Myocardial reperfusion injury exacerbation due to ALDH2 deficiency is mediated by neutrophil extracellular traps and prevented by leukotriene C4 inhibition.

BACKGROUND AND AIMS: The Glu504Lys polymorphism in the aldehyde dehydrogenase 2 (ALDH2) gene is closely associated with myocardial ischaemia/reperfusion injury (I/RI). The effects of ALDH2 on neutrophil extracellular trap (NET) formation (i.e. NETosis) during I/RI remain unknown. This study aimed to investigate the role of ALDH2 in NETosis in the pathogenesis of myocardial I/RI. METHODS: The mouse model of myocardial I/RI was constructed on wild-type, ALDH2 knockout, peptidylarginine deiminase 4 (Pad4) knockout, and ALDH2/PAD4 double knockout mice. Overall, 308 ST-elevation myocardial infarction patients after primary percutaneous coronary intervention were enrolled in the study. RESULTS: Enhanced NETosis was observed in human neutrophils carrying the ALDH2 genetic mutation and ischaemic myocardium of ALDH2 knockout mice compared with controls. PAD4 knockout or treatment with NETosis-targeting drugs (GSK484, DNase1) substantially attenuated the extent of myocardial damage, particularly in ALDH2 knockout. Mechanistically, ALDH2 deficiency increased damage-associated molecular pattern release and susceptibility to NET-induced damage during myocardial I/RI. ALDH2 deficiency induced NOX2-dependent NETosis via upregulating the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/leukotriene C4 (LTC4) pathway. The Food and Drug Administration-approved LTC4 receptor antagonist pranlukast ameliorated I/RI by inhibiting NETosis in both wild-type and ALDH2 knockout mice. Serum myeloperoxidase-DNA complex and LTC4 levels exhibited the predictive effect on adverse left ventricular remodelling at 6 months after primary percutaneous coronary intervention in ST-elevation myocardial infarction patients. CONCLUSIONS: ALDH2 deficiency exacerbates myocardial I/RI by promoting NETosis via the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/LTC4/NOX2 pathway. This study hints at the role of NETosis in the pathogenesis of myocardial I/RI, and pranlukast might be a potential therapeutic option for attenuating I/RI, particularly in individuals with the ALDH2 mutation.

A macrocyclic peptide inhibitor traps MRP1 in a catalytically incompetent conformation.

Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC4). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.

Bronchodilatory effect of higenamine as antiallergic asthma treatment.

Asthma is a complex airway disease that affects more than 350 million humans worldwide. Allergic asthma symptoms are induced by Th2 immune response with the release of cytokines and allegro-inflammatory mediators that amplify the inflammatory response, airway hyper-responsiveness (AHR) and hyperproduction of mucus. Higenamine, as a chemical compound, is a ß2 adrenoreceptor agonist and can be used as bronchodilator in allergic asthma.BALB/c mice were allocated in four groups and then allergic asthma was induced in three groups. One of the asthmatic groups was treated with albuterol and other one was treated with higenamine. At least, methacholine challenge to determine the AHR, measurement of cytokines, total immunoglobulin E (IgE), LTB4 and LTC4 levels, evaluation of gene expression of Muc5ac, Muc5b, Agr2 and Arg1, and histopathological study were done.Higenamine treatment reduced AHR, interleukin (IL)-4, IL-13 levels, mRNA expression of MUC5ac, MUC5b, Arg1 and Agr2, goblet cell hyperplasia and mucus hypersecretion. Higenamine had no significant effect on IL-5, interferon-γ (INF-γ), IgE, LTB4, LTC4 levels and eosinophilic inflammation in lung tissue.Higenamine treatment controls asthma acute attack and breathlessness and can be used as asthma treatment with control of AHR and decrease of airflow obstruction and mucus hypersecretion and had allegro-immune-regulatory effect. But higenamine treatment had no notable effect on the inflammation and inflammatory factors.

The Protective Role of Cirsilineol against Ovalbumin-Induced Allergic Rhinitis in Mice by Suppression of Inflammation and Oxidative Stress.

Allergic rhinitis (AR) is a common type of inflammatory disease with symptoms including rhinorrhea, fatigue, sneezing, and disturbed sleep. AR affects nearly 40% of peoples worldwide with the increased numbers of new cases. In this work, the study was conducted to disclose the anti-inflammatory and antiallergic properties of cirsilineol against the ovalbumin (OVA)-sensitized AR in mice. AR was provoked in BALB/c mice through the OVA challenge 30 days along with 10 and 20 mg/kg of cirsilineol treatment. The nasal symptoms, i.e., rubbing and sneezing was monitored after the final OVA challenge. The status of OVA-specific IgE, PGD2, and LTC4 was investigated using assay kits. The status of pro-inflammatory markers also examined using assay kits. The levels of oxidative markers, SOD activity, and pro-inflammatory markers in the spleen mononuclear cells (SMEs) were studied by using respective assay kits. The mRNA expression of TXNIP was assessed using RT-PCR study. The 10 and 20 mg/kg of cirsilineol treatment effectively decreased the sneezing and nasal rubbings in OVA-provoked mice. Cirsilineol also decreased the IgE, PGD2, and LTC4 status in the AR animals. The status of pro-inflammatory markers, i.e., IL-4, IL-5, IL-6, IL-33 and TNF-α was found to be decreased in the cirsilineol administered AR mice. Cirsilineol effectively reduced the ROS and MDA and improved SOD in the OVA-challenged SMCs. The mRNA expression of TXNIP was appreciably suppressed by the cirsilineol treatment. Altogether, these findings proved the beneficial actions of cirsilineol against the OVA-triggered AR in mice. The additional studies on the cirsilineol could lead to the development of new drug for AR management.

The CysLT2R receptor mediates leukotriene C4-driven acute and chronic itch.

Acute and chronic itch are burdensome manifestations of skin pathologies including allergic skin diseases and atopic dermatitis, but the underlying molecular mechanisms are not well understood. Cysteinyl leukotrienes (CysLTs), comprising LTC4, LTD4, and LTE4, are produced by immune cells during type 2 inflammation. Here, we uncover a role for LTC4 and its signaling through the CysLT receptor 2 (CysLT2R) in itch. Cysltr2 transcript is highly expressed in dorsal root ganglia (DRG) neurons linked to itch in mice. We also detected CYSLTR2 in a broad population of human DRG neurons. Injection of leukotriene C4 (LTC4) or its nonhydrolyzable form NMLTC4, but neither LTD4 nor LTE4, induced dose-dependent itch but not pain behaviors in mice. LTC4-mediated itch differed in bout duration and kinetics from pruritogens histamine, compound 48/80, and chloroquine. NMLTC4-induced itch was abrogated in mice deficient for Cysltr2 or when deficiency was restricted to radioresistant cells. Itch was unaffected in mice deficient for Cysltr1, Trpv1, or mast cells (WSh mice). CysLT2R played a role in itch in the MC903 mouse model of chronic itch and dermatitis, but not in models of dry skin or compound 48/80- or Alternaria-induced itch. In MC903-treated mice, CysLT levels increased in skin over time, and Cysltr2-/- mice showed decreased itch in the chronic phase of inflammation. Collectively, our study reveals that LTC4 acts through CysLT2R as its physiological receptor to induce itch, and CysLT2R contributes to itch in a model of dermatitis. Therefore, targeting CysLT signaling may be a promising approach to treat inflammatory itch.

Crystal structures of human MGST2 reveal synchronized conformational changes regulating catalysis.

Microsomal glutathione S-transferase 2 (MGST2) produces leukotriene C4, key for intracrine signaling of endoplasmic reticulum (ER) stress, oxidative DNA damage and cell death. MGST2 trimer restricts catalysis to only one out of three active sites at a time, but the molecular basis is unknown. Here, we present crystal structures of human MGST2 combined with biochemical and computational evidence for a concerted mechanism, involving local unfolding coupled to global conformational changes that regulate catalysis. Furthermore, synchronized changes in the biconical central pore modulate the hydrophobicity and control solvent influx to optimize reaction conditions at the active site. These unique mechanistic insights pertain to other, structurally related, drug targets.

Study on functional sites in human multidrug resistance protein 1 (hMRP1).

Human multidrug resistance protein 1 (hMRP1) is an important member of the ATP-binding cassette (ABC) transporter superfamily. It can extrude a variety of anticancer drugs and physiological organic anions across the plasma membrane, which is activated by substrate binding, and is accompanied by large-scale cooperative movements between different domains. Currently, it remains unclear completely about how the specific interactions between hMRP1 and its substrate are and which critical residues are responsible for allosteric signal transduction. To the end, we first construct an inward-facing state of hMRP1 using homology modeling method, and then dock substrate proinflammatory agent leukotriene C4 (LTC4) to hMRP1 pocket. The result manifests LTC4 interacts with two parts of hMRP1 pocket, namely the positively charged pocket (P pocket) and hydrophobic pocket (H pocket), similar to its binding mode with bMRP1 (bovine MRP1). Additionally, we use the Gaussian network model (GNM)-based thermodynamic method proposed by us to identify the key residues whose perturbations markedly alter their binding free energy. Here the conventional GNM is improved with covalent/non-covalent interactions and secondary structure information considered (denoted as sscGNM). In the result, sscGNM improves the flexibility prediction, especially for the nucleotide binding domains with rich kinds of secondary structures. The 46 key residue clusters located in different subdomains are identified which are highly consistent with experimental observations. Furtherly, we explore the long-range cooperation within the transporter. This study is helpful for strengthening the understanding of the work mechanism in ABC exporters and can provide important information to scientists in drug design studies.

Functional role of phosphatidylcholine-specific phospholipase C in regulating leukotriene synthesis and degranulation in human eosinophils.

Phosphatidylinositol-specific phospholipase C (PI-PLC) and cytosolic phospholipase A2 (cPLA2) regulate both eosinophil degranulation and leukotriene (LT) synthesis via PI-PLC-mediated calcium influx and cPLA2 activation. Phosphatidylcholine-specific phospholipase C (PC-PLC) likely plays a key role in cellular signaling, including the eosinophilic allergic inflammatory response. This study examined the role of PC-PLC in eosinophil LT synthesis and degranulation using tricyclodecan-9-yl-xanthogenate (D609), a PC-specific PLC inhibitor. D609 inhibited N-formyl-met-leu-phe + cytochalasin B (fMLP/B)-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) secretion. However, at concentrations that blocked both AA release and LTC4 secretion, D609 had no significant inhibitory effect on stimulated cPLA2 activity. D609 also partially blocked fMLP/B-induced calcium influx, indicating that inhibition of AA release and LTC4 secretion by D609 is due to inhibition of calcium-mediated cPLA2 translocation to intracellular membranes, not inhibition of cPLA2 activity. In addition, D609 inhibited fMLP/B-stimulated eosinophil peroxidase release, indicating that PC-PLC regulates fMLP/B-induced eosinophil degranulation by increasing the intracellular calcium concentration ([Ca2+]i). Overall, our results showed that PC-PLC is critical for fMLP/B-stimulated eosinophil LT synthesis and degranulation. In addition, degranulation requires calcium influx, while PC-PLC regulates LTC4 synthesis through calcium-mediated cPLA2 activation.

Identification of cysteinyl-leukotriene-receptor 1 antagonists as ligands for the bile acid receptor GPBAR1.

The cysteinyl leukotrienes (CysLTs), i.e. LTC4, LTD4 and LTE4, are a family of proinflammatory agents synthesized from the arachidonic acid. In target cells, these lipid mediators bind to the cysteinyl leukotriene receptors (CysLTR), a family of seven transmembrane G-protein coupled receptors. The CysLT1R is a validated target for treatment of pulmonary diseases and several selective antagonists for this receptor, including montelukast, zafirlukast and pranlukast, have shown effective in the management of asthma. Nevertheless, others CysLT1R antagonists, such as the alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV5901), have been extensively characterized without reaching sufficient priority for clinical development. Since drug reposition is an efficient approach for maximizing investment in drug discovery, we have investigated whether CysLT1R antagonists might exert off-target effects. In the report we demonstrate that REV5901 interacts with GPBAR1, a well characterized cell membrane receptor for secondary bile acids. REV5901 transactivates GPBAR1 in GPBAR1-transfected cells with an EC50 of 2.5 µM and accommodates the GPBAR1 binding site as shown by in silico analysis. Exposure of macrophages to REV5901 abrogates the inflammatory response elicited by bacterial endotoxin in a GPBAR1-dependent manner. In vivo, in contrast to montelukast, REV5901 attenuates inflammation and immune dysfunction in rodent models of colitis. The beneficial effects exerted by REV5901 in these models were abrogated by GPBAR1 gene ablation, confirming that REV5901, a shelved CysLT1R antagonist, is a GPBAR1 ligand. These data ground the basis for the development of novel hybrid ligands designed for simultaneous modulation of CysTL1R and GPBAR1.

Two- and three-color STORM analysis reveals higher-order assembly of leukotriene synthetic complexes on the nuclear envelope of murine neutrophils.

Over the last several years it has become clear that higher order assemblies on membranes, exemplified by signalosomes, are a paradigm for the regulation of many membrane signaling processes. We have recently combined two-color direct stochastic optical reconstruction microscopy (dSTORM) with the (Clus-DoC) algorithm that combines cluster detection and colocalization analysis to observe the organization of 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) into higher order assemblies on the nuclear envelope of mast cells; these assemblies were linked to leukotriene (LT) C4 production. In this study we investigated whether higher order assemblies of 5-LO and FLAP included cytosolic phospholipase A2 (cPLA2) and were linked to LTB4 production in murine neutrophils. Using two- and three-color dSTORM supported by fluorescence lifetime imaging microscopy we identified higher order assemblies containing 40 molecules (median) (IQR: 23, 87) of 5-LO, and 53 molecules (62, 156) of FLAP monomer. 98 (18, 154) molecules of cPLA2 were clustered with 5-LO, and 77 (33, 114) molecules of cPLA2 were associated with FLAP. These assemblies were tightly linked to LTB4 formation. The activation-dependent close associations of cPLA2, FLAP, and 5-LO in higher order assemblies on the nuclear envelope support a model in which arachidonic acid is generated by cPLA2 in apposition to FLAP, facilitating its transfer to 5-LO to initiate LT synthesis.

Nepetin, a natural compound from Inulae flos, suppresses degranulation and eicosanoid generation through PLCγ1 and Akt signaling pathways in mast cells.

Nepetin derived from the flowers of Inula japonica, Inulae flos, has been reported to exert several biological activities, including anti-inflammatory responses. In this study, we evaluated the anti-allergic property of nepetin with its molecular mechanisms in bone marrow-derived mast cells (BMMC) and mice. In this in vitro study, we investigated the inhibitory effects of nepetin on degranulation and generation of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) in IgE/antigen (Ag)-stimulated BMMC. The effect of nepetin on passive cutaneous anaphylaxis (PCA) reaction was also studied in mice. Nepetin reduced degranulation and LTC4 generation in BMMC. The IgE/Ag-mediated signaling pathway demonstrated that nepetin suppressed intracellular Ca2+ level and activation of PLCγ1 and cPLA2. However, MAPKs were not affected by nepetin in BMMC. In addition, nepetin treatment reduced PGD2 production and suppressed cyclooxygenase-2 protein expression via the inhibition of the Akt and nuclear factor-κB signaling pathways. With respect to the local allergic response in vivo, oral administration of nepetin suppressed mast cell-dependent PCA reaction in a dose-dependent manner. The results of this study suggest that nepetin might have an anti-allergic potential related to mast cell-mediated inflammatory diseases.

Immune recovery following bronchiolitis is linked to a drop in cytokine and LTC4 levels.

BACKGROUND: Bronchiolitis is the main cause of hospitalization of children younger than 1 year; however, the immune mechanism of bronchiolitis is not completely understood. The aim of this study was to analyze the recovery of immune response after a bronchiolitis episode. METHODS: Forty-nine infants hospitalized with bronchiolitis diagnosis were enrolled. Nasopharyngeal aspirates (NPAs) were processed. Twenty-seven pro-inflammatory biomarkers linked to innate immunity, inflammation, and epithelial damage, as well as nitrites and lipid mediators, were evaluated in the NPA supernatant by ELISA (enzyme-linked immunosorbent assay) and Luminex. Also, 11 genes were analyzed in NPA cells by quantitative PCR. RESULTS: A widespread statistically significant decline of multiple pro-inflammatory parameters and cytokines were detected in the recovery period after respiratory infection: interferon-α2 (IFNα2), IFNγ, interleukin-10 (IL-10), IL-1ß, IL-8, IFN-γ-inducible protein-10, vascular endothelial growth factor, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α (MIP-1α), and MIP-1ß. Supporting these results, a decreased nuclear factor-κB gene expression was observed (P = 0.0116). A significant diminution of cysteinyl leukotriene C4 (LTC4) soluble levels (P = 0.0319) and cyclooxygenase-2 (COX-2) gene expression were observed in the recovery sample. In children classified by post-bronchiolitis wheezing, LTC4 remains elevated in the NPA supernatant. CONCLUSIONS: After bronchiolitis, cytokines and biomarkers linked to innate immune response in NPA decrease significantly in the recovery period accompanied by a drop in LTC4 levels; however, this reduction was lower in infants with post-bronchiolitis wheezing.

Structure-guided probing of the leukotriene C4 binding site in human multidrug resistance protein 1 (MRP1; ABCC1).

The human multidrug resistance protein 1 (hMRP1) transporter is implicated in cancer multidrug resistance as well as immune responses involving its physiologic substrate, glutathione (GSH)-conjugated leukotriene C4 (LTC4). LTC4 binds a bipartite site on hMRP1, which a recent cryoelectron microscopy structure of LTC4-bound bovine Mrp1 depicts as composed of a positively charged pocket and a hydrophobic (H) pocket that binds the GSH moiety and surrounds the fatty acid moiety, respectively, of LTC4. Here, we show that single Ala and Leu substitutions of H-pocket hMRP1-Met1093 have no effect on LTC4 binding or transport. Estrone 3-sulfate transport is also unaffected, but both hMRP1-Met1093 mutations eliminate estradiol glucuronide transport, demonstrating that these steroid conjugates have binding sites distinct from each other and from LTC4. To eliminate LTC4 transport by hMRP1, mutation of 3 H-pocket residues was required (W553/M1093/W1246A), indicating that H-pocket amino acids are key to the vastly different affinities of hMRP1 for LTC4vs. GSH alone. Unlike organic anion transport, hMRP1-mediated drug resistance was more diminished by Ala than Leu substitution of Met1093. Although our findings generally support a structure in which H-pocket residues bind the lipid tail of LTC4, their critical and differential role in the transport of conjugated estrogens and anticancer drugs remains unexplained.-Conseil, G., Arama-Chayoth, M., Tsfadia, Y., Cole, S. P. C. Structure-guided probing of the leukotriene C4 binding site in human multidrug resistance protein 1 (MRP1; ABCC1).

Roles of cysteinyl leukotrienes and their receptors in immune cell-related functions.

The cysteinyl leukotrienes (cys-LTs), leukotriene C4, (LTC4), LTD4, and LTE4, are lipid mediators of inflammation. LTC4 is the only intracellularly synthesized cys-LT through the 5-lipoxygenase and LTC4 synthase pathway and after transport is metabolized to LTD4 and LTE4 by specific extracellular peptidases. Each cys-LT has a preferred functional receptor in vivo; LTD4 to the type 1 cys-LT receptor (CysLT1R), LTC4 to CysLT2R, and LTE4 to CysLT3R (OXGR1 or GPR99). Recent studies in mouse models revealed that there are multiple regulatory mechanisms for these receptor functions and each receptor plays a distinct role as observed in different mouse models of inflammation and immune responses. This review focuses on the integrated host responses to the cys-LT/CysLTR pathway composed of sequential ligands with preferred receptors as seen from mouse models. It also discusses potential therapeutic targets for LTC4 synthase, CysLT2R, and CysLT3R.

Inhibitory effect of taxifolin on mast cell activation and mast cell-mediated allergic inflammatory response.

The aim of the present study is to investigate the anti-inflammatory and anti-allergic effects of taxifolin on mast cells and mast cell-mediated allergic reaction. We assessed the effect of taxifolin on the activation of bone marrow-derived mast cells (BMMCs) and rat basophilic leukemia (RBL)-2H3 cells induced by immunoglobulin E (IgE)/antigen (Ag), and the activation of human mast cell line (HMC-1) induced by PMA plus A23187. Taxifolin inhibited degranulation, generation of leukotriene C4 (LTC4), production of interlukin-6 (IL-6), and expression of cyclooxygenase-2 (COX-2) through blocking intracellular Ca2+ mobilization, phosphorylation of phospholipase Cγ (PLCγ) and mitogen-activated protein kinases (MAPKs), translocation of cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO), and Akt/IKK/NF-κB pathway, in BMMC cells. Furthermore, taxifolin suppressed phosphorylation of Syk, but without effect on Fyn and Lyn. Taxifolin also inhibited activation of RBL-2H3 and HMC-1 cells via Akt/IKK/NF-κB and MAPKs/cPLA2 signal pathway. Treatment with taxifolin attenuated the mast cell-mediated passive cutaneous anaphylaxis (PCA) reaction. Our results suggest that taxifolin might become a potential drug candidate for the treatment of allergic and inflammatory diseases.

Cysteinyl leukotriene receptor 2 drives lung immunopathology through a platelet and high mobility box 1-dependent mechanism.

Cysteinyl leukotrienes (cysLTs) facilitate eosinophilic mucosal type 2 immunopathology, especially in aspirin-exacerbated respiratory disease (AERD), by incompletely understood mechanisms. We now demonstrate that platelets, activated through the type 2 cysLT receptor (CysLT2R), cause IL-33-dependent immunopathology through a rapidly inducible mechanism requiring the actions of high mobility box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE). Leukotriene C4 (LTC4) induces surface HMGB1 expression by mouse platelets in a CysLT2R-dependent manner. Blockade of RAGE and neutralization of HMGB1 prevent LTC4-induced platelet activation. Challenges of AERD-like Ptges-/- mice with inhaled lysine aspirin (Lys-ASA) elicit LTC4 synthesis and cause rapid intrapulmonary recruitment of platelets with adherent granulocytes, along with platelet- and CysLT2R-mediated increases in lung IL-33, IL-5, IL-13, and bronchoalveolar lavage fluid HMGB1. The intrapulmonary administration of exogenous LTC4 mimics these effects. Platelet depletion, HMGB1 neutralization, and pharmacologic blockade of RAGE eliminate all manifestations of Lys-ASA challenges, including increase in IL-33, mast cell activation, and changes in airway resistance. Thus, CysLT2R signaling on platelets prominently utilizes RAGE/HMGB1 as a link to downstream type 2 respiratory immunopathology and IL-33-dependent mast cell activation typical of AERD. Antagonists of HMGB1 or RAGE may be useful to treat AERD and other disorders associated with type 2 immunopathology.

Exosomes and cells from lung cancer pleural exudates transform LTC4 to LTD4, promoting cell migration and survival via CysLT1.

Tumor-derived exosomes can modulate the cancer microenvironment and induce metastatic spread. Exosomes may carry enzymes for leukotriene (LT) biosynthesis, but the role of exosomal LTs has not been studied in cancer. We isolated exosomes and malignant cells from pleura exudates from 14 patients with non-small cell lung cancer. Lipidomic profiles, migration and apoptosis were determined. Both exosomes and primary cancer cells contained γ-glutamyl transpeptidase 1 (GGT-1) and avidly transformed exogenous LTC4 to pro-tumorigenic LTD4, for the cells to levels 100-fold above their endogenous CysLT production. This suggests that cancer cells promote their own survival via LTD4 if supplied with LTC4, which in the exudates was produced by monocytic cells. Furthermore, exosomes promoted migration of cancer cells, which was counteracted by the CysLT1 antagonist montelukast. Montelukast also induced apoptosis of cancer cells, and this was partially inhibited by exosomes. Our results demonstrate how cancer cells and exosomes, together with monocytic cells in lung cancer tissue, can produce high amounts of LTD4, to stimulate cancer cell migration and survival. This suggests that part of the pro-metastatic effect of exosomes is mediated by the leukotriene machinery, further supporting the use of CysLT1 antagonists for lung cancer therapy.

Elevated Systemic Levels of Eosinophil, Neutrophil, and Mast Cell Granular Proteins in Strongyloides Stercoralis Infection that Diminish following Treatment.

Infection with the helminth parasite Strongyloides stercoralis (Ss) is commonly clinically asymptomatic that is often accompanied by peripheral eosinophilia. Granulocytes are activated during helminth infection and can act as immune effector cells. Plasma levels of eosinophil and neutrophil granular proteins convey an indirect measure of granulocyte degranulation and are prominently augmented in numerous helminth-infected patients. In this study, we sought to examine the levels of eosinophil, neutrophil, and mast cell activation-associated granule proteins in asymptomatic Ss infection and to understand their kinetics following anthelmintic therapy. To this end, we measured the plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, neutrophil elastase, myeloperoxidase, neutrophil proteinase-3, mast cell tryptase, leukotriene C4, and mast cell carboxypeptidase-A3 in individuals with asymptomatic Ss infection or without Ss infection [uninfected (UN)]. We also estimated the levels of all of these analytes in infected individuals following definitive treatment of Ss infection. We demonstrated that those infected individuals have significantly enhanced plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, elastase, myeloperoxidase, mast cell tryptase, leukotriene C4, and carboxypeptidase-A3 compared to UN individuals. Following the treatment of Ss infection, each of these granulocyte-associated proteins drops significantly. Our data suggest that eosinophil, neutrophil, and mast cell activation may play a role in the response to Ss infection.

ATP Binding Enables Substrate Release from Multidrug Resistance Protein 1.

The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellular substrates are recruited to a bipartite binding site when the transporter rests in an inward-facing conformation. A key question remains: how are high-affinity substrates transferred across the membrane and released outside the cell? Using electron cryomicroscopy, we show here that ATP binding opens the transport pathway to the extracellular space and reconfigures the substrate-binding site such that it relinquishes its affinity for substrate. Thus, substrate is released prior to ATP hydrolysis. With this result, we now have a complete description of the conformational cycle that enables substrate transfer in a eukaryotic ABC exporter.

Lipopolysaccharide and cytokines modulate leukotriene (LT)B4 and LTC4 production by porcine endometrial endothelial cells.

Uterine inflammatory response is mediated by inflammatory mediators including eicosanoids and cytokines produced by immune and endometrial cells. Interactions between lipopolysaccharide (LPS) and cytokines, and leukotrienes (LTs) in endothelium, important for the host defence during the inflammation, are unknown. We studied the effect of LPS, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-4 and IL-10 on 5-lipooxygenase (5-LO), LTA4  hydrolase (LTAH) and LTC4 synthase (LTCS) mRNA and protein expression, LTB4 and LTC4 release from porcine endometrial endothelial cells, and cell viability. For 24 hr, cells were exposed to LPS (10 or 100 ng/ml of medium) and cytokines (each 1 or 10 ng/ml). 5-LO mRNA/protein expression augmented after incubation with larger doses of LPS, TNF-α, IL-4 and IL-10 and smaller dose of IL-1ß. Larger dose of TNF-α, smaller doses of LPS and IL-1ß and both doses of IL-10 increased LTAH mRNA/protein expression. LTAH protein content was up-regulated by larger dose of LPS, but it was reduced in response to both doses of IL-4. LTCS mRNA expression was elevated by larger doses of LPS, IL-4 and IL-10 or both doses of TNF-α and IL-1ß. LTCS protein level increased after treatment with both doses of IL-1ß, IL-4 and IL-10, smaller dose of LPS and larger dose of TNF-α. Both doses of LPS and larger doses of TNF-α and IL-10 increased LTB4 release. LPS, IL-1ß and IL-10 at smaller doses, or TNF-α and IL-4 at larger doses stimulated LTC4 release. Smaller doses of TNF-α and IL-1ß or both doses of IL-4 enhanced the cell viability. This work provides new insight on the participation of LPS, TNF-α, IL-1ß, IL-4 and IL-10 in LTB4 and LTC4 production/release from porcine endometrial endothelial cells, and the effect of above factors on these cells viability. The used cellular model gives the possibility to further establish the interactions between inflammatory mediators.