Small-molecule lysophosphatidic acid receptor 5 (LPAR5) antagonists: versatile pharmacological tools to regulate inflammatory signaling in BV-2 microglia cells

Lysophosphatidic acid (LPA) species in the extracellular environment induce downstream signaling via six different G protein-coupled receptors (LPAR1-6). These signaling cascades are essential for normal brain development and function of the nervous system. However, in response to acute or chronic central nervous system (CNS) damage, LPA levels increase and aberrant signaling events can counteract brain function. Under neuro-inflammatory conditions signaling along the LPA/LPAR5 axis induces a potentially neurotoxic microglia phenotype indicating the need for new pharmacological intervention strategies. Therefore, we compared the effects of two novel small-molecule LPAR5 antagonists on LPA-induced polarization parameters of the BV-2 microglia cell line. AS2717638 is a selective piperidine-based LPAR5 antagonist (IC(50) 0.038 μM) while compound 3 is a diphenylpyrazole derivative with an IC(50) concentration of 0.7 μM in BV-2 cells. Both antagonists compromised cell viability, however, at concentrations above their IC(50) concentrations. Both inhibitors blunted LPA-induced phosphorylation of STAT1 and STAT3, p65, and c-Jun and consequently reduced the secretion of pro-inflammatory cyto-/chemokines (IL-6, TNFα, IL-1β, CXCL10, CXCL2, and CCL5) at non-toxic concentrations. Both compounds modulated the expression of intracellular (COX-2 and Arg1) and plasma membrane-located (CD40, CD86, and CD206) polarization markers yet only AS2717638 attenuated the neurotoxic potential of LPA-activated BV-2 cell-conditioned medium towards CATH.a neurons. Our findings from the present in vitro study suggest that the two LPAR5 antagonists represent valuable pharmacological tools to interfere with LPA-induced pro-inflammatory signaling cascades in microglia

Effectiveness of the capsaicin 8% patch in the management of peripheral neuropathic pain in European clinical practice: the ASCEND study

Background In randomised studies, the capsaicin 8% patch has demonstrated effective pain relief in patients with peripheral neuropathic pain (PNP) arising from different aetiologies. Methods ASCEND was an open-label, non-interventional study of patients with non-diabetes-related PNP who received capsaicin 8% patch treatment, according to usual clinical practice, and were followed for ≤52 weeks. Co-primary endpoints were percentage change in the mean numeric pain rating scale (NPRS) ‘average daily pain’ score from baseline to the average of Weeks 2 and 8 following first treatment; and median time from first to second treatment. The primary analysis was intended to assess analgesic equivalence between post-herpetic neuralgia (PHN) and other PNP aetiologies. Health-related quality of life (HRQoL, using EQ-5D), Patient Global Impression of Change (PGIC) and tolerability were also assessed. Results Following first application, patients experienced a 26.6% (95% CI: 23.6, 29.62; n = 412) reduction in mean NPRS score from baseline to Weeks 2 and 8. Equivalence was demonstrated between PHN and the neuropathic back pain, post-operative and post-traumatic neuropathic pain and ‘other’ PNP aetiology subgroups. The median time from first to second treatment was 191 days (95% CI: 147, 235; n = 181). Forty-four percent of all patients were responders (≥30% reduction in NPRS score from baseline to Weeks 2 and 8) following first treatment, and 86.9% (n = 159/183) remained so at Week 12. A sustained pain response was observed until Week 52, with a 37.0% (95% CI: 31.3, 42.7; n = 176) reduction in mean NPRS score from baseline. Patients with the shortest duration of pain (0–0.72 years) experienced the highest pain response from baseline to Weeks 2 and 8. Mean EQ-5D index score improved by 0.199 utils (responders: 0.292 utils) from baseline to Week 2 and was maintained until Week 52. Most patients reported improvements in PGIC at Week 2 and at all follow-up assessments regardless of number of treatments received. Adverse events were primarily mild or moderate reversible application site reactions. Conclusion In European clinical practice, the capsaicin 8% patch provided effective and sustained pain relief, substantially improved HRQoL, improved overall health status and was generally well tolerated in a heterogeneous PNP population

Combination Forecasts of Bond and Stock Returns: An Asset Allocation Perspective

We investigate the out-of-sample forecasting ability of the HML, SMB, momentum, short-term and long-term reversal factors along with their size and value decompositions on U.S. bond and stock returns for a variety of horizons ranging from the short run (1 month) to the long run (2 years). Our findings suggest that these factors contain significantly more information for future bond and stock market returns than the typically employed financial variables. Combination of forecasts of the empirical factors turns out to be particularly successful, especially from an an asset allocation perspective. Similar findings pertain to the European and Japanese markets

Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA

Human sperm DNA damage may have adverse effects on reproductive outcome. Infertile men possess substantially more spermatozoa with damaged DNA compared to fertile donors. Although the extent of this abnormality is closely related to sperm function, the underlying etiology of ensuing male infertility is still largely controversial. Both intra-testicular and post-testicular events have been postulated and different mechanisms have been proposed to explain the presence of damaged DNA in human spermatozoa. Three among them, i.e. abnormal chromatin packaging, oxidative stress and apoptosis, are the most studied and discussed in the present review. Furthermore, results from numerous investigations are presented, including our own findings on these pathological conditions, as well as the techniques applied for their evaluation. The crucial points of each methodology on the successful detection of DNA damage and their validity on the appraisal of infertile patients are also discussed. Along with the conventional parameters examined in the standard semen analysis, evaluation of damaged sperm DNA seems to complement the investigation of factors affecting male fertility and may prove an efficient diagnostic tool in the prediction of pregnancy outcome

Lucrative Promenades

The design proposal investigates the generation of a possible social and physical structure within the area of the former army campus of Kodra;thus a communal flexible space for people to meet, act, produce and socialize. The general result of this process was to investigate the implementation of computational techniques to generate a building enviroment to reflect social and spacial needs, within the context of experimental green strategies theme.Non Standard and interactive architecture - HyperbodyArchitectureArchitecture and The Built Environmen

Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

Abstract Background Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1–6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed “LPA” throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. Methods Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. Results LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors’ phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. Conclusions In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators

Additional file 2: Figure S2. of Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

PKD family inhibition abrogates LPA-mediated downstream signaling. (A) BV-2 microglia cells were cultured in 6-well plates, serum-starved overnight and preincubated with CRT0066101 (‘CRT’, 1 μM) for the indicated time periods before incubation with LPA (1 μM) or LPA (1 μM) plus CRT. Cells incubated only with 0.1% BSA or CRT (1 μM) were used as negative control. The phosphorylation states of PKDs, JNK, AKT, ERK1/2, and p38 were detected by immunoblotting and one representative blot for each protein is shown. (B) Densitometric analysis of western blots (N = 3). Results are presented as mean values + SEM (***p < 0.001; compared to control; ## p < 0.01, ### p < 0.001 LPA plus CRT versus LPA; one-way ANOVA with Bonferroni correction). (PPT 777 kb

Additional file 5: Figure S5. of Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

The phosphorylation of pro-inflammatory transcription factors is under PKD family control. BV-2 cells, serum-starved overnight and treated with LPA (1 μM) or LPA (1 μM) in the presence of (A) CRT0066101 (1 μM) for the indicated time periods. Cells incubated only with 0.1% BSA or CRT (1 μM) were used as negative control. The phosphorylation state of p65-NF-κB, STAT1, STAT3, and c-Jun was detected by western blotting. One representative blot is shown. (B) Densitometric analysis of western blots (N = 3). Results are presented as mean values + SEM (**p < 0.01, ***p < 0.001 compared to control; ## p < 0.01, ### p < 0.001 LPA plus CRT versus LPA; one-way ANOVA with Bonferroni correction). (PPT 500 kb

Additional file 6: Figure S6. of Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

PKD family members control the secretion of pro-inflammatory cytokines and chemokines. BV-2 cells were cultured on 12-well plates; serum-starved o/n and the supernatants were collected after incubation with DMSO, DMSO plus LPA (1 μM) or LPA (1 μM) plus CRT (1 μM). ELISAs were used to quantitate IL-6, IL-1β, CXCL10 (IP-10), TNF-α, CXCL2 (MIP-2), and CCL5 (RANTES) concentrations. Results shown represent mean + SD from three independent experiments performed in triplicate (*p < 0.05; **p < 0.01 compared to vehicle control; # p < 0.05, ## p < 0.01; CRT + LPA compared to LPA treated cells; one-way ANOVA with Bonferroni correction). (PPT 174 kb