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Swarming behavior of neutrophils has been noticed in both sterile injury and infection models and the mechanisms are being unveiled. So far, no in vitro model has been established to study neutrophil swarming to microbes. In the current study, using live-cell imaging, we observed in vitro neutrophil swarming toward Cryptococcus neoformans , a fungal pathogen causing human meningoencephalitis. Complement C3 and CD11b expression are essential for neutrophils to form cell swarms surrounding C. neoformans . Leukotriene B4 (LTB4) was quickly released by neutrophils during their interactions with C. neoformans . Blockade of LTB4 synthesis inhibited the swarming response to C. neoformans . Importantly, blockade of LTB4 synthesis also significantly reduced neutrophil recruitment in the lung vasculature of mice infected intravenously with C. neoformans , demonstrating a critical role of LTB4 in intravascular neutrophil swarming during infection. Together, this is the first report of neutrophil dynamics of swarming toward a microorganism in vitro , mediated by complement and LTB4. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate how β-naphthoflavone interacts with lipopolysaccharide (LPS) and polyinosinic acid: polycytidylic acid (poly I: C) induced innate immune parameters as well as phase I and phase II detoxification enzymes in head kidney cells isolated from Atlantic cod. β-naphthoflavone is a pure agonist of aryl hydrocarbon receptor (AhR) while LPS and poly I: C are not. β-naphthoflavone was added to head kidney leukocytes alone or together with LPS or poly I: C and the responses were evaluated in terms of protein and gene expression. The results showed that β-naphthoflavone (25 nM), with and without LPS, significantly induced cytochrome P450 ( cyp1c ) transcription in cod head kidney cells. β-naphthoflavone (100 nM) in the presence of the virus mimic, poly I: C, also increased cyp1c1 transcription. LPS induced cyp1c1 , cyclooxygenase 2 ( cox2 ), interleukin 1β (IL-1β ), interleukin 6 ( IL-6 ) and interleukin 8 ( IL-8 ) transcription, genes that were not affected by the tested β-naphthoflavone concentrations alone. However, β-naphthoflavone (25 and 50 nM) strengthened LPS induced cox2 and IL-8 transcription. Cod head kidney cells exposed to β-naphthoflavone concentrations ranging from 25 to 100 nM, with and without LPS or poly I: C, expressed AhR protein. LPS or β-naphthoflavone (5–50 nM) significantly induced leukotriene B 4 (LTB 4 ) secretion compared to control. In conclusion, this study suggests that β-naphthoflavone could interfere with LPS induced immune cell signaling in cod head kidney cells. [ABSTRACT FROM AUTHOR]

Background: Nasturtium (Indian cress, Tropaeolum majus) is known for its pharmacological value in the treatment of bacterial infections of the upper air tract and urinary bladder. However, scientific data on the anti-inflammatory potency in human-derived cells is missing.Purpose: The aim of this study was to investigate the potential of nasturtium to inhibit the lipopolysaccharide (LPS) induced inflammatory response in primary human cells of the immune system.Study Design: The anti-inflammatory activities of nasturtium and its fractions were evaluated via regulation of arachidonic acid (AA) pathway and MAPK kinase cascade. Fraction H4 which was responsible for the anti-inflammatory effects was further characterized.Methods: Human peripheral blood mononuclear cells (PBMC) were either treated with plant extracts or fractions thereof, stimulated with LPS and/or N-formyl-methionyl-leucyl-phenylalanine (fMLP) and analysed for COX and LOX, release of prostaglandin PGE2, leukotriene LTB4, TNF-alpha and ERK signaling pathway activation. The plant extracts were separated into four fractions by HPLC; fraction H4 was subjected to UHPLC-ToF/MS analysis to identify potential bioactive compounds.Results: We found that aqueous extracts of nasturtium did exert strong concentration dependent suppression of LPS-triggered TNF-alpha release and COX pathway signaling, including PGE2 synthesis. Whereas COX-1 protein expression was not impacted, LPS-triggered COX-2 protein expression was concentration dependently blocked by the plant extract but not COX-2 enzyme activity. These findings suggest a mechanism of action for the plant extract which is different from non-steroidal anti-inflammatory drugs (NSAIDs). Moreover, the plant extract blocked leukotriene LTB4 release, the major end product of the 5-LOX pathway from PBMC. Down-regulation of ERK1/2 and c-Jun activation preceded COX-2 suppression upon plant extract treatment in the presence of LPS. Using HPLC separation of the aqueous extract followed by metabolomic analysis we could limit the number of relevant bioactive compounds in the extract to about 50.Conclusions: This study provides a rationale for the anti-inflammatory efficacy of nasturtium observed in man and gives first insight into the underlying molecular mechanisms. [ABSTRACT FROM AUTHOR]

Aims: Restoration of coronary blood flow is crucial in the treatment of acute myocardial infarction. Reperfusion, however, induces ischaemia-reperfusion (IR) injury, which further deteriorates myocardial function. The innate immune system plays an important role in this process, mediating rapid influx of immune cells into the reperfused myocardium. Leukotriene B4 is an important leucocyte chemoattractant, performing its actions through binding to its specific receptor BLT1. We hypothesized that treatment with LSN2792613, a selective BLT1 antagonist, reduces infarct size (IS) in a mouse model of myocardial IR injury. Methods and results: Male C57Bl/6J mice were subjected to myocardial ischaemia for 30 min by surgical coronary artery ligation, followed by reperfusion. Mice received either LSN2792613 or vehicle, three times daily (orally) for up to 72 h after reperfusion. BLT1 inhibition with LSN2792613 reduced IS compared with vehicle treatment (26.9±2.7 vs. 34.9±2.2%, P = 0.030) at 24 h after reperfusion. The levels of IL-6 and keratinocyte chemoattractant were reduced in the infarcted tissue of LSN2792613-treated mice. Reduced apoptosis in LSN2792613-treated micewas suggested by increased levels of phosphorylated JNK and GSK3a/b, and confirmed by flow cytometric analysis showing less apoptotic and necrotic cardiomyocytes in the infarcted myocardium. Echocardiography at 4 weeks after myocardial IR showed a slightly higher ejection fraction and stroke volume in mice treated with LSN2792613 compared with vehicle-treated mice, whereas left ventricular volumes were comparable. Conclusion: Selective BLT1 inhibition with LSN2792613 reduces inflammation and apoptosis following IR, resulting in reduced IS, and therefore might be a promising strategy to prevent myocardial IR injury. [ABSTRACT FROM AUTHOR]

The effect of spermidine, a type of polyamine present in exudates from inflamed tissues and bacteria-infected sites in humans, on calcium mobilization of leukotriene B4 (LTB4)-stimulated chicken abdominal macrophages and heterophils was examined in vitro. The intracellular calcium ion concentration [Ca2+]i was elevated immediately and reached a maximum a few seconds after the addition of LTB4. The elevated [Ca2+]i returned to the basal (resting) level approximately 2min after LTB4 addition. Pretreatment of cells with spermidine significantly prolonged the duration of [Ca2+]i elevation in LTB4-treated chicken phagocytes. However, no change was induced in the level of [Ca2+]i elevation by pretreating the LTB4-treated cells with spermidine. These results suggest that polyamines would enhance the activities of chicken macrophages and heterophils in inflammation sites and bacteria-infected sites through delaying the return to resting levels of stimulated cells, as in the case of human polymorphonuclear leukocytes.

The objective of this study is to find out 1) sub-acute noise stress effects on neutrophil functions and 2) to examine the effect ofScoparia dulcisas an immunomodulatory and anti-stressor agent. Four groups of rats were used control, control fed with Scoparia, noise-stress exposed, noise-stress exposed fed with Scoparia. Blood was used for the assessment of Nitro blue tetrazolium reduction test (NBT), Neutrophil adhesion test (NAT), Phagocytic index (PI), avidity index (AI), corticosterone, LTB4. Respiratory burst enzymes (CAT, SOD), GSH, Vit-C, LPO, and NO measurement also done after the isolation of neutrophil from blood. Results1) Rats exposed to sub-acute noise stress showed a significant increase in NBT, corticosterone, LTB4, LPO, NO whereas NAT, PI, AI, antioxidant enzymes of neutrophils were markedly decreased. Hence, the administration ofScoparia dulcisprevents the sub-acute noise stress induced changes in neutrophil functions and antioxidant status and concludes that it protects the natural immunity. [ABSTRACT FROM AUTHOR]

Deregulation of the oxidative cascade of poly-unsaturated fatty acids (PUFAs) has been associated with several cancers, including chronic lymphocytic leukemia (B-CLL). Leukotriene B4 (LTB4), a metabolite of arachidonic acid (AA), is produced by B-CLL and contributes to their survival. The aim of the present study was to analyze the activity of the oxidative cascade of PUFAs in B-CLL. Purified B cells from patients and normal B CD5 positive cells were subjected to flow cytometry, Western-blot and RT-qPCR analyses. LTB4 plasma and intracellular concentrations were determined by ELISA. Our results showed that aggressive B-CLL tumor cells, i.e. cells with an annual proliferation index above 2, over-expressed calcium-dependent and calcium-independent phospholipases A2 (cPLA2-alpha and iPLA2-beta, respectively), 5-lipoxygenase (5LOX) and leukotriene A4 hydroxylase (LTA4H). Intracellular LTB4 levels were lower in the most aggressive cells than in cells with a smaller proliferation index, despite equivalent plasma levels, and lower expression of cytochrome P450 4F3A (CYP4F3A), one major enzyme involved in LTB4 inactivation. Since BLT2, a LTB4 membrane receptor was also more often expressed on aggressive tumor cells, and since a BLT2 inhibitor significantly impaired B-CLL viability in vitro, we propose that LTB4 was efficiently trapped onto BLT2 present on aggressive tumors, thereby eliciting an autocrine response. Taken together our results demonstrate a major deregulation of the pathway leading to LTB4 synthesis and degradation in B-CLL cells, and provide a framework for understanding how these modifications promote cell survival and proliferation, especially in the most aggressive BCLL. [ABSTRACT FROM AUTHOR]

The effects of fish oil, which is rich in n–3 fatty acids, on cytokine levels in a murine model of acquired immune deficiency syndrome (AIDS) were studied. Thirty-two C57BL/6 female mice were divided into two dietary groups and fed either a corn oil diet or a fish oil diet. After 4 weeks, each diet group was further divided into two subgroups, and mice in one subgroup were injected i.p. with LP-BM5 murine retrovirus (MAIDS) stock. After 4 weeks, all mice were killed, blood samples were collected, and the spleens and the livers were excised. Splenocytes were isolated immediately and cultured in RPMI-1640 medium and stimulated by either lipopolysaccharide (LPS) or Concanavalin A (ConA) for 24 h. The supernatant was collected for cytokine assays. The results showed that MAIDS infection increased the levels of tumor necrosis factor-α (TNF-α) and interleukin-1-beta (IL-1β), while fish oil partially prevented this elevation. MAIDS infection depressed interleukin-2 (IL-2) and interferon-gamma (IFNγ), while fish oil partially prevented the depression of IL-2. In addition, MAIDS infection depressed LPS- and ConA-stimulated cell proliferation, while fish oil partially prevented the depression. The results suggest that fish oil may slow down the progression of murine AIDS by modulating levels of cytokines including TNF-α, IL-1β, and IL-2.—Xi, S., D. Cohen, and L. H. Chen. Effects of fish oil on cytokines and immune functions of mice with murine AIDS.

Highlights: [•] Simple “supernatant-and-shoot” methodology. [•] Low sample volume (50μL plasma). [•] Efficient experimental design for validation purposes. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Human cytomegalovirus (HCMV) can cause congenital infection with risk of neurological disability. Maternal-fetal transmission is associated with placental inflammation. 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of Leukotrienes (LTs), which are proinflammatory mediators. This study investigated the effect of HCMV infection on 5-LO expression and Leukotriene-B4 (LTB4) induction in human placentae and umbilical vein endothelial cells (HUVEC). Methods: Seven placentae from fetuses with congenital HCMV infection and brain damage and six controls were stained with HCMV-immediate-early-antigen (HCMV-IEA) and 5-LO by immunohistochemistry. 5-hydroxyeicosatetraenoic acid (5-HETE) and LTB4 were measured in culture supernatant from ex vivo HCMV-infected placental histocultures by liquid chromatography. In vitro, HCMV infected HUVEC cells were analyzed for 5-LO mRNA and protein expression by real time PCR and immunofluorescence staining. Results: HCMV-IEA was abundant in all HCMV infected placentae but absent in control placentae. 5-LO expression was higher in endothelial and smooth muscle cells of HCMV-infected placentae, compared to control placentae. HCMV infection induced an up-regulation of LTB4 in ex vivo placental explants with higher levels of LTB4 at 72 h compared to controls (p = 0.002). In vitro, 5-LO transcript and protein expression were significantly induced in HCMV-infected HUVEC, compared to the control cultures (p = 0.036). Conclusion: The presence of HCMV coincided with high 5-LO expression in cells of in vivo HCMV infected placentae. HCMV induced up-regulation of 5-LO in both ex vivo HCMV-infected placental explants and HUVEC. HCMV induced LT-biosynthesis in congenitally infected placentae may have a role in pathogenesis of congenital HCMV disease. [Copyright &y& Elsevier]

Highlights: [•] The efficiency of various cytokines to recruit mast cells in vivo was examined. [•] Rats were injected with rrIL3, rrSCF, rrIL-3+rrSCF, rrRANTES and LTB4. [•] rrIL-3 and rrIL-3+rrSCF were most effective in recruiting mast cells. [•] Immature orthochromatic mast cells were recruited. [•] Mast cell recruitment was integrin dependent. [ABSTRACT FROM AUTHOR]

Garlic ( Allium sativum) has been used as a food as well as a component of traditional medicine. Aged black garlic (ABG) is known to have various bioactivities. However, the effect of ABG on allergic response is almost unknown. In the present study, we investigated whether ABG can inhibit immunoglobulin E-mediated allergic response in RBL-2H3 cells as well as in vivo passive cutaneous anaphylaxis (PCA). In in vitro tests, ethyl acetate extract (EBG) of ABG significantly inhibited the release of β-hexosaminidase (IC50, 1.53 mg/mL) and TNF-α (IC50, 0.98 mg/mL). Moreover, BG10, an active fraction of EBG, dramatically suppressed the release of β-hexosaminidase (IC50, 53.60 μg/mL) and TNF-α (IC50, 27.80 μg/mL). In addition, BG10 completely blocked the formation of prostaglandin E2 and leukotriene B4 at ≥25 μg/mL. When the effect of BG10 on FcɛRI receptor cascade was investigated, BG10 significantly inhibited the phosphorylation of Syk, but not Lyn. Furthermore, BG10 dose dependently decreased the phosphorylation of cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO) as well as the expression of cyclooxygenase-2 (COX-2). Consistent with what has been mentioned earlier, BG10 also significantly inhibited the PCA reaction in mice. In conclusion, these results indicate that ABG suppresses the allergic response, and the mechanism for its anti-allergic action may involve suppressions of Syk, cPLA2, 5-LO, and COX-2. The anti-allergic actions of ABG, EBG, or BG10 suggest that they may be useful as functional foods for allergic diseases. [ABSTRACT FROM AUTHOR]

Purpose: The changes of leukotriene B4 (LTB4) in the serum and the 5-lipoxygenase (5-LO) in the decidua in different stages of gestation period were investigated. Methods: The ELISA double antibody sandwich method was used to detect the concentration of LTB4 in the serum in 21 cases and its levels of the different stages of pregnancy were analyzed. The real-time PCR method was used to detect the mRNA expression level of 5-LO in the decidua tissue of 20 healthy women in early pregnancy and 21 healthy women in third trimester and its levels in the two stages of pregnancy were compared. Results: There were significantly different in the concentration of LTB4 in the serum during the four stages of healthy pregnancy. There were also markedly differences in the mRNA expression of 5-LO in the decidua during the two stages of healthy pregnancy. After pregnancy, the concentration of LTB4 in the serum of the subjects in early pregnancy and second trimester decreased, while in third trimester, its level increased. And after pregnancy, its level decreased. The 5-LO mRNA expression in the decidua in third trimester was obviously higher than the early pregnancy group. Conclusions: The changes of LTB4 concentration in serum and the mRNA expression level of 5-LO in decidua may play an important role in the success and maintenance of a healthy pregnancy. [ABSTRACT FROM AUTHOR]

Background: Cytochrome P450 4F3 (CYP4F3) is an ω-hydroxylase that oxidizes leukotriene B4 (LTB4), prostaglandins, and fatty acid epoxides. LTB4 is synthesized by leukocytes and acts as a chemoattractant for neutrophils, making it an essential component of the innate immune system. Recently, involvement of the LTB4 pathway was reported in various immunological disorders such as asthma, arthritis, and inflammatory bowel disease. We report a 26-year-old female with a complex immune phenotype, mainly marked by exhaustion, muscle weakness, and inflammation-related conditions. The molecular cause is unknown, and symptoms have been aggravating over the years., Methods: Whole exome sequencing was performed and validated; flow cytometry and enzyme-linked immunosorbent assay were used to describe patient's phenotype. Function and impact of the mutation were investigated using molecular analysis: co-immunoprecipitation, western blot, and enzyme-linked immunosorbent assay. Capillary electrophoresis with ultraviolet detection was used to detect LTB4 and its metabolite and in silico modelling provided structural information., Results: We present the first report of a patient with a heterozygous de novo missense mutation c.C1123 > G;p.L375V in CYP4F3 that severely impairs its activity by 50% (P < 0.0001), leading to reduced metabolization of the pro-inflammatory LTB4. Systemic LTB4 levels (1034.0 ± 75.9 pg/mL) are significantly increased compared with healthy subjects (305.6 ± 57.0 pg/mL, P < 0.001), and immune phenotyping shows increased total CD19+ CD27- naive B cells (25%) and decreased total CD19+ CD27+ IgD- switched memory B cells (19%). The mutant CYP4F3 protein is stable and binding with its electron donors POR and Cytb5 is unaffected (P > 0.9 for both co-immunoprecipitation with POR and Cytb5). In silico modelling of CYP4F3 in complex with POR and Cytb5 suggests that the loss of catalytic activity of the mutant CYP4F3 is explained by a disruption of an α-helix that is crucial for the electron shuffling between the electron carriers and CYP4F3. Interestingly, zileuton still inhibits ex vivo LTB4 production in patient's whole blood to 2% of control (P < 0.0001), while montelukast and fluticasone do not (99% and 114% of control, respectively)., Conclusions: A point mutation in the catalytic domain of CYP4F3 is associated with high leukotriene B4 plasma levels and features of a more naive adaptive immune response. Our data provide evidence for the pathogenicity of the CYP4F3 variant as a cause for the observed clinical features in the patient. Inhibitors of the LTB4 pathway such as zileuton show promising effects in blocking LTB4 production and might be used as a future treatment strategy., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Objective: Exposure to cigarette smoke complicates the treatment and management of asthma through a variety of inflammatory effects. This study aimed to investigate the differences between newly diagnosed cases of asthma in smokers and nonsmokers in terms of localized and systemic biomarkers following treatment with inhaled corticosteroids (ICS) or ICS in combination with a long-acting β2 agonist (LABA)., Methods: Specimens of exhaled breath condensate (EBC) from newly diagnosed patients with asthma were used to quantify inflammation in the airways, while blood samples were used to assess systemic inflammation. In both samples, the levels of IL-6, LTB4, LTD4, and 8-isoprostane were measured and these were repeated after 3 months of treatment with ICS or ICS + LABA., Results: Of the 20 patients, 10 (50%) were nonsmokers with asthma (NSA) and 10 (50%) smokers with asthma (SA). There was no statistically significant difference in the blood or EBC levels of IL-6, LTB4, LTD4, or 8-isoprostane between the groups prior to treatment. Only the decrease in 8-isoprostane level in the EBC samples was found to be significantly greater in the NSA group after treatment (for smokers, the change was 2.91 ± 23.22, while for nonsmokers it was -22.72 ± 33.12, p  = 0.022). Post-treatment asthma control was significantly better in the NSA group ( p  = 0.033)., Conclusion: Monitoring the alterations in 8-isoprostane levels in EBC in patients with asthma who smoke may be helpful in deciding on therapeutic management and switching treatments. Asthma control was better in nonsmokers than in smokers.

Abstract: Recently, some endocannabinoids were reported to show anti-inflammatory and anti-allergic activities. In this respect, various arachidonoyl endocannabinoids were screened for the inhibition of allergic response in IgE-activated RBL-2H3 cells. Among arachidonoyl endocannabinoids with a low cytotoxicity, only NA-5HT remarkably inhibited the release of β-hexosaminidase (IC50, 13.58μM), a marker of degranulation, and tumor necrosis factor-α (IC50, 12.52μM), a pro-inflammatory cytokine, in IgE-activated RBL-2H3 cells. Additionally, NA-5HT markedly suppressed the formation of prostaglandin D2 (PGD2) with IC50 value of 1.27μM and leukotriene B4 (LTB4) with IC50 value of 1.20μM, and slightly LTC4. When effect of NA-5HT on early stage of FcεRI cascade was investigated, it significantly inhibited phosphorylation of Syk, but not Lyn. Furthermore, NA-5HT suppressed phosphorylation of PLCγ1/2 and PKCδ, related to degranulation process, as well as phosphorylation of LAT, ERK1/2, p38, JNK, Gab2, PI3K and Akt, implicated in the expression of pro-inflammatory cytokines. Relative to its effect on the late stage, NA-5HT slightly reduced phosphorylation of 5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2). Additionally, NA-5HT significantly reduced the level of p40 phox , and partially inhibited the expression of p47 phox and p67 phox . From these results, it is suggested that NA-5HT expresses anti-allergic action by suppressing the activation of Syk, LAT, p38, JNK, PI3K and Akt, as well as the expression of ERK1/2 and NADPH oxidase subunits. Further, a strong inhibition of PGD2 or LTB4 biosynthesis by NA-5HT may be an additional mechanism for its anti-allergic action. Such anti-allergic actions of NA-5HT may contribute to further information about its biological functions. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Atropa 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 study: Our 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 methods: We 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. Results: Our 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. Conclusion: Our 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. [Copyright &y& Elsevier]

Abstract: The leukotriene A4 hydrolase enzyme is a dual functioning enzyme with the following two catalytic activities: an epoxide hydrolase function that transforms the lipid metabolite leukotriene A4 to leukotriene B4 and an aminopeptidase function that hydrolyzes short peptides. To date, all drug discovery efforts have focused on the epoxide hydrolase activity of the enzyme, because of extensive biological characterization of the pro-inflammatory properties of its metabolite, leukotriene B4. Herein, we have designed a small molecule, 4-methoxydiphenylmethane, as a pharmacological agent that is bioavailable and augments the aminopeptidase activity of the leukotriene A4 hydrolase enzyme. Pre-clinical evaluation of our drug showed protection against intranasal elastase-induced pulmonary emphysema in murine models. [Copyright &y& Elsevier]

Context: Aristolochia tagala Cham. (syn: Aristolochia acuminata Lam.) (Aristolochiaceae), known as Nallayishwari in Telugu, has been of interest to researchers because of its traditional uses for treating rheumatic pains and fever. Objective: The anti-inflammatory activity of the petroleum ether, ethyl acetate, and ethanol extracts of A. tagala roots were investigated for the first time. Materials and Methods: In vivo and in vitro anti-inflammatory effects were investigated employing the carrageenan-induced hind paw edema in rats and the macrophage cell line RAW264.7 stimulated with proinflammatory stimuli (lipopolysaccharide interferon γ or the calcium ionophore A23187) to determine PGE2 or LTB4 release, respectively. Results: All the extracts exhibited anti-inflammatory effects which were found to be significant ( p < 0.001) at 200 and 400 mg/kg, p.o, in rats tested and the ethyl acetate extract inhibited the induction of PGE2 with IC50 == 39.1 mg mL−1 and LTB4 with IC50 == 29.5 mg mL−1. Discussion and conclusion: These findings demonstrate that the A. tagala roots have excellent anti-inflammatory activity and validate the traditional indications of this plant in its origin country. [ABSTRACT FROM AUTHOR]

Abstract: Schistosomes (blood flukes) are long lived, intravascular parasites that afflict ~200 million people worldwide. Here we review the potential ability of these parasites to exert control on local vascular physiology. We examine schistosome kallikrein-like proteins that drive vasodilation. We review biogenic amine metabolism in the parasites that involve the vasodilator histamine and its receptors and the vasoconstrictor serotonin and its receptor. Schistosomes can trigger the release of histamine from host cells and can import serotonin. We consider the ability of schistosomes to generate and release the eicosanoid vasodilators PGD2 and PGE2 and the vasoconstrictors LTB4 and LTC4. The literature on nitric oxide metabolism in these blood flukes is assessed. Finally the potential impact of other schistosome metabolic processes (e.g. exogenous adenosine generation and acetylcholine degradation) on vascular function is appraised. An increased understanding of these processes could lead to novel anti-parasitics as well as new therapies to treat vascular dysfunction. [Copyright &y& Elsevier]

Abstract: Processed aconite drugs are widely used in Eastern medicine as painkillers and antirheumatic agents. It is known that the traditional processing of aconite drugs increases the amount of lipo-alkaloids. In order to obtain information about the pharmacological potential of these compounds, semisynthesis of 9 aconitine-derived lipo-alkaloids was carried out and their COX-1, COX-2 and LTB4 formation inhibitory activities were investigated. It was found that compounds esterified with unsaturated fatty acids demonstrated significant COX-2 inhibitory effects, while in the COX-1 assay only 14-benzoylaconine-8-O-eicosapentaenoate exerted remarkable activity. The inhibition of LTB4 formation was pronounced in cases of long chain fatty acid derivatives. [Copyright &y& Elsevier]

Larrea divaricata exerts a dual effect on the proliferation of BW5147 cells. The aim of this work was to analyze the effect of leukotrienes on the antiproliferative action of the extract in relation to cyclic adenosine monophosphate production and protein kinase C activation. The extracts induced cyclic adenosine monophosphate and inhibited protein kinase C but induced leukotriene B4 production, which exerted contrary effects. These results suggest the finding of an extract free of compounds, which can increase leukotriene B4 level, in order to obtain a more active extract to be used in the treatment of lymphomas, positively modulated by leukotrienes. [ABSTRACT FROM AUTHOR]

Abstract: Vascular injury leads to a local inflammatory response, characterized by endothelial damage, extracellular matrix exposition and aggregation/adhesion of platelets and circulating leukocytes. The release of inflammatory mediators amplifies the process, and can induce vascular smooth muscle cells (SMC) migration and proliferation. Released by leukocytes, leukotriene B4 (LTB4) induces reactive oxygen species production and SMC chemotaxis. This study was conducted to elucidate the molecular mechanisms involved in the effect of LTB4 on SMC migration, and a rat linage of vascular SMC (A7r5) were used throughout. The chemotactic effect of LTB4 was dependent on the concentration used, being comparable to AngII at 100nM. Migration induced by LTB4 was inhibited in the presence of pertussis toxin, CP-105696, a BLT1 receptor antagonist, and by LY294002 or PD98059, two inhibitors of PI3K and MEK1/2, respectively. Stimulation of SMC with LTB4 triggered integrin-associated signaling pathways, inducing focal adhesion kinase (FAK) phosphorylation, mobilization of actin cytoskeleton, association of FAK to PI3K, ERK-2 phosphorylation and nuclear translocation, and also NFκB pathway activation. Pretreatment of SMC with a selective ligand of αvβ3 integrin, kistrin, inhibited LTB4-induced chemotaxis, FAK phosphorylation, FAK-PI3K association, and also inhibited ERK-2 and NFκB pathways activation. Taken together, the data demonstrated, for the first time, that the effect of LTB4 on SMC migration is modulated by integrin signaling activation, suggesting that these adhesion molecules might be important target for therapeutic intervention in cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Abstract: We evaluated the in vivo pharmacological properties of AM803 3-[3-tert-butylsulfanyl-1-[4-(6-ethoxy-pyridin-3-yl)-benzyl]-5-(5-methyl-pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid, a selective five-lipoxygenase-activating protein (FLAP) inhibitor, using rat and mouse models of acute inflammation. Oral administration of AM803 (1mg/kg) resulted in sustained inhibition of ex vivo ionophore-challenged whole blood LTB4 biosynthesis with >90% inhibition for up to 12h and an EC50 of ∼7nM. When rat lungs were challenged in vivo with calcium-ionophore, AM803 inhibited LTB4 and cysteinyl leukotriene (CysLT) production with ED50s of 0.12mg/kg and 0.37mg/kg, respectively. The inhibition measured 16h following a single oral dose of 3mg/kg was 86% and 41% for LTB4 and CysLTs, respectively. In an acute inflammation setting, AM803 dose-dependently reduced LTB4, CysLTs, plasma protein extravasation and neutrophil influx induced by peritoneal zymosan injection. Finally, AM803 increased survival time in mice exposed to a lethal intravenous injection of platelet activating factor (PAF). The magnitude of effect was similar to that of an inhibitor of five-lipoxygenase (5-LO) and LTA4 hydrolase but superior to a leukotriene CysLT1 receptor antagonist. In summary, AM803 is a novel, potent and selective FLAP inhibitor that has excellent pharmacodynamic properties in vivo and is effective in animal models of acute inflammation and in a model of lethal shock. [Copyright &y& Elsevier]

Abstract: Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma. [Copyright &y& Elsevier]

Abstract: Previously, we have shown that electroacupuncture (EA) in rats decreased eosinophil infiltration into the pulmonary tissue (PT) and in the bronchoalveolar lavage (BAL) in an experimental model of asthma. Th2 cytokines, leukotriene B4 (LTB4) and nitric oxide (NO) are involved in the asthma inflammatory process. The aim of this study was to verify the effects of EA on these asthma mediators. Male Wistar rats were divided into control (C), immobilized (I), sham acupuncture (SA), and acupuncture (A) groups. All rats were sensitized, and EA treatment using clinical acupuncture points was started 24h after antigen priming. EA was done every other day for 2weeks. Subsequently, animals were challenged by inhalation and sacrificed 24h later. At this time, the BAL and lungs were collected and used to analyze cytokine production, LTB4 and NO. The EA increased IL-1 and IFN-γ and decreased IL-4, IL-10, NO and LTB4 in the BAL and PT compared to the C and SA groups. The presence of eosinophils in the BAL negatively correlated with IL-1 and IFN-γ production and positively correlated with IL-4 and IL-10 production. Our results show that the beneficial anti-inflammatory action of EA on asthma is related to the balance of the Thl/Th2 response and the reduction of LTB4 and NO. These results suggest that EA therapy could be an important complementary treatment for asthma. [Copyright &y& Elsevier]

The present study was undertaken to assess the activity/anti-inflammatory potential of Linum usitatissimum fixed oil against castor oil-induced diarrhoea, turpentine oil-induced joint oedema, formaldehyde and Complete Freund’s Adjuvant (CFA)-induced arthritis in Wistar albino rats. The oil intraperitoneally, significantly inhibited the castor oil-induced diarrhoea and turpentine oil-induced exudative joint oedema in a dose-dependent manner. Significant inhibitory effect of L. usitatissimum fixed oil was observed in formaldehyde-induced proliferative global oedematous arthritis when given intraperitoneally, with significant checking of the serum glutamic oxaloacetic acid transaminase and serum glutamic pyruvic acid transaminase. Further, L. usitatissimum fixed oil showed a significant dose-dependent protective effect against CFA-induced arthritis as well. Secondary lesions produced by CFA due to a delayed hypersensitivity reaction were also reduced in a significant manner. Anti-inflammatory activity of L. usitatissimum fixed oil can be attributed to the presence of alpha linolenic acid (57.38%, an omega-3 fatty acid, 18:3, n-3) having dual inhibitory effect on arachidonate metabolism resulting in suppressed production of proinflammatory n-6 eicosanoids (PGE2, LTB4) and diminished vascular permeability. These observations suggest possible therapeutic potential of L. usitatissimum fixed oil in inflammatory disorders like rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Abstract: BLT2, a low-affinity leukotriene B4 (LTB4) receptor, is a member of the G-protein coupled receptor (GPCR) family and is involved in the pathogenesis of inflammatory diseases such as asthma. Despite its clinical implications, however, no pharmacological inhibitors are available. In the present study, we screened for small molecules that interfere with the interaction between the third intracellular loop region of BLT2 (BLT2iL3) and the Gαi3 protein subunit (Gαi3), using a high-throughput screening (HTS) assay with a library of 1040 FDA-approved drugs and bioactive compounds. We identified two small molecules—purpurin [1,2,4-trihydroxy-9,10-anthraquinone; IC50 =1.6μM for BLT2] and chloranil [tetrachloro-1,4-benzoquinone; IC50 =0.42μM for BLT2]—as specific BLT2-blocking agents. We found that blockade of the BLT2iL3-Gαi3 interaction by these small molecules inhibited the BLT2-downstream signaling cascade. For example, BLT2-signaling to phosphoinositide-3 kinase (PI3K)/Akt phosphorylation was completely abolished by these molecules. Furthermore, we observed that these small molecules blocked LTB4-induced chemotaxis by inhibiting the BLT2-PI3K/Akt-downstream, Rac1-reactive oxygen species-dependent pathway. Taken together, our results show that purpurin and chloranil interfere with the interaction between BLT2iL3 and Gαi3 and thus block the biological functions of BLT2 (e.g., chemotaxis). The present findings suggest a potential application of purpurin and chloranil as pharmacological therapeutic agents against BLT2-associated inflammatory human diseases. [Copyright &y& Elsevier]

Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with a risk of serious adverse events. Now, the development of dual inhibitors of cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) has become a hot area in searching for safer NSAIDs. NNU-hdpa, 2-(4-hydroxylphenyl)-3-(3,5-dihydroxylphenyl) propenoic acid, a newly synthesized compound, is expected to have COX/5-LOX dual inhibition with an improved gastrointestinal profile. In this study, NNU-hdpa was subjected to in vitro and in vivo experiment protocols. In vitro COX/5-LOX inhibition assays showed that NNU-hdpa exhibits a dual inhibitory activity against the COX and 5-LOX enzymes. Anti-inflammatory activity in vivo was evaluated using two animal edema model tests. Pretreatment with NNU-hdpa (p.o.) dose-dependently inhibited the xylene-induced ear edema in mice and carrageenan-induced paw edema in rats respectively. In gastric lesion test, NNU-hdpa was gastric-sparing in that it elicited markedly fewer stomach lesions as compared to the stomach lesions caused by aspirin in rats. In further studies, NNU-hdpa was found to significantly inhibit the productions of PGE2 and LTB4 in LPS-challenged RAW 264.7, which is parallel to its prevention of the nuclear translocation of the NF-κB p50 and p65 subunits. These data indicate that NNU-hdpa comprises a novel class of dual inhibitors of COX and 5-LOX having therapeutic potential with an enhanced gastric safety profile. [Copyright &y& Elsevier]

Abstract: The d-mannose binding lectin ArtinM from Artocarpus integrifolia, previously known as KM+ and artocarpin, is considered a stimulant of Th1-type immunity, which is able to confer resistance to some intracellular pathogens. In addition, ArtinM induces neutrophil migration by haptotaxis through simultaneous interactions of its carbohydrate recognition domains (CRDs) with glycans expressed on the extracellular matrix and the neutrophil surface. In the present study, we have expanded the characterization of ArtinM as a neutrophil activator. Exposure of neutrophils to ArtinM for 15min resulted in tyrosine phosphorylation of intracellular proteins, a process that was selectively inhibited by d-mannose or mannotriose. Shortly after stimulation, neutrophils secreted high levels of LTB4 and underwent shedding of L-selectin from their surface. Exposure to ArtinM enhanced neutrophil functions, such as respiratory burst and zymozan and Listeria monocytogenes phagocytosis. In addition, ArtinM-stimulated neutrophils displayed increased CXCL-8 secretion and TLR2 gene transcription. These results demonstrate that ArtinM is able to induce potent neutrophil activation, a feature that should be strongly considered in the assessment of the lectin capacity to confer resistance against infections. [Copyright &y& Elsevier]

Abstract: Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB4, a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB4 was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025–25ng/mL for LTB4 and 0.1–25ng/mL for 5-HETE. The EIA method has a validated range covering 0.025–4ng/mL. When both assays were applied to analyze LTB4 from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r =0.9426, Pearson''s correlation coefficient). A high correlation was also observed between the levels of LTB4 and 5-HETE measured by LC/MS/MS after ionophore stimulation (r =0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis. [Copyright &y& Elsevier]

Abstract: Several reports have demonstrated an important role of leukotriene B4 (LTB4) in the immune system. We investigated whether leukemic blasts from acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients produced LTB4, 12- and 15-hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) and whether these compounds affected blast proliferation and apoptosis. Leukemic blasts from AML M0-2 and ALL patients expressed 5-LOX, 12-LOX and 15-LOX transcripts. Quantitative polymerase chain reaction indicated that 5-LOX transcripts were far more abundant than 12-LOX and 15-LOX ones. Leukemic blasts expressed 5-LOX activating protein (FLAP) transcripts and produced LTB4 in response to calcium ionophore. In contrast no 15-HETE production was found. Calcium ionophore-stimulated leukemic blasts produced 12-HETE but also released thromboxane A2 suggesting that contaminating platelets accounted for the release of these compounds. No significant effect of LTB4, 12-HETE or 15-HETE could be documented on leukemic blast growth and on their apoptose rate. Results of the present study indicate that immature form of leukemic blasts produce LTB4. However, the three major lipoxygenase metabolites of arachidonic acid; i.e., LTB4, 12-HETE or 15-HETE, had no evident effect on their growth and apoptosis. We may speculate that LTB4-derived blast cells might initiate, augment or prolong tissue inflammation and damages by affecting the marrow and blood cytokine network. [Copyright &y& Elsevier]

Summary: Background: Very long chain n-3 polyunsaturated fatty acids have modulating effects on inflammatory mechanisms. Seal and fish oils are rich in n-3 polyunsaturated fatty acids, and possibly therefore high doses of nasoduodenally administered seal oil rapidly relieved inflammatory bowel disease (IBD)-associated joint pain in two recent studies. In the present study, we compared the effects of short-term oral administration of seal oil and cod liver oil on IBD-related joint pain, leucotriene B4 level, serum fatty acid profile and IBD activity. Methods: Thirty-eight patients with IBD-related joint pain were included in the study; 21 had Crohn''s disease and 17 ulcerative colitis. Ten milliters of seal oil (n =18) or cod liver oil (n =20) was self-administered orally 3 times a day for 14 days before meals in a double-blind setting. Results: There were no significant differences between the two intervention groups or between Crohn''s disease and ulcerative colitis patients. There was a tendency toward improvement in several joint pain parameters after both seal oil and cod liver oil administration. Further, plasma leucotriene B4 concentration, serum Σ n-6 to Σ n-3, and arachidonic acid (20:4n-6) to eicosapentaenoic acid (20:5n-3) ratios were similarly reduced after administration of seal oil and cod liver oil. Conclusion: No significant differences in the two treatment groups were seen; in both groups, the changes in several joint pain parameters, leucotriene B4 level of plasma, and serum fatty acid profile were putatively favourable. [Copyright &y& Elsevier]

Abstract: The stem bark of Discopodium penninervium afforded a withanolide, 6α,7α-epoxy-1-oxo-5α,12α,17α-trihydroxywitha-2,24-dienolide (1) and a coloratane sesquiterpene, 7α,11α-dihydroxy-4(13),8-coloratadien-12,11-olide (4) along with five known compounds, withanone (2), 5α,17β-dihydroxy-6α,7α-epoxy-1-oxowitha-2,24-dienolide (3), 7α,11α-dihydroxy-8-drimen-12,11-olide (5), withasomnine (6), and (E,Z)-9-hydroxyoctadeca-10,12-dienoic acid (7). The identity of the compounds was established on the basis of spectroscopic data analysis. All compounds were assessed for inhibition of leukotriene metabolism in an in vitro bioassay using activated human neutrophile granulocytes, and for in vitro cycloxygenase-1 and -2 inhibition from sheep cotyledons and seminal vesicles, respectively. In the leukotriene biosynthesis assay all compounds tested at a concentration of 50μM exhibited activity with percentage inhibitions ranging from 11.5 to 36.6. The withanolide, 1, displayed a 46.4% inhibition of COX-2 and a 22.9% inhibition of LTB4 formation at 50μM concentration. Compounds 4 and 6 inhibited LTB4 biosynthesis but showed minor inhibition of COX-1 and COX-2. The remaining compounds, on the other hand, were found to be inactive on COX enzymes. [Copyright &y& Elsevier]

Abstract: The activation of monocytes/macrophages by several stimuli is an initial event in the inflammatory response. To ascertain the importance of LTB4 and 5-lypoxigenase in the inflammatory site, we isolated and stimulated rat adherent granuloma macrophages (RAGMs) with calcium ionophore in the presence or absence of regulated on activation, normal T expressed and secreted (RANTES) [CCL5] at different concentrations. We tested the hypothesis that RANTES may influence the production of LTB4 stimulated by calcium ionophore A23187 (2.5μM/ml) in rat adherent granuloma macrophages derived from granuloma induced by potassium permanganate diluted 1:40 saturated solution. To test this hypothesis, we measured LTB4 production, in rat granuloma macrophages stimulated with A23187 (2.5μM) alone and in combination with RANTES at different concentrations. In these studies, the cell-free supernatant of stimulated RAGMs with the ionophore A23187, resulted in a drastic increase of LTB4. However, when the cells were treated with the combination RANTES plus A23187 the stimulatory effect was more pronounced than A23187 alone. LTB4 production was quantitated. The calcium ionophore A23187 directly induced LTB4 in macrophages, this production was markedly enhanced when the cells were pretreated with RANTES. However, the addition of RANTES in the absence of calcium ionophore A23187 did not directly induce LTB4 release, nor was lypoxigenase expression augmented. Preincubation of RAGMs with NDGA (nordihydroguiaretic acid) (10−5 M) completely abolished the production of LTB4 on RAGMSs challenged with A23187 in combination with RANTES or A23187 alone in the supernatants. Similar effects were obtained when the cells were pretreated with dexamethasone. These data suggest, for the first time, that RANTES may stimulate the release of LTB4, only when it is associated to other stimuli and for this reason we conclude that RANTES modulates inflammatory diseases, and may require other stimuli to be effective in amplifying its spectrum of action(s). [Copyright &y& Elsevier]

Abstract: The anti-inflammatory properties of three flavanones isolated from Inula viscosa, sakuranetin, 7-O-methylaromadendrin, and 3-acetyl-7-O-methylaromadendrin, have been tested both in vitro and in vivo. Acute inflammation in vivo was induced by means of topical application of 12-O-tetradecanoylphorbol 13-acetate (TPA) to mouse ears or by subcutaneous injection of phospholipase A2 (PLA2) into mouse paws. The test compounds were evaluated in vitro for their effect on both the metabolism of arachidonic acid and on the release and/or activity of enzymes involved in the inflammatory response such as elastase, myeloperoxidase (MPO), and protein kinase C (PKC). The most active compounds in vivo against PLA2-induced paw oedema were 7-O-methylaromadendrin (ED50 =8 mg/kg) and sakuranetin (ED50 =18 mg/kg). In contrast, the most potent compound against TPA-induced ear oedema was 3-acetyl-7-O-methylaromadendrin (ED50 =185 μg/ear), followed by sakuranetin (ED50 =205 μg/ear). In vitro, the latter compound was the most potent inhibitor of leukotriene (LT) B4 production by peritoneal rat neutrophils (IC50 =9 μM) and it was also the only compound that directly inhibited the activity of 5-lipoxygenase (5-LOX). 3-Acetyl-7-O-methylaromadendrin also inhibited LTB4 production (IC50 =15 μM), but had no effect on 5-LOX activity. The only flavanone that inhibited the secretory PLA2 activity in vitro was 7-O-methylaromadendrin. This finding may partly explain the anti-inflammatory effect observed in vivo, although other mechanisms such as the inhibition of histamine release by mast cells may also be implicated. Sakuranetin at 100 μM was found to inhibit elastase release, although this result is partly due to direct inhibition of the enzyme itself. At the same concentration, 7-O-methylaromadendrin only affected the enzyme release. Finally, none of the flavanones exhibited any effect on MPO or PKC activities. Taken together, these findings indicate that sakuranetin may be a selective inhibitor of 5-LOX. [Copyright &y& Elsevier]

Murine leukotriene B4 (LTB4) receptor (mBLT1) cDNA was identified by searching the EST database using human LTB4 receptor as the query sequence. Expression of functional mBLT1 after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was demonstrated as LTB4-evoked, Ca2+-activated Cl- currents recorded by two-electrode voltage clamp. From mBLT1-expressing oocytes, a dose-dependent relationship between the Ca2+-activated Cl- current and LTB4 concentration was demonstrated with an apparent EC50 of 6.7 nM. Following LTB4 stimulation of mBLT1, we observed two transient, spatially distinct Ca2+-activated, inwardly directed Cl- currents in the oocytes: a fast peak current requiring relatively high LTB4 concentrations, and a slowly progressing Cl- current. Nucleotides, PGE2, 12R-hydroxy-5, 8, 14-cis-10-trans-eicosatetraenoic acid, and LTD4 did not activate mBLT1. U75302, specifically targeting BLT1, significantly reduced LTB4-evoked Cl- currents. Repetitive LTB4 administration desensitized the LTB4-evoked currents. Activation of protein kinase C (PKC) by PMA addition completely eliminated the LTB4-evoked currents, whereas down-regulation of PKC by prolonged PMA exposure (20 h) impaired mBLT1 desensitisation. In addition, Ser-127-Ala substitution of the PKC consensus phosphorylation site on the second intracellular loop prevented the mBLT1 desensitisation. These data indicate that PKC-mediated phosphorylation at Ser-127 leads to mBLT1 desensitisation. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Summary: Objective: To assess the therapeutic effects of oral zileuton tablets combined with low-dose beclomethasone compared to doubling the dose of beclomethasone, in improving lung function and reducing asthma symptoms. Methods: Randomized, active-control, double-blind, parallel, multi-center study of zileuton (400 or 600mg QID)+200μg beclomethasone dipropionate (BDP) BID versus placebo+BDP 400μg BID in asthmatics with baseline FEV1 percent predicted values between 40% and 80% following a single-blind ICS (BDP 200μg BID) 2-week run-in. During the 3-month double-blind treatment period, assessments included safety, daytime and nighttime symptoms, acute asthma exacerbations, β 2-agonist use, AM and PM peak expiratory flow (PEF) and FEV1. Results: The addition of a 5-lipoxygenase (5-LO) inhibitor added to a low-dose of BDP showed no significant difference in FEV1 compared to doubling the dose of BDP. FEV1 improved in all 3 treatment groups, with mean increases of 10% with zileuton 600mg QID+BDP 200μg BID, 12% with zileuton 400mg QID+BDP 200μg BID, and 11% with BDP 400μg BID by study end. Within each treatment group, there were significant improvements in asthma symptoms and AM and PM PEF compared to baseline. No significant differences were observed between groups with regards to salbutamol use, acute asthma exacerbations, the requirement for oral/parenteral corticosteroids and adverse clinical events. Conclusions: The addition of a 5-LO inhibitor added to low-dose beclomethasone may be an alternative to higher-doses of ICS in patients unable to achieve sufficient asthma control on low-dose ICS therapy. [Copyright &y& Elsevier]

Abstract: Wilbrandia ebracteata (WE), a Brazilian medicinal plant used in folk medicine for the treatment of rheumatic diseases, displays anti-inflammatory properties and constitutes a rich source of cucurbitacins and cucurbitacin-related compounds. The current study investigated the potential anti-inflammatory properties of Dihydrocucurbitacin B (DHCB), a cucurbitacin-derived compound isolated from roots of WE, in some in vivo and in vitro experimental models. Intraperitoneal treatment of mice with DHCB reduced both carrageenan-induced paw edema (0.3, 1 and 3 mg/kg caused inhibitions of 26, 44 and 56 % at 2 h after stimulation, respectively) and pleurisy (10 mg/kg inhibited leukocyte numbers and LTB4 levels in the pleural fluid by 51 and 75% at 6 h after cavity challenge, respectively). In vitro, DHCB (up to 10 μg/mL) failed to modify LTB4 production by human neutrophils or PGE2 production by COS-7 cells transfected with COX-1, but PGE2 production by COX-2 transfected COS-7 cells was markedly inhibited (by 72%). The levels of COX-1 or COX-2 proteins in IL-1α-stimulated NIH3T3 cells were unaffected by DHCB. The results corroborate the potential anti-inflammatory properties ascribed to W. ebracteata Cogn. in folk medicine and suggest that they might be attributed, at least in part, to the capacity of one of this plants main constituents, DHCB, to inhibit COX-2 activity (but not its expression) during inflammation. [Copyright &y& Elsevier]

Diesel exhaust particles (DEP) are associated with respiratory disease and exposure to diesel exhaust induces an inflammatory response associated with marked leukocytic infiltration in the lung. This study examined whether neutrophils are activated by the active component of DEP (methanol extract of DEP [me-DEP]). The authors demonstrated that neutrophils exposed to me-DEP had increased levels of the f-actin content, the surface expression of adhesion molecules, and the release of interleukin (IL-8) and leukotriene B4 (LTB4), superoxide, and matrix metalloproteinase (MMP-9). Thus, the author conclude that DEP exposure activates neutrophils and that these activated neutrophils could contribute to the adverse respiratory health effects associated with DEP and to the pathogenesis of chronic inflammatory lung diseases. [ABSTRACT FROM AUTHOR]

Abstract: Serum amyloid A (SAA) and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) have been reported as formyl peptide receptor-like 1 (FPRL1) ligands. WKYMVm but not SAA stimulated superoxide generation by human neutrophils. In terms of the downstream signalings triggered by WKYMVm and SAA, both agonists stimulated cytosolic phospholipase A2-mediated arachidonic acid release, a precursor of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2). WKYMVm also strongly stimulated LTB4 production in human neutrophils without affecting PGE2 production, whereas SAA strongly stimulates cyclooxygenase-2 (COX-2) expression and PGE2 production but not LTB4 production. In terms of the receptors responsible for the differential actions of these two agonists, we found that FPRL1 is involved in the production of LTB4 by WKYMVm and PGE2 production by SAA. This study demonstrates that the chemoattractant receptor, FPRL1, can be differentially regulated by distinct ligands to generate different lipid mediators, and thus, different immune responses. [Copyright &y& Elsevier]

Abstract: Tendinopathy often involves inflammation and matrix degeneration. The inflammatory mediators such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) are implicated in the development of tendinopathy. Therefore, the purpose of this study was to determine the effect of PGE2 and LTB4 on the proliferation of human patellar tendon fibroblasts (HPTFs), the gene expression of collagen type I, MMP-1 and MMP-3, as well as the protein secretion of these gene products by the cells. The results showed that LTB4 at low doses (0.1 and 1 nM) significantly increased cell proliferation compared to controls and LTB4 at 0.1 nM negated the PGE2-induced decrease in cell proliferation. In addition, PGE2 at 100 ng/ml significantly increased the expression of MMP-1 and MMP-3 at both mRNA and protein levels. These stimulatory effects were significantly diminished by co-treatment with LTB4 at 0.1 nM. Finally, neither PGE2 nor LTB4 treatment affected collagen type I gene expression. These results suggest that low levels of LTB4 counterbalance the negative effects mediated by PGE2 on tendon fibroblast proliferation and MMP production, which may lead to matrix degradation. Thus, our findings suggest that although LTB4 is generally thought to be pathogenic, low levels of LTB4 are actually beneficial in maintaining tendon tissue homeostasis. [Copyright &y& Elsevier]