnosis of illnesses that are not known to induce changes in serum tryptase levels. Seventy-two sera were from atopic and/or allergic infants, and

A cohort of 10 Hashimoto's encephalopathy (HE) patients, 33 patients with unrelated neurological symptoms, 12 Hashimoto's thyroiditis patients and 4 healthy adult donors was studied to explore the neurological targets of anti-thyroperoxidase (TPO) autoantibodies (aAb) in HE. High levels of anti-TPO aAb were only detected in HE group's cerebrospinal fluids. In immunofluorescence assays on monkey brain cerebellum sections, both HE patients' sera and anti-TPO monoclonal antibodies (mAb) were able to bind cerebellar cells expressing glial fibrillary acid protein. Normal human astrocytes from primary cultures also reacted with anti-TPO mAb. Specific astrocyte binding of anti-TPO aAb suggests a role of these aAb in the HE pathogenesis.

BackgroundIrritable bowel syndrome (IBS) has a complex pathology, high prevalence and large impact on patients’ quality of life. As conventional therapy may yield unsatisfactory results, a more holistic approach may be desirable. The current study assessed the effect of micro-physiotherapy on the severity of IBS symptoms.MethodsIn a double-blind study, 61 recurrent IBS patients were randomised to two sessions of micro-physiotherapy or sham micro-physiotherapy. Inclusion criteria were the presence of ≥1 IBS symptom from abdominal pain, constipation, diarrhoea or bloating. Exclusion criteria were previous major intestinal surgery and the presence of chronic diseases. The mean patient age was 53.5±15.3 years. Micro-physiotherapy consisted of micro-palpatory examination to identify osteopathic lesions, followed by micro-massage to stimulate self-healing. The control group underwent a sham procedure. The presence and severity of symptoms was assessed at baseline and at 1-month follow-up by the same gastroenterologist.ResultsTwo patients did not complete the study. There was a significant difference in percentage of patients that improved after the first session, at 74 % for the micro-physiotherapy group and 38 % for the sham group, respectively (p=0.005). After the second session, the initial improvement was maintained in both groups, although with no further gains, and the differences between the study groups remained significant (p=0.007).ConclusionsMicro-physiotherapy significantly improves IBS symptoms and should be explored further for use in mainstream healthcare.

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 γ1 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.

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.

Contexte : Le syndrome du colon irritable (SCI) presente une pathologie complexe, une prevalence elevee et un impact important sur la qualite de vie des patients. Comme la therapie conventionnelle n’apporte souvent que des resultats insatisfaisants, une approche plus holistique peut etre souhaitable. L’etude actuelle a evalue l’effet de la microkinesitherapie sur la severite des symptomes du SCI.Methodes : Dans cette etude en double aveugle, 61 patients recurrents du SCI ont ete randomises pour recevoir deux seances d’un traitement de microkinesitherapie ou d’un traitement simule de microkinesitherapie. Les criteres d’inclusion etaient la presence de ≥1 symptome SCI de douleurs abdominales, constipation, diarrhee ou ballonnements. Les criteres d’exclusion etaient une chirurgie intestinale majeure anterieure et la presence de maladies chroniques. L’âge moyen du patient etait de 53,5 ± 15,3 ans. La microkinesitherapie consistait en un examen micro-palpatoire pour identifier les lesions osteopathiques, suivi d’un micro-massage pour stimuler l’auto-guerison.Le groupe temoin a subi une procedure simulee. La presence et la gravite des symptomes ont ete evaluees lors du suivi par un meme gastro-enterologue au depart de l’etude et 1 mois apres les seances.Resultats : Deux patients n’ont pas termine l’etude. Il y a une difference significative entre le pourcentage de patients qui s’est ameliore apres la premiere seance, a 74 % pour le groupe de microkinesitherapie et a 38 % pour le groupe placebo (p = 0,005). Apres la deuxieme seance, l’amelioration initiale a ete maintenue dans les deux groupes, bien que sans plus de gains, et les differences entre les groupes d’etude sont demeurees significatives (p = 0,007).Conclusions : La microkinesitherapie ameliore considerablement les symptomes du SCI et devrait etre exploree plus avant pour une utilisation dans les soins de sante traditionnels.

Esophageal cancer is the most markedly increasing tumor entity in Western countries. Due to very poor 5-year-survival, new therapeutic approaches are mandatory. Peripheral benzodiazepine receptors (PBR) have been implicated in growth control of various tumor models, but they have not been studied yet in esophageal cancer. We used esophageal cancer cell lines and primary cell cultures of human esophageal cancers and evaluated (i) expression and localization of PBR; (ii) PBR-ligand-induced inhibition of cell growth; (iii) induction of apoptosis; and (iv) alterations in cell cycle. Expression of PBR was detected both in cell lines and in primary cell cultures of human esophageal cancers. PBR was localized in the mitochondria. The PBR-specific ligands FGIN-1-27 and PK 11195, but not the centrally acting benzodiazepine clonazepam or the indolacetamide FGIN-1-52, neither of which displaying any affinity to the PBR, inhibited cell proliferation. FGIN-1-27 and PK 11195, but not clonazepam, potently induced apoptosis. FGIN-1-27 was shown to sequentially decrease the mitochondrial membrane potential, then to activate caspase-3 and finally to cause DNA fragmentation. In addition, PBR-specific ligands induced cell cycle arrest in the G1/G0 phase. Our data qualify PBR-specific ligands as innovative proapoptotic and antiproliferative substances. They might prove suitable for the treatment of esophageal cancer.

Human thyroperoxidase (TPO) ectodomain is successively made of myeloperoxidase-, complement control protein repeat-, and epidermal growth factor-like gene modules. However, the TPO immunodominant region targeted by autoantibodies from patients with an autoimmune thyroid disease has not been mapped on the molecule. Here, we used two purified recombinant TPO peptides produced in eukaryotic cells, which correspond to the major first and the further two gene modules of TPO. We compared by ELISA their respective immunoreactivity with that of the recombinant soluble TPO containing all the three gene modules. We used well-characterized murine and human TPO monoclonal antibodies and human autoantibodies affinity-purified from a large pool of patients' sera. We found that the TPO immunodominant region was susceptible to denaturation and required the integrity of the molecule to be correctly expressed. We concluded that TPO B-cell autoepitopes are made by amino acids from the three gene modules, which fold in one highly conformational immunodominant region.

Thyrocyte expression of HLA class I and class II antigens and related accessory molecules would convert these epithelial cells into functional antigen-presenting cells. Here we show that whereas normal thyrocytes express FcRn, Graves' disease thyrocytes also express FcgammaRIIB2. We further find that expression of FcgammaRIIB2, but not FcRn, is repressed by dihydrotestosterone. By mediating the uptake and transport of autoantibodies, we suggest that these IgG Fc receptors contribute in various ways to the onset and/or progression of autoimmune thyroid diseases. The androgen-mediated decrease of FcgammaRIIB2 expression in Graves' disease thyrocytes also provides a rationale for the predominant susceptibility of women to develop an autoimmune thyroid disease. Our findings open up a new prospect to autoimmunity, linking the role of the target organ to the sex dependence in autoimmune disease.

Combined endoscopic ultrasonography and duodenal bile drainage for microscopic bile examination was prospectively evaluated in patients with unexplained acute biliopancreatic disorders defined as follows: isolated biliary-type pain (group 1), biliary-type pain and elevated transaminases (group 2), or acute pancreatitis (group 3). All patients had prior negative transabdominal ultrasonography. Endoscopic ultrasonography and bile drainage were combined under anesthesia during the same procedure. Combined endoscopic ultrasonography and bile drainage was feasible without any complication in the 48 patients studied. Biliary lithiasis was diagnosed in 20 patients (42%), 5 in group 1 (38%), 7 in group 2 (39%), and 8 in group 3 (47%). Endoscopic ultrasonography and microscopic bile examination were positive respectively in 17 (36%) and in 13 (27%) patients. In conclusion, combined endoscopic ultrasonography and bile drainage for microscopic bile examination is a simple procedure. Biliary lithiasis is diagnosed in a high proportion of patients with unexplained acute biliopancreatic disorders.

Les « discordances » relevees au cours de l’utilisation, dans un contexte medical, des techniques de biologie, sont au cœur de la pratique de ce qui fut denomme « Chimie clinique », « Biologie clinique » puis « Biologie medicale » et, maintenant, « Medecine de laboratoire ». Cette evolution dans la designation des pratiques de biologie humaines impliquees dans le diagnostic et le suivi therapeutique des patients, reflete une diversification des techniques utilisees [...]

We recently reported that, during in vitro thyroid-hormone synthesis, H2O2 stress cleaved thyroglobulin (Tg) into C-terminal peptides. These peptides were found to contain the immunodominant region of Tg recognized by Tg autoantibodies from patients with an autoimmune thyroid disease. To test the hypothesis that Tg fragmentation is an early upstream initiating event involved in Tg autoimmune response and the consequence of oxidative injuries, we studied the effect of H2O2 stress on human thyroid cells. In culture conditions allowing Tg synthesis and iodine organification by the cells, we found that bolus addition of increasing millimolar doses of H2O2 induced a dose–response appearance of floating cells in the culture medium. These cells apparently resulted from a necrotic process, and they bore iodinated Tg fragments. These fragments were found to be similar to those previously obtained in vitro from purified Tg. In both cases, Tg peptides were recognized by a well-defined monoclonal antibody directed to the immunodominant region of Tg. The smallest immunoreactive Tg peptide had a molecular mass of 40kDa and entered human thyrocytes more efficiently than the entire Tg. These data suggest that thyrocytes exposed to locally increased H2O2 doses accumulate fragmented Tg for further delivery into surrounding living thyrocytes in the course of an autoimmune response.

The process of thyroid hormone synthesis, which occurs in the lumen of the thyroid follicles, results from an oxidative reaction leading, as side effects, to the multimerization of thyroglobulin (TG), the prothyroid hormone. Although hormone synthesis is a continuous process, the amount of Tg multimers is relatively constant. Here, we investigated the role of two molecular chaperones, protein disulfide isomerase (PDI) and immunoglobulin heavy chain-binding protein (BiP), present in the follicular lumen, on the multimerization process due to oxidation using both native Tg and its N-terminal domain (NTD). In vitro, PDI decreased multimerization of Tg and even suppressed the formation of NTD multimers. Under the same conditions, BiP was able to bind to Tg and NTD multimers but did not affect the process of multimerization. Associating BiP with PDI did not enhance the ability of PDI to limit the formation of multimers produced by oxidation. However, when BiP and PDI were reacted together with the multimeric forms and for a longer time (48 h), BiP greatly increased the efficiency of PDI. Accordingly, these two molecular chaperones probably act sequentially on the reduction of the intermolecular disulfide bridges. In the thyroid, a similar process may also be effective and participate in limiting the amount of Tg multimers present in the colloid. These results suggest that extracellular molecular chaperones play a similar role to that occurring in the endoplasmic reticulum and, furthermore, take part in the control of multimerization and aggregation of proteins formed by oxidation.

The peripheral benzodiazepine receptor (PBR) has been implicated in growth control of various tumour models. Although colorectal cancers were found to overexpress PBR, the functional role of PBR in colorectal cancer growth has not been addressed to date. Using primary cell cultures of human colorectal cancers and the human colorectal carcinoma cell lines HT29, LS174T, and Colo320 DM we studied the involvement of PBR in the growth control and apoptosis of colorectal cancers. Both mRNA and protein expression of PBR were detected by RT-PCR and flow cytometry. Using confocal laser scanning microscopy and immunohistochemistry the PBR was localized in the mitochondria. The specific PBR ligands FGIN-1-27, PK 11195, or Ro5-4864 inhibited cell proliferation dose-dependently. FGIN-1-27 decreased the mitochondrial membrane potential, which indicates an early event in apoptosis. Furthermore, FGIN-1-27, PK 11195 or Ro5-4864 increased caspase-3 activity. In addition to their apoptosis-inducing effects, PBR ligands induced cell cycle arrest in the G 1/G 0-phase. Thus, our data demonstrate a functional involvement of PBR in colorectal cancer growth and qualify the PBR as a possible target for innovative therapeutic approaches in colorectal cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com

BACKGROUND While cytometry is widely used in the detection of cell proteins, its application to quantitative evaluation remains problematic when target proteins or receptors are weakly expressed in cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) is a technique whose sensitivity and specificity make it appropriate for analyzing nucleic acids and thus genes expressed in cells. Combining these two techniques, we developed a method to quantify the transcript expression of the peripheral cannabinoid receptor (CB2-r) in peripheral blood lymphocyte subpopulations and in tonsillar B-cell subpopulations. METHODS This strategy first involves quantitative RT-PCR performed kinetically, followed by enzyme detection of PCR products using an oligonucleotide probe sandwich-hybridization assay onto microplates. RESULTS B cells exhibit CB2-receptor mRNA levels 10 times higher than those of other lymphocyte subsets. Using this technique, we observed a modulation of CB2-r mRNA level following tonsillar B-cell differentiation. Lastly, this new technology was validated by comparing the mRNA levels of CB2-r with the expression of CB2-r proteins assayed by flow cytometry, using specific CB2-r antibody labelling. CONCLUSIONS This method allows precise measurement of the mRNA of CB2-r performed on cell numbers as low as 10(5) after sorting. Its performance, high accuracy, reproducibility, and reliability make it a valuable tool for assaying proteins weakly expressed in cells.

To get some insight on the in vitro effect of TSH on the expression of two thyroid specific antigens (thyroglobulin (Tg) and thyroid peroxidase (TPO)) on the cell surface of cultured human thyroid cells an indirect immunofluorescence-activated cell sorter (FACStar IV, Becton-Dickinson) was used. Only half of the cultures responded to TSH by increased surface expression of the thyroid specific antigen Tg. In these cells, TSH stimulation of TPO expression showed a difference in epitopes recognized by murine monoclonal antibodies. Epitopes of domain D, recognized by monoclonal antibody 1, 30, 40 and 53 which are not involved in autoimmunity, were unaffected by TSH stimulation, (n = 2-10). In contrast, TSH regulated the surface expression of the TPO epitopes recognized by monoclonal antibody 15, 18, 24 and 60, which are known to be related to the serum autoantibody binding domain of TPO, (n = 6-8; p < 0.05). This indicated that increased activity of the thyroid cells selectively stimulated the expression of autoantigenic epitopes on the cell surface and supports the concept that increased cellular activity might predispose to the autoimmune processes leading to autoimmune thyroid disease.

Using a cytoplasmic domain of the peripheral benzodiazepine receptor (PBR) as a bait in the yeast two-hybrid system, we have isolated a cDNA encoding a new protein that specifically interacts with PBR. We named it PRAX-1, for peripheral benzodiazepine receptor-associated protein 1. PRAX-1 is a 1857-amino acid protein, the sequence of which was structurally unrelated to any known proteins. The gene encoding PRAX-1 is located in the q22-q23 region of the long arm of the human chromosome 17. The PRAX-1 mRNA is 7.5 kilobase pairs, predominantly expressed in the central nervous system, pituitary gland, and thymus. At the protein level, we found the PRAX-1 as a single 220–250-kDa protein in the brain and in many different human cell lines tested using specific antibody raised against PRAX-1. Parallel analysis of the PRAX-1 mRNA and protein expression performed in mouse and rat gave similar results. Immunocytochemistry analysis carried out to define the distribution of the PRAX-1 protein in the rat brain showed that PRAX-1 was prevalent in the mesolimbic system, specially abundant in the CA1 subfield of the hippocampus. Exhibiting several domains involved in protein-protein interaction (three proline-rich domains, three leucine-zipper motifs, and an Src homology region 3-like domain), the PRAX-1 may be looked upon as a new adaptator protein. We show that both the Src homology region 3-like domain and a proline-rich domain in PRAX-1 are required for the interaction with PBR. PRAX-1 is a cytoplasmic protein that also partially colocalizes with PBR in the mitochondria, as determined by confocal microscopy and Western blotting. Altogether our observations support a model of interaction implicating PBR and this newly described protein, PRAX-1. As being the first cytoplasmic protein associated with PBR, PRAX-1 is a new tool that opens new fields for exploring PBR biological roles.

Antibodies (Ab) to thyroglobulin (Tg) are common in patients with autoimmune thyroid diseases, but it is currently unclear how Tg Ab are involved in the pathology of autoimmune thyroid disease. We have previously reported the isolation of immunoglobulin G (IgG)kappa and IgGlambda Fab from phage display combinatorial libraries from the cervical lymph node of a single Hashimoto's thyroiditis patient with a high anti-Tg titer. Sequence analysis of these Fab indicated a restricted heavy chain usage with a nonrestricted light chain usage, with Fab inhibiting the binding of patient Tg Ab by between 39% and 79%. Comparative mapping of nine each of these IgGkappa and IgGlambda Fab, and the patient serum from whom the Fab were derived, is described here, using a panel of 10 murine monoclonal antibodies (Mab) to human thyroglobulin (hTg). The Fab interacted principally with mAb defining the overlapping antigenic domains I and IV, previously characterized as the region recognized by the majority of patient serum Tg Ab. Tg Ab from serum of the patient from whom the Fab were derived were also directed at this region, suggesting that the Fab are representative of the Tg Ab present in this patient.

Thyroglobulin (Tg), the prothyroid hormone, is stored in the lumen of the thyroid follicles as soluble dimers and tetramers and insoluble multimers. Soluble Tg is well characterized with regards to structure and role, but insoluble Tg (i-Tg) is not. Here we show that i-Tg, multimerized through formation of disulfide and dityrosine bonds, has a higher iodine content than soluble Tg and no thyroid hormones. Furthermore, the size and the resistance of i-Tg to proteolytic enzymes implied a new mechanism by which thyrocytes may degrade this form of Tg. Using peroxidase and H2O2generating system, we found that about 80% of i-Tg was degraded and 24% of its iodine content was released. Our data point to a role for i-Tg in iodine storage and the involvement of TPO in i-Tg degradation and iodide release.

SR 31747A, defined as a sigma ligand, is a novel immunosuppressive agent that blocks proliferation of human and mouse lymphocytes. Using a radiolabeled chemical probe, we here purified a target of SR 31747A and called it SR 31747A-binding protein (SR-BP). Purified SR-BP retained its binding properties and migrated on SDS-polyacrylamide gel as a M r 28,000 protein. Cloning of the cDNA encoding human SR-BP shows an open reading frame for a 223-amino acid protein, which is homologous to the recently cloned sigma 1 receptor. Interestingly, the deduced amino acid sequence was found to be related to fungal C8-C7 sterol isomerase, encoded by the ERG2 gene. The ERG2 gene product has been identified recently as the molecular target of SR 31747A that mediates antiproliferative effects of the drug in yeast. Northern blot analysis of SR-BP gene expression revealed a single transcript of 2 kilobases which was widely expressed among organs, with the highest abundance in liver and the lowest abundance in brain. Subcellular localization analysis in various cells, using a specific monoclonal antibody raised against SR-BP, demonstrated that this protein was associated with the nuclear envelope. When studying the binding of SR 31747A on membranes from yeast expressing SR-BP, we found a pharmacological profile of sigma 1 receptors; binding was displaced by (+)-pentazocine, haloperidol, and (+)-SKF 10,047, with (+)-SKF 10,047 being a more potent competitor than (−)-SKF 10,047. Scatchard plot analysis revealed K d values of 7.1 nm and 0.15 nm for (+)-pentazocine and SR 31747A, respectively, indicating an affinity of SR-BP 50-fold higher for SR 31747A than for pentazocine. Additionally, we showed that pentazocine, a competitive inhibitor of SR 31747A binding, also prevents the immunosuppressive effect of SR 31747A. Taken together, these findings strongly suggest that SR-BP represents the molecular target for SR 31747A in mammalian tissues, which could be critical for T cell proliferation.

The lectin jacalin was shown to specifically stimulate CD4(+) lymphocytes. This lectin, which presents a peptide highly similar to a sequence of the HIV external glycoprotein, interacts with CD4 and is able to inhibit in vitro HIV infection. Since jacalin binds also CD8, its mitogenic specificity cannot exclusively be attributed to its interaction with CD4. We therefore hypothesized that the lectin could trigger signals specifically associated with CD4. Here we show that jacalin triggers IL2 gene transcription only in CD4(+) lymphocytes. In parallel, we show that numerous proteins are tyrosine phosphorylated in this cell subset while only a restricted number of them are phosphorylated in CD8(+) cells. Moreover, we show that the tyrosine kinase p56lck, which is associated with both CD4 and CD8, is activated only in CD4(+) lymphocytes, making this lectin a good model for the study of cell signaling triggered in this restricted subpopulation.

Patients with autoimmune thyroid disease frequently have high affinity antibodies to thyroid peroxidase (TPO), although the role they play in disease pathogenesis is not known. We have previously prepared 37 monoclonal anti-TPO IgGκ Fab fragments from two patients with Hashimoto’s thyroiditis and demonstrated the similarity of these Fab sequences to those published previously, mainly derived from patients with Graves’ disease. In this paper, we describe epitope mapping of these Fabs using a previously characterized panel of murine monoclonal antibody (mAb) and show that the Fabs bind to two neighboring epitopes on native TPO. Although the epitope-mapping method differs from that used to characterize previously published TPO-reactive Fab sequences, it indicates a similarly restricted response to neighboring epitopes in both Graves’ disease and Hashimoto’s thyroiditis. The epitope mapping included mAb 47, which binds to a linear TPO peptide of known sequence in addition to native TPO. Although TPO-reactive Fab did not inhibit the binding of mAb 47, mAb 47 did inhibit the binding of Fab, indicating the likely site of the immunodominant region on native TPO. These results confirm the restricted nature of TPO antibody and further delineate the immunodominant region of native TPO as defined by the mAb.

The purpose of this study was to evaluate the results of a fundoplication aimed to avoid complications of the Nissen procedure. The procedure combined a posterior hemifundoplication and a short circular fundoplication and their fixation to crura. A total of 67 consecutive patients were prospectively evaluated before and after surgery (median follow-up 24 months). Typical and atypical signs of reflux were present in 96.9% and 28.1% of cases, respectively, before operation versus 12.5% and 6.3% after. Patients alleged dysphagia in 26.6% of cases before surgery and 20.3% after. After operation belching and vomiting were impossible in 6.3% and 29.7% of cases, respectively. The pH test did not demonstrate any pathologic acid esophageal exposure in 93.3% cases after surgery. The mean duration of acid esophageal exposure was 45.1 ± 21.8% (10–100%) before operation versus 1.9 ± 4.9% (0–30%) after ( p < 0.001). The mean lower esophageal sphincter pressure increased from 12.4 ± 6.1 cm H 2 O (0–28 cm H 2 O) before operation to 20.5 ± 7.3 cm H 2 O (11–50 cm H 2 O) after ( p < 0.0001). The velocity of esophageal waves increased from 2.8 ± 1.1 cm/sec (1.2–5.5) before surgery to 3.1 ± 1.5 cm/sec (1.4–7.7 cm/sec) after ( p < 0.001). None of the Nissen complications were observed, and it was not necessary to reoperate any patient. In conclusion, this procedure is effective, improves esophageal motor activity, and prevents the occurrence of complications of the Nissen procedure. It does not, however, alleviate side effects.

This report describes the results of the third part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The aim of the present work was to establish the link between immunoreactivity and biological activity of human LH, thus allowing to determine the antigenic domains of the molecule involved in the induction of the biological effect. The relationship between immunoreactivity and electric charge of hLH was also studied. This work allowed to further apprehend hLH isomorphism and its role in discrepancies observed among hLH assays and clinical status. It also made the feasibility of measuring biologically active isoforms by an immunological method to be assessed. The effect of 36 mAb with known epitopic specificity, was evaluated on both hLH binding to rat membrane receptor and hLH induced production of testosterone by porcine Leydig cells. All the epitopes located on the beta subunit were found to be strongly involved in the biological activity whereas 4/9 and 10/18 epitopes present on the alpha subunit or specific for the holomolecule respectively appeared weakly involved. Assaying biological hLH using immunological method would require that mAb specific for all the epitopes involved in the receptor activation be tested, and thus appears presently unsuitable for routine clinical evaluation. In the previous work some LH immunoassays were found to underestimate LH concentrations (J. Endocrinol. Invest 1994, 17: 397-406 and 407-416). The mAb used in liquid phase in these kits were found in the present work to be directed against the domains of LH weakly involved in the activation of the receptor and would suggest that bioactive LH would be misevaluated by these kits. The immunoreactivity of hLH isoforms separated by isoelectric focusing (IEF) in liquid phase was also determined. IEF allowed to separate three groups of hLH isoforms but none of them exhibited a specific discriminating pattern of immunoreactivity when they were tested against a panel of mAb. It suggests that, in our experimental conditions, the electric charge and the immunoreactivity of hLH were not closely linked.

Based on the amino acid sequence deduced from the cloned human peripheral benzodiazepine receptor (PBR) gene, monoclonal antibody (Mab 8D7) was produced against the C-terminal fragment of the receptor. Immunoblot experiments, performed against purified PBR, indicated that the antipeptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence of the PBR. When mitochondrial membranes form PBR transfected yeast or from THP1 and U937 cells were used on immunoblot analysis, a high level of immunoreactivity was observed at 18 kDa, the PBR molecular mass deduced from cDNA, establishing the specificity of the antibody for the receptor. Moreover, binding experiments realized with intact mitochondria demonstrated that the immunogenic sequence was accessible to the antibody indicating that the C-terminal fragment of the PBR faces the cytosol. Using this Mab we developed a technique which allowed precise quantification of PBR density per cell. Furthermore, cellular localization studies by flow cytometric analysis and confocal microscopy on cell lines displaying different levels of PBR showed that Mab 8D7 was entirely colocalized with an antimitochondria Mab. 34 refs., 7 figs.

Two proteins with seven transmembrane-spanning domains typical of guanosine-nucleotide-binding-protein-coupled receptors have been identified as cannabinoid receptors; the central cannabinoid receptor, CB1, and the peripheral cannabinoid receptor, CB2, initially described in rat brain and spleen, respectively. Here, we report the distribution patterns for both CB1 and CB2 transcripts in human immune cells and in several human tissues, as analysed using a highly sensitive and quantitative PCR-based method. CB1 was mainly expressed in the central nervous system and, to a lower extent, in several peripheral tissues such as adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils. In contrast, the CB2 gene, which is not expressed in the brain, was particularly abundant in immune tissues, with an expression level 10-100-fold higher than that of CB1. Although CB2 mRNA was also detected in some other peripheral tissues, its level remained very low. In spleen and tonsils, the CB2 mRNA content was equivalent to that of CB1 mRNA in the central nervous system. Among the main human blood cell subpopulations, the distribution pattern of the CB2 mRNA displayed important variations. The rank order of CB2 mRNA levels in these cells was B-cells > natural killer cells >> monocytes > polymorphonuclear neutrophil cells > T8 cells > T4 cells. The same rank order was also established in human cell lines belonging to the myeloid, monocytic and lymphoid lineages. The prevailing expression of the CB2 gene in immune tissues was confirmed by Northern-blot analysis. In addition, the expression of the CB2 protein was demonstrated by an immunohistological analysis performed on tonsil sections using specific anti-(human CB2) IgG; this experiment showed that CB2 expression was restricted to B-lymphocyte-enriched areas of the mantle of secondary lymphoid follicles. These results suggest that (a) CB1 and CB2 can be considered as tissue-selective antigens of the central nervous system and immune system, respectively, and (b) cannabinoids may exert specific receptor-mediated actions on the immune system through the CB2 receptor.

International audience; Achieving an appropriate docosahexaenoic acid (DHA) status in the neonatal brain is an important goal of neonatal nutrition. We evaluated how different dietary fat matrices improved DHA content in the brains of both male and female rats. Forty rats of each gender were born from dams fed over gestation and lactation with a low a-linolenic acid (ALA) diet (0.4% of fatty acids) and subjected for 6 weeks after weaning to a palm oil blend-based diet (10% by weight) that provided either 1.5% ALA or 1.5% ALA and 0.12% DHA with 0.4% arachidonic acid or to an anhydrous dairy fat blend that provided 1.5% or 2.3% ALA. Fatty acids in the plasma, red blood cells (RBCs) and whole brain were determined by gas chromatography. The 1.5% ALA dairy fat was superior to both the 1.5% ALA palm oil blends for increasing brain DHA (14.4% increase, P

De Micco C, Zoro P, Garcia S, Skoog L, Tani EM, Carayon P, Henry J-F. Thyroid peroxidase immunodetection as a tool to assist diagnosis of thyroid nodules on fine-need aspiration biopsy. Eur J Endocrinol 1994;131:474–9. ISSN 0804–4643 In a previous work we have reported the presence in 96.9% of malignant and 4.2% of benign thyroid tumors of an immunological abnormality of the enzyme thyroid peroxidase, impeding the fixation of the anti-thyroid peroxidase monoclonal antibody termed "MoAb47". The present study has been designed to establish the ability of thyroid peroxidase immunodetection to assist the diagnosis of malignancy in fine-needle aspiration of thyroid nodules. The fixation of anti-thyroid peroxidase monoclonal antibody was investigated by immunohistochemistry on fine-needle aspirates of 150 surgically removed thyroid nodules (20 papillary carcinomas, five follicular carcinomas, 90 colloid adenomas, nine fetal adenoma, 13 atypical adenomas, five oncocytic adenomas, six Hashimoto's thyroiditis and two Graves' disease). The percentage of positive cells has been compared to the final histological diagnosis. In samples from 113/125 benign nodules 80–100% cells presented a positive immunoreaction, whereas all samples from malignant tumors yielded less than 80% positive cells. Benign nodules exhibiting less than 80% positive cells corresponded to three degenerative colloid nodules, five atypical follicular adenomas, two oncocytomas and two thyroiditis. According to results obtained in this series, with the value of 80% as the limit for discrimination between benign and highrisk nodules, the sensitivity of thyroid peroxidase staining for diagnosis of malignancy would be 100%, its specificity 90% and its overall accuracy 92%. Thyroid peroxidase staining with monoclonal antibody MoAb47 on fine-needle aspirates is a useful adjunct to conventional cytology for the investigation of patients with thyroid nodules. Catherine De Micco, Laboratoire d'Anatomie Pathologique, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France

Recent evidence indicates that thyroid autoimmune disorders are associated with the presence of circulating autoantibodies (aAb) with dual specificity for thyroglobulin (TG) and thyroperoxidase (TPO). The question of whether these aAb, called TGPO aAb, are of clinical relevance compared to TG and TPO aAb remains to be determined. The availability of purified preparations of human TG and TPO allowed the development of a specific and sensitive RIA for TGPO aAb in serum. In the present study, we compared levels of aAb that cross-react with both TG and TPO (TGPO aAb) and total TG and TPO aAb levels, respectively, in sera from 84 normal controls and 226 patients with various thyroid and autoimmune diseases, including nontoxic goiter (n = 50), toxic nodular goiter (n = 13), thyroid carcinoma (n = 20), primary idiopathic myxedema (n = 15), postpartum thyroiditis (n = 11), Hashimoto's thyroiditis (n = 38), pernicious anemia (n = 27), rheumatoid arthritis (n = 19), and insulin-dependent diabetes mellitus (n = 33). In addition, 16 patients with Hashimoto's thyroiditis were studied before therapy and after more than 3 months of treatment with L-T4. It was shown that TGPO aAb were generally, but not always, present in the serum of patients with Hashimoto's thyroiditis, which also contained TG and TPO aAb. In contrast, TGPO aAb were undetectable in normal controls (excepting a few cases reaching borderline levels) as well as in sera from the majority of the other patients tested. Selecting sera positive for TGPO and either TG or TPO aAb, a statistically significant correlation was found between TGPO and TG (n = 26; P < 0.005), but not TPO aAb. Interestingly, the TGPO aAb level significantly decreased in patients with Hashimoto's thyroiditis after hormonal therapy (P < 0.05), some of them shifting from TGPO aAb positive before treatment to negative after treatment. In conclusion, TGPO aAb determination distinguishes Hashimoto's patients from patients with either thyroid and/or autoimmune diseases. The specific presence of TGPO aAb in a subset of Hashimoto's patients and their variation during T4 therapy remain to be understood. This could give a clue to mechanisms of autoimmune thyroid disease.

Vascular smooth muscle cells (SMC) do not represent a homogeneous population (Schwartz et al., 1990, Am. J. Pathol. 136: 1417-1428). Cellular clones resistant to the antiproliferative activity of heparin were isolated from rat aortic SMC cultures (Pukac et al., 1990, Cell Regul., 1:435-443; San Antonio et al., 1993, Arterioscler. Thromb., 13:748-757) and from explant of human arterial restenotic lesions (Chan et al., 1993, Lancet, 341:341-342). We have shown in the present study that long-term treatment (growth medium supplemented with 200 micrograms/ml heparin, from the second to the tenth passage) of rat aortic SMC, without cell cloning, resulted in a significant loss of sensitivity to the growth inhibition by heparin and its derivatives. The heparin resistance was stable after growing cells for two passages in heparin-free medium, suggesting the selection of a particular phenotype. We tried to characterize these cells and to determine the causes of the resistance to the growth inhibition by heparin. Heparin-treated SMC (HT-SMC) were smaller than their control culture at the same passage, expressed less alpha-SM actin, and did not overgrow after reaching confluence. As in the heparin-resistant clones (San Antonio et al., 1993, Cell Regul., 1:435-443) expression of alpha-SM actin could be increased in HT-SMC by heparin addition before Western blotting. Heparin resistance was associated with a tenfold decrease in [3H]-heparin binding capacity (Bmax = 1.9 x 10(6) sites per cell) compared to control cultures (Bmax = 1.7 x 10(7) sites per cell), which was irreversible after growing the cells for two additional passages in heparin-free medium. We also investigated protein kinase C (PKC) in HT-SMC in terms of both enzymatic activity and protein expression (evaluated by [3H]-staurosporine and [3H]-phorbol-12,13-dibutyrate binding). We found that HT-SMC had only half the PKC activity and expression as control SMC. Therefore, long-term treatment of rat aortic SMC with heparin allowed the selection of a less differentiated subpopulation of cells, exhibiting low sensitivity to the growth inhibition by heparin, which could be related to the low capacity of binding heparin and to a lower PKC activity and/or expression.

This report describes the results of the second part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The whole study was designed to understand the causes of discrepancy among LH immunoassay methods. In the parent report we described the characteristics of LH monoclonal antibodies. In the present work we focused on the comparison of 11 commercially available monoclonal antibody based kits and one polyclonal antibody based RIA. Recovery experiments of the second IS 80/552 and of a related LH preparation showed that curve calibration differed among kits. The ratio between the LH recovery of the two most different kits was 2.30. LH concentrations were determined, using the 12 assay methods in the sera of prepubertal children (n = 46), normal women (n = 26) and men (n = 39) before and after LHRH stimulation, post-menopausal women (n = 29) and patients with renal failure (n = 71) or polycystic ovaries (n = 28). In children LH was detected in 0 to 80% of the sera depending on the kit. In adults, the mean LH concentration provided by the 12 kits varied from 6.0 to 13.55 IU/L showing a ratio of 2.26 between the two most different kits. A thorough statistical analysis allowed to distinguish two groups of kits. The first groups consisting of 6 kits provided results close to those obtained by the RIA. The 5 other kits misrecognized circulating LH in subjects with various clinical status. These 5 kits were the only ones to use, as labelled probes, monoclonal antibodies specific for the holohormone (anti-alpha beta) whereas the 6 other kits used, as labelled probes, monoclonal antibodies directed to the alpha (1 kit) or the beta-subunit (5 kits). In both groups, the coated monoclonal antibodies are directed to either the beta-subunit (1/6 and 2/5 kits) or the holohormone (5/6 and 3/5 kits). Taken together these data suggest that discrepancy among LH assay kits is related to variation in standard curve calibration and in epitope specificity of monoclonal antibodies used in the kits. These findings may prove to be instrumental to alleviate differences among LH assay methods.

This report describes the results of the first part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The whole study was designed to understand the causes of discrepancy among LH immunoassay methods. In the parent work, we studied the characteristics of 55 monoclonal antibodies to LH which allowed us to establish a detailed map of the antigenic surface of the hormone. In the present report we used this information to interpret the discrepancy in LH concentrations assayed with 12 different methods in 300 sera from subjects with various clinical conditions. The 55 monoclonal antibodies provided by 11 commercial companies were tested in various experiments: the apparent affinity of the antibodies was, generally but not always, higher for LH presented by a second antibody coated to plastic than for LH directly coated to plastic; 26% of the antibodies recognized the alpha subunit, 26% the beta subunit and 48% reacted only with the holomolecule (anti-alpha beta); only 28% of the antibodies were strictly specific for LH. Criss-cross experiments allowed us to distinguish 13 antigenic regions on the surface of LH: 6 were located on the alpha subunit, 3 on the beta subunit and 4 on the holomolecule. The monoclonal antibodies to the alpha beta regions further separated into 12 clusters of reactivity. Accordingly, LH appeared to exhibit at least 21 epitopes. Comparison of the immunoreactivity of various LH preparations indicated that highly purified pituitary LH and immunoaffinity purified urinary LH reacted similarly with the monoclonal antibodies and strongly differed from crude urinary LH. These data indicated that the immunoreactivity of an LH preparation depends mainly upon the degree of purification and not that much upon the origin of the preparation. The epitope specificity of the monoclonal antibodies used in 11 commercially available LH assay kits was also determined: 10 kits used at least one anti-alpha beta monoclonal antibody associated with an anti-beta monoclonal antibody in 7 cases or another anti-alpha beta monoclonal antibody in 3 cases; one kit used an anti-alpha monoclonal antibody associated with an anti-beta monoclonal antibody. None of the kits were strictly identical with regards to the epitope specificity of the monoclonal antibodies used.

A highly sensitive two-site enzyme immunoassay (Capcellia) was developed to determine the concentration of CD4 and CD8 molecules expressed on the surface of human T lymphocytes. This assay, performed in one step (20 min), involves the specific immunocapture of T lymphocytes and reaction of the CD4 or CD8 molecules with an enzyme-labeled monoclonal antibody (mAb). The results were expressed as molar concentrations of the T-cell markers on the basis of results obtained with calibrated CD4 and CD8 standards. The assay was sensitive enough to detect 0.4 pmol/L CD4 or 0.8 pmol/L CD8, which corresponded to approximately 20 x 10(6) CD4+ or CD8+ T cells per liter of blood. Mean concentrations in healthy adults were 17.2 pmol/L for CD4 and 22.1 pmol/L for CD8. The CD4 concentration was < 8 pmol/L in 50% of HIV-1-infected patients and in 95% of AIDS patients. Given the epitopic specificity of the mAb to CD4 we used, these values correspond to the concentration of CD4 molecules free of envelope glycoprotein (gp)120.

Thyroid-cytotoxic antibodies (thyroid-cytotoxic Abs) have been described in patients with autoimmune thyroiditis, but their role in the development of hypothyroidism remains to be clarified. In this study, we evaluated the pathogenetic role of thyroid-cytotoxic Abs in 20 patients with atrophic thyroiditis (idiopathic myxedema; AT) and 94 patients with goitrous Hashimoto's thyroiditis (HT). Among patients with HT, 27 were euthyroid (HT-E), 27 had subclinical hypothyroidism (HT-SH), and 40 had overt hypothyroidism (HT-H). Seventeen normal subjects and 8 patients with nonthyroidal illnesses were used as controls (C). To detect thyroid-cytotoxic Abs, human thyroid cells expressing thyroid peroxidase (TPO) were labeled with 51Cr and challenged with the immunoglobulin G (IgG) fraction of serum plus rabbit complement. The cytotoxic effect of IgGs was calculated as the percent specific lysis (% SL), taking into account the lytic effect of complement alone and the maximal lysis produced by a detergent. Most C-IgGs decreased the cytotoxic effect of complement (median % SL, -3.3). IgGs from hypothyroid patients with thyroiditis had a greater cytotoxic effect than C-IgGs, either as a whole group (P0.001), or when subdivided according to clinical diagnosis: HT-SH (median % SL, 4.8; P0.005), HT-H (%SL, 2.2; P0.0001), or AT (%SL, 0.9; P0.01). Among patients with HT, the lytic activity of IgGs from patients with subclinical and overt hypothyroidism was higher than that of IgGs from euthyroid patients (P0.05). The results of IgGs from euthyroid patients with HT (median % SL, -0.9) did not significantly differ from those of C-IgGs. By taking a cut-off over the upper range of % SL produced by C-IgGs (2), the prevalence of thyroid-cytotoxic Abs was 30% in AT, 59% in HT-SH, and 55% in HT-H. However, 37% of euthyroid patients with HT also had thyroid-cytotoxic Abs. No IgG containing TPO antibodies (TPOAb) at low titer (40(2)) was cytotoxic. However, the levels of thyroid-cytotoxic Abs did not correlate with TPOAb titers, and preabsorption with TPO only partially abolished the lytic effect of some HT-IgG. These findings suggest that TPO is a target of thyroid-cytotoxic Abs, but other thyroid antigens are also involved in the cytotoxic reaction.(ABSTRACT TRUNCATED AT 400 WORDS)

In order to study the activation of suppressor T lymphocytes by thyroid-specific antigens in autoimmune thyroid disease (AITD), we have investigated the effects of the organ-specific antigens, thyroperoxidase (TPO), thyroglobulin (Tg), and thyroid microsomal antigen (TMc), as well as renal microsomes (RMc) as a control antigen, on the activation of suppressor T lymphocytes; this was accomplished by measuring major histocompatibility complex class II (HLA-DR) expression on their surfaces by flow cytometric analysis. Peripheral blood mononuclear cells (PBMC), obtained from 33 patients with Graves' disease (GD), 26 with Hashimoto's thyroiditis (HT), 5 with nontoxic nodular goiter (NTG), and 30 normal persons (N), were cultured for 7 days in the presence or absence of TPO, Tg, or RMc at final concentration of 10, 100, and 1000 ng/ml. Cultured cells were stained with fluorescent-conjugated monoclonal antibodies (anti-CD8, anti-CD11b, and anti-HLA-DR), and the activation of CD8+ and CD8+CD11b+ (pure suppressor) T cells by the antigens was analyzed on a flow cytometer. In the absence of antigen, i.e., the autologous mixed lymphocyte reaction (AMLR), CD8+ and CD8+CD11b+ T lymphocytes from patients with GD and HT showed significantly lower activation as compared to N. We measured the Stimulation Index (Sl) of activated T lymphocytes to compare antigen-specific activation between CD8+ and CD8+CD11b+ cells from normal persons and patients. With stimulation of 100 and/or 1000 ng/mL of TPO or Tg, Sl of activated CD8+ cells was significantly (p < 0.05 to 0.01) lower in GD and HT as compared with N.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of thyroglobulin (TG) was investigated by immunohistochemistry with one polyclonal and three distinct monoclonal anti-TG antibodies in 19 primary and 15 metastatic medullary thyroid carcinoma (MTC) cases. In two primary tumors possessing a mixed trabecular and follicular growth pattern, a significant number of tumor cells were positive with the four antibodies in all parts of the tumor; these tumors were considered to be genuine mixed follicular and parafollicular tumors. The 17 other primary tumors looked like "classical" (ie, predominantly compact) MTC. Eight contained tubular or microfollicle-like structures that were consistently TG-negative. Eight contained residual entrapped normal follicles positive with the four anti-TG antibodies. In six cases isolated foci of tumor cells reacted with polyclonal antibody or monoclonal antibody 11. In 10 cases circulating TG stained positively with polyclonal antibody in vascular channels, five cases stained positively for TG with monoclonal antibody 11, four cases stained positively for TG with monoclonal antibody 7, and three cases stained positively for TG with monoclonal antibody 1. In metastases TG was found in tumor cells in only one instance, but positive reactions were obtained in vascular channels and macrophages in several cases. We conclude from this study that there is no genuine secretion of TG in classical, compact MTC, which differs morphologically and functionally from mixed follicular and parafollicular tumors secreting both TG and thyrocalcitonin.

To investigate T-cell sensitization to thyroid autoantigens in autoimmune thyroid disease (AITD), purified soluble human thyroglobulin (Tg) and thyroid peroxidase (TPO) were used. Peripheral blood mononuclear cells (PBMC) as well as CD8-depleted, CD4-enriched PBMC ("selected" PBMC) from 9 patients with Graves' disease (GD), 13 Hashimoto's thyroiditis (HT) and 10 healthy subjects, were cultured for 6 days with or without varying concentrations (0.1, 1.0 and 5.0 micrograms/ml, respectively) of Tg or TPO and their responses were evaluated using the 3H-thymidine incorporation assay. Total PBMC as well as selected PBMC from GD and HT responded to both TPO and Tg, but normal PBMC did not. This induction was more marked in "selected" PBMC; on the other hand, CD8 depletion did not permit normal PBMC to respond to either antigen. However, reactivity of selected AITD PBMC to Tg differed from that of TPO. Two way analysis of variance showed that the proliferative response was significantly greater with Tg than with TPO, (again particularly notable with the "selected" PBMC) in both GD and HT. There was no difference between control and AITD preparations when an irrelevant (renal microsomal) antigen was employed. Taken together with our previous report that CD4 cells were induced by TPO even when cultured with CD8 cells, it is evident that suppressor CD8 cells do play a role in CD4 cells from proliferating against Tg and TPO; however their function alone or in combination with suppressor-inducer CD4 cells is partially disturbed, so that T cell sensitization to Tg and TPO can be identified in the AITD PBMC.

The present work was aimed at studying the interaction of autoantibodies (aAb) and monoclonal antibodies (mAb) with the N-terminal thyroid hormone forming site of human thyroglobulin (TG). Obtained by CNBr treatment of TG, the peptide (22 kDa) containing the complete major hormonogenic site of human TG was purified in three forms according to the degree of iodination and iodotyrosine coupling: the native, poorly iodinated form (n-22K), the iodinated form containing iodotyrosine but not hormone residues (i-22K) and the form containing thyroid hormone (t-22K). We report that aAb from some patients with autoimmune thyroid diseases showed significant binding to both iodinated 22 kDa forms. Furthermore, a detailed study using mAb evidenced that iodination and coupling induced changes in the antigenicity of the molecule, some occurring without direct implication of iodine or thyroid hormones. The 22 kDa peptide appears as an interesting model to study the antigenic changes induced by the structural modifications in the course of thyroid hormone synthesis. This observation could be relevant to the etiopathogenic process of thyroid autoimmune diseases.