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In Brief: In many mammals, the lipid platelet-activating factor (PAF) has important functions in female reproduction and fertility. This study shows that PAF is present in the reproductive tissues of mares and is involved in processes related to ovulation and early pregnancy., Abstract: Platelet-activating factor (PAF) has been implicated in a number of reproductive processes ranging from ovulation to embryo motility but has not been widely explored in the mare. To identify the presence and examine the role of PAF in the equine periconception processes, targeted mass spectrometry coupled with chromatographic separation was performed on equine follicular fluid (FF), and PAF was quantitatively detected. Subsequently, untargeted high-resolution mass spectrometry-based lipidomic analysis was carried out to quantify PAF in different-sized pre-ovulatory follicles, whereby different molecular species of PAF, PAF (14:0) and PAF (16:1), were both seen to be increasing with follicle diameter. These findings suggest that PAF within FF is increasing as preovulatory follicles approach ovulation. Additionally, immunofluorescence staining identified the PAF receptor in the luminal pericellular, apical, and basal aspect of equine oviductal epithelial cells. Lastly, an equine oviductal epithelial organoid model was generated and showed that the addition of PAF significantly increased the ciliary beat frequency (CBF) (Hz), an action consistent with a role for PAF in embryo migration. It is proposed that the local action of PAF on the ciliated cells of the oviduct propels both the oocyte and the conceptus towards the uterus. In the mare, it appears that PAF is a contributor during the periconception period, potentially being a mediator in the mechanisms of ovulation and in the dialogue of very early pregnancy.

Platelet-activating factor (PAF) plays a significant role in several leucocyte functions, including platelet aggregation and inflammation. Additionally, PAF has a role in the behavioral and physiological changes in mammals. However, the effect of PAF has not been well studied in birds. Therefore, the study aimed to determine if PAF affects feeding behavior, voluntary activity, cloacal temperature, and feed passage through the digestive tract in chicks (Gallus gallus). We also studied the involvement of PAF in the innate immune system induced by lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. Both intraperitoneal (IP) and intracerebroventricular (ICV) injections of PAF significantly decreased food intake. IP injection of PAF significantly decreased voluntary activity and slowed the feed passage from the crop, whereas ICV injection had no effect. Conversely, ICV injection of PAF significantly increased the cloacal temperature, but IP injection had no effect. The IP injection of LPS significantly reduced the mRNA expression of lysophosphatidylcholine acyltransferase 2, an enzyme responsible for PAF production in the heart and pancreas. On the other hand, LPS significantly increased the mRNA expression of the PAF receptor in the peripheral organs. The present study shows that PAF influences behavioral and physiological responses and is related to the response against bacterial infections in chicks., Competing Interests: Declaration of Competing Interest The authors declare that we have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Platelet-activating factor (PAF) is expected to increase esophageal motility. However, to the best of our knowledge, this has not been examined. Thus, we investigated the contractile effects of PAF on guinea pig (GP) esophageal muscularis mucosae (EMM) and the extracellular Ca 2+ influx pathways responsible. PAF (10 -9 -10 -6  M) contracted EMM in a concentration-dependent manner. PAF (10 -6  M)-induced contractions were almost completely suppressed by apafant (a PAF receptor antagonist, 3 × 10 -5  M). In EMM strips, PAF receptor and PAF-synthesizing/degrading enzyme mRNAs were detected. PAF (10 -6  M)-induced contractions were abolished by extracellular Ca 2+ removal but were not affected by diltiazem [a voltage-dependent Ca 2+ channel (VDCC) inhibitor, 10 -5  M]. PAF (10 -6  M)-induced contractions in the presence of diltiazem were significantly suppressed by LOE-908 [a receptor-operated Ca 2+ channel (ROCC) inhibitor, 3 × 10 -5  M], SKF-96365 [an ROCC and store-operated Ca 2+ channel (SOCC) inhibitor, 3 × 10 -5  M], and LOE-908 plus SKF-96365. Among the tested ROCC/SOCC-related mRNAs, Trpc3, Trpc6, and Trpv4/Orai1, Orai3, and Stim2 were abundantly expressed in EMM strips. These results indicate that PAF potently induces GP EMM contractions that are dependent on extracellular Ca 2+ influx through ROCCs/SOCCs, and VDCCs are unlikely to be involved., Competing Interests: Declaration of competing interest The authors indicated no potential conflicts of interest., (Copyright © 2024 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Since its first discovery as a bioactive phospholipid inducing potent platelet aggregation, platelet-activating factor (PAF) has been shown to be involved in a wide variety of inflammatory and allergic disease states. Many pharmacological studies in the 1980s and 1990s also showed that PAF induces endothelium-dependent vascular relaxation and contraction of various smooth muscles (SMs), including those in the airway, gastrointestinal organs, and uterus. However, since the late 1990s, there have been few reports on the SM contractions induced by PAF. The lower urinary tract (LUT), particularly the urinary bladder (UB) has attracted recent attention in SM pharmacology research because patients with LUT dysfunctions including overactive bladder are increasing as the population ages. In addition, recent clinical studies have implicated the substantial role of PAF in the inflammatory state in LUT because its production increases with smoking and with cancer. However, the effects of PAF on mechanical activities of LUT SMs including UBSM have not been investigated to date. Recently, we found that PAF very strongly increased mechanical activities of UBSM in guinea pigs and mice, and partly elucidated the possible mechanisms underlying these actions of PAF. In this review, we describe the effects of PAF on LUT SMs by introducing our recent findings obtained in isolated UBSMs and discuss the physiological and pathophysiological significance. We also introduce our data showing the effects of PAF on the SM mechanical activities of genital tissues (prostate and vas deferens).

Inflammatory mediators constitute a recently coined term in the field of metal-based complexes with antiplatelet activities. Our strategy targets Platelet-Activating Factor (PAF) and its receptor, which is the most potent lipid mediator of inflammation. Thus, the antiplatelet (anti-PAF) potency of any substance could be exerted by inhibiting the PAF-induced aggregation in washed rabbit platelets (WRPs), which internationally is a well-accepted methodology. Herein, a series of mononuclear ( mer -[Cr(pqx)Cl 3 (H 2 O]) ( 1 ), [Co(pqx)Cl 2 (DMF)] ( 2 ) (DMF = N , N '-dimethyl formamide), [Cu(pqx)Cl 2 (DMSO)] ( 3 ) (DMSO = dimethyl sulfoxide), [Zn(pqx)Cl 2 ] ( 4 )) and dinuclear complexes ([Mn(pqx)(H 2 O) 2 Cl 2 ] 2 ( 5 ), [Fe(pqx)Cl 2 ] 2 ( 6 ) and [Ni(pqx)Cl 2 ] 2 ( 7 )) incorporating the 2-(2'-pyridyl)quinoxaline ligand (pqx), were biologically evaluated as inhibitors of the PAF- and thrombin-induced aggregation in washed rabbit platelets (WRPs). The molecular structure of the five-co-ordinate analog ( 3 ) has been elucidated by single-crystal X-ray diffraction revealing a trigonal bipyramidal geometry. All complexes are potent inhibitors of the PAF-induced aggregation in WRPs in the micromolar range. Complex ( 6 ) displayed a remarkable in vitro dual inhibition against PAF and thrombin, with IC 50 values of 1.79 μM and 0.46 μM, respectively. Within the series, complex ( 5 ) was less effective (IC 50 = 39 μM) while complex ( 1 ) was almost 12-fold more potent against PAF, as opposed to thrombin-induced aggregation. The biological behavior of complexes 1 , 6 and 7 on PAF's basic metabolic enzymatic pathways reveals that they affect key biosynthetic and catabolic enzymes of PAF underlying the anti-inflammatory properties of the relevant complexes. The in vitro cytotoxic activities of all complexes in HEK293T (human embryonic kidney cells) and HeLa cells (cervical cancer cells) are described via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results reveal that complex 3 is the most potent within the series.

Background: Excess polymorphonuclear neutrophil (PMN) recruitment or excessive neutrophil extracellular trap (NET) formation can lead to the development of multiple organ dysfunction during sepsis. M2 macrophage-derived exosomes (M2-Exos) have exhibited anti-inflammatory activities in some inflammatory diseases to mediate organ functional protection, but their role in treating sepsis-related acute lung injury (ALI) remains unclear. In this study, we sought to investigate whether M2-Exos could prevent potentially deleterious inflammatory effects during sepsis-related ALI by modulating abnormal PMN behaviours., Methods: C57BL/6 wild-type mice were subjected to a caecal ligation and puncture (CLP) mouse model to mimic sepsis in vivo, and M2-Exos were administered intraperitoneally 1 h after CLP. H&E staining, immunofluorescence and immunohistochemistry were conducted to investigate lung tissue injury, PMN infiltration and NET formation in the lung. We further demonstrated the role of M2-Exos on PMN function and explored the potential mechanisms through an in vitro coculture experiment using PMNs isolated from both healthy volunteers and septic patients., Results: Here, we report that M2-Exos inhibited PMN migration and NET formation, alleviated lung injury and reduced mortality in a sepsis mouse model. In vitro, M2-Exos significantly decreased PMN migration and NET formation capacity, leading to lipid mediator class switching from proinflammatory leukotriene B4 (LTB4) to anti-inflammatory lipoxin A4 (LXA4) by upregulating 15-lipoxygenase (15-LO) expression in PMNs. Treatment with LXA4 receptor antagonist attenuated the effect of M2-Exos on PMNs and lung injury. Mechanistically, prostaglandin E2 (PGE2) enriched in M2-Exos was necessary to increase 15-LO expression in PMNs by functioning on the EP4 receptor, upregulate LXA4 production to downregulate chemokine (C-X-C motif) receptor 2 (CXCR2) and reactive oxygen species (ROS) expressions, and finally inhibit PMN function., Conclusions: Our findings reveal a previously unknown role of M2-Exos in regulating PMN migration and NET formation through lipid mediator class switching, thus highlighting the potential application of M2-Exos in controlling PMN-mediated tissue injury in patients with sepsis., (© 2023. The Author(s).)

Background: The underlying mechanisms of an immediate food-induced allergic reaction involve mast cell degranulation and recruitment of other effector cells, such as lymphocytes, eosinophils, and basophils. How the interaction of various mediators and cells results in anaphylaxis is not fully understood., Objective: To evaluate changes in platelet-activating factor (PAF), platelet-activating factor acetylhydrolase (PAF-AH), tryptase, eosinophils, basophils, and eosinophil cationic protein (ECP) in cashew nut-induced anaphylaxis., Methods: Open cashew nut challenges were performed on 106 children (aged 1-16 years), sensitized to cashew nut, with earlier allergic reaction to cashew nut or no known exposure. PAF, PAF-AH, tryptase, ECP, eosinophils, and basophils were measured at 4 time points., Results: Of 72 challenges with positive results, 34 were defined as anaphylactic. Eosinophil count decreased progressively during an anaphylactic reaction at all 4 time points (P < .005*) compared with baseline. Although significant PAF elevation was observed 1 hour from moderate-to-severe reaction (P = .04*), PAF seemed to peak especially in anaphylaxis but did not achieve statistical significance. PAF peak ratio (peak PAF/baseline PAF) was significantly greater in anaphylactic reactions compared with the no-anaphylaxis group (P = .008*). Maximal percentage change in eosinophils revealed negative correlation to severity score and PAF peak ratio (Spearman's rho -0.424 and -0.516, respectively). Basophils decreased significantly in moderate-to-severe reactions and in anaphylaxis (P < .05*) compared with baseline. Delta-tryptase (peak tryptase minus baseline) did not differ significantly between anaphylaxis and the no-anaphylaxis subgroups (P = .05)., Conclusion: PAF is a specific anaphylaxis biomarker. Marked decline of eosinophils during anaphylaxis may be related to robust secretion of PAF reflecting migration of eosinophils to target tissues., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

We investigated the extracellular Ca 2+ influx pathways involved in platelet-activating factor (PAF)-enhanced guinea pig detrusor smooth muscle (DSM) contractile activities. One micromolar PAF-enhanced DSM contractile activities were completely inhibited by extracellular Ca 2+ removal and strongly suppressed by voltage-dependent Ca 2+ channel (VDCC) inhibitors. PAF-enhanced DSM contractile activities remaining in the presence of verapamil (10 μM) were not inhibited by LOE-908 (30 μM, an inhibitor of receptor-operated Ca 2+ channels (ROCCs)), but were almost completely inhibited by SKF-96365 (30 μM, an inhibitor of store-operated Ca 2+ channels (SOCCs) and ROCCs). These results suggest that VDCCs and SOCCs are responsible for PAF-enhanced DSM contractile activities., Competing Interests: Declaration of competing interest The authors indicated no potential conflicts of interest., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Microvascular hyperpermeability is a hallmark of inflammation. Many negative effects of hyperpermeability are due to its persistence beyond what is required for preserving organ function. Therefore, we propose that targeted therapeutic approaches focusing on mechanisms that terminate hyperpermeability would avoid the negative effects of prolonged hyperpermeability while retaining its short-term beneficial effects. We tested the hypothesis that inflammatory agonist signaling leads to hyperpermeability and initiates a delayed cascade of cAMP-dependent pathways that causes inactivation of hyperpermeability. We applied platelet-activating factor (PAF) and vascular endothelial growth factor (VEGF) to induce hyperpermeability. We used an Epac1 agonist to selectively stimulate exchange protein activated by cAMP (Epac1) and promote inactivation of hyperpermeability. Stimulation of Epac1 inactivated agonist-induced hyperpermeability in the mouse cremaster muscle and in human microvascular endothelial cells (HMVECs). PAF induced nitric oxide (NO) production and hyperpermeability within 1 min and NO-dependent increased cAMP concentration in about 15-20 min in HMVECs. PAF triggered phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in a NO-dependent manner. Epac1 stimulation promoted cytosol-to-membrane eNOS translocation in HMVECs and in myocardial microvascular endothelial (MyEnd) cells from wild-type mice, but not in MyEnd cells from VASP knockout mice. We demonstrate that PAF and VEGF cause hyperpermeability and stimulate the cAMP/Epac1 pathway to inactivate agonist-induced endothelial/microvascular hyperpermeability. Inactivation involves VASP-assisted translocation of eNOS from the cytosol to the endothelial cell membrane. We demonstrate that hyperpermeability is a self-limiting process, whose timed inactivation is an intrinsic property of the microvascular endothelium that maintains vascular homeostasis in response to inflammatory conditions. NEW & NOTEWORTHY Termination of microvascular hyperpermeability has been so far accepted to be a passive result of the removal of the applied proinflammatory agonists. We provide in vivo and in vitro evidence that 1 ) inactivation of hyperpermeability is an actively regulated process, 2 ) proinflammatory agonists (PAF and VEGF) stimulate microvascular hyperpermeability and initiate endothelial mechanisms that terminate hyperpermeability, and 3 ) eNOS location-translocation is critical in the activation-inactivation cascade of endothelial hyperpermeability.

The ability of ultraviolet radiation to suppress the immune system is thought to be central to both its beneficial (protection from autoimmunity) and detrimental (carcinogenic) effects. Previous work revealed a key role for lipids particularly platelet-activating factor and sphingosine-1-phosphate in mediating UV-induced immune suppression. We therefore hypothesized that there may be other UV-induced lipids that have immune regulatory roles. To assess this, mice were exposed to an immune suppressive dose of solar-simulated UV (8 J/cm 2 ). Lipidomic analysis identified 6 lipids (2 acylcarnitines, 2 neutral lipids, and 2 phospholipids) with significantly increased levels in the skin-draining lymph nodes of UV-irradiated mice. Imaging mass spectrometry of the lipids in combination with imaging mass cytometry identification of lymph node cell subsets indicated a preferential location of UV-induced lipids to T cell areas. In vitro co-culture of skin-draining lymph node lipids with lymphocytes showed that lipids derived from UV-exposed mice have no effect on T cell activation but significantly inhibited T cell proliferation, indicating that the lipids play an immune regulatory role. These studies are important first steps in identifying novel lipids that contribute to UV-mediated immune suppression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tse, Ferguson, Koay, Grau, Don and Byrne.)

Platelet-activating factor (PAF) not only acts as a mediator of platelet aggregation, inflammation, and allergy responses but also as a constrictor of various smooth muscle (SM) tissues, including gastrointestinal, tracheal/bronchial, and pregnancy uterine SMs. Previously, we reported that PAF induces basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder SM (UBSM). In this study, we examined the Ca 2+ influx pathways involved in PAF-induced BTI and OC in the mouse UBSM. PAF (10 -6  M) induced BTI and OC in mouse UBSM. However, the PAF-induced BTI and OC were completely suppressed by extracellular Ca 2+ removal. PAF-induced BTI and OC frequencies were markedly suppressed by voltage-dependent Ca 2+ channel (VDCC) inhibitors (verapamil (10 -5  M), diltiazem (10 -5  M), and nifedipine (10 -7  M)). However, these VDCC inhibitors had a minor effect on the PAF-induced OC amplitude. The PAF-induced OC amplitude in the presence of verapamil (10 -5  M) was strongly suppressed by SKF-96365 (3 × 10 -5  M), an inhibitor of receptor-operated Ca 2+ channel (ROCC) and store-operated Ca 2+ channel (SOCC), but not by LOE-908 (3 × 10 -5  M) (an inhibitor of ROCC). Overall, PAF-induced BTI and OC in mouse UBSM depend on Ca 2+ influx and the main Ca 2+ influx pathways in PAF-induced BTI and OC may be VDCC and SOCC. Of note, VDCC may be involved in PAF-induced BTI and OC frequency, and SOCC might be involved in PAF-induced OC amplitude.

Thrombin is the most potent platelet aggregator. To discover the selective inhibitor of thrombin that is important to curing platelet aggregation-related diseases, docking experiments were performed to dock (1R,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1R,3S)-THCCA], and (1S,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1S,3S)-THCCA], into the p pocket of bovine thrombin. The ideal match supported that (1R,3S)-THCCA could be used as a potential lead compound. In this case 20 natural amino acids were theoretically introduced into the 3-carboxyl of (1R,3S)-THCCA and 20 derivatives, (1R,3S)-THCCA-amino acids, were docked into p pocket of bovine thrombin to perform virtual screening. The screening revealed that comparing to (1R,3S)-THCCA itself the DockScores of 16 derivatives were higher, and (1R,3S)-THCCA-Asn (4j) got the highest DockScore. Thus, 16 derivatives were synthesized for experimental study. The in vitro anti-platelet aggregation assay showed that at 100 μM of concentration the 16 derivatives failed to inhibit the platelet aggregation induced by both adenosine diphosphate and arachidonic acid. On the other hand, however, the IC 50 value of the 16 derivatives inhibiting the platelet aggregation induced by platelet activating factor and thrombin ranged from 9.44 μM to 194.64 μM and from 0.07 μM to 9.56 μM, respectively. The in vitro anti-platelet aggregation assay suggested that the 16 derivatives selectively inhibited the platelet aggregation induced by thrombin. In particular, the IC 50 of (1R,3S)-THCCA-Asn (4j) had the lowest value. On rat model at 1 nmol/kg of dosage the 16 derivatives effectively prevented thrombus formation. It is worth pointing out that even at 0.01 nmol/kg of dosage, 4j still effectively prevented thrombus formation. 4j hardly has effects on the proliferation of mammalian cells and rat tail bleeding time. In conclusion, the combination of virtual screening and biological assays successfully lead to the discovery of 4j as a promising candidate of selective inhibitor of thrombin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

G protein-coupled receptors (GPCRs) are important drug targets that mediate various signaling pathways by activating G proteins and engaging β-arrestin proteins. Despite its importance for the development of therapeutics with fewer side effects, the underlying mechanism that controls the balance between these signaling modes of GPCRs remains largely unclear. Here, we show that assembly into dimers and oligomers can largely influence the signaling mode of the platelet-activating factor receptor (PAFR). Single-particle analysis results show that PAFR can form oligomers at low densities through two possible dimer interfaces. Stabilization of PAFR oligomers through cross-linking increases G protein activity, and decreases β-arrestin recruitment and agonist-induced internalization significantly. Reciprocally, β-arrestin prevents PAFR oligomerization. Our results highlight a mechanism involved in the control of receptor signaling, and thereby provide important insights into the relationship between GPCR oligomerization and downstream signaling., (© 2022. The Author(s).)

5-fluorouracil (5-FU) is a widely used chemotherapeutic drug, but its hepatotoxicity challenges its clinical use. Thus, searching for a hepatoprotective agent is highly required to prevent the accompanied hepatic hazards. The current study aimed to investigate the potential benefit and mechanisms of action of rupatadine (RU), a Platelet-activating factor (PAF) antagonist, in the prevention of 5-FU-related hepatotoxicity in rats. Hepatotoxicity was developed in male albino rats by a single 5-FU (150 mg/kg) intra-peritoneal injection on the 7th day of the experiment. RU (3 mg/kg/day) was orally administrated to the rodents for 10 days. Hepatic toxicity was assessed by measuring both liver and body weights, serum alanine aminotransferase and aspartate aminotransferase (ALT and AST), hepatic oxidative stress parameters (malondialdehyde (MDA), nitric oxide levels (NOx), reduced glutathione (GSH), superoxide dismutase (SOD)), and heme oxygenase-1 (HO-1). Inflammatory markers expressions (inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNFα), interleukins; IL-1B, IL-6), the apoptotic marker (caspase-3), and PAF were measured in the hepatic tissue. 5-FU-induced hepatotoxicity was proved by the biochemical along with histopathological assessments. RU ameliorated 5-FU-induced liver damage as proved by the improved serum ALT, AST, and hepatic oxidative stress parameters, the attenuated expression of hepatic pro-inflammatory cytokines and PAF, and the up-regulation of HO-1. Therefore, it can be concluded that RU pretreatment exerted a hepatoprotective effect against 5-FU-induced liver damage through both its powerful anti-inflammatory, antioxidant, and anti-apoptotic effect., (© 2022. The Author(s).)

Experiments were undertaken to identify the nature of a previously identified inhibitor of PAF-induced platelet aggregation (PA) in human saliva. Human saliva fractionated by preparative thin layer chromatography (TLC) yielded a fraction that co-migrated with fatty acids (FAs) and inhibited PAF-induced aggregation of platelets. Synthetic FAs tested for their capacities to inhibit 0.1 nM PAF-induced PA showed that only the cis- unsaturated compounds were inhibitory with activities of some of the polyunsaturated FAs (PUFA) reaching almost 100% at 20 μM. Eicosapentanoic acid (EPA) and 8,11,14-eicosatrienoic acid also deaggregated the PAF-induced aggregates. With the exception of oleic acid (OLA), cis- monounsaturated FAs, and elaidic acid, the trans isomer of OLA, were poor inhibitors. In a direct comparison with other platelet agonists, ADP, thrombin, and ionophore A23187, the active saliva fraction and selected individual FAs inhibited, to greater or lesser extent, PA induced by each of the agonists. EPA, OLA, linoleic acid (LNA), and the active saliva fraction were potent inhibitors of ADP-induced PA, EPA completely inhibited thrombin-induced PA and the saliva fraction showed only weak - moderate inhibitory activity to both thrombin- and ionophore A23187-induced PA. Other reports of endogenous PAF inhibitors in mammalian tissues are compared to the present results. PAF can trigger and amplify inflammatory cascades suggesting a possible modulation role for cis-unsaturated FAs in some diseases.

In this study, we investigated the effects of platelet-activating factor (PAF) on the basal tone and spontaneous contractile activities of guinea pig (GP) and mouse urinary bladder (UB) smooth muscle (UBSM) tissues to determine whether PAF could induce UBSM tissue contraction. In addition, we examined the mRNA expression of the PAF receptor, PAF-synthesizing enzyme (lysophosphatidylcholine acyltransferase, LPCAT), and PAF-degrading enzyme (PAF acetylhydrolase, PAF-AH) in GP and mouse UB tissues using RT-qPCR. PAF (10 -9 -10 -6  M) strongly enhanced the basal tone and spontaneous contractile activities (amplitude and frequency) of GP and mouse UBSM tissues in a concentration-dependent manner. The enhancing effects of PAF (10 -6  M) on both GP and mouse UBSM contractile activities were strongly suppressed by pretreatment with apafant (a PAF receptor antagonist, GP: 10 -5  M; mouse: 3 × 10 -5  M). The PAF receptor (Ptafr), LPCAT (Lpcat1, Lpcat2), and PAF-AH (Pafah1b3, Pafah2) mRNAs were detected in GP and mouse UB tissues. These findings reveal that PAF strongly enhances the contractile mechanical activities of UBSM tissues through its receptor and suggest that the PAF-synthesizing and -degrading system exists in UBSM tissues. PAF may serve as both an endogenous UBSM constrictor and an endogenous mediator leading to detrusor overactivity., (© 2022. The Author(s).)

In the present work the entomopathogenic fungus B. bassiana lipids were studied against the potent pro-inflammatory and thrombotic mediators implicated in several disorders, platelet-activating factor (PAF) and thrombin. Bioactivities of lipid extracts from B. bassiana cells and culture supernatants and of their lipid fractions separated by a one-step HPLC-analysis ere assessed against the PAF/Thrombin-induced aggregation of washed rabbit platelets. Lipid extracts from both cell-biomass and supernatant inhibited strongly PAF/Thrombin-activities and platelet-aggregation, exhibiting higher specificity against PAF. Similarly, HPLC-derived lipid-fractions of phenolics/glycolipids, Sphingomyelins and Phosphatidylcholines (PC) showed strong anti-PAF potency. PC PAF-like molecules exhibited the strongest antagonistic anti-PAF effects, while in higher amounts they agonistically inhibited PAF-activities. Some bioactive lipids with strong anti-PAF effects are exo-cellularly secreted in the culture media during fungal growth, while others are not. The presence of such lipid bioactives in B. bassiana with strong anti-inflammatory and anti-thrombotic properties, provide new perspectives and putative future applications for this entomopathogenic fungus., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Forsyqinlingines C (1) and D (2), two C 9 -monoterpenoid alkaloids bearing a rare skeleton, were isolated from the ripe fruits of Forsythia suspensa. Their structures, including absolute configurations, were fully elucidated by extensive spectroscopic data and ECD experiments. The plausible biogenetic pathway for compounds 1 and 2 was also proposed. In vitro, two C 9 -monoterpenoid alkaloids showed anti-inflammatory activity performed by the inhibitory effect on the release of β-glucuronidase in rat polymorphonuclear leukocytes (PMNs), as well as antiviral activity against influenza A (H1N1) virus and respiratory syncytial virus (RSV)., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Neutrophils are essential for innate immunity as the first line of defence. Neutrophils act as phagocytic white blood cells to kill bacteria and other microorganisms. A strong respiratory burst of neutrophils, dependent on reactive oxygen species, is produced during phagocytosis. Platelet-activating factor (PAF) is a signalling molecule with several prominent roles in tissue injury, inflammation, and platelet aggregation. However, the detailed mechanisms and intracellular signalling pathways involved in PAF-mediated neutrophil activation remain unclear. Here, we investigated the effect of PAF on changes in calcium concentration ([Ca 2+ ] i ) and oxygen radical (O 2 - ) generation in activating canine neutrophils. We further evaluated these effects of PAF with inhibition of G protein-coupled receptors using the specific inhibitor suramin. Blood samples were collected from a total of five dogs and neutrophils were isolated. PAF stimulation of canine neutrophils caused an increase in [Ca 2+ ] i as well as the generation of O 2 - , and the PAF receptor was sensitive to suramin. The results suggested that PAF stimulation of canine neutrophils may cause Ca 2+ influx from the endoplasmic reticulum into the cytoplasm (as the first wave) and then trigger store-operated Ca 2+ entry (as the second wave), which is an important intracellular signal transduction pathway for neutrophil activation. Furthermore, O 2 - generation by PAF stimulation may depend on the intracellular signalling pathway, with increasing inositol trisphosphate levels and [Ca 2+ ] i via G protein-coupled receptors. The finding that PAF-activating platelet aggregation is involved in canine neutrophil activation suggests a close relationship between haemostasis and neutrophil activation in dogs, offering new insight into the response to infection., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12-20 different glycoforms. We isolated a glycoform secreted from platelet-activating factor (PAF)-stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behavior, electrophoretic mobility, and pattern of glycosylation. The function of these 2 glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration, and neutrophil extracellular traps (NETosis) involving myeloperoxidase, peptidylarginine deiminase 4, and phosphorylation of ERK in a dose-dependent fashion, whereas nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. Interestingly, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by PAF or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and phosphorylation of the protein kinase A substrate vasodilator-stimulated phosphoprotein and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity, whereas nAGP-1 glycoform also limits proinflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on 2 critical cells of acute inflammation., (©2020 Society for Leukocyte Biology.)

Platelet-activating factor (PAF) is an important mediator of the systemic inflammatory response. In the case of sepsis, proper activation and function of neutrophils as the first line of cellular defense are based on a well-balanced physiological response. However, little is known about the role of PAF in cellular changes of neutrophils during sepsis. Therefore, this study investigates the reaction patterns of neutrophils induced by PAF with a focus on membrane potential (MP), intracellular pH, and cellular swelling under physiological and pathophysiological conditions and hypothesizes that the PAF-mediated response of granulocytes is altered during sepsis. The cellular response of granulocytes including MP, intracellular pH, cellular swelling, and other activation markers were analyzed by multiparametric flow cytometry. In addition, the chemotactic activity and the formation of platelet-neutrophil complexes after exposure to PAF were investigated. The changes of the (electro-)physiological response features were translationally verified in a human ex vivo whole blood model of endotoxemia as well as during polymicrobial porcine sepsis. In neutrophils from healthy human donors, PAF elicited a rapid depolarization, an intracellular alkalization, and an increase in cell size in a time- and dose-dependent manner. Mechanistically, the alkalization was dependent on sodium-proton exchanger 1 (NHE1) activity, while the change in cellular shape was sodium flux- but only partially NHE1-dependent. In a pathophysiological altered environment, the PAF-induced response of neutrophils was modulated. Acidifying the extracellular pH in vitro enhanced PAF-mediated depolarization, whereas the increases in cell size and intracellular pH were largely unaffected. Ex vivo exposure of human whole blood to lipopolysaccharide diminished the PAF-induced intracellular alkalization and the change in neutrophil size. During experimental porcine sepsis, depolarization of the MP was significantly impaired. Additionally, there was a trend for increased cellular swelling, whereas intracellular alkalization remained stable. Overall, an impaired (electro-)physiological response of neutrophils to PAF stimulation represents a cellular hallmark of those cells challenged during systemic inflammation. Furthermore, this altered response may be indicative of and causative for the development of neutrophil dysfunction during sepsis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hug, Bernhard, Stratmann, Erber, Wohlgemuth, Knapp, Bauer, Vidoni, Fauler, Föhr, Radermacher, Hoffmann, Huber-Lang and Messerer.)

Epidemiological data from the SARS-CoV-2 outbreak suggest sex differences in mortality and vulnerability; however, sex-dependent incidence of acute respiratory distress syndrome (ARDS) remains controversial and the sex-dependent mechanisms of endothelial barrier regulation are unknown. In premenopausal women, increased signalling of angiotensin (Ang)(1-7) via the Mas receptor has been linked to lower cardiovascular risk. Since stimulation of the Ang(1-7)/Mas axis protects the endothelial barrier in acute lung injury (ALI), we hypothesised that increased Ang(1-7)/Mas signalling may protect females over males in ALI/ARDS.Clinical data were collected from Charité inpatients (Berlin) and sex differences in ALI were assessed in wild-type (WT) and Mas-receptor deficient ( Mas -/- ) mice. Endothelial permeability was assessed as weight change in isolated lungs and as transendothelial electrical resistance (TEER) in vitro In 734 090 Charité inpatients (2005-2016), ARDS had a higher incidence in men as compared to women. In murine ALI, male WT mice had more lung oedema, protein leaks and histological evidence of injury than female WT mice. Lung weight change in response to platelet-activating factor (PAF) was more pronounced in male WT and female Mas -/- mice than in female WT mice, whereas Mas-receptor expression was higher in female WT lungs. Ovariectomy attenuated protection in female WT mice and reduced Mas-receptor expression. Oestrogen increased Mas-receptor expression and attenuated endothelial leakage in response to thrombin in vitro This effect was alleviated by Mas-receptor blockade.Improved lung endothelial barrier function protects female mice from ALI-induced lung oedema. This effect is partially mediated via enhanced Ang(1-7)/Mas signalling as a result of oestrogen-dependent Mas expression., Competing Interests: Conflict of interest: L. Erfinanda has nothing to disclose. Conflict of interest: K. Ravindran has nothing to disclose. Conflict of interest: F. Kohse has nothing to disclose. Conflict of interest: K. Gallo has nothing to disclose. Conflict of interest: R. Preissner has nothing to disclose. Conflict of interest: T. Walther reports grants from the Deutsche Forschungsgemeinschaft (WA 1441/22), during the conduct of the study; is a scientific advisor to Constant Pharmaceuticals Ltd; and has a patent “Use of an Ang-(1–7) receptor agonist in acute lung injury” pending. Conflict of interest: W.M. Kuebler reports grants from the Deutsche Forschungsgemeinschaft (KU 1218/7), during the conduct of the study; and has a patent “Use of an Ang-(1–7) receptor agonist in acute lung injury” pending., (Copyright ©ERS 2021.)

Aim: To examine the effects of platelet-activating factor (PAF) on the barrier functions of cultured retinal pigment epithelial (RPE) cells., Methods: A human RPE cell line (ARPE-19) was cultured on microporous filter supports and treated with PAF and WEB 2086, a specific PAF-receptor (PAF-R) antagonist. The permeability of the RPE monolayer was measured using transepithelial electrical resistance (TER) and sodium fluorescein flux. The expression of the tight junction protein zonula occludens (ZO)-1 and the adherens junction protein N-cadherin was assessed using immunohistochemistry and Western blotting. We also measured the vascular endothelial growth factor (VEGF) concentrations in PAF-treated cultures and re-measured RPE monolayer permeability in the presence of VEGF-neutralizing antibodies., Results: PAF significantly decreased the TER and enhanced the sodium fluorescein flux of the RPE monolayer and downregulated the expression of ZO-1 and N-cadherin. These effects were abolished by WEB 2086-mediated blockage of the PAF-R. PAF stimulation increased VEGF expression in RPE cells, and the antibody-mediated neutralization of VEGF caused a partial recovery of the barrier properties., Conclusion: The barrier functions of ARPE-19 cells were altered by PAF, and these effects were partly mediated by an upregulation of VEGF expression in these cells. Our results contribute to the growing body of evidence supporting the role of PAF in choroidal neovascularization. Our findings suggest that PAF is a novel target in the development of therapies for increased permeability of the RPE monolayer., Competing Interests: The authors have no conflicts of interest to declare., (© 2020 Zhang et al.)

G protein-coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor-activating peptide, an increased maximum response to adenosine 5'-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10-/- platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18-/- mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18-/- and RGS10-/-18-/- mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival., (© 2020 by The American Society of Hematology.)

Platelet-activating factor (PAF) affects capacitation, acrosome reaction and fertilisation potential of spermatozoa. This study investigated the underlying mechanism(s) through which PAF regulated sperm function. Our data demonstrated that PAF dose-dependently induced, whilst lyso-PAF (PAF precursor) showed no effect on acrosome reaction of capacitated human spermatozoa. Treatment with PAF for 90 min enhanced tyrosine phosphorylation and expression of extracellular signal-regulated protein kinases (ERK) 1 and 2 in human spermatozoa. Moreover, pre-treatment with the ERK inhibitor U0126 significantly and dose-dependently suppressed PAF-induced acrosome reaction. Therefore, PAF may be actively involved in the modulation of sperm acrosome reaction by interacting with ERK. The role of PAF in fertilisation warrants further investigation., (© 2020 Blackwell Verlag GmbH.)

To act on tissues, circulating insulin must perfuse the relevant organ and then leave the bloodstream by crossing the endothelium-a process known as insulin delivery. It has been postulated that the continuous endothelium is a rate-limiting barrier to insulin delivery but existing data are contradictory. This conflict is in part due to the limitations of current models, including the inability to maintain a constant blood pressure in animals and the absence of shear stress in cultured cells. We developed a murine cardiac ex vivo perfusion model that delivers insulin to the heart in situ at a constant flow. We hypothesized that if the endothelial barrier were rate-limiting to insulin delivery, increasing endothelial permeability would accelerate insulin action. The kinetics of myocardial insulin action were determined in the presence or absence of agents that increased endothelial permeability. Permeability was measured using Evans Blue, which binds with high affinity to albumin. During our experiments, the myocardium remained sensitive to insulin and the vasculature retained barrier integrity. Perfusion with insulin induced Akt phosphorylation in myocytes but not in the endothelium. Infusion of platelet-activating factor or vascular endothelial growth factor significantly increased permeability to albumin without altering insulin action. Amiloride, an inhibitor of fluid-phase uptake, also did not alter insulin action. These data suggest that the endothelial barrier is not rate limiting to insulin's action in the heart; its passage out of the coronary circulation is consistent with diffusion or convection. Modulation of transendothelial transport to overcome insulin resistance is unlikely to be a viable therapeutic strategy., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Myelosupression resulting from chemotherapy has been widely described in veterinary medicine; however, there is limited information relating to alterations in neutrophil function after chemotherapy in dogs with cancer. The aim of this study was to determine the non-proliferative effects of vincristine, carboplatin, and cisplatin on canine neutrophils by evaluating activation of oxidative and non-oxidative responses. Neutrophils were isolated from venous blood. Levels of reactive oxygen species (ROS) and metalloproteinase 9 (MMP-9) were measured in vitro during neutrophil exposure to these chemotherapeutic agents for 15 min followed by stimulation with platelet activating factor (PAF). ROS production was detected via luminescence, and MMP- 9 liberation was determined by zymography. The chemotherapeutic agents caused an increase in PAF-induced ROS production, but no change in the non-oxidative response was observed. These results suggest that these chemotherapeutic agents may act as priming agents by increasing the oxidative response. These effects could be beneficial for dogs with cancer by supporting their immune systems; however, excessive ROS liberation has been associated with inflammation, neutrophil-mediated cell injury, carcinogenesis, and metastasis. Clinical studies are necessary to evaluate the significance of these findings., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aim: A UPLC-MS/MS method was developed to determine LBPT as well as its four metabolites in human plasma to support the clinical study aiming to evaluate the efficacy of LBPT tablet in patients undergoing hip/knee replacement. Methodology: Plasma samples were prepared by protein precipitation and then separated on a C 18 analytical column using (A) acetonitrile (B) 0.1% formic acid and 10 mM ammonium formate in water. The detection was performed on a triple quadrupole tandem mass spectrometer in positive electrospray ionization using multiple reactions monitoring mode. Results & conclusion: The method has been validated in accordance with the US FDA guidelines and was applied to the measurement of five analytes in human plasma samples from a Phase II clinical trial.

Rationale: Specialized pro-resolving mediators (SPM-lipoxins, resolvins, protectins, and maresins) are produced via the enzymatic conversion of essential fatty acids, including the omega-3 fatty acids docosahexaenoic acid and n-3 docosapentaenoic acid. These mediators exert potent leukocyte directed actions and control vascular inflammation. Supplementation of animals and humans with essential fatty acids, in particular omega-3 fatty acids, exerts protective actions reducing vascular and systemic inflammation. Of note, the mechanism(s) activated by these supplements in exerting their protective actions remain poorly understood., Objective: Given that essential fatty acids are precursors in the biosynthesises of SPM, the aim of the present study was to establish the relationship between supplementation and peripheral SPM concentrations. We also investigated the relationship between changes in plasma SPM concentrations and peripheral blood platelet and leukocyte responses., Methods and Results: Healthy volunteers were enrolled in a double-blinded, placebo-controlled, crossover study, and peripheral blood was collected at baseline, 2, 4, 6, and 24 hours post administration of placebo or one of 3 doses of an enriched marine oil supplement. Assessment of plasma SPM concentrations using lipid mediator profiling demonstrated a time- and dose-dependent increase in peripheral blood SPM concentration. Supplementation also led to a regulation of peripheral blood cell responses. Here we found a dose-dependent increase in neutrophil and monocyte phagocytosis of bacteria and a decrease in the diurnal activation of leukocytes and platelets, as measured by a reduction in adhesion molecule expression. In addition, transcriptomic analysis of peripheral blood cells demonstrated a marked change in transcript levels of immune and metabolic genes 24 hours post supplementation when compared with placebo., Conclusions: Together, these findings demonstrate that supplementation with an enriched marine oil leads to an increase in peripheral blood SPM concentrations and reprograms peripheral blood cells, indicating a role for SPM in mediating the immune-directed actions of this supplement., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT03347006.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD + ) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD + adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD + using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λ max  = 326 nm) in the presence of NAD + . This adduct was confirmed to be INH-NAD + using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD + , was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD + adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Platelet-activating factor (PAF) is a potent lipid mediator whose involvement in the onset and progression of atherosclerosis is mediated by, among others, the modulation of cytokine expression patterns. The presence of multiple potential protein-tyrosine phosphatase (PTP) 1B substrates in PAF receptor signaling pathways brought us to investigate its involvement in PAF-induced cytokine expression in monocyte-derived dendritic cells (Mo-DCs) and to study the pathways involved in this modulation., Methods: We used in-vitro-matured human dendritic cells and the HEK-293 cell line in our studies. PTP1B inhibition was though siRNAs and a selective inhibitor. Cytokine expression was studied with RT-PCR, luciferase assays and ELISA. Phosphorylation status of kinases and transcription factors was studied with western blotting., Results: Here, we report that PTP1B was involved in the modulation of cytokine expression in PAF-stimulated Mo-DCs. A study of the down-regulation of PAF-induced IL-8 expression, by PTP1B, showed modulation of PAF-induced transactivation of the IL-8 promoter which was dependent on the presence of the C/EBPß -binding site. Results also suggested that PTP1B decreased PAF-induced IL-8 production by a glycogen synthase kinase (GSK)-3-dependent pathway via activation of the Src family kinases (SFK). These kinases activated an unidentified pathway at early stimulation times and the PI3K/Akt signaling pathway in a later phase. This change in GSK-3 activity decreased the C/EBPß phosphorylation levels of the threonine 235, a residue whose phosphorylation is known to increase C/EBPß transactivation potential, and consequently modified IL-8 expression., Conclusion: The negative regulation of GSK-3 activity by PTP1B and the consequent decrease in phosphorylation of the C/EBPß transactivation domain could be an important negative feedback loop by which cells control their cytokine production after PAF stimulation.

Purpose: Previous studies have identified alkyl-phospholipids as promising compounds for cancer therapy by targeting constituents of the cell membrane and different signaling pathways. We previously showed that the alkylphospholipid Inositol-C2-PAF inhibits the proliferation and migration of immortalized keratinocytes and the squamous carcinoma-derived cell line SCC-25. Here, we investigated the effect of this compound on growth and motility as well as its mode of action in mammary carcinoma-derived cell lines., Methods: Using BrdU incorporation and haptotactic cell migration assays, we assessed the effects of Inositol-C2-PAF on MCF-7 and MBA-MB-231 cell proliferation and migration. The phosphorylation status of signaling molecules was investigated by Western blotting as well as indirect immunofluorescence analysis and capillary isoelectric focusing., Results: We found that Inositol-C2-PAF inhibited the growth as well as the migration in MCF-7 and MBA-MB-231 cells. Furthermore, we found that this compound inhibited phosphorylation of the protein kinase Akt at serine residue 473, but had no impact on phosphorylation at threonine 308. Phosphorylation of other kinases, such as Erk1/2, FAK and Src, which are targeted by Inositol-C2-PAF in other cells, remained unaffected by the compound in the mammary carcinoma-derived cell lines tested. In MCF-7 cells, we found that IGF-1-induced growth, as well as phosphorylation of Akt S473 , mTOR and the tumor suppressor pRB, was inhibited in the presence of Inositol-C2-PAF. Moreover, we found that in these cells IGF-1 had no impact on migration and did not seem to be linked to full Akt activity. Therefore, MCF-7 cell migration appears to be inhibited by Ino-C2-PAF in an Akt-independent manner., Conclusion: The antagonistic effects of Inositol-C2-PAF on cell migration and proliferation are indicative for its potential for breast cancer therapy, alone or in combination with other cytostatic drugs.

It is widely hypothesized that menstrual pain is triggered by prostaglandin synthesis that evokes high-pressure uterine contractions and ischemia. However, the effects of molecules implicated in menstrual pain on uterine contractility, perfusion, and oxygenation in vivo have been rarely demonstrated. Studies in women that do not respond to nonsteroidal anti-inflammatory drugs (NSAIDs) have reported elevated levels of platelet-activating factor (PAF). To establish in vivo evidence of PAF's capability to impair uterine homeostasis and to elicit visceral pain, we examined the effects of the PAF receptor agonist (carbamyl PAF [CPAF]) in comparison to other molecules hypothesized to play a role in uterine pain in mice. Uterine pressure was increased by oxytocin, prostaglandin F2α (PGF2α), and CPAF. Even in the absence of inflammatory molecules, uterine contractions reduced uterine oxygenation by 38%. CPAF reduced uterine perfusion by 40% ± 8% and elicited further oxygen desaturation approaching hypoxia (9.4 ± 3.4 mm Hg Pao 2 ). Intraperitoneal injections of CPAF and PGF2α evoked visceral pain and pelvic hyperalgesia in awake wild-type mice. However, pain was not observed in identically injected PAF-receptor knockout mice. Thus, our model provides a demonstration that a molecule implicated in NSAID-resistant dysmenorrhea has a detrimental effect on uterine homeostasis and is capable of causing visceral pain. Our results support the general hypothesis that menstrual cramps are caused by uterine contractions, impaired perfusion, and reduced oxygenation. Since this study was limited to mice, confirmation of these results in humans would be valuable for development of novel therapeutics targeted at inflammatory precursors, contractility, perfusion, and tissue oxygenation.

The amyloid hypothesis of Alzheimer's disease suggests that synaptic degeneration and pathology is caused by the accumulation of amyloid-β (Aβ) peptides derived from the amyloid precursor protein (APP). Subsequently, soluble Aβ oligomers cause the loss of synaptic proteins from neurons, a histopathological feature of Alzheimer's disease that correlates with the degree of dementia. In this study, the production of toxic forms of Aβ was examined in vitro using 7PA2 cells stably transfected with human APP. We show that conditioned media from 7PA2 cells containing Aβ oligomers caused synapse degeneration as measured by the loss of synaptic proteins, including synaptophysin and cysteine-string protein, from cultured neurons. Critically, conditioned media from 7PA2 cells treated with valproic acid (2-propylpentanoic acid (VPA)) or propylisopropylacetic acid (PIA) did not cause synapse damage. Treatment with VPA or PIA did not significantly affect total Aβ 42 concentrations; rather these drugs selectively reduced the concentrations of Aβ 42 oligomers in conditioned media. In contrast, treatment significantly increased the concentrations of Aβ 42 monomers in conditioned media. VPA or PIA treatment reduced the concentrations of APP within lipid rafts, membrane compartments associated with Aβ production. These effects of VPA and PIA were reversed by the addition of platelet-activating factor, a bioactive phospholipid produced following activation of phospholipase A 2 , an enzyme sensitive to VPA and PIA. Collectively these data suggest that VPA and PIA reduce Aβ oligomers through inhibition of phospholipase A 2 and suggest a novel therapeutic approach to Alzheimer's treatment., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

The aim of the study is to identify possible mechanisms that could provoke a recurrence of chronic obstructive pyelonephritis (COP) in eldery women, thus to find out - why age and sex can be risk factors for acute pyelonephritis in urolithiasis. The results of 43 women over 65 years of age (mean age 71±1,5 years) are presented in study. The 26 patients were examined in remission phase of COP; 17 patients were examined in recurrence phase. For platelet stimulation epinephrine and platelet activating factor (PAF) were used at an effective concentration (EC50). The formation of platelet-leukocyte aggregates (PLA) was modeled in vitro by incubation of stimulated platelets (epinephrine and PAF) and intact leukocytes. The number of intact PLA was examined after blood smears staining according to Romanovsky-Giemsa method. In women older than 65 years realization of acute inflammation in urinary tract with urolithiasis occurs on the background of normal sensitivity of α2-adrenergic receptors and hyperreactivity of the PAF-receptors of platelets. In remission phase of COP the hyporeactivity of α2-adrenergic and PAF receptors of platelets was revealed but under interaction of epinephrine and PAF activation of platelets are possible which accompanied by formation of PLA. In women over 65 years hypoactivity of platelets in remission phase of COP can be a risk factor for the acute pyelonephritis development, since the simultaneous action of epinephrine and PAF increases the number of PLAs that recruit leukocytes for the realization of acute inflammatory reaction in urolithiasis.

Background: Neutrophil (polymorphonuclear) granulocytes (PMN) have been shown to contribute to the pathogenesis of psoriasis by releasing interleukin-17 and LL37-DNA complexes via neutrophil extracellular traps (NETs), webs of chromatin strands decorated with antimicrobial peptides, in psoriatic skin. Fumaderm ® , a fumaric acid ester (FAE) formulation consisting of different FAE salts, has been successfully used to treat psoriasis for decades. Most recently, FAE treatment was reported to inhibit NET formation in murine epidermolysis bullosa acquisita., Objectives: To elucidate the effect of FAE treatment on human psoriasis and healthy donor NET formation., Results: Among the compounds present in the FAE formulation, dimethyl fumarate (DMF) pretreatment of human psoriasis and healthy donor PMN resulted in a consistent inhibitory effect on NET formation in response to phorbol 12-myristate 13-acetate but not to platelet activating factor and ionomycin. This effect was l-glutathione (GSH) dependent and involved a decrease in reactive oxygen species (ROS) production, a key event in NET formation. In contrast, G-protein-coupled signalling and protein synthesis were not involved. Monomethyl fumarate (MMF) was found to slightly reduce ROS production without affecting NET formation., Conclusions: We report DMF as a potent, stimulus-specific, GSH- and ROS-dependent modulator of NET formation. Our results support the notion that modulation of NET formation contributes to the beneficial effects of FAEs in a variety of inflammatory conditions., (© 2017 British Association of Dermatologists.)

Capillary leakage syndrome, vasomotor disturbances and gut atony are common clinical problems in intensive care medicine. Various inflammatory mediators and signalling pathways are involved in these pathophysiological alterations among them platelet-activating factor (PAF). The related signalling mechanisms of the PAF-induced dysfunctions are only poorly understood. Here we used the model of the isolated perfused rat small intestine to analyse the role of calcium (using calcium deprivation, IP-receptor blockade (2-APB)), cAMP (PDE-inhibition plus AC activator), myosin light chain kinase (inhibitor ML-7) and Rho-kinase (inhibitor Y27632) in the following PAF-induced malfunctions: vasoconstriction, capillary and mucosal leakage, oedema formation, malabsorption and atony. Among these, the PAF-induced vasoconstriction and hyperpermeability appear to be governed by similar mechanisms that involve IP3 receptors, extracellular calcium and the Rho-kinase. Our findings further suggest that cAMP-elevating treatments - while effective against hypertension and oedema - bear the risk of dysmotility and reduced nutrient uptake. Agents such as 2-APB or Y27632, on the other hand, showed no negative side effects and improved most of the PAF-induced malfunctions suggesting that their therapeutic usefulness should be explored.

Platelet-activating factor (PAF) is produced by macrophages during inflammation and infections. We evaluated whether PAF is able to modulate the infection of human macrophages by Leishmania braziliensis, the main Leishmania sp. in Brazil. Monocyte-derived macrophages were incubated with promastigote forms in absence or presence of exogenous PAF. We observed that the treatment of macrophages with low concentrations of PAF prior to infection increased the phagocytosis of L. braziliensis. More importantly, exogenous PAF reduced the parasitism when it was added before, during or after infection. In addition, treatment with a PAF antagonist (PCA 4248) resulted in a significant increase of macrophage infection in a concentration-dependent manner, suggesting that endogenous PAF is important to control L. braziliensis infection. Mechanistically, while exogenous PAF increased production of reactive oxygen species (ROS) treatment with PCA 4248 reduced oxidative burst during L. braziliensis infection. The microbicidal effects of exogenous PAF were abolished when macrophages were treated with apocynin, an NADPH oxidase inhibitor. The data show that PAF promotes the production of ROS induced by L. braziliensis, suggesting that this lipid mediator may be relevant to control L. braziliensis infection in human macrophages., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytoplasmic pH homeostasis is required for an appropriate response in polymorphonuclear neutrophils (PMNs). In these cells, chemotaxis and reactive oxygen species (ROS) production are reduced by the use of Na + /H + exchanger (NHE-1) inhibitors, but these results are mainly obtained using amiloride, a non-selective NHE-1 inhibitor. In bovine PMNs, the role of NHE-1 in functional responses has not been confirmed yet. The aim of this study was to determine the role of NHE-1 using amiloride and zoniporide in pH regulation, ROS production, matrix metalloproteinase 9 (MMP-9) release and calcium flux in bovine PMNs induced by the platelet activation factor (PAF), additionally we evaluated the presence of NHE-1 and NHE-2 mRNA Our data show the presence only of NHE-1 but not NHE-2 in bovine PMNs. Amiloride or zoniporide inhibited the intracellular alkalization induced by PAF without affecting calcium flux. Amiloride diminished ROS production and MMP-9 release, while zoniporide enhanced ROS production without change the MMP-9 release induced by PAF. Our work led us to conclude that changes in intracellular pH induced by PAF are regulated by NHE-1 in bovine neutrophils, but the effects of amiloride on ROS production and MMP-9 release induced by PAF are not NHE-1 dependent., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The effect of variation in platelet function in platelet donors on patient outcome following platelet transfusion is unknown. This trial assessed the hypothesis that platelets collected from donors with highly responsive platelets to agonists in vitro assessed by flow cytometry (high-responder donors) are cleared more quickly from the circulation than those from low-responder donors, resulting in lower platelet count increments following transfusion. This parallel group, semirandomized double-blinded trial was conducted in a single center in the United Kingdom. Eligible patients were those 16 or older with thrombocytopenia secondary to bone marrow failure, requiring prophylactic platelet transfusion. Patients were randomly assigned to receive a platelet donation from a high- or low-responder donor when both were available, or when only 1 type of platelet was available, patients received that. Participants, investigators, and those assessing outcomes were masked to group assignment. The primary end point was the platelet count increment 10 to 90 minutes following transfusion. Analysis was by intention to treat. Fifty-one patients were assigned to receive platelets from low-responder donors, and 49 from high-responder donors (47 of which were randomized and 53 nonrandomized). There was no significant difference in platelet count increment 10 to 90 minutes following transfusion in patients receiving platelets from high-responder (mean, 21.0 × 10 9 /L; 95% confidence interval [CI], 4.9-37.2) or low-responder (mean, 23.3 × 10 9 /L; 95% CI, 7.8-38.9) donors (mean difference, 2.3; 95% CI, -1.1 to 5.7; P = .18). These results support the current policy of not selecting platelet donors on the basis of platelet function for prophylactic platelet transfusion., (© 2017 by The American Society of Hematology.)

Purpose: Altered platelet aggregability has been implicated in the pathogenesis of glaucoma. This study aims to investigate the anti-platelet potential of intraocular pressure lowering drops, with the possibility of establishing it as an additional mechanism of anti-glaucomatous action., Materials and Methods: The anti-aggregating effects of a series of anti-glaucomatous eye drops were determined on human platelets in the platelet aggregation model, using four known aggregating factors (platelet activating factor [PAF], adenosine diphosphate [ADP], thrombin receptor-activating peptide [TRAP], and arachidonic acid [AA])., Results: Almost all of the tested samples inhibited platelet aggregation induced by PAF, ADP, TRAP, and AA, except for Alphagan, which did not demonstrate inhibition of ADP- and TRAP-induced aggregation at a wide range of concentrations. Trusopt, Betoptic, and Azarga eye drops were the most potent inhibitors of all four aggregating factors, while Alphagan was the least potent ( P <0.05)., Conclusion: This study shows that anti-glaucomatous eye drops possess anti-platelet effects, and this was shown for the first time by experimenting on human platelets., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The aim of this study was to investigate the effect of PAF supplementation in semen extender on ATP content in cryopreserved bull spermatozoa used for artificial insemination at different time intervals. Cryopreserved semen was treated with different concentrations of PAF: 1×10-5M, 1×10-6M, 1×10-7M, 1×10-8M and 1×10-9M at 37°C. In the present work we showed that content of ATP in cryopreserved semen supplemented with 1×10-9M PAF was statistically significantly higher at 90 and 120 minutes of incubation in comparison to the control group (p≤0.05). Present study indicates the potential influence of PAF on ATP content in male spermatozoa via it's protective role towards mitochondria metabolic activity.

Ultraviolet (UV)-irradiated keratinocytes secrete the lipid mediator of inflammation, platelet-activating factor (PAF). PAF plays an essential role in UV-induced immune suppression and skin cancer induction. Dermal mast cell migration from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced PAF activates mast cell migration by up-regulating mast cell CXCR4 surface expression. Recent findings indicate that PAF up-regulates CXCR4 expression via histone acetylation. UV-induced PAF also activates cell cycle arrest and disrupts DNA repair, in part by increasing p21 expression. Do epigenetic alterations play a role in p21 up-regulation? Here we show that PAF increases Acetyl-CREB-binding protein (CBP/p300) histone acetyltransferase expression in a time and dose-dependent fashion. Partial deletion of the HAT domain in the CBP gene, blocked these effects. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the p21 promoter. PAF-treatment had no effect on other acetylating enzymes (GCN5L2, PCAF) indicating it is not a global activator of histone acetylation. This study provides further evidence that PAF activates epigenetic mechanisms to affect important cellular processes, and we suggest this bioactive lipid can serve as a link between the environment and the epigenome., Competing Interests: The authors declare no competing financial interests.

Two new pyranoflavonoids, morustralins A (1) and B (2), a new natural benzene derivative, one benzenoid (Z)-1-hydroxy-4-(2-nitroethenyl)benzene (3), and thirty known compounds were isolated and characterized from the root bark of Morus australis. The structures of the new compounds were established from spectroscopic and spectrometric analyses. Ten isolates (1-10) were examined for inhibitory effects on adenosine diphosphate (ADP)-, arachidonic acid (AA)-, and platelet-aggregating factor (PAF)-induced platelet aggregation. Among the tested compounds, compound 3 displayed the most significant inhibition of ADP- and AA-induced platelet aggregation with IC 50 values of 9.76±5.54 and 9.81±2.7μM, respectively. In addition, eight purified compounds (3-10) were examined for inhibition of nitric oxide (NO) production in RAW 264.7 cells and six compounds (3-8) displayed significant inhibitory effects with IC 50 values ranging from 2.1±0.3 to 6.3±0.6μM., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP). BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

Background and Objective: Platelet-activating factor (PAF) is a lipid mediator involved in the pathophysiology of several allergic diseases, for example, in the amplification of mast cell (MC) activation in anaphylaxis. Rupatadine is an antihistamine with a demonstrated anti-PAF effect, although its capacity to inhibit PAF-induced MC degranulation has not been fully evaluated. Objectives: To compare the ability of rupatadine to inhibit PAF-induced MC degranulation with that of desloratadine and levocetirizine and to confirm the dual anti-H1 and anti-PAF activity of rupatadine., Methods: The human MC line LAD2 and primary MCs (human lung tissue MCs [hLMCs]) were used. MC mediator release was evaluated using the b-hexosaminidase and histamine release assay. The effects of rupatadine (H1 antagonist + PAF receptor antagonist), desloratadine, and levocetirizine (H1 antagonists) on LAD2 and hLMCs were compared. The PAF receptor antagonists WEB2086, BN52021, and CV6209 were also tested. PAF receptor protein expression was evaluated in both LAD2 and hLMCs., Results: CV6209 and rupatadine inhibited PAF-induced MC degranulation in both LAD2 and hLMCs. In LAD2, rupatadine (5 and 10 µM) and levocetirizine (5 µM), but not desloratadine, inhibited PAF-induced b-hexosaminidase release. Rupatadine (1-10 µM), levocetirizine (1-10 µM), and desloratadine (10 µM) inhibited PAF-induced histamine release. Rupatadine at 10 µM had an inhibitory effect on hLMC degranulation, but levocetirizine and desloratadine did not., Conclusions: This study shows that rupatadine and, to a lesser extent, levocetirizine, but not desloratadine, inhibit PAF-induced degranulation in both LAD2 and hLMCs. These findings support the dual antihistamine and anti-PAF effect of rupatadine in allergic disorders.

This study analysed the impact of anandamide, cannabidiol (CBD), and WIN55,212-2 on platelet activity and thrombogenesis for the first time. The effects of the cannabinoids on venular thrombosis were studied in the ear of hairless mice. Cannabinoid treatment was performed either once or repetitive by a once-daily administration for three days. To assess the role of cyclooxygenase metabolites in the putative action of anandamide, in vivo studies likewise included a combined administration of anandamide with indomethacin. In vitro, the effect of the cannabinoids on human platelet activation was studied by means of P-selectin expression using flow cytometry. Platelets were analysed under resting or thrombin receptor activating peptide (TRAP)-stimulated conditions, both after cannabinoid treatment alone and after TRAP stimulation and subsequent cannabinoid exposure. Finally, platelet count was assessed after treatment with high concentrations of anandamide. Anandamide, but not CBD and WIN55,212-2, significantly accelerated thrombus growth after one-time treatment as compared to vehicle control. Co-administration with indomethacin neutralized this effect. However, thrombogenesis was not altered by repeated treatment with the cannabinoids. In vitro, anandamide was shown to elicit a concentration-dependent activation of resting human platelets. However, at higher concentrations anandamide reduced the response to TRAP activation associated with a decrease of platelet count. CBD and WIN55,212-2 neither increased nor reduced activation of platelets. Acute exposure to anandamide elicits a cyclooxygenase-dependent prothrombotic effect in vivo. Anandamide seems to affect human platelet activation by a concentration-dependent toxic effect. By contrast, CBD and WIN55,212-2 were not associated with induction of thrombosis or activation of platelets. © 2016 BioFactors, 42(6):581-590, 2016., (© 2016 International Union of Biochemistry and Molecular Biology.)

Background: The inflamed bronchial mucosal surface is a profoundly hypoxic environment. Neutrophilic airway inflammation and neutrophil-derived proteases have been linked to disease progression in conditions such as COPD and cystic fibrosis, but the effects of hypoxia on potentially harmful neutrophil functional responses such as degranulation are unknown., Methods and Results: Following exposure to hypoxia (0.8% oxygen, 3 kPa for 4 h), neutrophils stimulated with inflammatory agonists (granulocyte-macrophage colony stimulating factor or platelet-activating factor and formylated peptide) displayed a markedly augmented (twofold to sixfold) release of azurophilic (neutrophil elastase, myeloperoxidase), specific (lactoferrin) and gelatinase (matrix metalloproteinase-9) granule contents. Neutrophil supernatants derived under hypoxic but not normoxic conditions induced extensive airway epithelial cell detachment and death, which was prevented by coincubation with the antiprotease α-1 antitrypsin; both normoxic and hypoxic supernatants impaired ciliary function. Surprisingly, the hypoxic upregulation of neutrophil degranulation was not dependent on hypoxia-inducible factor (HIF), nor was it fully reversed by inhibition of phospholipase C signalling. Hypoxia augmented the resting and cytokine-stimulated phosphorylation of AKT, and inhibition of phosphoinositide 3-kinase (PI3K)γ (but not other PI3K isoforms) prevented the hypoxic upregulation of neutrophil elastase release., Conclusion: Hypoxia augments neutrophil degranulation and confers enhanced potential for damage to respiratory airway epithelial cells in a HIF-independent but PI3Kγ-dependent fashion., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

OBJECTIVE To characterize platelet-activating factor (PAF)-induced edema and erythema in the skin of dogs and compare those reactions with histamine-induced cutaneous reactions. ANIMALS 6 healthy Beagles. PROCEDURES Experiments were performed at ≥ 2-week intervals. Each dog received ID injections (5 μg/site) of PAF C16, PAF C18, lyso-PAF, and histamine. Edema (mean diameter) and erythema scores (none, mild, moderate, or severe) were assessed 30 minutes after the injections. Dogs received ID injections of PAF and histamine each with various concentrations of WEB 2086 (PAF receptor antagonist) or underwent ID testing with PAF and histamine before and 3 hours after oral administration of cetirizine hydrochloride or prednisolone (at 2 doses each). RESULTS ID injections of PAF C16 and PAF C18, but not lyso-PAF, induced comparable levels of edema and erythema. The PAF-induced edema and erythema peaked at 30 minutes and lasted for 6 hours after the injection; histamine-induced edema and erythema peaked at 30 minutes and lasted for 3 hours after the injection. Edema sizes and erythema scores were significantly smaller and lower, respectively, for PAF than for histamine. The WEB 2086 inhibited PAF-induced but not histamine-induced edema and erythema. Cetirizine slightly, but significantly, repressed PAF-induced edema and erythema as well as histamine-induced cutaneous reactions. Prednisolone suppressed both PAF-induced and histamine-induced edema and erythema. CONCLUSIONS AND CLINICAL RELEVANCE In canine skin, the duration of PAF-induced inflammation was longer than that of histamine-induced inflammation. The PAF- and histamine-induced cutaneous reactions were effectively suppressed by oral administration of prednisolone. The importance of PAF in dogs with anaphylaxis and allergic disorders warrants further investigation.