Design and characterization of a triazole-based growth hormone secretagogue receptor modulator inhibiting the glucoregulatory and feeding actions of ghrelin.

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor that regulates essential physiological functions. In particular, activation of GHSR in response to its endogenous agonist ghrelin promotes food intake and blood glucose increase. Therefore, compounds aimed at blocking GHSR signaling constitute potential options against obesity-related metabolic disorders. We have previously developed potent ligands of GHSR based on a triazole scaffold. Here, we report a new 3,4,5-trisubstituted 1,2,4-triazole compound, named JMV 6616, that potently blocks GHSR activity in vitro and in vivo. Specifically, in HEK293T cells JMV 6616 behaves as an inverse agonist since it binds to GHSR and inhibits its ghrelin-independent signaling. Accordingly, using purified labeled GHSR assembled into lipid nanodiscs we found that JMV 6616 decreases GHSR-catalyzed G protein activation and stabilizes an inactive receptor conformation. Importantly, JMV 6616 also acts on native GHSR since it blocks the insulinostatic effect of ghrelin in pancreatic islets. In mice, JMV 6616 inhibits blood glucose-raising effects of ghrelin treatment and the orexigenic actions of acute ghrelin administration. Together, our data suggest that this triazole-derived modulator of GHSR holds promise to mitigate several pathological features associated with eating and metabolic disorders.

Reduced production of isoprostanes by peri-pancreatic adipose tissue from Zucker fa/fa rats as a new mechanism for ß-cell compensation in insulin resistance and obesity.

During early stages of type 2 diabetes, named prediabetes, pancreatic ß-cells compensate for insulin resistance through increased insulin secretion in order to maintain normoglycemia. Obesity leads to the development of ectopic fat deposits, among which peri-pancreatic white adipose tissue (pWAT) can communicate with ß-cells through soluble mediators. Thus we investigated whether pWAT produced oxygenated lipids, namely isoprostanes and neuroprostanes and whether they can influence ß-cell function in obesity. In the Zucker fa/fa rat model, pWAT and epididymal white adipose tissue (eWAT) displayed different inflammatory profiles. In obese rats, pWAT, but not eWAT, released less amounts of 5-F2t-isoprostanes, 15-F2t-isoprostanes, 4-F4t-neuroprostanes and 10-F4t-neuroprostane compared to lean animals. These differences could be explained by a greater induction of antioxidant defenses enzymes such as SOD-1, SOD-2, and catalase in pWAT of obese animals compared to eWAT. In addition, sPLA2 IIA, involved in the release of isoprostanoids from cellular membranes, was decreased in pWAT of obese animals, but not in eWAT, and may also account for the reduced release of oxidized lipids by this tissue. At a functional level, 15-F2t-isoprostane epimers, but not 5-F2t-isoprostanes, were able to decrease glucose-induced insulin secretion in pancreatic islets from Wistar rats. This effect appeared to be mediated through activation of the thromboxane A2 receptor and reduction of cAMP signaling in pancreatic islets. In conclusion, through the removal of an inhibitory tone exerted by isoprostanes, we have shown, for the first time, a new mechanism allowing ß-cells to compensate for insulin resistance in obesity that is linked to a biocommunication between adipose tissue and ß-cells.

Liver-Expressed Antimicrobial Peptide 2 antagonizes the insulinostatic effect of ghrelin in rat isolated pancreatic islets.

The hormone ghrelin is the endogenous agonist of the G protein-coupled receptor (GPCR) termed growth hormone secretagogue receptor (GHSR). Ghrelin inhibits glucose-stimulated insulin secretion by activating pancreatic GHSR. Recently, Liver-Expressed Antimicrobial Peptide 2 (LEAP2) was recognized as an endogenous GHSR ligand that blocks ghrelin-induced actions. Nonetheless, the effect of LEAP2 on glucose-stimulated insulin secretion from pancreatic islets is unknown. We aimed at exploring the activity of LEAP2 on glucose-stimulated insulin secretion. Islets of Langerhans isolated from rat pancreas were exposed to glucose in the presence or in the absence of LEAP2 and ghrelin and then insulin secretion was assayed. LEAP2 did not modulate glucose-stimulated insulin secretion. However, LEAP2 blocked the insulinostatic action of ghrelin. Our data show that LEAP2 behaves as an antagonist of pancreatic GHSR.

Development of Nonpeptidic Inverse Agonists of the Ghrelin Receptor (GHSR) Based on the 1,2,4-Triazole Scaffold.

GHSR controls, among others, growth hormone and insulin secretion, adiposity, feeding, and glucose metabolism. Therefore, an inverse agonist ligand capable of selectively targeting GHSR and reducing its high constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In this context, we present a study that led to the development of several highly potent and selective inverse agonists of GHSR based on the 1,2,4-triazole scaffold. We demonstrate that, depending on the nature of the substituents on positions 3, 4, and 5, this scaffold leads to ligands that exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. Thanks to an in vivo evaluation, we also show that one of the most promising ligands not only exerts an effect on insulin secretion in rat pancreatic islets but also affects the orexigenic effects of ghrelin in mice.

N-Terminal Liver-Expressed Antimicrobial Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the Ghrelin Receptor.

The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.

Adipose tissue derived-factors impaired pancreatic ß-cell function in diabetes.

Inflammatory factors produced and secreted by adipose tissue, in particular peri-pancreatic adipose tissue (P-WAT), may influence pancreatic ß-cell dysfunction. Using the ZDF Rat model of diabetes, we show the presence of infiltrating macrophage (ED1 staining) on pancreatic tissue and P-WAT in the pre-diabetes stage of the disease. Then, when the T2D is installed, infiltrating cells decreased. Meanwhile, the P-WAT conditioned-medium composition, in terms of inflammatory factors, varies during the onset of the T2D. Using chemiarray technology, we observed an over expression of CXCL-1, -2, -3, CCL-3/MIP-1α and CXCL-5/LIX and TIMP-1 in the 9 weeks old obese ZDF pre-diabetic rat model. Surprisingly, the expression profile of these factors decreased when animals become diabetic (12 weeks obese ZDF rats). The expression of these inflammatory proteins is highly associated with inflammatory infiltrate. P-WAT conditioned-medium from pre-diabetes rats stimulates insulin secretion, cellular proliferation and apoptosis of INS-1 cells. However, inhibition of conditioned-medium chemokines acting via CXCR2 receptor do not change cellular proliferation apoptosis and insulin secretion of INS-1 cells induced by P-WAT conditioned-medium. Taken together, these results show that among the secreted chemokines, increased expression of CXCL-1, -2, -3 and CXCL-5/LIX in P-WAT conditioned-medium is concomitant with the onset of the T2D but do not exerted a direct effect on pancreatic ß-cell dysfunction.

Possible protective effect of membrane lipid rafts against interleukin-1ß-mediated anti-proliferative effect in INS-1 cells.

We recently reported that pancreatic islets from pre-diabetic rats undergo an inflammatory process in which IL-1ß takes part and controls ß-cell function. In the present study, using the INS-1 rat pancreatic ß-cell line, we investigated the potential involvement of membrane-associated cholesterol-enriched lipid rafts in IL-1ß signaling and biological effects on insulin secretion, ß-cell proliferation and apoptosis. We show that, INS-1 cells exposure to increasing concentrations of IL-1ß leads to a progressive inhibition of insulin release, an increase in the number of apoptotic cells and a dose-dependent decrease in pancreatic ß-cell proliferation. Disruption of membrane lipid rafts markedly reduced glucose-stimulated insulin secretion but did not affect either cell apoptosis or proliferation rate, demonstrating that membrane lipid raft integrity is essential for ß-cell secretory function. In the same conditions, IL-1ß treatment of INS-1 cells led to a slight further decrease in insulin secretion for low concentrations of the cytokine, and a more marked one, similar to that observed in normal cells for higher concentrations. These effects occurred together with an increase in iNOS expression and surprisingly with an upregulation of tryptophane hydroxylase and protein Kinase C in membrane lipid rafts suggesting that compensatory mechanisms develop to counteract IL-1ß inhibitory effects. We also demonstrate that disruption of membrane lipid rafts did not prevent cytokine-induced cell death recorded after exposure to high IL-1ß concentrations. Finally, concerning cell proliferation, we bring strong evidence that membrane lipid rafts exert a protective effect against IL-1ß anti-proliferative effect, possibly mediated at least partly by modifications in ERK and PKB expression/activities. Our results 1) demonstrate that IL-1ß deleterious effects do not require a cholesterol-dependent plasma membrane compartmentalization of IL-1R1 signaling and 2) confer to membrane lipid rafts integrity a possible protective function that deserves to be considered in the context of inflammation and especially T2D pathogenesis.

IL-1ß and TSH disturb thyroid epithelium integrity in autoimmune thyroid diseases.

Pro-inflammatory cytokines such as IL-1ß and TNFα are known to affect thyroid function. They stimulate IL-6 secretion and modify epithelium integrity by altering junction proteins. To study the role of cytokines on thyroid epithelia tightness in autoimmune thyroid diseases (AITD), we analyzed the expression profiles of junction proteins (ZO-1, Claudin, JAM-A) and cytokines in human thyroid slices and also investigated the effect of IL-1ß on the epithelium integrity in primary cultures of human thyrocytes. Junction proteins expression (ZO-1, Claudin, JAM-A) has been analyzed by immunohistochemistry on thyroid slices and by Western blot on membrane proteins extracted from thyrocytes of patients suffering from Graves and Hashimoto diseases. The high expression of junction proteins we found on Graves' disease thyroid slices as well as in cell membrane extracts acknowledges the tightness of thyroid follicular cells in this AITD. In contrast, the reduced expression of JAM and ZO-1 in thyroid cells from patients suffering from Hashimoto thyroiditis is in agreement with the loss of thyroid follicular cell integrity that occurs in this pathology. Concerning the effects on epithelium integrity of TSH and of the pro-inflammatory cytokine IL-1ß in primary cultures of human thyroid cells, TSH appeared able to modify JAM-A localization but without any change in the expression levels of JAM-A, Claudin and ZO-1. Inversely, IL-1ß provoked a decrease in the expression of- and a redistribution of both, Claudin and ZO-1 without modifying the expression and sub-cellular distribution patterns of JAM-A in thyroid cells. These results demonstrate (i) that Hashimoto's- and Graves' diseases display different junction proteins expression patterns with a loss of epithelium integrity in the former and (ii) that IL-1ß modifies thyroid epithelial tightness of human thyrocytes by altering the expression and localization of junction proteins. Therefore, IL-1ß could play a role in the pathogenesis of thyroid autoimmunity.

IL-1ß a potential factor for discriminating between thyroid carcinoma and atrophic thyroiditis.

Interactions between cytokines and others soluble factors (hormones, antibodies...) can play an important role in the development of thyroid pathogenesis. The purpose of the present study was to examine the possible correlation between serum cytokine concentrations, thyroid hormones (FT4 and TSH) and auto-antibodies (Tg and TPO), and their usefulness in discriminating between different thyroid conditions. In this study, we investigated serum from 115 patients affected with a variety of thyroid conditions (44 Graves' disease, 17 Hashimoto's thyroiditis, 11 atrophic thyroiditis, 28 thyroid nodular goitre and 15 papillary thyroid cancer), and 30 controls. Levels of 17 cytokines in serum samples were measured simultaneously using a multiplexed human cytokine assay. Thyroid hormones and auto-antibodies were measured using ELISA. Our study showed that IL-1ß serum concentrations allow the discrimination between atrophic thyroiditis and papillary thyroid cancer groups (p = 0.027).

Nonviral delivery of small interfering RNA into pancreas-associated immune cells prevents autoimmune diabetes.

The development of small interfering RNA (siRNA) for the treatment of human disorders has been often hampered by their low transfection efficiency in vivo. In order to overcome this major drawback, various in vivo siRNA transfection methods have been developed. However, their capacity to transfect immune or insulin-producing ß-cells within the pancreas for the treatment of autoimmune diabetes remains undetermined. We found that lipid- or polyethylenimine-based delivery agents were efficient to address siRNA molecules within pancreas-associated antigen-presenting cells (APCs) (but not ß-cells) and particularly a CD11b(+) cell population comprising both CD11b(+)CD11c(neg) macrophages and CD11b(+)CD11c(+) dendritic cells. However, the route of administration and the carrier composition greatly affected the transfection efficacy. Therapeutically, we showed that early (starting at 6-week-old) short-course treatment with lipid/Alox15-specific siRNA complex promoted long-term protection from type 1 diabetes (T1D) in wild-type (WT) nonobese diabetic (NOD) mice. Alox15 downregulation in pancreas-associated CD11b(+) cells significantly upregulated a variety of costimulatory molecules and particularly the programmed death 1 ligand 1 (PD-L1) pathway involved in tolerance induction. Concomitantly, we found that regulatory T cells were increased in the pancreas of lipid/Alox15 siRNA-treated NOD mice. Collectively, our data provide new insights into the development of siRNA-based therapeutics for T1D.

Human in-cell scFv library from infiltrating B cell.

The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Although useful for the production of recombinant antibodies, this method is not totally adapted to the study of the antibody repertoire formed in vivo during, for example, autoimmune diseases.We describe here, the use of the in-cell PCR together with Cre-recombination applied to human B cells to obtain in situ pairing of the variable (V) region genes of the immunoglobulin heavy (H) and light (L) chains. Our method is based on amplification and recombination of the VH and VL genes within CD19+ B cells isolated from human tissue. Nested primers were designed to amplify the known major human VH and VL gene families. After reverse transcription PCR and three rounds of PCR including recombination between VH and VL using the Cre-loxP system, the 800-bp band corresponding to scFv was cloned and human scFv fragments selected.This in-cell amplification, association, and scFv selection procedure is a potentially useful tool for the study of antibody repertoire and the VH/VL pairing that occurs during the diseases' process.

Excessive food intake, obesity and inflammation process in Zucker fa/fa rat pancreatic islets.

Inappropriate food intake-related obesity and more importantly, visceral adiposity, are major risk factors for the onset of type 2 diabetes. Evidence is emerging that nutriment-induced ß-cell dysfunction could be related to indirect induction of a state of low grade inflammation. Our aim was to study whether hyperphagia associated obesity could promote an inflammatory response in pancreatic islets leading to ß-cell dysfunction. In the hyperphagic obese insulin resistant male Zucker rat, we measured the level of circulating pro-inflammatory cytokines and estimated their production as well as the expression of their receptors in pancreatic tissue and ß-cells. Our main findings concern intra-islet pro-inflammatory cytokines from fa/fa rats: IL-1ß, IL-6 and TNFα expressions were increased; IL-1R1 was also over-expressed with a cellular redistribution also observed for IL-6R. To get insight into the mechanisms involved in phenotypic alterations, abArrays were used to determine the expression profile of proteins implicated in different membrane receptors signaling, apoptosis and cell cycle pathways. Despite JNK overexpression, cell viability was unaffected probably because of decreases in cleaved caspase3 as well as in SMAC/DIABLO and APP, involved in the induction and amplification of apoptosis. Concerning ß-cell proliferation, decreases in important cell cycle regulators (Cyclin D1, p35) and increased expression of SMAD4 probably contribute to counteract and restrain hyperplasia in fa/fa rat islets. Finally and probably as a result of IL-1ß and IL-1R1 increased expressions with sub-cellular redistribution of the receptor, islets from fa/fa rats were found more sensitive to both stimulating and inhibitory concentrations of the cytokine; this confers some physiopathological relevance to a possible autocrine regulation of ß-cell function by IL-1ß. These results support the hypothesis that pancreatic islets from prediabetic fa/fa rats undergo an inflammatory process. That the latter could contribute to ß-cell hyperactivity/proliferation and possibly lead to progressive ß-cell failure in these animals, deserves further investigations.

SLC26A4 expression among autoimmune thyroid tissues.

CONTEXT: The PDS gene (SLC26A4) is responsible for Pendred syndrome (PS). Genetic analysis of PDS using Tunisian samples showed evidence for linkage and association with autoimmune thyroid diseases (AITD) emergence. In addition, the PDS gene product, pendrin, was recently identified as a novel autoantigen in Graves' disease (GD) or Hashimoto thyroiditis (HT) patients' sera. OBJECTIVE: The aim of this study was to quantify the PDS gene expression and to evaluate the pendrin in vivo and in vitro immunolocalisation. PATIENTS: A total of 52 thyroid gland tissue samples (22 GD, 11 HT, 5 multinodular goiter (MNG), 3 normal thyroid tissues, 8 papillary thyroid carcinoma (PTC), 1 follicular thyroid carcinoma (FTC) and 2 medullar thyroid carcinoma (MTC)) were explored. METHOD: PDS and pendrin expression levels were determined using quantitative RT-PCR and immuno-detection methods. TSH and thyroglobulin (Tg) effects on pendrin expression were investigated by immunofluorescence on primary cell culture from GD thyroid tissues. RESULTS: The relative quantification using PDS transcript level among GD thyroid tissues was increased compared to normal thyroid tissues used as calibrator (mean: 27.17-fold higher than normal thyroid tissues). However, thyroids with HT, carcinoma and MNG showed a decrease expression level (means: 92.05-, 77.68-, 14.3-fold lower than normal thyroid tissues, respectively). These results were confirmed by immunoanalysis. Immunofluorescence results showed an apical and a cytoplasmic pendrin localisation on GD thyroid tissues and a marked pendrin expression reduction on HT thyroid tissues. GD primary cell cultures under TSH and Tg stimulation showed a trafficking improvement of pendrin apical localisation. CONCLUSIONS: Our data point to the presence of a relation between SLC26A4 expression in AITD and thyroid function.

Absence of anti-pendrin auto-antibodies in the sera of Tunisian patients with autoimmune thyroid diseases.

BACKGROUND: We previously demonstrated that the PDS gene is involved in the genetic susceptibility to autoimmune thyroid diseases (AITD) in Tunisia. In the same population, we now investigated the presence of anti-pendrin auto-antibodies (aAbs) in AITD patients' sera. METHODS: Thirty seven Tunisian AITD patients and 19 healthy subjects from families previously linked to the PDS gene, 75 unrelated patients and 20 healthy unrelated subjects were included in our study. The detection of anti-pendrin aAbs in patients' sera was performed by ELISA using membrane protein extracts of CHO cells expressing pendrin (CHO-hPDS) and by immunofluorescence using transient COS-7 cells expressing a GFP tagged pendrin. CHO cells transfected with human TPO in the same ELISA conditions were used as positive control. RESULTS: The majority of AITD patients' sera were positive for the presence of anti-TPO aAbs. In contrast, no reactivity was detected with CHO-hPDS membrane protein extracts. Likewise, no significant immunostaining was found on transfected COS-7cells upon exposure to patients' and controls' sera. CONCLUSIONS: Our data point to the absence of anti-pendrin aAbs in Tunisian AITD patients' sera.

Antithyroperoxidase antibody-dependent cytotoxicity in autoimmune thyroid disease.

CONTEXT: Thyroid antibody-dependent cytotoxicity has been reported in autoimmune thyroid disease (AITD). Indeed, the role of thyroperoxidase (TPO) autoantibodies (aAbs) in complement-mediated damage by binding to TPO expressed on the surface of human thyroid cells was demonstrated, whereas their activity in antibody-dependent cell cytotoxicity (ADCC) is not well established. OBJECTIVE: The aim of this study was to define the partners involved in antibody and complement-dependent cytotoxicity (CDC) in AITD and characterize which effector cells are involved in cytotoxicity mediated by anti-TPO aAbs using a chromium release assay. RESULTS: The relative capability of anti-TPO aAbs to mediate ADCC using human thyroid cells in culture varies from 11 to 74.5%, depending on the effectors cells used. The human monocyte cell line HL60 gives a better lysis than the THP-1 cell line as effector cells. It seems obvious that the mechanism of ADCC is mediated quite exclusively by FcgammaRI. Indeed, the two effector cell lines differ by the level of the FcgammaRI expression (91.83% for HL-60 cells and 22.55%t for the THP-1). In addition to ADCC, the anti-TPO aAbs mediate the destruction of thyrocytes by CDC (56%). CONCLUSIONS: These results demonstrate that anti-TPO aAbs can damage cultured thyroid cells by ADCC and CDC mechanisms. The monocytes, via their FcgammaRI, are important effector cells in ADCC mediated by anti-TPO aAbs and may contribute with T cells to the destruction of thyroid gland in AITD.

Association analysis of interleukin gene polymorphisms in autoimmune thyroid diseases in the Tunisian population.

Autoimmune thyroid diseases (AITDs), including Graves' disease (GD) and autoimmune hypothyroidism (AH), are inherited as complex traits. Among the genes contributing to AITDs susceptibility are genes of the IL-1 family. IL-1 regulates T and B lymphocyte maturation, including the induction of several cytokines and cytokine receptors. Therefore, disturbances of this balance may not only play a role in inflammation but also in the pathogenesis of autoimmunity. In order to investigate genetic association of IL-1 gene polymorphisms with AITDs, we performed both a familial study in a large Tunisian pedigree with high prevalence of AITDs (64 patients and 176 controls), and a case-control study (131 GD unrelated patients and 225 healthy controls). PCR and PCR-RFLP methods were used to analyse respectively a VNTR in the IL-1RN gene and three SNPs in both IL-1B genes (-511 C/T and +3954 C/T) and IL-1A (-889 C/T). The family-based association study showed an association of the IL-1B+3954 C/T polymorphism (p=0.02) and two haplotypes IL-1RN*3/C/T/T and IL-1RN*1/C/T/T (p=0.009 and p=0.047 respectively) with AITDs. The case-control study is the first study revealing a significant association of the IL-1A-889 C/T polymorphism (chi2=10.23; p=0.0014) with susceptibility to GD. Our data suggest that the IL-1 gene cluster may harbour susceptibility genes for AITDs and GD pathogenesis in the Tunisian population.

Protein inhibitor of neuronal nitric oxide synthase (PIN) is a new regulator of glucose-induced insulin secretion.

We previously showed that pancreatic beta-cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now provide evidence that the endogenous protein inhibitor of nNOS (PIN) is expressed in rat pancreatic islets and INS-1 cells. Double-immunofluorescence studies showed a colocalization of PIN with both nNOS and myosin Va in insulin-secreting beta-cells. Electron microscopy studies confirmed that PIN is mainly associated with insulin secretory granules and colocated with nNOS in the latter. In addition, PIN overexpression in INS-1 cells enhanced glucose-induced insulin secretion, which is only partly reversed by addition of an NO donor, sodium nitroprusside (SNP), and unaffected by the inhibitor of cytochrome c reductase activity, miconazole. In contrast, the pharmacological inhibitor of nNOS, Nomega-nitro-l-arginine methyl ester, amplified glucose-induced insulin secretion, an effect insensitive to SNP but completely normalized by the addition of miconazole. Thus, PIN insulinotropic effect could be related to its colocalization with the actin-based molecular motor myosin Va and as such be implicated in the physiological regulation of glucose-induced insulin secretion at the level of the exocytotic machinery.

The key residues in the immunodominant region 353-363 of human thyroid peroxidase were identified.

Auto-antibodies (aAbs) to thyroid peroxidase (TPO) interact with a restricted immunodominant region (IDR) divided into two overlapping regions A and B. Among the five major regions structuring the IDR/B, regions 210-225, 353-363, 549-563, 713-720 and 766-775, region 353-363 constitutes an important anchor point for the binding of TPO-specific aAbs in sera from Hashimoto's and Graves' patients. We combined site-directed mutagenesis and expression of TPO mutants in stably transfected CHO cells to precisely define the critical residues in that region. By using flow cytometry and ELISA, we identified four amino acid residues, H353, D358, S359 and R361, that contribute to the interaction between human TPO and anti-TPO aAbs. This identification of these contributing amino acid residues in the IDR allowed us to more precisely depict contours of the IDR.

Localization of the immunodominant region on human thyroid peroxidase in autoimmune thyroid diseases: an update.

Recent studies in the field of autoimmune thyroid diseases have largely focused on the delineation of B-cell auto-epitopes recognized by the main autoantigens to improve our understanding of how these molecules are seen by the immune system. Among these autoantigens which are targeted by autoantibodies during the development of autoimmune thyroid diseases, thyroid peroxidase is a major player. Indeed, high amounts of anti-thyroid peroxidase autoantibodies are found in the sera of patients suffering from Graves' disease and Hashimoto's thyroiditis, respectively hyper and hypothyroidism. Since anti-thyroid peroxidase autoantibodies from patients'sera mainly recognize a discontinuous immunodominant region on thyroid peroxidase and due to the complexity of the three dimensional structure of human thyroid peroxidase, numerous investigations have been necessary to closely localize this immunodominant region. The aim of the present review is to summarize the current knowledge regarding the localization of the immunodominant region recognized by human thyroid peroxidase-specific autoantibodies generated during the development of autoimmune thyroid diseases.

New insights into the conformational dominant epitopes on thyroid peroxidase recognized by human autoantibodies.

Human anti-thyroperoxidase (TPO) autoantibodies (aAbs) are a major hallmark of autoimmune thyroid diseases. Their epitopes are discontinuous and mainly restricted to an immunodominant region (IDR) consisting of two overlapping regions (IDR/A and B). To shed light on the relationship between these regions, we first performed competitive studies using all available reference anti-TPO antibodies. Interestingly, we showed that human IDR/A- and B-specific anti-TPO aAbs recognized essentially the same regions on the TPO molecule. However, our data also indicated that IDR/A-specific human aAbs strongly recognized the region containing residues 599-617, whereas the IDR/B-specific aAbs bind to several regions as well as region 599-617. Next, we scanned this key region to identify the residues involved in the immunodominant autoepitope. Using peptide spot technology together with competitive ELISA experiments, we demonstrated that residues (604)ETP-DL(609) play a major role in the anti-peptide P14 epitope and that IDR/A-specific human anti-TPO aAbs, either expressed as recombinant Fab or obtained from Graves' disease patients, specifically recognize the sequences (597)FCGLPRLE(604) and (611)TAIASRSV(618). All together our data emphasize that both the IDRs involve the same surface area on human TPO, but the differential usage of one or the other regions leads to different inhibition patterns in competitive experiments. In conclusion, our data help to resolve the long-sought issue on the molecular immunology of the two IDRs on TPO and provide new clues to design efficient peptides that may be part of a combinatorial treatment aiming at delaying development of autoimmune thyroiditis when used prophylactically.