A unique neuroendocrine cell model derived from the human foetal neural crest.

PURPOSE: Nowadays, no human neuroendocrine cell models derived from the neural crest are available. In this study, we present non-transformed long-term primary Neural Crest Cells (NCCs) isolated from the trunk region of the neural crest at VIII-XII gestational weeks of human foetuses obtained from voluntary legal abortion. METHODS AND RESULTS: In NCC, quantitative real-time RT PCR demonstrated the expression of neural crest specifier genes, such as Snail1, Snail2/SLUG, Sox10, FoxD3, c-Myc, and p75NTR. Moreover, these cell populations expressed stemness markers (such as Nanog and nestin), as well as markers of motility and invasion (TAGLN, MMP9, CXCR4, and CXCR7), and of neuronal/glial differentiation (MAP2, GFAP, SYP, and TAU). Functional analysis demonstrated that these cells not only possessed high migration properties, but most importantly, they expressed markers of sympatho-adrenal lineage, such as ASCL1 and tyrosine hydroxylase (TH). Moreover, the expression of TH increased after the induction with two different protocols of differentiation towards neuronal and sympatho-adrenal phenotypes. Finally, exposure to conditioned culture media from NCC induced a mature phenotype in a neuronal cell model (namely SH-SY5Y), suggesting that NCC may also act like Schwann precursors. CONCLUSION: This unique human cell model provides a solid tool for future studies addressing the bases of human neural crest-derived neuroendocrine tumours.

Therapeutic effects of the selective farnesoid X receptor agonist obeticholic acid in a monocrotaline-induced pulmonary hypertension rat model.

BACKGROUND: Activation of the farnesoid X receptor (FXR), a member of the nuclear receptor steroid superfamily, leads to anti-inflammatory and anti-fibrotic effects in several tissues, including the lung. We have recently demonstrated a protective effect of the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) in rat models of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and bleomycin-induced pulmonary fibrosis. The aim of the present study was to investigate whether the positive effects of OCA treatment could be exerted also in established MCT-induced PAH, i.e., starting treatment 2 weeks after MCT administration. METHODS: Rats with MCT-induced PAH were treated, 2 weeks after MCT administration, with OCA or tadalafil for two additional weeks. Pulmonary functional tests were performed at week 2 (before treatment) and four (end of treatment). At the same time points, lung morphological features and expression profile of genes related to smooth muscle relaxation/contraction and tissue remodeling were also assessed. RESULTS: 2 weeks after MCT-induced injury, the treadmill resistance (a functional parameter related to pulmonary hypertension) was significantly decreased. At the same time point, we observed right ventricular hypertrophy and vascular remodeling, with upregulation of genes related to inflammation. At week 4, we observed a further worsening of the functional and morphological parameters, accompanied by dysregulation of inflammatory and extracellular matrix markers mRNA expression. Administration of OCA (3 or 10 mg/kg/day), starting 2 weeks after MCT-induced injury, significantly improved pulmonary function, effectively normalizing the exercise capacity. OCA also reverted most of the lung alterations, with a significant reduction of lung vascular wall thickness, right ventricular hypertrophy, and restoration of the local balance between relaxant and contractile pathways. Markers of remodeling pathways were also normalized by OCA treatment. Notably, results with OCA treatment were similar, or even superior, to those obtained with tadalafil, a recently approved treatment for pulmonary hypertension. CONCLUSIONS: The results of this study demonstrate a significant therapeutic effect of OCA in established MCT-induced PAH, improving exercise capacity associated with reduction of right ventricular hypertrophy and lung vascular remodeling. Thus, OCA dosing in a therapeutic protocol restores the balance between relaxant and contractile pathways in the lung, promoting cardiopulmonary protective actions in MCT-induced PAH.

Therapeutic effects of obeticholic acid (OCA) treatment in a bleomycin-induced pulmonary fibrosis rat model.

PURPOSE: We recently demonstrated a protective effect of the farnesoid X receptor agonist obeticholic acid (OCA) in rat models of bleomycin-induced pulmonary fibrosis (PF). Aim of the present study was to investigate whether the positive effects of OCA treatment are apparent also on ongoing bleomycin-induced PF, i.e., after 2 weeks of bleomycin administration. METHODS: Bleomycin-induced PF rats were treated 2 weeks after bleomycin administration with OCA or pirfenidone for two additional weeks. Pulmonary function test was performed at 2 and 4 weeks in all experimental groups. At the same time points, lung morphological features and mRNA expression profile of genes related to fibrosis, inflammation and epithelial-mesenchymal transition were also assessed. RESULTS: After 2 weeks, bleomycin significantly increased the pressure at the airway opening (PAO), a functional parameter related to fibrosis-induced lung stiffness, and induced diffuse lung interstitium fibrosis, with upregulation of inflammation (IL1ß, MCP1) and tissue remodeling (COL1A1, COL3A1, ET1, MMP7, PDGFa, αSMA, SNAI1) markers. At week four, a further increase of lung fibrosis and PAO was observed, accompanied by upregulation of extracellular matrix-related mRNA expression. OCA administration, even after the establishment of PF, significantly improved pulmonary function, normalizing PAO, and reverted the bleomycin-induced lung alterations, with significant reduction of markers of inflammation (CD206, COX2, HIF1, IL1ß, MCP1), epithelial proliferation (CTGF, PDGFa) and fibrosis (COL1A1, COL3A1, ET1, FN1, MMPs, αSMA, SNAIs, TGFß1, TIMPs). Results with OCA were similar or superior to those obtained with pirfenidone. CONCLUSIONS: In conclusion, our results demonstrate a significant therapeutic effect of OCA in already established PF.

The phosphodiesterase 5 inhibitor tadalafil regulates lipidic homeostasis in human skeletal muscle cell metabolism.

PURPOSE: Tadalafil seems to ameliorate insulin resistance and glucose homeostasis in humans. We have previously reported that tadalafil targets human skeletal muscle cells with an insulin (I)-like effect. We aim to evaluate in human fetal skeletal muscle cells after tadalafil or I: (i) expression profile of I-regulated genes dedicated to cellular energy control, glycolitic activity or microtubule formation/vesicle transport, as GLUT4, PPARγ, HK2, IRS-1, KIF1C, and KIFAP3; (ii) GLUT4, Flotillin-1, and Caveolin-1 localization, all proteins involved in energy-dependent cell trafficking; (iii) activation of I-targeted paths, as IRS-1, PKB/AKT, mTOR, P70/S6K. Free fatty acids intracellular level was measured. Sildenafil or a cGMP synthetic analog were used for comparison; PDE5 and PDE11 gene expression was evaluated in human fetal skeletal muscle cells. METHODS: RTq-PCR, PCR, western blot, free fatty acid assay commercial kit, and lipid stain non-fluorescent assay were used. RESULTS: Tadalafil upregulated I-targeted investigated genes with the same temporal pattern as I (GLUT4, PPARγ, and IRS-1 at 3 h; HK2, KIF1C, KIFAP3 at 12 h), re-localized GLUT4 in cell sites positively immune-decorated for Caveolin-1 and Flotillin-1, suggesting the involvement of lipid rafts, induced specific residue phosphorylation of IRS-1/AKT/mTOR complex in association with free fatty acid de novo synthesis. Sildenafil or GMP analog did not affect GLUT4 trafficking or free fatty acid levels. CONCLUSION: In human fetal skeletal muscle cells tadalafil likely favors energy storage by modulating lipid homeostasis via IRS-1-mediated mechanisms, involving activation of I-targeted genes and intracellular cascade related to metabolic control. Those data provide some biomolecular evidences explaining, in part, tadalafil-induced favorable control of human metabolism shown by clinical studies.

Testosterone insulin-like effects: an in vitro study on the short-term metabolic effects of testosterone in human skeletal muscle cells.

PURPOSE: Testosterone by promoting different metabolic pathways contributes to short-term homeostasis of skeletal muscle, the largest insulin-sensitive tissue and the primary site for insulin-stimulated glucose utilization. Despite evidences indicate a close relationship between testosterone and glucose metabolism, the molecular mechanisms responsible for a possible testosterone-mediated insulin-like effects on skeletal muscle are still unknown. METHODS: Here we used undifferentiated proliferating or differentiated human fetal skeletal muscle cells (Hfsmc) to investigate the short-term effects of testosterone on the insulin-mediated biomolecular metabolic machinery. GLUT4 cell expression, localization and the phosphorylation/activation of AKT, ERK, mTOR and GSK3ß insulin-related pathways at different time points after treatment with testosterone were analyzed. RESULTS: Independently from cells differentiation status, testosterone, with an insulin-like effect, induced Glut4-mRNA expression, GLUT4 protein translocation to the cytoplasmic membrane, while no effect was observed on GLUT4 protein expression levels. Furthermore, testosterone treatment modulated the insulin-dependent signal transduction pathways inducing a rapid and persistent activation of AKT, ERK and mTOR, and a transient inhibition of GSK3ß. T-related effects were shown to be androgen receptor dependent. CONCLUSION: All together our data indicate that testosterone through the activation of non-genomic pathways, participates in skeletal muscle glucose metabolism by inducing insulin-related effects.

Fibroblast growth factor and endothelin-1 receptors mediate the response of human striatal precursor cells to hypoxia.

Fetal striatal transplantation has emerged as a new therapeutic strategy in Huntington's disease (HD). Hypoxia is one of the microenvironmental stress conditions to which fetal tissue is exposed as soon as it is isolated and transplanted into the diseased host brain. Mechanisms that support neuroblast survival and replenishment of damaged cells within the HD brain in the hypoxic condition have yet to be fully elucidated. This study is aimed at investigating the molecular pathways associated with the hypoxic condition in human fetal striatal neuroblasts (human striatal precursor (HSP) cells), using the hypoxia-mimetic agent cobalt chloride (CoCl2). We analyzed the effect of CoCl2 on HSP cell proliferation and on the expression of hypoxia-related proteins, such as hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Moreover, we evaluated fibroblast growth factor 2 (FGF2; 50ng/ml) and endothelin-1 (ET-1; 100nM) proliferative/survival effects in HSP cells in normoxic and hypoxic conditions. Dose-response experiments using increasing concentrations of CoCl2 (50-750µM) showed that the HSP cell growth was unaffected after 24h, while it increased at 48h, with the maximal effect observed at 400µM. In contrast, cell survival was impaired at 72h. Hypoxic conditions determined HIF-1α protein accumulation and increased gene and protein expression of VEGF, while FGF2 and ET-1 significantly stimulated HSP cell proliferation both in normoxic and hypoxic conditions, thus counteracting the apoptotic CoCl2 effect at 72h. The incubation with selective receptor (FGFR1, endothelin receptor A (ETA) and endothelin receptor B (ETB)) inhibitors abolished the FGF2 and ET-1 neuroprotective effect. In particular, ET-1 stimulated HSP cell survival through ETA in normoxic conditions and through ETB during hypoxia. Accordingly, ETA expression was down-regulated, while ETB expression was up-regulated by CoCl2 treatment. Overall, our results support the idea that HSP cells possess the machinery for their adaptation to hypoxic conditions and that neurotrophic factors, such as FGF2 and ET-1, may sustain neurogenesis and long-term survival through complex receptor-mediated mechanisms.

Opposite effects of tamoxifen on metabolic syndrome-induced bladder and prostate alterations: a role for GPR30/GPER?

BACKGROUND: BPH and LUTS have been associated to obesity, hypogonadism, and metabolic syndrome (MetS). MetS-induced prostate and bladder alterations, including inflammation and tissue remodeling, have been related to a low-testosterone and high-estrogen milieu. In addition to ERs, GPR30/GPER is able to mediate several estrogenic non-genomic actions. METHODS: Supplementing a subgroup of MetS rabbits with tamoxifen, we analyzed the in vivo effects on MetS-induced prostate and bladder alterations. The effects of selective ER/GPER ligands and GPER silencing on prostate inflammation were also studied in vitro using hBPH cells. RESULTS: ERα, ERß, and PR expression was upregulated in MetS bladder, where tamoxifen decreased ERα and PR expression, further stimulating ERß. In addition, tamoxifen-dosing decreased MetS-induced overexpression of inflammatory and tissue remodeling genes. In prostate, sex steroid receptors, pro-inflammatory and pro-fibrotic genes were upregulated in MetS. However, tamoxifen did not affect them and even increased COX-2. In hBPH cells, 17ß-estradiol increased IL-8 secretion, an effect blunted by co-treatment with GPER antagonist G15 but not by ER antagonist ICI 182,780, which further increased it. GPER agonist G1 dose-dependently (IC50 = 1.6 nM) induced IL-8 secretion. In vitro analysis demonstrated that GPER silencing reverted these stimulatory effects. CONCLUSIONS: GPER can be considered the main mediator of estrogen action in prostate, whereas in bladder the mechanism appears to rely on ERα, as indicated by in vivo experiments with tamoxifen dosing. Limiting the effects of the MetS-induced estrogen action via GPER could offer new perspectives in the management of BPH/LUTS, whereas tamoxifen dosing showed potential benefits in bladder.

Expression and localization of VEGF receptors in human fetal skeletal tissues.

During development the vertebrate skeleton is the product of deriving cells from distinct embryonic lineages. The craniofacial skeleton is formed by migrating cranial neural crest cells, whereas the axial and limb skeletons are derived from mesodermal cells. The Vascular Endothelial Growth Factors (VEGFs) / receptors (VEGFRs) system plays an important role in angiogenesis, as well as osteogenesis, during bone development, growth, and remodeling, attracting endothelial cells and osteoclasts and stimulating osteoblast differentiation. Recent evidence has shown that during development VEGFR-3 is also expressed in neural and glial precursors of forebrain and cerebellum, as well as in the eye. In this study, we found that VEGFR-1, VEGFR-2 and VEGFR-3 are expressed in human bone both in fetal and adult life. The gene expression levels were significantly higher in fetal samples especially in mandibles. In addition, higher levels of VEGFR-3 in orofacial district were confirmed by western blotting analysis. We also observed that in fetal mandibular samples VEGFRs colocalized in several osteoblasts, osteoclasts and osteoprogenitor cells. Furthermore, some cells coexpressed VEGFR-3 and ET-1, a marker of neural crest cells. The results demonstrated different expression of VEGFRs in human mandibular and femoral bones which could be correlated to their different structure, function and development during organogenesis. VEGFR-3 might represent a specific signal for ectomesenchymal lineage differentiation during early human development.

Relationship between the neuroprotective effects of insulin-like growth factor-1 and 17ß-oestradiol in human neuroblasts.

Insulin-like growth factor-1 (IGF-1) and oestrogens interact with each other as neuroprotective factors. We have previously demonstrated that 17ß-oestradiol protects against ß-amyloid and oxidative stress toxicity and increases the amount of cell cholesterol in human foetal neuroblasts (FNC). The present study aimed: (i) to assess the protective effects of IGF-1 in FNC cells; (ii) to investigate the relationship between IGF-1 and 17ß-oestradiol; and (iii) to determine whether cholesterol was a major mediator of the effects of IGF-1, similarly to 17ß-oestradiol. We found that IGF-1 effectively exerts neuroprotective effects in FNC cells. We also demonstrated that the IGF-1 receptor (IGF-1R) pathway is needed to maintain oestrogen-mediated neuroprotection. Finally, we found that, opposite to 17ß-oestradiol, IGF-1 did not cause a significant increase in cell cholesterol. These findings indicate that a cross-talk between IGF-1 and 17ß-oestradiol occurs in FNC cells. In particular, the activation of the IGF-1R cascade appears to be fundamental to warrant 17ß-oestradiol-mediated neuroprotection, even though cell cholesterol does not play a major role as an effector of this pathway.

Progress in restorative neurosurgery: human fetal striatal transplantation in Huntington's disease. Review.

The purpose of this paper was to offer a review of the rationale, methods, biological and clinical results of human fetal striatal transplantation (HFST) in the treatment of Huntington's disease (HD). HD is a heritable neurodegenerative disease in which degeneration of neurons in the striatum leads to motor, psychiatric and cognitive deficits. The disease is progressive and inexorably lethal. At present there are no curative treatments for HD. A restorative therapy based on the intrastriatal transplantation of striatal neuroblasts taken from human fetus is currently being explored as potential treatment in selected HD patients. Pilot clinical trials of HFST have been started in few neurosurgery restorative centres. Results demonstrated that HFST is feasible and safe without relevant adverse effects; grafted neuroblasts survive, grow without evidence of neoplasia or teratoma, build new tissue with striatal-like imaging features, and move into the host brain towards short and long-distance cortical and sub-cortical targets. HFST delays disease progression and provides a period of improvement and stability. Even though larger-scale studies are still necessary to establish the true value of such a treatment, at this time, HFST represents a promising experimental therapy for patients with HD and one of the most interesting clinical application of restorative neurosurgery.

Human prostatic urethra expresses vitamin D receptor and responds to vitamin D receptor ligation.

BACKGROUND: Chronic inflammation is now considered a determinant of benign prostatic hyperplasia (BPH), promoting, together with the hormonal milieu, prostate overgrowth and lower urinary tract symptoms (LUTS). Prostatic urethra actively participates in determining progression of LUTS associated with BPH. AIM: To investigate the expression of the vitamin D receptor (VDR) and the ability of the VDR agonist elocalcitol to reduce inflammatory responses in human prostatic urethra (hPU) cells. MATERIALS AND METHODS: Human prostatic urethra, prostate and bladder neck were obtained from patients affected by BPH. Immunohistochemical studies for VDR expression were performed in tissue samples, from which primary cell cultures were also derived. In hPU cells, proliferation and chemiotaxis were studied, along with Rho kinase (ROCK) activity (MYPT-1 phosphorylation) by western blot. Quantitative RT-PCR was performed for VDR, cyclooxygenase (COX-2), and interleukin (IL)-8 expression. RESULTS: Urethra displays higher VDR expression compared to prostate and bladder neck tissues. The VDR agonist elocalcitol partially reverts COX-2 and IL-8 mRNA upregulation induced by a pro-inflammatory cytokine mixture (IL-17, interferon-γ, tumor necrosis factor-α) and inhibits cell migration in urethral cells. Elocalcitol prevents activation of ROCK, as previously demonstrated in bladder and prostate cell cultures. CONCLUSIONS: Our results suggest that prostatic urethra is, within the lower urinary tract, a novel target for VDR agonists, as shown by the capacity of elocalcitol to inhibit ROCK activity and to limit inflammatory responses in human primary urethra cells.

Sialic acid in human testis and changes with aging.

The aim of the present study was to investigate the distribution of the glycoconjugates sialoderivatives in the human testis. Orchidectomy specimens from men aged 18-30 years (Group 1) and from men aged 70-93 years (Group 2) were obtained at autopsy. The study was performed using digoxigenin-labelled lectins, namely Maackia amurensis II lectin (MAA), Sambucus nigra agglutinin (SNA) and Arachis hypogaea lectin (PNA), in addition to enzymatic and chemical treatments (neuraminidase, KOH-neuraminidase, mild oxidation-neuraminidase, mild oxidation-KOH-neuraminidase, strong oxidation-neuraminidase, strong oxidation-KOH-neuraminidase), to characterise the different glycosidic linkages of the sialoderivatives and to obtain information regarding their structure. In all Group 2 samples, sialic acids linked alpha-2,3 to galactose and alpha-2,6 to galactose/N-acetyl-D-galactosamine (Gal/GalNAc), revealed by MAA and SNA, respectively, were observed in testicular interstitial tissue and in the lamina propria. Sialic acid linked alpha-2,6 to Gal/GalNAc was detected in only some samples from Group 1. After treatment, PNA showed structural changes and/or the gradual disappearance of sialic acid linked to D-galactose-beta(1-3)-N-acetyl-D-galactosamine in testicular components with aging. These findings indicate that changes in the metabolism of sialoderivatives in the testis could be related to morphofunctional changes in various testicular components typical of this organ during aging. This suggests that sialoderivatives are important in the functionality of the mature testis in men, as well as its involution.

Immunomodulatory effects of BXL-01-0029, a less hypercalcemic vitamin D analogue, in human cardiomyocytes and T cells.

Current immunosuppressive protocols have reduced rejection occurrence in heart transplantation; nevertheless, management of heart transplant recipients is accompanied by major adverse effects, due to drug doses close to toxic range. In allograft rejection, characterized by T-helper 1 (Th1) cell-mediated response, the CXCL10-CXCR3 axis plays a pivotal role in triggering a self-promoting inflammatory loop. Indeed, CXCL10 intragraft production, required for initiation and development of graft failure, supports organ infiltration by Th1 cells. Thus, targeting the CXCL10-CXCR3 axis while avoiding generalized immunosuppression, may be of therapeutic significance. Based on preclinical evidence for immunoregulatory properties of vitamin D receptor agonists, we propose that a less hypercalcemic vitamin D analogue, BXL-01-0029, might have the potential to contribute to rejection management. We investigated the effect of BXL-01-0029 on CXCL10 secretion induced by proinflammatory stimuli, both in human isolated cardiomyocytes (Hfcm) and purified CD4+ T cells. Mycophenolic acid (MPA), the active agent of mycophenolate mofetil, was used for comparison. BXL-01-0029 inhibited IFNgamma and TNFalpha-induced CXCL10 secretion by Hfcm more potently than MPA, impairing cytokine synergy and pathways. BXL-01-0029 reduced also CXCL10 protein secretion and gene expression by CD4+ T cells. Furthermore, BXL-01-0029 did not exert any toxic effect onto both cell types, suggesting its possible use as a dose-reducing agent for conventional immunosuppressive drugs in clinical transplantation.

Intermittent high glucose concentrations reduce neuronal precursor survival by altering the IGF system: the involvement of the neuroprotective factor DHCR24 (Seladin-1).

The exposure of neurons to high glucose concentrations is considered a determinant of diabetic neuropathy, whereas members of the IGF system are neurotropic factors. Here, we investigated the effects of constant and intermittent high glucose concentrations on IGF1 and IGF-binding proteins (IGFBPs) in human neuroblast long-term cell cultures fetal neuroepithelial cells (FNC). These cells express the IGF1 receptor, and express and release in the culture medium IGFBP2, IGFBP4, and IGF1. The release of IGF1 was significantly increased by 17beta-estradiol (10 nM). IGF1 (100 nM) treatment determined a significant increase of IGFBP2 and a decrease of IGFBP4 release. In addition, IGF1 (1-100 nM) stimulated FNC cell proliferation in a dose-dependent manner. We hypothesized that this effect may be, at least partially, due to IGF1-induced up-regulation of the expression of the Alzheimer's disease related gene SELADIN-1 (now known as DHCR24 ), which acts as a pro-survival factor for neuronal cells. Conversely, the exposure to intermittent (20/10 mM), but not stable (20 mM), high glucose concentrations decreased the release of IGF1 and IGFBP2 in the culture medium and inhibited FNC growth by inducing apoptosis. The latter was prevented by the addition of IGF1 to the culture medium. Furthermore, high glucose concentrations reduced the expression of DHCR24. In conclusion, our results indicate for the first time that intermittent high glucose concentrations, similar to those observed in poorly controlled diabetic patients, may contribute to the development of diabetic neuropathy by interfering with the tropic effects exerted by the IGF system, and suggest the involvement of the neuroprotective factor DHCR24.

Thyroid hormones promote cell differentiation and up-regulate the expression of the seladin-1 gene in in vitro models of human neuronal precursors.

Thyroid hormones (TH) play an important role in the development of human brain, by regulating the expression of specific genes. Selective Alzheimer's disease indicator-1 (seladin-1) is a recently discovered gene with neuroprotective properties, which has been found to be down-regulated in brain regions affected by Alzheimer's disease. Seladin-1 has anti-apoptotic properties mainly due to the inhibition of the activation of caspase 3. The aim of this study was to determine whether seladin-1 may be regarded as a new mediator of the effects of TH in the developing brain. In order to demonstrate this hypothesis, the effects of TH both on cell differentiation and on the expression of seladin-1 were assessed in two different cell models, i.e. fetal human neuroepithelial cells (FNC) and human mesenchymal stem cells (hMSC), which can be differentiated into neurons. 3,3',5-Triiodothyronine (T3) determined different biological responses (inhibition of cell adhesion, induction of migration, and increase in the expression of the neuronal marker neurofilament-M and Na+ and Ca2+ channel functionality) in both FNC and hMSC, which express TH receptors. Then, we showed that TH significantly increase the expression levels of seladin-1, and that T3 effectively prevents camptothecin-induced apoptosis. However, in hMSC-derived neurons the expression of seladin-1 was not affected by TH. Our results demonstrated for the first time that seladin-1 is a novel TH-regulated gene in neuronal precursors. In view of its anti-apoptotic activity, it might be hypothesized that one of the functions of the increased seladin-1 levels in the developing brain may be to protect neuronal precursor cells from death.

Elocalcitol inhibits inflammatory responses in human thyroid cells and T cells.

T-helper 1 (Th1) cell-mediated inflammatory responses predominate in the early pathogenesis of Graves' disease (GD), whereas Th2 cell-mediated immunity may play a role in later stages. The chemokine CXCL10 and its receptor CXCR3 are expressed in most thyroid glands of early GD patients. Circulating CXCL10 levels inversely correlate with disease duration; CXCL10 maximal expression also correlates with interferon (IFN)gamma levels in recent GD onset. Methimazole (MMI) reduces CXCL10 secretion by isolated thyrocytes, decreases serum CXCL10 levels, and promotes a transition from Th1 to Th2 dominance in patients in GD active phase. Vitamin D receptor agonists exhibit antiinflammatory properties and promote tolerance induction. We investigated the effects and the mechanism of action of a nonhypercalcemic vitamin D receptor agonist, elocalcitol (BXL-628), compared with MMI on CXCL10 secretion induced by proinflammatory cytokines. Furthermore, we studied the effects of both drugs on Th1, Th17, and Th2 cytokine secretion in CD4+ T cells. ELISA, cytometry, immunocytochemistry, Western blot, and quantitative real-time PCR were used for protein and gene analysis. In human thyrocytes, elocalcitol inhibited IFNgamma and TNFalpha-induced CXCL10 protein secretion more potently than MMI. Elocalcitol impaired both cytokine intracellular pathways, whereas MMI was effective only on the IFNgamma pathway. In CD4+ T cells, elocalcitol decreased Th1- and Th17-type cytokines, and promoted Th2-type cytokine secretion. Elocalcitol and MMI inhibited Th1 cytokine-mediated responses in thyrocytes and CD4+ T cells. In addition, elocalcitol promoted a shift toward a Th2 response. In conclusion, elocalcitol could represent a novel pharmacological tool in the treatment of autoimmune thyroid diseases.

Effect of sildenafil administration on penile hypoxia induced by cavernous neurotomy in the rat.

Hypoxia is a normal, physiological condition in penile tissue, which is interrupted by reoxygenation associated to sleep-related erections. We previously described in the rat that a penile fibrosis and overexpression of the pro-fibrotic endothelin-1 type B receptor (ETB) are associated to prolonged (3 months) hypoxia induced by the bilateral surgical resection of the cavernous nerves (bilateral cavernous neurotomy (BCN)). The aim of the present study was to define the time frame in which BCN induces hypoxia and ETB overexpression in the penile tissue. In addition, we studied the time-dependency of the rescuing effect of an acute administration of the phosphodiesterase type 5 inhibitor, sildenafil. We found that BCN induced penile hypo-oxygenation (immunohistochemistry for Hypoxyprobe), penile ETB mRNA overexpression (quantitative real-time reverse transcriptase polymerase chain reaction) and hypersensitivity to the ETB agonist IRL-1620 (in vitro contractility study). Sildenafil treatment was able to counteract all these alterations (penile hypoxygenation, hyper-sensitivity to IRL-1620 and ETB overexpression), with its effect being more evident the earlier it was administered.

Methimazole inhibits CXC chemokine ligand 10 secretion in human thyrocytes.

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)alpha demonstrates a potent synergistic effect on interferon (IFN)gamma-induced CXCL10 secretion. We investigated the mechanism underlying the synergism between IFNgamma and TNFalpha and the effect of MMI on CXCL10 secretion in human thyrocytes. A peroxisome proliferator-activated receptor gamma 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 IFNgamma membrane receptor expression. Specific mRNA analysis was performed by Taqman real-time PCR. Immunofluorescence was performed to detect nuclear translocation of nuclear factor-kappaB (NF-kappaB). In human thyrocytes, the synergistic effect of TNFalpha with IFNgamma on CXCL10 secretion is due to the upregulation of IFNgamma receptor expression. MMI decreased cytokine-induced CXCL10 secretion by reducing TNFalpha-induced upregulation of the IFNgamma receptor. RGZ decreased the cytokine-induced CXCL10 secretion by impairing NF-kappaB translocation, without affecting IFNgamma 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.

Characterization of M540 bodies in human semen: evidence that they are apoptotic bodies.

Our group has recently identified, in human semen, round bodies of different size and density which were termed M540 bodies due to their staining with the fluorochrome merocyanine 540. Here, we investigate the hypothesis that such structures represent apoptotic bodies. To this aim, by both fluorescence-activated cell sorting (FACS) and fluorescence microscopy, we examined the occurrence of apoptotic markers such as caspase activity, Fas, p53 and Bcl-x in M540 bodies. In addition, we evaluated their ultrastructure by transmission electron microscopy. We found that M540 bodies express all the investigated markers, strongly supporting our hypothesis. We also found that M540 bodies contain fragmented DNA, another evidence of their apoptotic derivation. We investigated also the presence of M540 bodies in the different categories of patients. With respect to normozoospermic subjects, a higher content of M540 bodies was found in oligoasthenoteratozoospermic and asthenoteratozoospermic, but not in asthenozoospermic and teratozoospermic men. Interestingly, these subjects are those whose semen shows the highest levels of apoptotic signs. The variable occurrence of apoptotic bodies in semen may thus be considered a sign of abortive apoptosis in male reproductive organs. Of interest, since M540 bodies exhibit a similar size and density to sperm, they represent a confounding factor in FACS studies on ejaculated sperm.

Identification, localization and functional in vitro and in vivo activity of oxytocin receptor in the rat penis.

We recently found that the oxytocin receptor (OTR) is expressed in the human and rabbit corpus cavernosum and mediates contractility in vitro. The present study extended our investigations to the rat, and explored whether OTR regulates penile detumescence in vivo. Real-time RT-PCR quantitatively characterized the distribution of OTR mRNA in the male genital tract. Specific transcripts for OTR were expressed in all the tissues investigated. Penile expression of OTR was comparable to that observed in testis and prostate. Western blot analysis detected a single band of the expected molecular mass for OTR in all tissues examined, including rat penis. Expression of OTR protein in rat penile extracts was further confirmed by binding studies, using the OTR selective radiolabeled ligand 125I-OTA (K(d) = 17 +/- 6.5 pM, B(max)=15.7 +/- 5 fmoles/mg protein). OTR was immunolocalized to the endothelial and smooth muscle compartments of cavernous spaces and blood vessels. In rat corpus cavernosum strips, oxytocin (OT) and an OTR selective agonist ([Thr4,Gly7]OT) induced identical increases in tension, while different vasopressin agonists were less active. In vivo, OT intra-cavernous injection (ICI) dose-dependently inhibited intracavernous pressure (ICP) increase elicited by either electrical stimulation of the cavernous nerve or ICI of papaverine with similar IC(50)s (117.7 +/- 37 mU). The OTR antagonist, atosiban, counteracted the contractile effect of OT both in vitro and in vivo. Atosiban alone significantly increased ICP at lower stimulation frequencies (2 Hz = P<0.001 and 4 Hz = P<0.05 vs control), but not at the maximal frequency (16 Hz). Our data showed that OTR is present in the rat penis and mediates contractility both in vitro and in vivo, therefore suggesting a role for OT in maintaining penile detumescence.