Search

BACKGROUND: Altered biosynthesis of eicosanoids is linked to type 2 inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP), but their role in recalcitrant NPs is unclear. OBJECTIVES: We sought to identify endotypes that are linked to recalcitrant CRSwNP, based on eicosanoids, their biosynthetic enzymes, and receptors as well as cytokines and the presence of eosinophils and mast cells in recurrent NPs. METHODS: Mucosal tissue collected at the time of sinus surgery from 54 patients with CRSwNP and 12 non-CRS controls were analysed for leukotriene (LT) E4, prostaglandin (PG) D2, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and 17 cytokines with ELISAs and Bio-Plex immunoassays. Patient subgroups were identified by cluster analysis and the probability of NP recurrence were tested with logistic regression analyses. Gene expressions were analysed with qPCR. Tryptase and eosinophil-derived neurotoxin (EDN) were measured with ELISAs as indications of the presence of mast cells and eosinophils, respectively. RESULTS: Clustering of patients showed that an inflammatory signature characterised by elevated LTE4, PGD2, 15(S)-HETE and IL-13 was associated with NP recurrence. Previous NP surgery as well as aspirin-exacerbated respiratory disease were significantly more common among these patients. Expression of cyclooxygenase 1 was the only gene associated with NP recurrence. Levels of EDN, but not tryptase, were significantly higher in patients with recurrent NPs. CONCLUSION: Distinguishing endotypes that include LTE4, PGD2, 15HETE and conventional biomarkers of type 2 inflammation could help predict recurrent nasal polyposis and thus identify cases of recalcitrant CRSwNP.

Innate lymphoid cells (ILC) promote lung inflammation in asthma through cytokine production. RNA-binding proteins (RBPs) are critical post-transcriptional regulators, although less is known about RBPs in ILC biology. Here, we demonstrate that RNA-binding motif 3 (RBM3) is highly expressed in lung ILCs and is further induced by alarmins TSLP and IL-33. Rbm3-/- and Rbm3-/-Rag2-/- mice exposed to asthma-associated Alternaria allergen develop enhanced eosinophilic lung inflammation and ILC activation. IL-33 stimulation studies in vivo and in vitro show that RBM3 suppressed lung ILC responses. Further, Rbm3-/- ILCs from bone marrow chimeric mice display increased ILC cytokine production suggesting an ILC-intrinsic suppressive function of RBM3. RNA-sequencing of Rbm3-/- lung ILCs demonstrates increased expression of type 2/17 cytokines and cysteinyl leukotriene 1 receptor (CysLT1R). Finally, Rbm3-/-Cyslt1r-/- mice show dependence on CysLT1R for accumulation of ST2+IL-17+ ILCs. Thus, RBM3 intrinsically regulates lung ILCs during allergen-induced type 2 inflammation that is partially dependent on CysLT1R.

Background: Leukotrienes are pro-inflammatory vasoactive lipid mediators implicated in the pathophysiology of atherosclerotic cardiovascular disease. We studied the effect of the 5-lipoxygenase-activating protein inhibitor AZD5718 on leukotriene biosynthesis and coronary microvascular function in a single-blind, phase 2a study. Methods: Patients 7-28 days after myocardial infarction (+/- ST elevation), with < 50% left anterior descending coronary artery stenosis and Thrombolysis in Myocardial Infarction flow grade >= 2 after percutaneous coronary intervention, were randomized 2:1:2 to once-daily AZD5718 200 mg or 50 mg, or placebo, in 4-and 12-week cohorts. Change in urine leukotriene E-4 (uLTE(4)) was the primary endpoint, and coronary flow velocity reserve (CFVR; via echocardiography) was the key secondary endpoint. Results: Of 129 randomized patients, 128 received treatment (200 mg, n = 52; 50 mg, n = 25; placebo, n = 51). Statistically significant reductions in uLTE4 levels of > 80% were observed in both AZD5718 groups versus the placebo group at 4 and 12 weeks. No significant changes in CFVR were observed for AZD5718 versus placebo. Adverse events (AEs) occurred in 12/18, 3/6 and 6/13 patients receiving 200 mg, 50 mg and placebo, respectively, in the 4-week cohort, and in 27/34, 14/19 and 24/38 patients, respectively, in the 12-week cohort. Serious AEs in seven patients receiving AZD5718 and four receiving placebo were not treatment-related, and there were no deaths. Conclusions: In patients with recent myocardial infarction, AZD5718 was well tolerated, and leukotriene biosynthesis was dose-dependently inhibited. No significant changes in CFVR were detected.

In the pharmacotherapy of bronchial asthma, a "step-by-step" approach is recommended, which includes an increase or decrease in the volume of therapy, depending on the severity of clinical symptoms. In complex therapy, it is possible to use non-drug methods of treatment, although the effectiveness of some of them is debatable and needs further study. To achieve this goal, children over the age of 3 years (the group of patients consisted of 26 children) with different forms and degrees of severity of bronchial asthma were included in the study. Patients of the main group took L-Montus (montelukast), children of the ketotifen comparison group. Leukotriene receptor antagonists (montelukast, zafirlukast) are the first mediator-specific therapy of AD and represent a new therapeutic class of drugs in the treatment of the disease. The effectiveness of leukotriene receptor antagonists has been proven in randomized clinical trials among adults and children with bronchial asthma. Leukotriene receptor blockers, in particular L-Montus relieves the symptoms of AD and is a drug for the basic therapy of this disease, leads to a significant improvement in control, reduces the frequency of exacerbations of AD caused by viral infection in children aged 3-5 years. It has an anti-inflammatory effect, complementary to the action of glucocorticosteroids.

Background: Leukotrienes are pro-inflammatory vasoactive lipid mediators implicated in the pathophysiology of atherosclerotic cardiovascular disease. We studied the effect of the 5-lipoxygenase-activating protein inhibitor AZD5718 on leukotriene biosynthesis and coronary microvascular function in a single-blind, phase 2a study. Methods: Patients 7–28 days after myocardial infarction (±ST elevation), with <50% left anterior descending coronary artery stenosis and Thrombolysis in Myocardial Infarction flow grade ≥ 2 after percutaneous coronary intervention, were randomized 2:1:2 to once-daily AZD5718 200 mg or 50 mg, or placebo, in 4- and 12-week cohorts. Change in urine leukotriene E4 (uLTE4) was the primary endpoint, and coronary flow velocity reserve (CFVR; via echocardiography) was the key secondary endpoint. Results: Of 129 randomized patients, 128 received treatment (200 mg, n = 52; 50 mg, n = 25; placebo, n = 51). Statistically significant reductions in uLTE4 levels of >80% were observed in both AZD5718 groups versus the placebo group at 4 and 12 weeks. No significant changes in CFVR were observed for AZD5718 versus placebo. Adverse events (AEs) occurred in 12/18, 3/6 and 6/13 patients receiving 200 mg, 50 mg and placebo, respectively, in the 4-week cohort, and in 27/34, 14/19 and 24/38 patients, respectively, in the 12-week cohort. Serious AEs in seven patients receiving AZD5718 and four receiving placebo were not treatment-related, and there were no deaths. Conclusions: In patients with recent myocardial infarction, AZD5718 was well tolerated, and leukotriene biosynthesis was dose-dependently inhibited. No significant changes in CFVR were detected. ClinicalTrials.gov identifier: NCT03317002.

Objective: Malassezia furfur is a lipid-dependent yeast that is part of the human skin microbiota. Mechanisms underlying growth on skin including its lipid metabolism are largely unknown. Lipid droplets (LDs), also known as lipid particles, lipid bodies, oil bodies, or oleosomes, are highly dynamic organelles that can be produced in nearly all cells. The presence of LDs in Malassezia (Pityrosporum orbiculare) have been reported, but their role has not been studied. LDs contribute to a variety of processes including lipid storage, cell signaling, temporary protein storage, cell lipid homeostasis, prevention of lipotoxicity, biosynthesis and secretion of inflammatory mediators such as prostaglandins and leukotriene's, interferon responses, and antigen cross presentation. Lipid droplets are expected to play an important role in Malassezia spp during their growth and/or adaptation on the skin yet this has not been investigated. The purpose of this study is to characterize LD of M. furfur in complex medium (mDixon) and minimal medium supplemented with sources of palmitic acid (PA; C16:0) and/or oleic acid (OA; C18:1). We addressed the presence of LD during starvation for fatty acids, the composition of the lipid droplets using lipidomics and we used genomic analysis to identify the pathways involved in neutral lipid synthesis in this yeast. Methods: Lipid droplets (LDs) were identified in M. furfur growing in mDixon broth or MM with or without PA and/or OA through confocal microscopy using nile red staining. LD dynamics and cell viability was monitored upon transferring M. furfur cells that had been pre-grown in MM with PA and / or OA to MM without fatty acids. Lipidomic analyses were performed on chloroform/methanol extracted lipids from LD isolated via density gradient centrifugation. Results: CFUs strongly decreased upon lipid starvation irrespective of the type(s) of fatty acid present in the pre-culture and no CFUs were found after 160 h. Interestingly, cells observ

The 5th Scientific Conference of the International Pemphigus and Pemphigoid Foundation (IPPF), "Pemphigus and Pemphigoid: A New Era of Clinical and Translational Science" was held in Orlando, Florida, on May 15-16, 2018. Scientific sessions covered recent, ongoing, and future clinical trials in pemphigus and bullous pemphigoid, disease activity and quality of life instruments, and the IPPF Natural History Study. Furthermore, the meeting provided an opportunity to hear firsthand from patients, investigators, and industry about their experience enrolling for clinical trials.

BackgroundCritical examination of the quality and validity of available allergic rhinitis (AR) literature is necessary to improve understanding and to appropriately translate this knowledge to clinical care of the AR patient. To evaluate the existing AR literature, international multidisciplinary experts with an interest in AR have produced the International Consensus statement on Allergy and Rhinology: Allergic Rhinitis (ICAR:AR).MethodsUsing previously described methodology, specific topics were developed relating to AR. Each topic was assigned a literature review, evidence-based review (EBR), or evidence-based review with recommendations (EBRR) format as dictated by available evidence and purpose within the ICAR:AR document. Following iterative reviews of each topic, the ICAR:AR document was synthesized and reviewed by all authors for consensus.ResultsThe ICAR:AR document addresses over 100 individual topics related to AR, including diagnosis, pathophysiology, epidemiology, disease burden, risk factors for the development of AR, allergy testing modalities, treatment, and other conditions/comorbidities associated with AR.ConclusionThis critical review of the AR literature has identified several strengths; providers can be confident that treatment decisions are supported by rigorous studies. However, there are also substantial gaps in the AR literature. These knowledge gaps should be viewed as opportunities for improvement, as often the things that we teach and the medicine that we practice are not based on the best quality evidence. This document aims to highlight the strengths and weaknesses of the AR literature to identify areas for future AR research and improved understanding.

In the work the questions of automation of radicular ischemia diagnosis according to the results of clinical and laboratory diagnostics of patients with pain in the lumbar-sacral region are considered. Automation of the process of determining radicular ischemia at an early stage of development will increase the efficiency of the specialist, unify the data necessary to differentiate this pathology, as well as improve the workflow

Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL(+)CD34(+)CD38(-) cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34(+)CD38(-) cells from 7 CML patients. The majority of the single leukemic BCR-ABL(+)CD34(+)CD38(-) cells expressed cysteinyl leukotriene receptors CYSLTI and CYSLT2. However, montelukast, an inhibitor of CYSLTI, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLTI signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients. (C) 2017 The Author(s). Published by Elsevier Inc.

Background. For many women, the monthly suffering induced by menstrual “cramps” is severe enough to profoundly disrupt their quality of life. In the case of primary dysmenorrhea, a condition related to premenstrual syndrome (PMS), intense uterine contractions are thought to trigger moderate to intense pain despite the absence of an underlying infection or other medically-identifiable disease states. The associated uterine hyper-contractility is reminiscent of labor, and associated pain is likely to be mediated by the release of prostaglandins, leukotrienes and the infiltration of leukocytes that normally accompany the breakdown of the endometrial lining.Standardized extracts of Vitex agnus-castus berries (VAC extracts of chaste tree, or chaste berries) are clinically effective in treating the symptoms of PMS, yet the mechanisms of how the chemically complex mixture acts are largely unknown.Methods. Using an in vivo dysmenorrhea model rats were treated with 10 mg/kg estradiol-benzoate i.p. once daily for 12 days and with 2.1, 10.3 or 20.7 mg/kg VAC dry extract p.o. once daily for 7 days prior to induction of convulsions. Uterine contractions where induced with 2 IU/kg oxytocin i.p., followed by monitoring of abdominal convulsions and signs of pain on the last day of the experiment. Moreover, in vitro methods were applied that are described in the methods section.Results. Here, we show that the VAC herbal dry extract BNO 1095 (commercially available as Cyclodynon®) targets the uterine myometrial tissue and inflammatory signaling molecules of associated migratory/inflammatory cells. Specifically, BNO 1095 dose-dependently inhibited oxytocin-induced uterine contractions in a rat dysmenorrhea model in vivo and drug-induced contractions in isolated human and rat uterine tissue in vitro. Furthermore, BNO 1095 showed a promising anti-inflammatory capacity by potently inhibiting 5-lipoxygenase activity and leukotriene production and by reducing the production of reactive oxy

Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R-/- mice, but not CysLT2R-/- mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R-/- mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata Thus, CysLT1R promotes LTC4- and Alternaria-induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses.

5-Lipoxygenase (5-LOX) catalyzes two steps in conversion of arachidonic acid to proinflammatory leukotrienes. Lipoxygenases, including human 5-LOX, consist of an N-terminal C2-like beta-sandwich and a catalytic domain. We expressed the 5-LOX domains separately, these were found to interact in the yeast two-hybrid system. The 5-LOX structure suggested association between Arg(101) in the beta-sandwich and Asp(166) in the catalytic domain, due to electrostatic interaction as well as hydrogen bonds. Indeed, mutagenic replacements of these residues led to loss of two-hybrid interaction. Interestingly, when Arg(101) was mutated to Asp in intact 5-LOX, enzyme activity was increased. Thus, higher initial velocity of the reaction (v(init)) and increased final amount of products were monitored for 5-LOX-R101D, at several different assay conditions. In the 5-LOX crystal structure, helix alpha 2 and adjacent loops (including Asp(166)) of the 5-LOX catalytic domain has been proposed to form a flexible lid controlling access to the active site, and lid movement would be determined by bonding of lid residues to the C2-like beta-sandwich. The more efficient catalysis following disruption of the R101-D166 ionic association supports the concept of such a flexible lid in human 5-LOX. (C) 2014 Elsevier Inc. All rights reserved.

The research in this dissertation describes the allosteric activation and inhibition of 5-LOX human lipoxygenase as well as the substrate preferences for all 3 main LOX isozymes, 5-LOX, 12-LOX, and 15-LOX-1, to generate higher-order lipid mediators. 5-LOX is both a hydroperoxidase of arachidonic acid (AA) and an epoxidase of 5(S)-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5(S)-HpETE) to form leukotrienes from a single polyunsaturated fatty acid (PUFA). This dissertation investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE has a significantly lower rate of substrate capture (Vmax/Km) than AA hydroperoxidation, however, hyperbolic kinetic parameters for ATP activation suggest a larger activation with 5(S)-HpETE. Solvent isotope effect (SIE) results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly due a change in the dependency of the rate-limiting step on hydrogen-bond rearrangement toward substrate rearrangement. The products of 5-LOX, leukotrienes and resolvins, both promote and inhibit inflammation, respectively, and therefore changes in ATP concentration in the cell could have wide implications in their relative concentrations and ultimately the regulation of cellular inflammation. 5-LOX inhibition was also profiled with the discovery of a novel dual inhibitor targeting fungal sterol 14alpha-demethylase (CYP51 or Erg11) and 5-LOX. A phenylenediamine core was translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Docking of ketaminazole confirmed kinetic results showing that the drug binds in the active site presenting the phenylenediamine core for effective reduction of the 5-LOX catalytic iron. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have ther

BackgroundExercise-induced bronchoconstriction (EIB) is a prototypical feature of indirect airway hyperresponsiveness. Mast cells are implicated in EIB, but the characteristics, regulation, and function of mast cells in patients with EIB are poorly understood.ObjectivesWe sought to examine mast cell infiltration of the airway epithelium in patients with EIB and the regulation of mast cell phenotype and function by epithelially derived cytokines.MethodsEndobronchial biopsy specimens, epithelial brushings, and induced sputum were obtained from asthmatic patients with and without EIB and healthy control subjects. Mast cell proteases were quantified by using quantitative PCR, and mast cell density was quantified by using design-based stereology. Airway epithelial responses to wounding and osmotic stress were assessed in primary airway epithelial cells and ex vivo murine lung tissue. Mast cell granule development and function were examined in cord blood-derived mast cells.ResultsTryptase and carboxypeptidase A3 expression in epithelial brushings and epithelial mast cell density were selectively increased in the asthma group with EIB. An in vitro scratch wound initiated the release of thymic stromal lymphopoietin, which was greater in epithelial cells derived from asthmatic patients. Osmotic stress induced the release of IL-33 from explanted murine lungs, which was increased in allergen-treated mice. Thymic stromal lymphopoietin combined with IL-33 increased tryptase and carboxypeptidase A3 immunostaining in mast cell precursors and selectively increased cysteinyl leukotriene formation by mast cells in a manner that was independent of in vitro sensitization.ConclusionsMast cell infiltration of the epithelium is a critical determinant of indirect airway hyperresponsiveness, and the airway epithelium might serve as an important regulator of the development and function of this mast cell population.

Background: The effect of aspirin on the release of key arachidonic acid metabolites in activated eosinophils from subjects with aspirin-intolerant asthma (AIA) has not been investigated previously, despite the characteristic eosinophilia in AIA. Methods: Peripheral blood eosinophils were isolated from four groups of subjects: healthy volunteers (HV; n = 8), mild asthma (MA; n = 8), severe asthma (SA; n = 9) and AIA (n = 7). In the absence or presence of lysine-aspirin, eosinophils were stimulated with arachidonic acid or calcium ionophore to trigger the 15-lipoxygenase-1 (15-LO) and 5-lipoxygenase (5-LO) pathways, respectively. 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and eoxin C4 (EXC4) were measured as 15-LO products and leukotriene (LT)C4 as a product of the 5-LO pathway. Results: Activated eosinophils from patients with SA and AIA produced approximately five times more 15-HETE than eosinophils from HV or MA patients. In the presence of lysine-aspirin, eosinophils from AIA, MA and SA patients generated higher levels of 15-HETE than in the absence of lysine-aspirin. Furthermore, in the presence of lysine-aspirin, formation of EXC4 was also significantly increased in eosinophils from AIA patients, and LTC4 synthesis was increased both in AIA and SA patients. Conclusions: Taken together, this study shows an increased release of the recently discovered lipid mediator EXC4, as well as the main indicator of 15-LO activity, 15-HETE, in activated eosinophils from severe and aspirin-intolerant asthmatics, and also elevated EXC4 and LTC4 formation in eosinophils from AIA patients after cellular activation in the presence of lysine-aspirin. The findings support a pathophysiological role of the 15-LO pathway in SA and AIA.

Background: Cysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT(1)R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT(2)R) is lost. Further, our previous data indicate that patients with high CysLT(1)R and low CysLT(2)R expression have a poor prognosis. In this study, we examined whether the balance between CysLT(1)R and CysLT(2)R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA). Methods: To determine the effect of ATRA on CysLT(2)R promoter activation, mRNA level, and protein level, we performed luciferase gene reporter assays, real-time polymerase chain reactions, and Western blots in colon cancer cell lines under various conditions. Results: ATRA treatment induces CysLT(2)R mRNA and protein expression without affecting CysLT(1)R levels. Experiments using siRNA and mutant cell lines indicate that the up-regulation is retinoic acid receptor (RAR) dependent. Interestingly, ATRA also up-regulates mRNA expression of leukotriene C-4 synthase, the enzyme responsible for the production of the ligand for CysLT(2)R. Importantly, ATRA-induced differentiation of colorectal cancer cells as shown by increased expression of MUC-2 and production of alkaline phosphatase, both of which could be reduced by a CysLT(2)R-specific inhibitor. Conclusions: This study identifies a novel mechanism of action for ATRA in colorectal cancer cell differentiation and demonstrates that retinoids can have anti-tumorigenic effects through their action on the cysteinyl leukotriene pathway.

Leukotrienes (LTs) are pro-inflammatory lipid mediators that belong to the group of eicosanoids, which are oxygenated metabolites of one common precursor, the aracidonic acid (AA). This polyunsaturated fatty acid is esterified at the sn-2 position of cellular membrane phospholipids and can be released by cytosolic phospholipase A2 alpha (cPLA2alpha) enzymatic deacylation. AA can be converted into LTs by the catalytic reaction of 5-lipoxygenase (5-LO). Enzymatic activation of cPLA2alpha as well as of 5-LO is regulated by similar determinants. In response to cellular stimuli that elevate the intracellular Ca2+ level and/or activate MAP kinase pathways, cPLA2alpha and 5-LO comigrate from a soluble cell compartment (mainly the cytosol) to the nuclear membrane, where AA is released und converted into LTs. LTs play a significant role in promoting inflammatory reactions and immune processes. They have been shown to be released from leukocytes in response to bacterial and viral infections and substantially contribute to an effective immune reaction for host defense. Innate immune pathogen recognition is mediated to a substantial part by the Toll-like receptor (TLR) family. So far, 10 human TLR subtypes have been identified, all of which detect distinct highly conserved microbial structures and trigger the induction of signaling pathways that lead to the expression of numerous immune and inflammatory genes. TLR signaling culminates in the activation NF-kappaB and/or MAP kinases, which as well are known to be involved in the regulation of cellular LT biosynthesis. In this regard, it seemed conceivable that the release of LTs might be regulated by TLR activation. Present studies were undertaken in order to verify and characterize a possible influence of TLR activation on the LT biosynthesis, and furthermore to identify the involved signaling pathways and underlying mechanisms. First experiments revealed that pre-incubation of differentiated Mono Mac 6 (MM6) cells with a TLR4 l, Leukotriene (LTs) sind Entzündungsmediatoren aus der Gruppe der Eikosanoide, welche sich von der Arachidonsäure (AA) als ihre gemeinsame Vorstufe ableiten. Diese mehrfach ungesättigte Fettsäure ist in der Zelle in Membranphospholipiden verestert, und kann enzymatisch durch die zytosolische PLA2alpha (cPLA2alpha) freigesetzt werden. Weiterhin wird die AA durch die 5-Lipoxygenase (5-LO) zu LTs umgesetzt. 5-LO und cPLA2alpha sind sehr ähnlich reguliert. Zelluläre Stimuli, die zu einer Erhöhung der intrazellulären Ca2+-Konzentration und/oder Aktivierung von Mitogen-aktivierten Proteinkinasen (MAPKs) führen, lösen eine Aktivierung, und die konzertierte Translokation beider Enzyme aus dem Zytosol zur Kernmembran aus, wo die Freisetzung der AA und die LT-Biosynthese erfolgt. LTs spielen eine wichtige Rolle in Entzündungsreaktionen und Immunprozessen. Sie werden von Leukozyten bei bakteriellen und viralen Infektionen gebildet, und tragen zur Aktivierung entsprechender Immunabwehrmechanismen bei. Toll-like Rezeptoren (TLRs) sind Rezeptoren des angeborenen Immunsystems, die eine Schlüsselrolle für die Detektion von Pathogenen im Organismus spielen. Sie erkennen hoch konservierte pathogen-assoziierte molekulare Strukturen und aktivieren Signaltransduktionswege, die zur Expression entzündungsrelevanter Proteine und somit zur Entwicklung einer Immunantwort führen. Die TLR-Aktivierung führt im Allgemeinen zur Aktivierung des Transkriptionsfaktors NF-kappaB, und zur Aktivierung von MAP-Kinasen, deren Rolle in der Regulation der LT-Biosynthese bereits erwähnt wurde. Vor diesem Hintergrund schien es denkbar, dass im Zuge einer TLR-Aktivierung auch eine Regulation der LT-Bildung erfolgen könnte. Ziel dieser Arbeit war es, diesen Zusammenhang zu verifizieren, den Einfluss von TLR-Liganden auf die Biosynthese von LTs zu charakterisieren und die zugrundeliegenden Mechanismen aufzuklären. Erste Versuche zeigten, dass die Vorbehandlung differenzierter Mono Mac 6 (MM6)-Zellen mit einem TLR

Recent reports demonstrate a role for nucleotides as inflammatory modulators. Uridine, for example, reduces oedema formation and leucocyte infiltration in a Sephadex-induced lung inflammation model. Tumour necrosis factor (TNF) concentration was also reduced. Previous in vivo observations indicated that 4-thiouridine might have similar effects on leucocyte infiltration and TNF release. The aim of this study was thus to investigate the effects of 4-thiouridine in greater detail. We used a Sephadex-induced acute lung inflammation model in Sprague-Dawley rats. The dextran beads were instilled intratracheally into the lungs, which were excised and examined after 24 h. Sephadex alone led to massive oedema formation and infiltration of macrophages, neutrophils and eosinophils. Microgranulomas with giant cell formations were clearly visible around the partially degraded beads. A significant increase in bronchoalveolar lavage fluid (BALF) content of TNF and leukotrienes was also seen. 4-Thiouridine co-administration affected all variables investigated in this model, i.e. oedema, microscopic and macroscopic appearance of lung tissue, total leucocyte and differential leucocyte counts in BALF, TNF and leukotrienes C-4 (LTC4), LTD4 and LTE4 in BALF, indicating a reproducible anti-inflammatory effect. In conclusion, we have demonstrated that 4-thiouridine has anti-inflammatory effects similar to those of uridine. To our knowledge, this is the first demonstration of pharmacological 4-thiouridine effects in vivo. The results suggest nucleoside/nucleotide involvement in inflammatory processes, warranting further studies on nucleoside analogues as attractive new alternatives in the treatment of inflammatory diseases., The definitive version is available at www.blackwell-synergy.com:Chamilly Evaldsson, Ingvar Rydén, Anders Rosén and Srinivas Uppugunduri, 4-Thiouridine induces dose-dependent reduction of oedema, leucocyte influx and tumour necrosis factor in lung inflammation, 2009, CLINICAL AND EXPERIMENTAL IMMUNOLOGY, (155), 2, 330-338.http://dx.doi.org/10.1111/j.1365-2249.2008.03795.xCopyright: Blackwell Publishing Ltdhttp://www.blackwellpublishing.com

Eicosanoids have been implicated in a vast number of devastating inflammatory conditions, including arthritis, atherosclerosis, pain, and cancer. Currently, over a hundred different eicosanoids have been identified, with many having potent bioactive signaling capacity. These lipid metabolites are synthesized de novo by at least 50 unique enzymes, many of which have been cloned and characterized. Due to the extensive characterization of eicosanoid biosynthetic pathways, this field provides a unique framework for integrating genomics, proteomics, and metabolomics toward the investigation of disease pathology. To facilitate a concerted systems biology approach, this review outlines the proteins implicated in eicosanoid biosynthesis and signaling in human, mouse, and rat. Applications of the extensive genomic and lipidomic research to date illustrate the questions in eicosanoid signaling that could be uniquely addressed by a thorough analysis of the entire eicosanoid proteome.

BACKGROUND Increased peritoneal eicosanoid concentrations have been reported in endometriosis patients and might be important in disease-associated pain and inflammation. Here, we evaluated the expression of key biosynthetic and catabolic enzymes involved in this abnormal eicosanoid production in peritoneal macrophages and endometriotic lesions. METHODS Peritoneal macrophages, endometriotic lesions and matched eutopic endometrium were collected from endometriosis patients (n = 40). Peritoneal macrophages and eutopic endometrium samples were also collected from disease-free women (n = 25). Expression of type IIA secretory phospholipase A(2) (sPLA(2)-IIA), cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 5-lipoxygenase (5-LO) was quantified by real-time PCR, and these five key enzymes were localized by immunohistochemistry. RESULTS sPLA(2)-IIA, COX-2 and mPGES-1 mRNA was significantly increased in peritoneal macrophages of endometriosis patients compared with controls (P = 0.006, P = 0.016 and P = 0.025, respectively). In endometriosis patients, sPLA(2)-IIA, mPGES-1 and 15-PGDH mRNA was significantly enhanced in peritoneal lesions compared with matched eutopic endometrium (P < 0.001, P < 0.001 and P = 0.005, respectively). In eutopic endometrium, a significant decrease in 15-PGDH mRNA was found in the endometriosis group compared with controls (P = 0.023). Finally, sPLA(2)-IIA, COX-2, mPGES-1 and 15-PGDH immunostaining was found mainly in endometrial glands, whereas 5-LO was distributed throughout the glands and stroma. CONCLUSIONS Our study highlights an imbalance between eicosanoid biosynthesis and degradation in endometriosis patients. Both peritoneal macrophages and endometriotic lesions may be involved. Research into new molecules inhibiting biosynthetic enzymes (such as sPLA(2)-IIA and mPGES-1) and/or activating catabolic enzymes (such as 15-PGDH) may prove to be a major field of inves

Colorectal cancer is the third most common type of cancer, and the second leading cause of cancer-related deaths in the Western world. Chronic inflammation, such as inflammatory bowel diseases (IBD), increases the risk of cancer. Cysteinyl leukotrienes are pro-inflammatory mediators that are involved in IBD. They are ligands to the G-protein coupled receptors CysLT1R and CysLT2R, of which CysLT1R is associated with poor prognosis and CysLT2R with good prognosis for colorectal cancer patients. Signaling through CysLT1R results in increased proliferation, survival and migration of intestinal epithelial cells, whereas signaling through CysLT2R leads to differentiation of colorectal cancer cells. My main findings have been that 1) Leukotriene D4 (LTD4) induces intestinal epithelial cell proliferation by activating the mitogenic JNK / AP-1 pathway; 2) The mitogen EGF, involved in cancer progression, down-regulates CysLT2R expression and is inversely correlated with CysLT2R in a colorectal cancer tissue array, while 3) the anti-tumorigenic agents all-trans retinoic acid and interferon alpha up-regulate CysLT2R expression in colorectal cancer cells; 4) LTC4, a ligand for CysLT2R, inhibits migration of colorectal cancer cells; 5) Intestinal epithelial and colorectal cancer cells express enzymes and receptors of the cysteinyl leukotriene and prostaglandin pathways; and 6) The pro-tumorigenic cytokine TNF-alpha up-regulates CysLT1R while down-regulating CysLT2R. These studies provide more evidence that CysLT1R and CysLT2R have opposite functions in colon cancer progression, and we now know how to regulate their expression. This thesis provides a basic understanding on the role and regulation of the cysteinyl leukotriene receptors, which is useful for designing future therapies of colorectal cancer.

Background: We have produced hypertrophic and metaplastic changes of goblet cells in nasal epithelium of ovalbumin (OVA)-sensitized rats by intranasal challenge with OVA. A variety of allergic mediators and inflammatory cells are capable of stimulating goblet cell degranulation (epithelial mucus secretion); however, little is known about the mechanism by which antigen induces mucus hypersecretion. Objective: Our aim was to explain the mechanism of goblet cell degranulation in allergic inflammation. Methods: Antigen-induced goblet cell degranulation was evaluated by the transient decrease of epithelial mucosubstance 1 to 6 hours after intranasal challenge with OVA in nasal epithelium of OVA-sensitized rats. The effects of the H1-antagonist (d -chlorpheniramine malate), H2-antagonist (cimetidine), atropine, indomethacin, cysteinyl leukotriene (cysLT) antagonist (ONO1078), and antirat PMN antiserum on OVA-induced goblet cell degranulation were examined. Results: Goblet cell secretion 1 hour after OVA challenge was significantly inhibited by H1-antagonist, atropine, and cysLT antagonist, whereas the secretion 6 hours after the challenge was significantly inhibited by cysLT antagonist and antirat PMN antiserum. Circulating PMN cells and mucosal infiltrating eosinophils were completely abolished by antirat PMN antiserum. Conclusions: These results indicate the different mechanisms of goblet cell secretion between early-phase (1 hour after OVA challenge) and late-phase (6 hours after the challenge) reactions. Histamine stimulates early-phase secretion through the H1-receptor of cholinergic nerve terminals, and infiltrating inflammatory cells (eosinophils and/or neutrophils) play a role in late-phase secretion. CysLTs (leukotrienes C4, D4, and E4) are important for both early-phase and late-phase secretion.

5-Oxo-ETE ist ein chemotaktischer Faktor für Granulozyten, der von der NADP+-abhängigen Dehydrogenase 5h-dh aus dem 5-Lipoxygenaseprodukt 5-HETE gebildet wird. Ziel dieser dreiteiligen Studie war es, die der 5-oxo-ETE-Produktion zugrunde liegenden Regulationsmechanismen aufzuklären. I. Einfluß von myeloider Zelldifferenzierung auf die Expression von 5h-dh in HL-60 und U-937 Zellen. Undifferenzierte HL-60 und U-937 Zellen produzieren vergleichbare Mengen von 5-oxo-ETE wie Monozyten oder Granulozyten. Differenzierung von U-937 Zellen mit PMA verdreifacht die Enzymaktivtät von 5h-dh, während die Behandlung von HL-60 Zellen mit dh-VitD3 diese verdoppelte. Der Einfluß von PMA auf 5h-dh wurde darüber hinaus in Mikrosomen von U-937 Zellen untersucht. Die Behandlung PMA verdreifachte Vmax, liess aber KM unbeeinflußt. II. Regulation der 5-oxo-ETE-Produktion durch oxidativen Stress und Glukose. Da der GSH-Redoxzyklus die Produktion von NADP+ zur Folge hat, stimulierten die Hydroperoxide H2O2 und tBOOH die Synthese von 5-oxo-ETE in U-937 Zellen. Aufgrund seiner Verarbeitung durch den Pentosephosphat Zyklus, der NAD+ in NADPH umwandelt, inhibierte Glucose diesen Effekt von H2O2. Die Synthese von 5-oxo-ETE wurde durch H2O2 auch in humanem Monozyten, Lymphocyten und Thrombozyten, aber nicht in Neutrophilen angeregt. Im Gegensatz zu Monozyten zeigten sich Thrombozyten und Lymphozyten allerdings glukose-resistent. T-BOOH ehöhte auch die Produktion von 5-oxo-ETE nach Zugabe von Ionophore und Arachidonsäure zu mononukleären Blutzellen. III. 5h-dh-Expression in human Strukturzellen. Zunächst rasterten wir mehrere sekundäre Epithelzelllinien und fanden 5h-dh in allen Zellen. Drei Indizien lassen vermuten, daß die epithele 5h-dh der myeloiden entspricht: (i) die enzymatische Aktivtät liegt vor allem in der mikrosomalen Fraktion vor, (ii) bei dem Kofaktor handelt es sich um NADP+ und nicht um NAD+, und (iii) 5S-HETE ist das bevorzugte Substrat. Weitere Studien zeigten, daß auch primäre h, 5-Oxo-ETE is a highly potent granulocyte chemoattractant that is formed by the NADP+-dependent dehydrogenase 5h-dh by oxidation of the 5-lipoxygenase product 5-HETE. The objective of this study was to investigate underlying regulatory mechanisms of 5-oxo-ETE production in human cells. This matter was addressed from three directions. I. Expression of 5h-dh in HL-60 and U-937 cells and its activity changes during myeloid cell differentiation. Undifferentiated U-937 and HL-60 cells produce similar amounts of 5-oxo-ETE compared to monocytes or neutrophils. Differentiation of U-937 cells with PMA resulted in a 3-fold increase in 5-oxo-ETE production. Similarly, incubation of HL-60 cells with dh-VitD3 induced a 2-fold increase in 5-oxo-ETE production. The impact of PMA on 5h-dh was also investigated in the microsomal fraction of U-937 cells and compared to neutrophil microsomes. PMA treatment leads to a increase of Vmax but does not affect KM. II. Regulation of 5-oxo-ETE by oxidative stress and glucose levels. We found that H2O2 and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U-937 cells through the GSH redox cycle by providing NADP+. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, which converts NADP+ back to NADPH. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-BOOH. III. Expression of 5h-dh in human structural cells. We screened several secondary epithelial cell lines and detected 5h-dh in all cell lines. Epithelial 5h-dh and the inflammatory cell 5h-dh are identical: (i) the enzymatic activity is localized in microsomes, (ii) the cofactor is NADP+, and (iii) 5S

The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 µmol·min−1·mg−1 in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequenc

IL-13 ist ein Polypeptidhormon, welches von aktivierten TH2-Helferlymphozyten gebildet wird. Es besitzt eine Reihe immunmodulierender Eigenschaften auf B-Lymphozyten, Makrophagen, Monozyten, PMN und Endothelzellen. Es tritt neben anderen anti-inflammatorischen Zytokinen wie dem IL-10 und IL-4 während der immunparalytischen Phase der Sepsis auf. IL-13 wird in die Gruppe der anti-inflammatorischen Zytokine eingeordnet, weil es in vitro die Eigenschaft besitzt, die Produktion von pro-inflammatorischen Zytokinen wie TNFα, IL-1, IL-6 sowie IL-8 zu unterdrücken. Wenige und teilweise widersprüchlich erscheinende Informationen sind bisher über die direkte und die primende Wirkung von IL-13 auf PMN bekannt. In dieser Arbeit wurde experimentell unter inflammatorischen Bedingungen die Wirkung von IL-13 auf den granulozytären Oxidativen Burst, die Degranulationsreaktion sowie den Lipidmediatorstoffwechsel untersucht. Durch LPS ließ sich der fmlp-induzierte Oxidative Burst steigern. Eine weitere Steigerung war durch die Zugabe von Serum zum Inkubationspuffer möglich. Die zusätzliche Inkubation der PMN mit IL-13 führte tendenziell zu einer weiteren Verstärkung des Oxidativen Burstes. Der Einsatz des PKC Inhibitors Bisindolylmaleimide führte sowohl in der Versuchsreihe mit IL-13 als auch ohne IL-13 zu einer Hemmung des Oxidativen Burstes. Nach der Inkubation mit IL-13 zeigte sich, daß im Vergleich eine höhere IC50 nötig war. Diese Beobachtung läßt die Schlußfolgerung zu, daß IL-13 auf einer Stufe der Signaltransduktionskette bis hin zur PKC Einfluß auf deren Aktivierung nimmt. Weiterhin konnten wir zeigen, daß IL-13 zu einer Hemmung der Degranulationsreaktion fähig ist. IL-13 Konzentrationen bis einschließlich 1ng/ml führten zu einer dosisabhängigen Hemmung der Elastase-Ausschüttung, die bei ansteigenden IL-13 Konzentrationen wieder auf das Niveau des Referenzwertes zurückkehrte. Eine Erklärung für diesen Sachverhalt könnte die vermehrte Bildung von Sauerstoffradikalen bei hö, IL-13 is a polypeptide hormone which derives from activated TH2-lymphocytes. B-lymphocytes, macrophages, monocytes, PMN and endothelial cells respond to its range of immunomodulating properties. IL-13 appears during the immunoparalytic phase of sepsis, along with other anti-inflammatory cytokines such as IL-10 and IL-4. It is placed in the group of anti-inflammatory cytokines as it has the in vitro property of suppressing the production of pro-inflammatory cytokines such as TNFα, IL-1, IL-6 and IL-8. At present, little and seemingly partially contradictory information is known about the direct and the priming effect of IL-13 on PMN. In this experimental research, the effect of IL-13 under inflammatory conditions on the PMN oxidative burst, degranulation, as well as the lipid mediator metabolism were examined. The fmlp-triggered oxidative burst was enhanced by LPS. It was possible to further enhance the LPS response through the addition of serum to the incubation buffer. Additional incubation of PMN with IL-13 tended to further amplify the oxidative burst. The use of PKC inhibitor bisindolymaleimide in a series of experiments, both with and without IL-13, led to the inhibition of the oxidative burst. In the series with IL-13, a higher IC50 was necessary to inhibit the oxidative burst in comparison to the series without. This observation led to the conclusion that IL-13 influences the activation of PKC during stages in the signal transduction chain. Furthermore we were able to show that IL-13 is capable of inhibiting degranulation. IL-13 concentrations up to and including 1ng/ml led to a dose-dependent inhibition of the elatase release which, with increasing IL-13 concentrations, returned once again to the level of reference. An explanation for this fact could be the increased formation of oxygen radicals with higher IL-13 concentrations which influences degranulation through autocrine and paracrine mechanisms. The examined effects of IL-13 on the lipid mediator

We investigated the signal transduction pathway(s) of leukotriene D(4) (LTD(4)) in the human promonocytic U937 cells, a cell line known to constitutively express CysLT(1) receptors. Herein, we demonstrate that LTD(4) specifically acts on a CysLT(1) receptor to dose-dependently increase (three to five-fold over basal) RasGTP through a G(i/o) protein. In fact, while cytosolic Ca(2+) ([Ca(2+)](i)) increase was only partially sensitive to pertussis toxin (PTx), Ras activation was almost completely inhibited by the same toxin. Furthermore, the phospholipase C (PLC) inhibitor U73122 completely inhibited both [Ca(2+)](i) and RasGTP increase, suggesting that in these cells PLC is the point of convergence for both PTx insensitive and sensitive pathways leading to [Ca(2+)](i) release and Ras activation. Indeed, chelating intracellular Ca(2+) strongly (>70%) prevented LTD(4)-induced Ras activation, indicating that this ion plays an essential role for CysLT(1)-induced downstream signaling in differentiated U937 (dU937) cells. In addition, while Src did not appear to be substantially involved in CysLT(1)-induced signaling, genistein was able to partially inhibit LTD(4)-induced [Ca(2+)](i) transient ( approximately 34%) and almost completely prevented Ras activation (>90%), suggesting a potential role for other Ca(2+)-dependent tyrosine kinases in LTD(4)-induced signaling. Finally, agonist-induced CysLT(1) stimulation was followed by a specific extracellular regulated kinase (ERK) 1/2 phosphorylation, an event with a pharmacological profile similar to that of Ras activation, partially ( approximately 40%) sensitive to Clostridium sordellii lethal toxin and totally blocked by PTx. In conclusion, LTD(4)-induced CysLT(1) receptor activation in dU937 cells leads to Ras activation and ERK phosphorylation mostly through a PTx-sensitive G(i/o) protein, PLC, and Ca(2+)-dependent tyrosine kinase(s).

The synthesis of leukotriene B4 by A23187-stimulated rat peritoneal leukocytes was studied in the presence of 0.1% normal human serum, serum from patients treated with NSAIDs for either an inflammatory (rheumatoid arthritis, RA) or a non-inflammatory condition (lumbar disc protrusion, LDP), and serum from RA patients drawn one week after withdrawal from NSAID treatment. The capacity for LTB4 synthesis was significantly lower in the presence of serum from NSAID treated patients: thirty per cent less than observed in presence of normal serum in the RA group, and fifty per cent in the LDP group. When NSAIDs were withdrawn from RA patients, the LTB4 production in presence of serum increased, but was not completely normalized after one week. These results indicate that NSAID treatment may down-regulate the capacity for leukotriene synthesis by an indirect effect.

Previous studies have indicated that thromboxane (Tx) and leukotriene (LT) B sub 4 act synergistically to induce neutrophil (PMN) adhesion in the microvasculature. This study tests the ability of Tx to induce LTB sub 4 synthesis which then leads to activation of PMN and endothelial adhesion receptors. Tx-mimic (U 46619, 1 micrograms/ml) was administered into abraded skin chambers placed on the back of rabbits (n=6). After 3 h LTB sub 4 was synthesized in the blister fluid, 385 pg/ml, a value higher than levels in saline treated blisters 10 pg/ml (p<0.05). The LTB sub 4 generation following Tx-mimic was correlated (p<0.05, r=0.70) with neutrophil diapedesis. These averaged 645 PMN/mm(exp 3), higher than saline values of 20 PMN/mm(exp 3) (p<0.05). Intravenous (IV) treatment of other rabbits (n=4) with the lipoxygenase inhibitor diethylcarbamazine 60 mg/kg followed by 40 mg/kg/h prevented Tx-mimic induced LTB sub 4 synthesis (10 pg/ml) and diapedesis (19 PMN/mm(exp 3) (both p<0.05). IV treatment of yet other rabbits (n=4) with the anti-CD 18 monoclonal antibody R 15.7, 1 mg/kg abolished Tx-induced diapedesis (3 PMN/mm(exp 3)) (p<0.05). In contrast, local administration of the protein synthesis inhibitor actinomycin D 3 ng, to prevent expression of endothelial adhesion proteins, limited TNF but not Tx-induced diapedesis. The data indicate that Tx-induced diapedesis is mediated by the generation of LTB sub 4 and activation of neutrophil CD 18 but not endothelial adhesion receptors.

Background: Polymorphisms spanning genes involved in the production of leukotriene B4 (LTB4) e.g. ALOX5AP and LTA4H are associated with asthma susceptibility, suggesting a role for LTB4 in disease. The contribution of LTB4 receptor polymorphism is currently unknown. The aim of this study was to characterise the genes for the two pivotal LTB4 receptors, LTB4R1 and LTB4R2 in lung tissue and determine if polymorphisms spanning these genes are associated with asthma and disease severity. Methods: Rapid amplification of cDNA ends (RACE) was used to characterise the LTB4R1 and LTB4R2 gene structure in lung. The LTB4R1/2 locus on chromosome 14q11.2 was screened for polymorphic variation. Six LTB4R single nucleotide polymorphisms (SNPs) were genotyped in 370 Caucasian asthma families and 299 Adult Asthma Individuals (n=1877 total) and were evaluated for association with asthma and severity (BTS) outcome measures using Family Based Association Test, linear regression and chi square. Results: LTB4R1 has complex mRNA arrangement including multiple 50 -untranslated exons, suggesting additional levels of regulation. Three potential promoter regions across the LTB4R1/2 locus were identified with some airway cell specificity. 22 SNPs (MAF>0.01) were validated across the LTB4R locus in the Caucasian population. LTB4R1 and LTB4R2 SNPs were not associated with asthma susceptibility, FEV1 or severity. Conclusions: LTB4R1 and LTB4R2 shows splice variation in the 50 -untranslated region and multiple promoter regions. The functional significance of this is yet to be determined. Both receptor genes were shown to be polymorphic. LTB4R polymorphisms do not appear to be susceptibility markers for the development of asthma in Caucasian subjects

Leukotrienes (LT) are potent pro-inflammatory mediators formed from arachidonic acid (AA) in reactions catalyzed by 5-lipoxygenase and either leukotriene A4 hydrolase or leukotriene C4 synthase. 5-lipoxygenase activating protein (FLAP) is also required. We have previously reported expression of FLAP in the hematopoietic compartment of the fetal liver raising questions regarding the role of leukotrienes in hematopoietic regulation. Here we report evidence from in situ hybridization, immunohistochemistry and qRT-PCR experiments that the complete LT biosynthesis machinery is abundantly expressed in hematopoietic cells of the fetal mouse liver from e11.5 until birth. FACS sorting of hematopoietic cells from e15.5 liver and adult bone marrow into different subpopulations followed by quantitative RT-PCR analysis showed that expression was confined mainly to myeloid cells but also detected in hematopoietic stem and progenitor cells. Analysis of FLAP knockout mice showed that a lack of this gene abolished LT and reduced 5(S)- hydroxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid (HETE) production. Furthermore, decreased relative numbers of B-lymphocytes and increased numbers of T-lymphocytes were observed in peripheral blood and increased numbers of common lymphoid progenitor cells were observed in BM. Taken together these findings suggest that production of LTs can occur in cells of the fetal and adult hematopoietic compartments and that deficiency of the FLAP gene (and leukotrienes) may affect lymphocyte maturation.