Oestrogen deficiency modulates particle-induced osteolysis

International audienceABSTRACT: INTRODUCTION: Postmenopausal osteoporosis may modulate bone response to wear debris. In this article, we evaluate the influence of oestrogen deficiency on experimental particle-induced osteolysis. METHODS: Polyethylene (PE) particles were implanted onto the calvaria of normal controls, sham-ovariectomized (OVX), OVX mice and OVX mice supplemented with oestrogen (OVX+E). After 14 days, seven skulls per group were analyzed using a high-resolution micro-computed tomography (micro-CT) and histomorphometry, and for tartrate-specific alkaline phosphatase. Five calvariae per group were cultured for the assay of IL-1β, IL-6, TNF-α and receptor activator of the nuclear factor κB (RANKL) secretion using quantitative ELISA. Serum IL-6 concentrations were obtained. The expression of RANKL and osteoprotegerin (OPG) mRNA were evaluated using real-time PCR. RESULTS: As assessed by μCT and by histomorphometry, PE particles induced extensive bone resorption and an intense inflammatory reaction in normal controls, sham-OVX and OVX+E mice, but not in the OVX mice group. In normal controls, sham-OVX and OVX+E mice, PE particles induced an increase in serum IL-6, in TNF-α and RANKL local concentrations, and resulted in a significant increase in RANKL/OPG messenger RNA (mRNA) ratio. Conversely, these parameters remained unchanged in OVX mice after PE implantation. CONCLUSIONS: Oestrogen privation in the osteolysis murine model ultimately attenuated osteolytic response to PE particles, suggesting a protective effect. This paradoxical phenomenon was associated with a down-regulation of pro-resorptive cytokines. It is hypothesized that excessive inflammatory response was controlled, illustrated by the absence of increase of serum IL-6 in OVX mice after PE implantation

Metastatic Melanomas Express Inhibitory Low Affinity Fc Gamma Receptor and Escape Humoral Immunity

Our research, inspired by the pioneering works of Isaac Witz in the 1980s, established that 40% of human metastatic melanomas express ectopically inhibitory Fc gamma receptors (FcγRIIB), while they are detected on less than 5% of primary cutaneous melanoma and not on melanocytes. We demonstrated that these tumoral FcγRIIB act as decoy receptors that bind the Fc portion of antimelanoma IgG, which may prevent Fc recognition by the effector cells of the immune system and allow the metastatic melanoma to escape the humoral/natural immune response. The FcγRIIB is able to inhibit the ADCC (antibody dependent cell cytotoxicity) in vitro. Interestingly, the percentage of melanoma expressing the FcγRIIB is high (70%) in organs like the liver, which is rich in patrolling NK (natural killer) cells that exercise their antitumoral activity by ADCC. We found that this tumoral FcγRIIB is fully functional and that its inhibitory potential can be triggered depending on the specificity of the anti-tumor antibody with which it interacts. Together these observations elucidate how metastatic melanomas interact with and potentially evade humoral immunity and provide direction for the improvement of anti-melanoma monoclonal antibody therapy

Liver function abnormalities, clinical profile, and outcome in acute decompensated heart failure

AIMS: The aim of this study was to assess the prevalence of abnormal liver function tests (LFTs) and the associated clinical profile and outcome(s) in acute decompensated heart failure (ADHF) patients. Alteration in LFTs is a recognized feature of ADHF, but prevalence and outcomes data from a broad contemporary cohort of ADHF are scarce and the mechanism(s) of ADHF-induced cholestasis is unknown. METHODS AND RESULTS: We conducted a post hoc analysis of SURVIVE, a large clinical trial including ADHF patients treated with levosimendan or dobutamine. All LFTs were available in 1134 patients at baseline. Abnormal LFTs were seen in 46% of ADHF patients: isolated abnormal alkaline phosphatase (AP) was noted in 11%, isolated abnormal transaminases in 26%, and a combination of abnormal AP and transaminases in 9%. Abnormal AP was associated with marked signs of systemic congestion and elevated right-sided filling pressure. Abnormal AP had no relationship with 31-day mortality but was associated with worse 180-day mortality (23.5 vs. 34.9%, P = 0.001 vs. patients with normal AP). Abnormal transaminases were associated with clinical signs of hypoperfusion and with greater 31-day and 180-day mortality compared with normal transaminase profiles (17.6 vs. 8.4% and 31.6 vs. 22.4%, respectively; both P < 0.001). There was no additive value of abnormal AP plus abnormal transaminase on a long-term outcome. CONCLUSION: Abnormal LFTs were present in about a half of patients presenting with ADHF treated with inotropes. Abnormal AP and abnormal transaminases were associated with specific clinical, biological, and prognostic features, including a short-term overmortality with increased transaminases but not with biological signs of cholestasis, in ADHF patient

Étude des structures et fonctions des récepteurs de faible affinité pour la portion Fc des immunoglobulines G

Les RFcgIIIb cristallisent sous forme de dimères. In vivo ils sont physiquement distants donc non dimériques. L'étude de mutants confirme le rôle essentiel du domaine 2 dans la fixation des IgG et révèle le rôle inattendu du domaine 1 dans la modulation de leur affinité pour les IgG. Par ailleurs, l'étude de sa glycosylation révèle une forte présence de Mannose, absente du mRFcgII, qui pourrait rendre compte de l'interaction RFcgIIIb-CD11b. Le hRFcgIIb, d'expression hématopoïétique, est un récepteur inhibiteur. Les mélanomes métastatiques humains l'expriment ectopiquement. Ce hRFcgIIb tumoral induit l'inhibition de la croissance des mélanomes in vitro ou greffés aux souris immunodéficientes. Pour étudier la relation "hôte-tumeur" dans un hôte immunocompétent, nous avons inoculé aux souris le mélanome B16F0 exprimant le mRFcgIIb1. Ce modèle révèle le rôle de "leurre" du mRFcgIIb1 tumoral qui permet aux mélanomes d'échapper à la réponse antitumorale humoraleFcgRIIIb are dimeric in the crystal lattice but not in vivo where they are physically distant as shown by chemical crosslinking. Mutants study confirmed the essential role of domain 2 in IgG binding and enlightened the role of domain1 in the modulation of IgG binding affinity. Glycosylations study revealed high mannose sugar, which are absent from mFcgRII and could be implicated in the specificity of FcgRIIIb - CD11b interaction. hFcgRIIb expressed specifically on hematopoietic cells is an inhibitory receptor. Human metastatic melanoma express it ectopicaly. These tumoral hFcgRIIb inhibit melanoma growth in vitro as well as in vivo for tumor grafted in immunodeficient mice. To study the "Tumor-host" immunological relationship, the murin B16 melanoma expressing mFcgRIIb have been inoculated into immunocompetent mice. This model shown the decoy role of tumoral mFcgRIIb expression which allow the melanoma to escape antitumor humoral responsePARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Developmental switch from LTD to LTP in low frequency-induced plasticity.

International audienceThe stimulation of the Schaffer collateral/commissural fibers at low frequency (1 Hz) for 3-5 min can trigger a slow-onset form of low-frequency stimulation (LFS)-long-term potentiation (LTP) (LFS-LTP) in the CA1 area of the adult rat hippocampus. Here we have examined the developmental profile of this plasticity. In 9-15 day-old rats, the application of 1 Hz for 5 min induced long-term depression (LFS-LTD). In 17-21 day-old rats, 1 Hz stimulation had no effect when applied for 5 min but mediated LTD when stimulus duration was increased to 15 min. Over 25 day-old, 1 Hz stimulation mediated LFS-LTP. LFS-LTD was dependent on both N-methyl-D-aspartate (NMDA) and mGlu5 receptor activation. Antagonists of mGlu1alpha and cannabinoid type 1 receptor were ineffective to block LTD induction. LFS-LTD was not associated with a change in paired-pulse facilitation ratio, suggesting a postsynaptic locus of expression of this plasticity. Next, we examined whether LFS-LTD was related to 'chemical' LTDs obtained by the direct stimulation of mGlu5 and NMDA receptors. The saturation of LFS-LTD completely occluded NMDA- and (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG)-induced LTD. CHPG-LTD and NMDA-LTD occluded each other. In addition, we observed that NMDA-LTD was dependent on mGlu5 receptor activation in 9-12 day old rats while it was not in animals older than 15 day-old. Therefore we postulate that during LFS application, NMDA and mGlu5 receptor could interact to trigger LTD. Low-frequency-mediated synaptic plasticity is subject to a developmental switch from NMDA- and mGlu5 receptor-dependent LTD to mGlu5 receptor-dependent LTP with a transient period (17-21 day-old) during which LFS is ineffective

Neuroprotection induced by vitamin E against oxidative stress in hippocampal neurons: involvement of TRPV1 channels.

International audiencePretreatment of cultured hippocampal neurons with a low concentration of alpha-tocopherol (alpha-TP), the major component of vitamin E, results in a long-lasting protection against oxidative damages, via genomic effects. This neuroprotection is associated with the attenuation of a calcium influx triggered by oxidative agents such as Fe(2+) ions. This Ca(2+) influx is supported by a TRP-like channel, also partly involved in capacitive calcium entry within neurons. Here, we evidence the contribution of TRPV1 channels in this mechanism. TRPV1 channels are activated by various agents including capsaicin, the pungent component of hot chili peppers and blocked by capsazepine (CPZ) or 5'-iodo-resiniferatoxin. Both TRPV1 inhibitors strongly reduced Fe(2+) ion-mediated toxicity and Ca(2+) influx, in the same way as to alpha-TP pretreatment. Moreover, CPZ also decreased capacitive calcium entry in hippocampal neurons. Finally, both CPZ and 5'-iodo-resiniferatoxin reduced spontaneous excitatory synaptic transmission; this depression of synaptic transmission being largely occluded in alpha-TP-pretreated neurons. In conclusion, in our experimental model, TRPV1 channels are involved in the Fe(2+) ion-induced neuronal death and a negative modulation of this channel activity by alpha-TP pretreatment may account, at least in part, for the long-lasting neuroprotection against oxidative stress

Gliotoxicity in hippocampal cultures is induced by transportable, but not by nontransportable, glutamate uptake inhibitors.

International audienceExtracellular glutamate is kept below a toxic level by glial and neuronal glutamate transporters. Here we show that the transportable glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (t-PDC) induced cell death in mature, but not in immature, hippocampal neuron-enriched cultures. The cell death produced by a 24-hr treatment with t-PDC was dose-dependent and reached 85% of the cell population at a 250 microM concentration at 23 days in vitro (DIV). Immunocytochemistry experiments showed that, under these experimental conditions, t-PDC killed not only neurons as expected but also glial cells. The N-methyl-D-aspartate (NMDA) antagonist D-2-aminophosphonovalerate (D-APV; 250 microM) only partially reversed this toxicity, completely protecting the neuronal cell population but not the glial population. The antioxidant compounds alpha-tocopherol or Trolox, used at concentrations that reverse the oxidative stress-induced toxicity, did not block the gliotoxicity specifically produced by t-PDC in the presence of D-APV. The nontransportable glutamate uptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) elicited cell death only in mature, but not in immature, hippocampal cultures. The TBOA toxic effect was dose dependent and reached a plateau at 100 microM in 23-DIV cultures. About 50% of the cell population died. TBOA affected essentially the neuronal population. D-APV (250 microM) completely reversed this toxicity. It is concluded that nontransportable glutamate uptake inhibitors are neurotoxic via overactivation of NMDA receptors, whereas transportable glutamate uptake inhibitors induce both an NMDA-dependent neurotoxicity and an NMDA- and oxidative stress-independent gliotoxicity, but only in mature hippocampal cultures

Potent activation of FGF-2 IRES-dependent mechanism of translation during brain development

Fibroblast growth factor-2 (FGF-2) plays a fundamental role in brain functions. This role may be partly achieved through the control of its expression at the translational level via an internal ribosome entry site (IRES)-dependent mechanism. Transgenic mice expressing a bicistronic mRNA allowed us to study in vivo and ex vivo where this translational mechanism operates. Along brain development, we identified a stringent spatiotemporal regulation of FGF-2 IRES activity showing a peak at post-natal day 7 in most brain regions, which is concomitant with neuronal maturation. At adult age, this activity remained relatively high in forebrain regions. By the enrichment of this activity in forebrain synaptoneurosomes and by the use of primary cultures of cortical neurons or cocultures with astrocytes, we showed that this activity is indeed localized in neurons, is dependent on their maturation, and correlates with endogenous FGF-2 protein expression. In addition, this activity was regulated by astrocyte factors, including FGF-2, and spontaneous electrical activity. Thus, neuronal IRES-driven translation of the FGF-2 mRNA may be involved in synapse formation and maturation

Synthesis and characterization of a cyclooctapeptide analogue of ω-agatoxin IVB enhancing the activity of CaV2.1 calcium channels activity in cultured hippocampal neurons.

International audienceThe structure of the toxin ω-agatoxin IVB, extracted from the venom of funnel-web spider Agelenopsis aperta, is an important lead structure when considering the design of modulators of synaptic transmission which largely involves P/Q-type (CaV2.1) voltage gated calcium channels (VGCC) at central synapses. Focusing on the loop 2 of the ω-agatoxin IVB that seems to be the most preeminent interacting domain of the toxin with the CaV2.1 VGCC, cyclooctapeptides mimicking this loop were synthesized. While (14)Trp is essential for the binding of the neurotoxin to the CaV2.1 VGCC, the substitution of the (12)Cys for a glycidyl residue led to a cyclooctapeptide named EP14 able to enhance CaV2.1 VGCC-associated currents measured with patch-clamp recordings and to evoke ω-agatoxin IVA-sensitive intracellular Ca(2+) increase as measured by fura-2 spectrofluoroimaging. Furthermore, this cyclooctapeptide was able to potentiate spontaneous excitatory synaptic transmission in a network of cultured hippocampal neurons, consistent with the activation of presynaptic VGCC by EP14. In addition, this peptide did not affect cell survival measured with the MTT assay. Therefore, such new cyclopeptidic structures are potential good candidates for synthesis of new agents aimed at the restoration deficient excitatory synaptic transmission