Serum antimüllerian hormone levels remain stable through the menstrual cycle and after oral or vaginal administration of synthetic sex steroids

Rapport de synthèse : Objectif de l'étude : étudier si l'administration orale ou vaginale d'hormones contraceptives influence les concentrations sériques d'hormone antimüllérienne (AMH). Design : essai prospectif chez des femmes recrutées par annonce. Les femmes désireuses d'avoir une contraception ont été randomisées entre une contraception orale et une contraception vaginale. Celles qui ne souhaitaient pas de contraception ont été incluses dans le groupe témoin. Cadre de l'étude : unité de médecine de la reproduction d'un hôpital universitaire. Patientes : vingt-quatre jeunes femmes en bonne santé avec des cycles menstruels réguliers qui n'avaient pas utilisé de contraception hormonale pendant les trois mois précédant l'étude. Intervention : contraception orale ou vaginale du 5ème au 25ème jour du cycle menstruel dans les groupes contraception versus pas de contraception dans le groupe témoin. Mesure d'issue : variations inter et intra-cycle des concentrations sériques d'AMH dans les trois groupes: groupe témoin en cycle spontané et groupes sous contraception oestroprogestative orale ou vaginale. Résultats : les fluctuations d'AMH observées pendant le cycle menstruel (variations intra-cycle) restent dans les valeurs des variations entre deux cycles (variations inter-cycles) tant chez les femmes en cycle spontané que chez les femmes sous contraception orale ou vaginale. Conclusions : nos résultats confirment que les concentrations sériques d'AMH restent stables pendant le cycle menstruel et indiquent qu'elles ne sont pas influencées par l'administration exogène de stéroïdes sexuels contraceptifs, que ce soit par voie orale ou vaginale.

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha.

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

Probing the interaction between feline immunodeficiency virus and CD134 by using the novel monoclonal antibody 7D6 and the CD134 (Ox40) ligand.

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumor necrosis factor receptor superfamily, and all primary viral strains tested to date use CD134 for infection. We examined the expression of CD134 in the cat using a novel anti-feline CD134 monoclonal antibody (MAb), 7D6, and showed that as in rats and humans, CD134 expression is restricted tightly to CD4+, and not CD8+, T cells, consistent with the selective targeting of these cells by FIV. However, FIV is also macrophage tropic, and in chronic infection the viral tropism broadens to include B cells and CD8+ T cells. Using 7D6, we revealed CD134 expression on a B220-positive (B-cell) population and on cultured macrophages but not peripheral blood monocytes. Moreover, macrophage CD134 expression and FIV infection were enhanced by activation in response to bacterial lipopolysaccharide. Consistent with CD134 expression on human and murine T cells, feline CD134 was abundant on mitogen-stimulated CD4+ T cells, with weaker expression on CD8+ T cells, concordant with the expansion of FIV into CD8+ T cells with progression of the infection. The interaction between FIV and CD134 was probed using MAb 7D6 and soluble CD134 ligand (CD134L), revealing strain-specific differences in sensitivity to both 7D6 and CD134L. Infection with isolates such as PPR and B2542 was inhibited well by both 7D6 and CD134L, suggesting a lower affinity of interaction. In contrast, GL8, CPG, and NCSU were relatively refractory to inhibition by both 7D6 and CD134L and, accordingly, may have a higher-affinity interaction with CD134, permitting infection of cells where CD134 levels are limiting.

Receptor activator for NF-κB ligand in acute myeloid leukemia: expression, function, and modulation of NK cell immunosurveillance.

The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.

Gene transfer of programmed death ligand-1.Ig prolongs cardiac allograft survival.

BACKGROUND: The CD28 homologue programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2 (which are homologous to B7), constitute an inhibitory pathway of T cell costimulation. The PD-1 pathway is of interest for immune-mediated diseases given that PD-1-deficient mice develop autoimmune diseases. We have evaluated the effect of local overexpression of a PD-L1.Ig fusion protein on cardiac allograft survival. METHODS: Adenovirus-mediated PD-L1.Ig gene transfer was performed in F344 rat donor hearts placed in the abdominal position in Lewis recipients. Inflammatory cell infiltrates in the grafts were assessed by immunohistochemistry. RESULTS: Allografts transduced with the PD-L1.Ig gene survived for longer periods of time compared with those receiving noncoding adenovirus or virus dilution buffer alone: median survival time (MST), 17 (range: 16-20) days vs. 11 (8-14) and 9 (8-13) days, respectively (P < 0.001). PD-L1.Ig gene transfer combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone: MST, 25 (15-42) vs. 15 (13-19) days (P < 0.05). PD-L1.Ig gene transfer was associated with decreased numbers of CD4 cells and monocytes/macrophages infiltrating the graft (P < 0.05). CONCLUSIONS: Localized PD-L1.Ig expression in donor hearts attenuates acute allograft rejection in a rat model. The effect is additive to that of a subtherapeutic regimen of CsA. These results suggest that targeting of PD-1 by gene therapy may inhibit acute cardiac allograft rejection in vivo.

Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover.

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.

Effects of intranasal administration of synthetic (4-28)human atrial natriuretic peptide to normal volunteers.

The effects of intranasal administration of increasing doses of synthetic human natriuretic peptide (4-28 hANP) were studied in six healthy volunteers. The peptide was administered as a nasal spray at doses of 50, 100, 200, and 500 micrograms in ascending order at 48-h intervals. Vehicle was administered by the same route randomly between any two of the doses. Intranasal hANP administration had no effect on either blood pressure, heart rate (HR), or hematocrit. Diuresis did not change consistently, whereas natriuresis tended to rise with vehicle as well as with hANP administration. This was attributed to the infusion of isotonic saline during the experiment. There was no significant increase in plasma ANP levels after intranasal administration of any of the different doses. Thus, no evidence that the atrial natriuretic peptide tested (4-28 hANP) can cross the nasal mucosal barrier was found.

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides.

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.

Chemical probes that differentially modulate peroxisome proliferator-activated receptor alpha and BLTR, nuclear and cell surface receptors for leukotriene B(4).

Peroxisome proliferator-activated receptor alpha (PPARalpha)is a nuclear receptor for various fatty acids, eicosanoids, and hypolipidemic drugs. In the presence of ligand, this transcription factor increases expression of target genes that are primarily associated with lipid homeostasis. We have previously reported PPARalpha as a nuclear receptor of the inflammatory mediator leukotriene B(4) (LTB(4)) and demonstrated an anti-inflammatory function for PPARalpha in vivo (Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39-43). LTB(4) also has a cell surface receptor (BLTR) that mediates proinflammatory events, such as chemotaxis and chemokinesis (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624). In this study, we report on chemical probes that differentially modulate activity of these two LTB(4) receptors. The compounds selected were originally characterized as synthetic BLTR effectors, both agonists and antagonists. Here, we evaluate the compounds as effectors of the three PPAR isotypes (alpha, beta, and gamma) by transient transfection assays and also determine whether the compounds are ligands for these nuclear receptors by coactivator-dependent receptor ligand interaction assay, a semifunctional in vitro assay. Because the compounds are PPARalpha selective, we further analyze their potency in a biological assay for the PPARalpha-mediated activity of lipid accumulation. These chemical probes will prove invaluable in dissecting processes that involve nuclear and cell surface LTB(4) receptors and also aid in drug discovery programs.

Physiological ligands of PPARs in inflammation and lipid homeostasis

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that are involved in many physiological processes, such as inflammation and energy homeostasis. In general, PPARs must be activated by ligands to stimulate the expression of their target genes. These agonists can be synthetic molecules, such as drugs used to treat hypertriglyceridemia and insulin resistance, or natural physiological ligands, such as fatty acids and eicosanoids. Although recent work has uncovered a surprisingly broad variety of natural molecules capable of activating PPARs, relatively little is known about their mode of action in an in vivo physiological context. The action of physiological ligands in situations of food deprivation and abundance, especially with respect to their intervention in the inflammatory response, and in both lipid homeostasis and inflammation resolution will be reviewed.

Medicinal application of long synthetic peptide technology.

This review covers the latest developments of long synthetic peptide technology for the rapid identification and development of malaria vaccine candidates and immunological modulators. A brief description of the two most common solid-phase synthetic procedures, together with the latest advances in optimisation of peptide chain assembly and analytical instrumentation, is given, with special attention to non-specialists. Several examples of vaccine candidates developed in the authors' or their collaborators' laboratories are also provided.

Long synthetic peptides for the production of vaccines and drugs: a technological platform coming of age.

Long synthetic peptides (LSPs) have a variety of important clinical uses as synthetic vaccines and drugs. Techniques for peptide synthesis were revolutionized in the 1960s and 1980s, after which efficient techniques for purification and characterization of the product were developed. These improved techniques allowed the stepwise synthesis of increasingly longer products at a faster rate, greater purity, and lower cost for clinical use. A synthetic peptide approach, coupled with bioinformatics analysis of genomes, can tremendously expand the search for clinically relevant products. In this Review, we discuss efforts to develop a malaria vaccine from LSPs, among other clinically directed work.

Influence of the diketonato ligand on the cytotoxicities of [Ru(eta(6)-p-cymene)-(R(2)acac)(PTA)](+) complexes (PTA=1,3,5-triaza-7-phosphaadamantane)

A series of compounds of general formula [Ru(eta(6)-p-cymene) (R(2)acac)(PTA)][X] (R(2)acac = Me(2)acac, tBu(2)acac, Ph(2)acac, Me(2)acac-Cl; PTA = 1,3,5-triaza-7-phosphaadamantane; X = BPh4, BF4), and the precursor to the Me2acac-Cl derivative [Ru(eta(6)-p-cymene)(Me(2)acac-Cl)Cl], have been prepared and characterised spectroscopically. Five of the compounds have also been characterised in the solid state by X-ray crystallography. The tetrafluoroborate salts are water-soluble, quite resistant to hydrolysis, and have been evaluated for cytotoxicity against A549 lung carcinoma and A2780 human ovarian cancer cells. The compounds are cytotoxic towards the latter cell line, and relative activities are discussed in terms of hydrolysis (less important) and lipophilicity, which appears to exert the dominating influence.

International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors.

The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand- and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPARalpha is expressed at high levels in organs with significant catabolism of fatty acids. PPARbeta/delta has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPARgamma is expressed as two isoforms, of which PPARgamma2 is found at high levels in the adipose tissues, whereas PPARgamma1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipid-lowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPARalpha and PPARgamma agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.