LTB4 Production Research Articles

Characterization of a Salmonella Typhimurium ghost carrying an adjuvant protein as a vaccine candidate for the protection of chickens against virulent challenge

In this study we describe the generation of a safe, immunogenic, genetically inactivated Salmonella Typhimurium ghost vaccine candidate carrying the Escherichia coli heat-labile enterotoxin B subunit (LTB) protein as an adjuvant molecule. An asd+ p15A ori– plasmid pJHL187-LTB harbouring the E lysis gene cassette and a foreign antigen delivery cassette containing the eltB gene was used to transform a Δasd Salmonella Typhimurium (JOL1311) strain to construct the ghost strain, JOL1499. Incubation of mid-logarithmic phase JOL1499 cultures at 42°C resulted in co-expression of the eltB and E lysis genes, leading to the generation of Salmonella Typhimurium ghost cells carrying the LTB protein (Salmonella Typhimurium-LTB ghost). The production of LTB in Salmonella Typhimurium-LTB ghost preparations was confirmed by western blot analysis, and functional activity of the LTB protein to bind with GM1 receptors was determined by means of GM1 enzyme-linked immunosorbent assay. Efficacy of the Salmonella Typhimurium-LTB ghost as a vaccine candidate was evaluated in a chicken model using 56 chickens at 5 weeks old, which were divided into four groups (n = 14): group A was designated the non-vaccinated control group, whereas the birds in groups B, C, and D were immunized intramuscularly with 109, 108, and 107 ghost cells, respectively. Compared with the non-immunized chickens (group A), immunized chickens (groups B, C and D) exhibited increased titres of plasma IgG and intestinal secretory IgA antibodies. After oral challenge with 109 colony-forming units of a virulent Salmonella Typhimurium strain, the vaccinated group B birds showed a decrease in internal organ colonization with the challenge strain.

Protective effects of garlic extract, PMK-S005, against nonsteroidal anti-inflammatory drugs-induced acute gastric damage in rats.

PMK-S005 is synthetic s-allyl-L-cysteine (SAC), a sulfur-containing amino acid, which was initially isolated from garlic. The antioxidant and anti-inflammation activities of SAC have been demonstrated in diverse experimental animal models. The purpose of this study was to investigate the gastroprotective effects of PMK-S005 against NSAIDs-induced acute gastric damage in rats. Eight-week SD rats were pretreated with PMK-S005 (1, 5, or 10mg/kg) or rebamipide (50mg/kg) 1h before administration of NSAIDs including aspirin (200mg/kg), diclofenac (80mg/kg), and indomethacin (40mg/kg). After 4h, the gross ulcer index, histological index, and gastric mucus level were determined. Myeloperoxidase (MPO), TNF-α, IL-1β, PGE2, and LTB4 levels were estimated in the gastric mucosal tissue by ELISA. Protein expressions of cPLA2, COX-1, and COX-2 were assessed by Western blot analysis. Pretreatment with PMK-S005 significantly attenuated the NSAIDs-induced gastric damage and increased the gastric mucus level. In addition, PMK-S005 attenuated increases in MPO, TNF-α, and IL-1β production. The expressions of cPLA2 and COX-2 induced by NSAIDs were decreased by PMK-S005 pretreatment. PMK-S005 did not cause suppression of PGE2 synthesis induced by NSAIDs, but LTB4 production was significantly suppressed by PMK-S005. The effects of PMK-S005 were consistently maximized at a concentration of 5mg/kg, which were frequently superior to those of rebamipide. These results strongly suggest that PMK-S005 can be a useful gastroprotective agent against acute gastric mucosal damage by suppressing proinflammatory cytokines, down-regulating cPLA2, COX-2 and LTB4 expression, and increasing the synthesis of mucus.

Low leukotriene B4 receptor 1 leads to ALOX5 downregulation at diagnosis of chronic myeloid leukemia.

ALOX5 is implicated in chronic myeloid leukemia development in mouse leukemic stem cells, but its importance in human chronic myeloid leukemia is unknown. Functional ALOX5 was assessed using an LTB4 ELISA and ALOX5, and LTB4R1 mRNA expression was determined via a TaqMan gene expression assay. LTB4R1 and 5-LOX protein levels were assessed by cell surface flow cytometry analysis. At diagnosis ALOX5 was below normal in both blood and CD34(+) stem cells in all patients. On treatment initiation, ALOX5 levels increased in all patients except those who were destined to progress subsequently to blast crisis. LTB4 levels were increased despite low ALOX5 expression, suggesting that the arachidonic acid pathway is functioning normally up to the point of LTB4 production. However, the LTB4 receptor (BLT1) protein in newly diagnosed patients was significantly lower than after a period of treatment (P<0.0001). The low level of LTB4R1 at diagnosis explains the downregulation of ALOX5. In the absence of LTB4R1, the arachidonic acid pathway intermediates (5-HEPTE and LTA4) negatively regulate ALOX5. ALOX5 regulation is aberrant in chronic myeloid leukemia patients and may not be important for the development of the disease. Our data suggest caution when extrapolating mouse model data into human chronic myeloid leukemia.

Functional characterization of histamine H4 receptor on human mast cells

Among the four different types of histamine receptors (H1-H4), H4R is predominantly expressed in immune cells and involved in immunomodulatory response. Here, in this study we determined the expression of H4R in human mast cells (HMC-1, LAD-2 and primary cord blood derived CD34+ human mast cells) and characterized its functional properties. Interestingly, we found that human mast cells responded to both histamine (natural ligand) and 4-methylhistamine (selective H4R agonist) for sustained intracellular calcium mobilization, degranulation and cytokine production. However, only histamine induced the release of cAMP, but 4-methylhistamine down regulates cAMP indicating that H4R mediates its effect through Gαi/o protein and H1R via Gαq protein. Furthermore, both histamine and 4-methylhistamine induced the production of cysteinyl leukotrienes and LTB4. Using human inflammation antibody array membrane, we found that H4R induced the expression of various inflammatory proteins, involving pro-inflammatory cytokines and chemokines and these are TGF-β1, TNF-α, TNF-β, PDGF-BB, TIMP-2, M-CSF, IP-10, IL-16, IL-6, IL-3, IL-10, MIP-1α, IL-1α, ICAM-1, Eotaxin-2, RANTES, IL-8, MCP-1, and IL-6sR. We also quantified the level of various inflammatory cytokines produced by human mast cells through H4R. It was observed that, the production level of Th2 cytokines IL-4(401.34pg/ml), IL-5 (64.21pg/ml) and IL-13 (1044pg/ml) and classical proinflammatory cytokines IL-6 (221.27pg/ml) and IL-1β (34.24pg/ml) and chemokines MCP-1(106pg/ml) and IL-8 (818.32pg/ml). Furthermore, activation of H4R caused the phosphorylation of ERK and PI3K in a time dependent manner. Taken together these data demonstrate that, the activation of H4R in human mast cells produced not only inflammatory mediators that are associated with allergic reactions but also other inflammatory conditions.

Add‐on therapy with montelukast in the treatment of Henoch–Schönlein purpura

Previous studies suggested that leukotrienes (LT) were involved in the pathogenesis of Henoch-Schönlein purpura (HSP). This study investigated the efficacy of an add-on therapy with montelukast in the treatment of HSP. In this four-center, double-blind, placebo-controlled, parallel paired comparative study, 130 children with HSP were divided into two large groups: 84 patients without nephritis and 46 patients with nephritis. For each pair of patients with the same severity of disease, one subject was randomly allocated to one subgroup and the other allocated to the other subbroup; one subgroup received routine treatment plus placebo treatment, while the other subgroup received routine treatment plus montelukast treatment for 3 months. The efficacy was determined using Severity Scale Score (SSS). Blood eosinophil count, eosinophil cationic protein (ECP), IgE, interleukin (IL)-4, IL-5, IL-6, IL-8, IL-17, LTB4 , and urinary LTE4 were measured. Add-on therapy with montelukast alleviated the symptoms of HSP including purpura, abdominal pain, stool occult blood, arthritis, proteinuria and hematuria, and, accordingly, shortened the length of hospital stay, and lowered blood eosinophil count, ECP, IgE, IL-4, IL-5, IL-6, IL-8, IL-17, LTB4 , and urinary LTE4 production, and also lowered the HSP relapse rate during the 3 months of treatment, but did not alter the outcome of nephritis at the end of follow up. Add-on therapy with montelukast alleviated the symptoms of HSP. HSP may be improved by add-on therapy with a leukotriene receptor antagonist.

De Novo Design of Multitarget Ligands with an Iterative Fragment-Growing Strategy

The discovery of multitarget drugs has recently attracted much attention. Most of the reported multitarget ligands have been serendipitous discoveries. Although a few methods have been developed for rational multitarget drug discovery, there is a lack of elegant methods for de novo multitarget drug design and optimization, especially for multiple targets with large differences in their binding sites. In this paper, we report the first de novo multitarget ligand design method, with an iterative fragment-growing strategy. Using this method, dual-target inhibitors for COX-2 and LTA₄H were designed, with the most potent one inhibiting PGE₂ and LTB₄ production in the human whole blood assay with IC₅₀ values of 7.0 and 7.1 μM, respectively. Our strategy is generally applicable in rational and efficient multitarget drug design, especially for the design of highly integrated inhibitors for proteins with dissimilar binding pockets.

Human neutrophils produce IL-12, IL-10, PGE2 and LTB4 in response to Paracoccidioides brasiliensis. Involvement of TLR2, mannose receptor and dectin-1

The functions of phagocytic cells against pathogens are initiated by the interaction between membrane receptors and molecular structures which compose the cell wall of these microorganisms. Thus our study aimed to identify the neutrophil receptors involved in the recognition of different strains of Paracoccidioides brasiliensis and the consequent modulation of immune response through the production of cytokines and inflammatory mediators. Neutrophils did not produce TNF-alfa in response to both strains. However, these cells produce IL-12, mainly in response to Pb 265, with participation of TLR2 and dectin-1. These cells also produce L-10, whose levels were higher for Pb 18 with involvement of TLR2 and MR and only TLR2 for Pb 265. The production of PGE2 and LTB4 was detected similarly for the two strains. For PGE2, MR and dectin-1 were involved, while in relation to LTB4, none of them. In summary, we demonstrated that neutrophils have a dynamic role during host immune response to P. brasiliensis, since in addition to their role as effector cells of innate immunity; they have the capacity to modulate innate and adaptative immune response against this fungus by producing cytokines and lipidic mediators. This modulation may be toward a pró- or anti-inflammatory pattern in a dependence of P. brasiliensis strains and PRR involved in fungus recognition by these cells.

Homoisoflavanone prevents mast cell activation and allergic responses by inhibition of Syk signaling pathway

Mast cells play important roles in allergic inflammatory responses because they produce leukotrienes (LTs), prostaglandins (PGs), and a variety of inflammatory cytokines. Thus, pharmacological interventions for allergies have focused on inhibiting mast cell activation. Homoisoflavanone (HIF), isolated from Cremastra appendiculata Makino, has anti-angiogenic activities; however, its effects on allergic reactions have not been determined. The aim of this study was to assess the inhibitory effects of HIF on mast cell activation, which is critical for anti-allergic reaction and the underlying mechanisms. Enzyme-linked immunosorbent assays, quantitative real-time PCR, western blot analyses, and degranulation assay were performed to measure pro-inflammatory and allergic mediators in PMA/A23187- or IgE/antigen-stimulated mouse bone marrow-derived mast cells (BMMCs), HMC-1, RBL-1, or human PBMC-derived mast cells treated with or without HIF. The anti-allergic effects of HIF were determined in mouse models using dinitrophenol-immunoglobulin E-induced passive cutaneous anaphylaxis (PCA) and compound 48/80-induced ear swelling. Homoisoflavanone down-regulated PGD2 , LTB4 , and LTC4 production and inhibited the production of pro-inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α in PMA/A23187- or IgE/antigen-stimulated mast cells. The molecular mechanisms by which HIF caused these inhibitory effects were determined to be the inactivation of spleen tyrosine kinase (Syk) signaling and the concurrent suppression of cPLA2 . HIF inhibited IgE-mediated PCA and compound 48/80-induced ear swelling in mouse. Homoisoflavanone inhibited mast cell activation through the suppression of Syk pathway together with the inhibition of cPLA2 . Thus, it might be a good candidate molecule for allergic diseases.

A remarkable Activity of Steroid Biosynthesis in Captopril Preserved Leydig Cells of Mice Implicated Leukotriene B4 and Gonadotropin Releasing Hormones in vitro

In this study, investigated whether captopril inhibited steroidogenesis and components of the Leukotriene B4 pathways are involved in GnRH agonist (GnRH)-induced testis steroidogenesis in mice Leydig cells. Primary cultures of mice Leydig cells were established. Purified Leydig cells from adult albino mice were incubated with gradual various concentrations of GnRH with and without Captopril, Luteinizing hormone (LH); LTB4, steroidogenic (testosterone) activity and LTB4 concentration were measured after various time intervals and Leydig cell viability. The maneuvers of Leydig cells treated media was covered the singular and dual actions of antisteroidogenic of captopril and the reversible activity by GnRH-LTB4 as well as contribution of LTB4 in Leydig cells testosterone production endpoint. The different treatment media are Medium alone; Medium plus captopril 60 μM, 65 μM, 70 μM, 75 μM and 80 μM 100 μM; Medium plus 2.5mU/ml leukotriene B4; Medium plus 0.1 mM LH; Medium plus 0.1 μM GnRH; Medium plus 65 μM captopril plus 2.5 mU/ml leukotriene B4;Medium plus 65 μM captopril plus 0.1 mM LH; Medium plus 65 μM captopril plus 0.1 mM GnRH; Medium plus 0.1 mM GnRH plus 2.5 mU/ml leukotriene B4 and Medium plus 65 μM captopril plus 0.1 mM GnRH plus 2.5 mU/ml leukotriene B4. Basal testosterone levels were maximal at 0.1 μM GnRH concentration and superior testosterone yield in Leydig cells incubated 0.1 μM GnRH media than without GnRH media, and the activity profile LTB4 flow up. That comparable result led to highly correlated approved the contribution of LTB4 in GnRH stimulated Leydig cell steroidogenic end point. Furthermore captopril had an abolishment effect partially of testosterone yield and recovered and improved by GnRH and LTB4. The Leydig cells viability results suggest that the major effect of GnRH is probably beyond the LTB4. The entire key; GnRH induced testosterone production and upregulated LTB4 Levels at both the captopril inhibitory LTB4-testesteron Leydig cells culture media and captopril abolished LTB4 levels; it also activated endogenous LTB4, but not LH motivated testosterone pathway. Our data show that GnRH positively regulates steroidogenesis via LTB4 signaling in mice Leydig cells. LTB4 activation by GnRH may be responsible for the induction of Ca++ signaling indirect. Possibility improve the captopril steroidogenic disruption in Leydig cells via LTB4 and/or GnRH induction of endogenous LTB4, likewise the positive maintenance of Leydig cells viability matched induce testosterone synthesis. The LTB4 production, which may ultimately modulate steroidogenesis in mice Leydig cells, and promise new antidotal and preventative of captopril adverse effects.

Inhibition of LOX by flavonoids: a structure–activity relationship study

The lipoxygenase (LOX) products have been identified as mediators of a series of inflammatory diseases, namely rheumatoid arthritis, inflammatory bowel disease, psoriasis, allergic rhinitis, atherosclerosis and certain types of cancer. Hence, LOX inhibitors are of interest for the modulation of these phenomena and resolution of the inflammatory processes. During LOX activity, peroxyl radical complexes are part of the reaction and may function as sources of free radicals. Thus antioxidants, such as flavonoids, capable of inhibiting lipid peroxidation and scavenging free radicals, may act as LOX inhibitors. The aim of this work was to assess the structure–activity relationship among a series of flavonoids concerning 5-LOX inhibition, through a systematic study of the inhibition of the formation of LTB4 in human neutrophils. The type of inhibition of the flavonoids was further studied using soybean LOX, type I, and Saturation Transfer Difference 1H NMR (STD-1H NMR) was used to characterize the binding epitopes of the compounds to LOX-1. The obtained results reinforce flavonoids as effective inhibitors of LTB4 production in human neutrophils. It was also possible to establish a structure/activity relationship for the inhibitory activity and the type of inhibition.

Growth inhibitory effect of KYKZL-1 on Hep G2 cells via inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the inhibitory activity test on Hep G2 growth. We found that KYKZL-1 inhibited the growth of Hep G2 cells via inducing apoptosis. Further studies showed that KYKZL-1 activated caspase-3 through cytochrome c release from mitochondria and down regulation of Bcl-2/Bax ratio and reduced the high level of COX-2 and 5-LOX. As shown in its anti-inflammatory effect, KYKZL-1 also exhibited inhibitory effect on the PGE2 and LTB4 production in Hep G2 cells. Accordingly, exogenous addition of PGE2 or LTB4 reversed the decreases in cell viability. In addition, KYKZL-1 caused cell cycle arrest at the S–G2 checkpoint via the activation of p21CIP1 protein and down-regulation of cyclin A expression.These data indicate that the growth inhibitory effect of KYKZL-1 is associated with inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest.Combined with our previous findings, KYKZL-1 exhibiting COX/5-LOX inhibition may be a promising potential agent not only for inflammation control but also for cancer prevention/therapy with an enhanced gastric safety profile.

AB0613 Omega-3 polyunsaturated fatty acids for the treatment of rheumatoid arthritis: A meta-analysis

BackgroundOmega-3 polyunsaturated fatty acids (PUVAs) may lead to a reduction of pain and inflammation via competitively inhibiting the production of PGE2 and LTB4 and inhibiting the release of interleukin-1 beta...

Protective Role of Acetylsalicylic Acid in Experimental Trypanosoma cruzi Infection: Evidence of a 15-epi-Lipoxin A4-Mediated Effect

Chagas' disease, produced by Trypanosoma cruzi, affects more than 8 million people, producing approximately 10,000 deaths each year in Latin America. Migration of people from endemic regions to developed countries has expanded the risk of infection, transforming this disease into a globally emerging problem. PGE2 and other eicosanoids contribute to cardiac functional deficits after infection with T. cruzi. Thus, the inhibition of host cyclooxygenase (COX) enzyme emerges as a potential therapeutic target. In vivo studies about the effect of acetylsalicylic acid (ASA) upon T. cruzi infection are controversial, and always report the effect of ASA at a single dose. Therefore, we aimed to analyze the effect of ASA at different doses in an in vivo model of infection and correlate it with the production of arachidonic acid metabolites. ASA decreased mortality, parasitemia, and heart damage in T. cruzi (Dm28c) infected mice, at the low doses of 25 and 50 mg/Kg. However, this effect disappeared when the high ASA doses of 75 and 100 mg/Kg were used. We explored whether this observation was related to the metabolic shift toward the production of 5-lipoxygenase derivatives, and although we did not observe an increase in LTB4 production in infected RAW cells and mice infected, we did find an increase in 15-epi-LXA4 (an ASA-triggered lipoxin). We also found high levels of 15-epi-LXA4 in T. cruzi infected mice treated with the low doses of ASA, while the high ASA doses decreased 15-epi-LXA4 levels. Importantly, 15-epi-LXA4 prevented parasitemia, mortality, and cardiac changes in vivo and restored the protective role in the treatment with a high dose of ASA. This is the first report showing the production of ASA-triggered lipoxins in T. cruzi infected mice, which demonstrates the role of this lipid as an anti-inflammatory molecule in the acute phase of the disease.

Retinoid derivatives offer anti‐inflammatory benefits by promoting neutrophil apoptosis and inhibiting proinflammatory mediator production in a model of bovine respiratory disease

Clearance of apoptotic neutrophils (PMN) following infection is critical for the resolution of inflammation. Despite demonstrating immunomodulatory properties, the effects of retinoids in PMN in the context of an inflammatory response remain unknown.Objectiveto evaluate the immunomodulatory properties of two retinoids, oxidatively‐transformed carotene‐β (OxC‐β) and retinoic acid (RA) in a model of Mannheimia haemolytica‐induced bovine respiratory disease, which is characterized by severe inflammation.Resultsin vitro, RA and OxC‐β induced caspase‐and time‐dependent apoptosis, but not necrosis, in bovine PMN, but not epithelial cells or fibroblasts. Neither RA nor OxC‐β affected PMN function. In M. haemolytica‐infected calves (2×107 CFU), animals that received a 28‐day dietary OxC‐β treatment (10 mg/kg) had elevated apoptotic leukocytes and reduced LTB4 levels in their lower airways 3h post‐infection vs infected‐untreated calves.ConclusionRA and OxC‐β promote cell‐specific apoptosis and inhibit the production of pro‐inflammatory LTB4, both mechanisms that promote the resolution of inflammation, thereby suggesting an anti‐inflammatory role for retinoid derivatives. Supported by NSERC and AIHS.

Astaxanthin decreases inflammatory biomarkers associated with cardiovascular disease in human umbilical vein endothelial cells

As a potent antioxidant, astaxanthin may downregulate inflammation‐associated factors involved in endothelial dysfunction and cardiovascular disease progression. We studied the possible protective effects of astaxanthin against endothelial dysfunction in human umbilical vein endothelial cells (HUVEC) induced with H2O2. Cells were pre‐incubated with 0, 0.01, 0.1, or 1.0 μmol/L astaxanthin for 48 h, then oxidative stress induced with 100 μmol/L H2O2 overnight. Uptake kinetics showed a time‐dependent accumulation of astaxanthin (1.0 μmol/L) by HUVEC over the 48 h period. Oxidative stress induction with H2O2 decreased intracellular antioxidant activity (SOD and GPx) and increased the production of reactive oxygen species (ROS), inflammatory cytokines and mediators. In contrast, pre‐incubation of HUVEC with astaxanthin prior to H2O2 stress increased (P &lt; 0.05) SOD, but not GPx activity, and decreased the production of ROS, PGE2, LTB4, NO, IL‐8, and IFN‐γ. Pre‐treatment with astaxanthin also downregulated transcriptional activation of NF‐κB and activator protein‐1, thereby inhibiting downstream production of inflammatory cytokines and mediators. Therefore, astaxanthin inhibits inflammatory biomarkers upregulated by H2O2, likely by scavenging ROS required for transcriptional activation. Supported by the University of Washington and Washington State University.

Atropa acuminata Royle Ex Lindl. blunts production of pro-inflammatory mediators eicosanoids., leukotrienes, cytokines in vitro and in vivo models of acute inflammatory responses

Ethnopharmacological relevanceAtropa acuminata Royle Ex Lindl. has been widely used in folk medicine for several inflammatory disorders such as arthritis, asthma, conjunctivitis, encephalitis, pancreatitis, peritonitis, acute infections and neuroinflammatory disorders. Aim of the studyOur aim was to evaluate Atropa acuminata for its anti-inflammatory properties and to delineate its possible mechanism of action on the modulation of the inflammatory mediators. Materials and methodsWe investigated the inhibitory action of ethanolic extract of Atropa acuminata (AAEE) on production of NO, TNF-α and IL-1β in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and also assayed it for COX 1/2 and 5-LOX inhibitory activities. Next AAEE was tested in acute inflammatory animal models., carragenean induced rat paw edema, carragenean induce pleurisy in rats and vascular permeability in mice and the effects on NO, PGE2 and LTB4 production in the pleural fluid and paw exudates were evaluated. In addition the effects on leukocyte migration and exudation and vascular permeability were also observed. ResultsOur findings summarized novel anti-inflammatory mechanisms for Atropa acuminata based on dual in vitro cyclooxygenase 1/2/ and 5-Lipoxygenase inhibitory activities and also significant downregulation of nitric oxide (NO) and pro-inflammatory cytokin (TNF-α and Il-1 β) release in LPS-stimulated RAW 246.7 macrophage cell line. In acute inflammatory models in vivo (carragenean induced edema, carragenean induced pleurisy in rats and vascular permeability in mice), AAEE exhibited an extensive diverse mechanism for anti-inflammatory properties. This was indicated on the basis of dose dependent suppression of multi targeted inflammatory mediators., NO, TNF-α and IL-1β, eicosanoids., PGE2 and leukotrienes., LTB4 along with significantly decreased leucocyte migration, exudation and decreased vascular permeability. These effects were more potent and prolonged than traditional NSAIDS, thereby indicating fewer side effects. AAEE was found to be safe for long term administration, as confirmed by the results of acute toxicity studies and MTT assay. The complex mode of action of the herbs was attributed possibly due to the high polyphenolic, flavanol and flavonoid content present in the extracts as observed by means of quantitative screening for phytochemicals. ConclusionOur study provides scientific evidence to support the traditional anti-inflammatory uses of Atropa acuminata and is probably due to inhibitory effects on multiple inflammatory mediators which indicates a promising potential for the development of a strong anti-inflammatory agent from this plant.

Inhibition of PGE2 and LTB4 production by dendritic cells induced by Paracoccidioides brasiliensis is associated with the non maturation of these cells

Event Abstract Back to Event Inhibition of PGE2 and LTB4 production by dendritic cells induced by Paracoccidioides brasiliensis is associated with the non maturation of these cells Reginaldo K. Fernandes1*, Tatiana F. Bachiega1, Daniela R. Rodrigues1, Márjorie A. Golim2, Luciane A. Dias-Melicio3, Helanderson A. Balderramas1, Ramon Kaneno4 and Ângela M. Soares1* 1 Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP) - Câmpus de Botucatu, Microbiologia e Imunologia - Instituto de Biociências, Brazil 2 Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Hemocentro, Brazil 3 Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Depto. Patologia - FMB - Botucatu, Brazil 4 Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Microbiologia e Imunologia, Brazil The fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic mycosis endemic in Latin America. Dendritic cells (DCs) play a crucial role in the detection of pathogens, triggering an initial response from the host as well as the instruction of the adaptive immune response. Mediators released by DCs, by an autocrine way, modulate their functions. Among these, we highlight the prostaglandins and leukotrienes. We therefore assessed whether human DCs produce PGE2 and LTB4 in response to fungus, the participation of recognition receptors of molecular patterns (PRRs) in this production, as well as the modulating role of these eicosanoids on phenotypic maturation of these cells. Human immature DCs (CD14-/ CD1ahigh/CD83low) derived from the differentiation of monocytes cultured with GM-CSF and IL-4 (7 days) released substantial concentrations of PGE2 and LTB4 which increased significantly in the case of PGE2, with incubation with LPS for 1h, 2h, 4h, 8h, 12h, 18h, 24h or 48h. However, the levels of PGE2 and LTB4 were significantly inhibited after challenge with two different strains of the fungus. Assays using monoclonal antibodies to block PRRS showed the involvement of mannose receptor in PGE2 inhibition by the fungus. Additionally, phenotyping assays showed that after challenge with the fungus, DCs did not change their phenotype of immature cells to mature ones. Accordingly, we detected a clear association between inhibition of the production of PGE2 and LTB4 fungus and inability to induce maturation of DCs. Assays using exogenous PGE2 and LTB4 confirmed this association. Acknowledgements Financial Support: FAPESP 2011/12477-0 / 2011/04556-8 Keywords: Eicosanoids, Dendritic Cells, prostaglandin, leukotriene, Paracoccidioides brasiliensis Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Fernandes RK, Bachiega TF, Rodrigues DR, Golim MA, Dias-Melicio LA, Balderramas HA, Kaneno R and Soares ÂM (2013). Inhibition of PGE2 and LTB4 production by dendritic cells induced by Paracoccidioides brasiliensis is associated with the non maturation of these cells. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00536 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Apr 2013; Published Online: 22 Aug 2013. * Correspondence: Mr. Reginaldo K Fernandes, Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP) - Câmpus de Botucatu, Microbiologia e Imunologia - Instituto de Biociências, Botucatu-SP, Brazil, regiskeller@msn.com Prof. Ângela M Soares, Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP) - Câmpus de Botucatu, Microbiologia e Imunologia - Instituto de Biociências, Botucatu-SP, Brazil, acsoares@ibb.unesp.br Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Reginaldo K Fernandes Tatiana F Bachiega Daniela R Rodrigues Márjorie A Golim Luciane A Dias-Melicio Helanderson A Balderramas Ramon Kaneno Ângela M Soares Google Reginaldo K Fernandes Tatiana F Bachiega Daniela R Rodrigues Márjorie A Golim Luciane A Dias-Melicio Helanderson A Balderramas Ramon Kaneno Ângela M Soares Google Scholar Reginaldo K Fernandes Tatiana F Bachiega Daniela R Rodrigues Márjorie A Golim Luciane A Dias-Melicio Helanderson A Balderramas Ramon Kaneno Ângela M Soares PubMed Reginaldo K Fernandes Tatiana F Bachiega Daniela R Rodrigues Márjorie A Golim Luciane A Dias-Melicio Helanderson A Balderramas Ramon Kaneno Ângela M Soares Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Leukotriene B4 Activation of Arterial Smooth Muscle BK Channels

Blood circulation depends on the myogenic tone of small arteries. Tone is increased by a rise in overall cytosolic calcium [Ca2+]i in the arterial smooth muscle cell (SMC). This rise leads to activation of large-conductance, Ca2+/voltage-gated K+ (BK) channels. This generates outward currents that hyperpolarize the membrane and limit Ca2+ entry. Thus, BK channel activation opposes SMC contraction.Several negatively charged endogenous lipids, such as PIP2, fatty and bile acids have been shown to directly activate SMC BK channels. The chemical structure of leukotrienes (LTs) includes a long hydrophobic backbone and a carboxylic end group, which is largely ionized at physiological pH=6.8-7.4. Thus, we hypothesized that LTs were BK channel activators.We tested LTA4, LTB4, LTC4, LTD4, and LTE4 on heterologously expressed BK channels conformed by cbv1 (AY330293) and rβ1 (FJ154955) subunits cloned from rat cerebral artery SMC. LTs were applied at 1 nM to the cytosolic side of inside-out (I/O) membrane patches from Xenopus oocytes; Vm=-80 to −20 mV, [Ca2+]i=10 μM. LTB4 was the only LT that significantly increased BK channel activity (130% from pre-LT values), with a ≈330% increase in activity in response to 2 μM LTB4. LTB4-induced BK channel activation was also observed in I/O patches from freshly isolated cerebral artery SMCs. Therefore, we identified LTB4 as a potent and effective SMC BK channel activator that works independently of cell signaling. using pressurized, de-endothelized cerebral arteries from rats, we blocked LTB4 production by 50 μM bestatin. This treatment reduced artery diameter by ≈5%, which is expected to cause up to 10-20% decrease in cerebral blood flow. This result suggests that LTB4 production within arterial SMC exerts a tonic effect on arterial tone, favoring SMC relaxation and opposing vasoconstriction.

5-lipoxygenase deficiency reduces acetaminophen-induced hepatotoxicity and lethality.

5-Lipoxygenase (5-LO) converts arachidonic acid into leukotrienes (LTs) and is involved in inflammation. At present, the participation of 5-LO in acetaminophen (APAP)-induced hepatotoxicity and liver damage has not been addressed. 5-LO deficient (5-LO−/−) mice and background wild type mice were challenged with APAP (0.3–6 g/kg) or saline. The lethality, liver damage, neutrophil and macrophage recruitment, LTB4, cytokine production, and oxidative stress were assessed. APAP induced a dose-dependent mortality, and the dose of 3 g/kg was selected for next experiments. APAP induced LTB4 production in the liver, the primary target organ in APAP toxicity. Histopathological analysis revealed that 5-LO−/− mice presented reduced APAP-induced liver necrosis and inflammation compared with WT mice. APAP-induced lethality, increase of plasma levels of aspartate aminotransferase and alanine aminotransferase, liver cytokine (IL-1β, TNF-α, IFN-γ, and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-β-D-glucosaminidase activity, Nrf2 and gp91phox mRNA expression, and decrease of reduced glutathione and antioxidant capacity measured by 2,2′-azinobis(3-ethylbenzothiazoline 6-sulfonate) assay were prevented in 5-LO−/− mice compared to WT mice. Therefore, 5-LO deficiency resulted in reduced mortality due to reduced liver inflammatory and oxidative damage, suggesting 5-LO is a promising target to reduce APAP-induced lethality and liver inflammatory/oxidative damage.

The role of PAF/PAFR signaling in zymosan-induced articular inflammatory hyperalgesia

Platelet-activating factor (PAF) and its receptor (PAFR) have been shown to be involved in several inflammatory events, including neutrophil chemoattraction and nociception. The present study addressed the role of PAF in the genesis of articular hyperalgesia in a model of joint inflammation. Zymosan-induced articular hyperalgesia, oedema and neutrophil migration were dose-dependently reduced following pretreatment with selective PAFR antagonists, UK74505 (5, 10 and 20mg/kg) and PCA4248 (3, 10, 30mg/kg). These parameters were also reduced in PAF receptor-deficient mice (PAFR(-/-)). The hyperalgesic action of PAF was further confirmed by the demonstration that joint injection of PAF induces a dose- (0.3, 1 and 3μg/joint), time- and PAFR-dependent articular hyperalgesia and oedema. The PAF hyperalgesic mechanisms were dependent on prostaglandins, leukotrienes and neutrophils, as PAF-induced articular hyperalgesia was inhibited by indomethacin (COX inhibitor), MK886 (leukotrienes synthesis inhibitor) or fucoidan (leukocyte rolling inhibitor). Furthermore, PAF-induced hyperalgesia was reduced in 5-lypoxigenase-null mice. In corroboration of these findings, intra-articular injection of PAF promotes the production of LTB(4) as well as the recruitment of neutrophils to the joint. These results suggest that PAF may participate in the cascade of events involved in the genesis of articular inflammatory hyperalgesia via stimulation of prostaglandins, leukotrienes and neutrophil migration. Finally, targeting PAF action (e.g., with a PAFR antagonist) might provide a useful therapeutic approach to inhibit articular inflammatory hyperalgesia.