Aims/hypotheses: Chronic hyperglycaemia in-

Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmann's syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time-and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dosedependent increase in migration (up to 3-4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.

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Introduction The G-protein-coupled receptor 54 (GPR54) and its ligand kisspeptin, encoded by the KiSS-1 gene, have been involved in the molecular mechanisms underlying the reawakening of gonadotropin-releasing hormone (GnRH) neurons at puberty. GPR54 mutations cause hypogonadotropic hypogonadism in human and mice. Aim Our aim was to study regulation of the KiSS-1/GPR54 system using a previously characterized primary culture of human fetal GnRH-secreting neuroblasts, FNC-B4. Methods KiSS-1/GPR54 gene and protein expressions in FNC-B4 were evaluated by quantitative reverse transcription–polymerase chain reaction (qRT–PCR), immunocytochemistry, and Western blot. Expression of kisspeptin and GPR54 in fetal olfactory mucosa (OM), from which FNC-B4 cells were derived, was analyzed with confocal microscopy. Main Outcome Measures Regulation of KiSS-1/GPR54 expression in FNC-B4 was evaluated in response to sexual steroids and leptin. Effect of kisspeptin on GnRH secretion and migration in FNC-B4 was also investigated. Results Kisspeptin and GPR54 were immunolocalized and co-expressed with GnRH in OM and FNC-B4 cells. Kisspeptin (1 µM, 24 hours) induced GnRH secretion, but not gene expression, and inhibited migration (IC50 = 6.28 ± 3.71 nM) in FNC-B4. The 24-hour exposure to increasing concentrations of 17-β-estradiol (0.01–1 nM) significantly and dose-dependently decreased, whereas androgens (dihydrotestosterone [DHT], 0.01–1 nM) significantly stimulated KiSS-1/GPR54 mRNA. Testosterone (1 nM) showed a stimulatory effect only after blocking its aromatization with letrozole. In addition, leptin (1 nM, 24 hours), an adipocyte-derived hormone acting on the reproductive axis, significantly increased KiSS-1/GPR54 expression in FNC-B4. Immunocytochemistry and Western blot analysis confirmed the regulatory effects found with qRT–PCR. Interestingly, leptin (1 nM, 24 hours) also significantly increased both leptin receptor (LEPR) and androgen receptor (AR) mRNA. DHT (0.01–1 nM) also up-regulated LEPR and AR genes, suggesting a synergistic action between leptin and androgens aimed to up-regulate the KiSS-1/GPR54 system, which, in contrast, was inhibited by estrogens. Conclusion Our results indicate that an interplay between metabolic and sexual hormones may trigger the KiSS-1/GPR54 signaling to GnRH neurons suggesting new mechanisms which regulate puberty onset.

CXC chemokine ligand 10 (CXCL10) plays a pivotal role in the self-perpetuation of the inflammatory processes in patients with autoimmune thyroid disease. Treatment with methimazole (MMI) reduces serum CXCL10 in patients with Graves' disease. In isolated human thyrocytes, tumor necrosis factor (TNF)a demonstrates a potent synergistic effect on interferon (IFN)g-induced CXCL10 secretion. We investigated the mechanism underlying the synergism between IFNg and TNFa and the effect of MMI on CXCL10 secretion in human thyrocytes. A peroxisome proliferatoractivated receptor g agonist, rosiglitazone (RGZ), a known inhibitor of T helper 1 (Th1)-mediated responses, was also studied for comparison. Experiments were carried out in human thyrocytes isolated from internodular parenchyma of thyroid tissues derived from patients who had undergone surgery for multinodular goiter. ELISA was used to measure CXCL10 levels in culture supernatant. Flow cytometry was used to assess IFNg membrane receptor expression. Specific mRNA analysis was performed by Taqman real-time PCR. Immunofluorescence was performed to detect nuclear translocation of nuclear factor-kB (NF-kB). In human thyrocytes, the synergistic effect of TNFa with IFNg on CXCL10 secretion is due to the upregulation of IFNg receptor expression. MMI decreased cytokine-induced CXCL10 secretion by reducing TNFa-induced upregulation of the IFNg receptor. RGZ decreased the cytokine-induced CXCL10 secretion by impairing NF-kB translocation, without affecting IFNg receptor. MMI and RGZ targeted thyrocytes with the same pharmacological potency, likely acting throughout different mechanisms. Targeting T helper 1-mediated autoimmune thyroid disease with drugs that impair different intracellular pathways could be a novel pharmacological tool.