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Women and men, female and male animals and cells are biologically different, and acknowledgement of this fact is critical to advancing medicine. However, incorporating concepts of sex-specific analysis in basic research is largely neglected, introducing bias into translational findings, clinical concepts and drug development. Research funding agencies recently approached these issues but implementation of policy changes in the scientific community is still limited, probably due to deficits in concepts, knowledge and proper methodology. This expert review is based on the EUGenMed project (www.eugenmed.eu) developing a roadmap for implementing sex and gender in biomedical and health research. For sake of clarity and conciseness, examples are mainly taken from the cardiovascular field that may serve as a paradigm for others, since a significant amount of knowledge how sex and oestrogen determine the manifestation of many cardiovascular diseases (CVD) has been accumulated. As main concepts for implementation of sex in basic research, the study of primary cell and animals of both sexes, the study of the influence of genetic vs. hormonal factors and the analysis of sex chromosomes and sex specific statistics in genome wide association studies (GWAS) are discussed. The review also discusses methodological issues, and analyses strength, weaknesses, opportunities and threats in implementing sex-sensitive aspects into basic research.

1 Substance P (SP) is deeply involved in lung pathophysiology and plays a key role in the modulation of inflammatory-immune processes. We previously demonstrated that SP activates guinea-pig alveolar macrophages (AMs) and human monocytes, but a careful examination of its effects on human AMs is still scarce. 2 This study was undertaken to establish the role of SP in human AM isolated from healthy smokers and non-smokers, by evaluating the presence of tachykinin NK 1 receptors (NK-1R) and SP's ability to induce superoxide anion (O 2 À ) production and cytokine release, as well as activation of the nuclear factor-kB (NF-kB) pathway.3 By Western blot analysis and immunofluorescence, we demonstrate that authentic NK-1R are present on human AMs, a three-fold enhanced expression being observed in healthy smokers. These NK-1R are functional, as SP and NK 1 agonists dose-dependently induce O 2 À production and cytokine release. In AMs from healthy smokers, SP evokes an enhanced respiratory burst and a significantly increased release of tumor necrosis factor-a as compared to healthy non-smokers, but has inconsistent effects on IL-10 release. The NK 1 selective antagonist CP 96,345 ((2S,3S)-cis-2-diphenylmethyl-N[(2-methoxyphenyl)-methyl]-1-azabicyclo-octan-3-amine)) competitively antagonized SP-induced effects. 4 SP activates the transcription factor NF-kB, a three-fold increased nuclear translocation being observed in AMs from healthy smokers. This effect is receptor-mediated, as it is reproduced by the NK 1 selective agonist [Sar 9 Met(O 2 ) 11 ]SP and reverted by CP 96,345. 5 These results clearly indicate that human AMs possess functional NK-1R on their surface, which are upregulated in healthy smokers, providing new insights on the mechanisms involved in tobacco smoke toxicity.

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Background and purpose: Phenolic compounds exert cytoprotective effects; our purpose was to investigate whether the isosteric polyphenolic compounds clovamide and rosmarinic acid are neuroprotective. Experimental approach: Three in vitro models of neuronal death were selected: (i) differentiated SH-SY5Y human neuroblastoma cells exposed to tert-butylhydroperoxide (t-BOOH), for oxidative stress; (ii) differentiated SK-N-BE(2) human neuroblastoma cells treated with L-glutamate, for excitotoxicity; and (iii) differentiated SH-SY5Y human neuroblastoma cells exposed to oxygen-glucose deprivation/reoxygenation, for ischaemia-reperfusion. Cell death was evaluated by lactate dehydrogenase measurements in the cell media, while the mechanisms underlying the effects by measuring: (i) t-BOOH-induced glutathione depletion and increase in lipoperoxidation; and (ii) L-glutamate-induced intracellular Ca 2+ overload (fura-2 method) and inducible gene expression (c-fos, c-jun), by reverse transcriptase-PCR. The ability of compounds to modulate nuclear factor-kB and peroxisome proliferator-activated receptor-g activation was evaluated by Western blot in SH-SY5Y cells not exposed to harmful stimuli. Key results: Both clovamide and rosmarinic acid (10-100 mmol·L -1 ) significantly protected neurons against insults with similar potencies and efficacies. The EC50 values were in the low micromolar range (0.9-3.7 mmol·L -1 ), while the maximal effects ranged from 40% to -60% protection from cell death over untreated control at 100 mmol·L -1. These effects are mediated by the prevention of oxidative stress, intracellular Ca 2+ overload and c-fos expression. In addition, rosmarinic acids inhibited nuclear factor-kB translocation and increased peroxisome proliferator-activated receptor-g expression in SH-SY5Y cells not exposed to harmful stimuli. Conclusion and implications: Clovamide and rosmarinic acid are neuroprotective compounds of potential use at the nutritional/pharmaceutical interface.

High plasma levels of nicotinamide phosphoribosyltransferase (NAMPT), traditionally considered an intracellular enzyme with a key role in NAD synthesis, have been reported in several oncological, inflammatory and metabolic diseases. We now show that eNAMPT can be actively released by melanoma cells in vitro. We analysed the mechanisms of its release, and we found both classical and non-classical pathway involvement. eNAMPT released by melanoma cells, in our hands, has paracrine and autocrine effects: it activates MAPK, AKT and NF-κB pathways and increases colony formation in anchorage-independent conditions. eNAMPT also induces M1 polarization in human monocytes. Last, we demonstrate, for the first time in any cancer type, that eNAMPT levels in plasma of tumour-bearing mice increase and that this increase can be reconducted to the tumour itself. This provides an important cue on previous observations that eNAMPT is increased in patients with cancer. Moreover, silencing NAMPT in melanoma cells leads to a reduction in the tumour growth rate. Our findings extend the basis to consider eNAMPT as a cytokine involved in tumour progression.

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Microparticles (MP) are phospholipid vesicles shed by cells upon activation or apoptosis. Monocyte-derived MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells; the molecular bases of such activity are unknown. Peroxisome proliferatoractivated receptors (PPAR) have been demonstrated to be involved in the modulation of nuclear factor (NF)-kB transcriptional activity and inflammation.We investigated whether the upregulation of the synthesis of proinflammatory cytokines by human lung epithelial cells induced by monocyte/macrophage-derived MP involves NF-kB activation and is modulated by PPAR-c.MP were generated by stimulation of human monocytes/macrophages with the calcium ionophore, A23187. MP were incubated with human lung epithelial cells. NF-kB translocation was assessed by electrophoretic mobility shift assay. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 synthesis was assessed by ELISA and RT-PCR.Stimulation of A549 alveolar cells with monocyte/macrophage-derived MP caused an increase in NF-kB activation and IL-8 and MCP-1 synthesis that was inhibited by pre-incubation with the PPAR-c agonists, rosiglitazone and 15-deoxy-D 12,14 -prostaglandin-J 2 . Parallel experiments with normal human bronchial epithelial cells largely confirmed the results. The effects of PPAR-c agonists were reversed by the specific antagonist, GW9662.Upregulation of the synthesis of proinflammatory mediators by human lung epithelial cells induced by monocyte/macrophage-derived MP is mediated by NF-kB activation through a PPAR-c dependent pathway.