Leukotriene B4 Research Articles

Calcium-dependent cytosolic phospholipase A2 activation is implicated in neuroinflammation and oxidative stress associated with ApoE4

BackgroundApolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer’s disease (AD), but the mechanisms for this association are not clear. The activation of calcium-dependent cytosolic phospholipase A2 (cPLA2) is involved in inflammatory signaling and is elevated within the plaques of AD brains. The relation between APOE4 genotype and cPLA2 activity is not known.MethodsMouse primary astrocytes, mouse and human brain samples differing by APOE genotypes were collected for measuring cPLA2 expression, phosphorylation, and activity in relation to measures of inflammation and oxidative stress.ResultsGreater cPLA2 phosphorylation, cPLA2 activity and leukotriene B4 (LTB4) levels were identified in ApoE4 compared to ApoE3 in primary astrocytes, brains of ApoE-targeted replacement (ApoE-TR) mice, and in human brain homogenates from the inferior frontal cortex of persons with AD dementia carrying APOE3/4 compared to APOE3/3. Higher phosphorylated p38 MAPK but not ERK1/2 was found in ApoE4 primary astrocytes and mouse brains than that in ApoE3. Greater cPLA2 translocation to cytosol was observed in human postmortem frontal cortical synaptosomes with recombinant ApoE4 than ApoE3 ex vivo. In ApoE4 astrocytes, the greater levels of LTB4, reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) were reduced after cPLA2 inhibition.ConclusionsOur findings implicate greater activation of cPLA2 signaling system with APOE4, which could represent a potential drug target for mitigating the increased neuroinflammation with APOE4 and AD.

Toxoplasma gondii Infection Decreases Intestinal 5-Lipoxygenase Expression, while Exogenous LTB4 Controls Parasite Growth.

5-Lipoxygenase (5-LO) is an enzyme required for the production of leukotrienes and lipoxins and interferes with parasitic infections. In vitro, Toxoplasma gondii inhibits leukotriene B4 (LTB4) production, and mice deficient in 5-LO are highly susceptible to infection. The aim of this study was to investigate the effects of the pharmacological inhibition of the 5-LO pathway and exogenous LTB4 supplementation during experimental toxoplasmosis. For this purpose, susceptible C57BL/6 mice were orally infected with T. gondii and treated with LTB4 or MK886 (a selective leukotriene inhibitor through inhibition of 5-LO-activating protein [FLAP]). The parasitism, histology, and immunological parameters were analyzed. The infection decreased 5-LO expression in the small intestine, and treatment with MK886 reinforced this reduction during infection; in addition, MK886-treated infected mice presented higher intestinal parasitism, which was associated with lower local interleukin-6 (IL-6), interferon gamma (IFN-γ), and tumor necrosis factor (TNF) production. In contrast, treatment with LTB4 controlled parasite replication in the small intestine, liver, and lung and decreased pulmonary pathology. Interestingly, treatment with LTB4 also preserved the number of Paneth cells and increased α-defensins expression and IgA levels in the small intestine of infected mice. Altogether, these data demonstrated that T. gondii infection is associated with a decrease in 5-LO expression, and on the other hand, treatment with the 5-LO pathway product LTB4 resulted in better control of parasite growth in the organs, adding to the knowledge about the pathogenesis of T. gondii infection.

Silicone hydrogel contact lenses retain and document ocular surface lipid mediator profiles

ABSTRACT Clinical relevance A leading reason for patients to abandon their contact lenses is discomfort. Mechanisms and biomarkers for lens discomfort remain to be elucidated. Background Physical stress and tear film interaction are likely factors for lens discomfort. Lipid mediators are generated from polyunsaturated fatty acids. They regulate ocular surface physiology and pathophysiology, are constituents of human tears and may interact with contact lenses. This study set out to determine if hydrogel lenses and silicone hydrogel lenses interact with tear film polyunsaturated fatty acids and polyunsaturated fatty acids-derived mediators. Methods In vitro incubations, rat experiments and analysis of worn human lenses assessed polyunsaturated fatty acids and lipid mediator interactions with lenses. Silicone hydrogel and hydrogel lenses were incubated with lipid mediators and polyunsaturated fatty acids up to 24 hours. Rats were fitted with custom silicone hydrogel lenses and basal tears collected. Silicone hydrogel lenses worn for 2 weeks were obtained from 57 human subjects. Tear and lens lipidomes were quantified by mass spectrometry. Results Silicone hydrogel lenses retained polyunsaturated fatty acids and lipid mediators within 15 minutes in vitro. Lenses contained 90% of total polyunsaturated fatty acids and 83–89% of total monohydroxy fatty acids by 12 hours. Retention correlated with polarity of lipid mediators and lipophilic properties of silicone hydrogel lenses. Polyunsaturated fatty acids and lipid mediators such as lipoxygenase- and cyclooxygenase-derived eicosanoids were present in tears and worn lenses from rats. Worn silicone hydrogel lenses from human subjects established robust and lens-type specific lipidomes with high levels of polyunsaturated fatty acids, lipoxygenase-pathway markers and subject-specific differences in lipoxin A4 and leukotriene B4. Conclusion Worn silicone hydrogel lenses rapidly retain and accumulate tear polyunsaturated fatty acids and lipid mediators. Marked subject and lens type differences in the lipidome may document changes in ocular surface physiology, cell activation or infection that are associated with lens wear. If contact lens discomfort and adverse events induce specific tear and lens fatty acid and lipid mediator profiles warrants further studies.

Dietary supplements in lymphedema.

Lymphedema is a chronic inflammatory disorder resulting from ineffective fluid uptake by the lymphatic system, and the effects are principally felt in the lower limbs. The condition is said to be primary when caused by genetic mutations and secondary when caused by injuries, infections, or surgery. Lymphedema, a worldwide pathology, does not have an effective therapy so far. Leukotriene B4 has recently been identified as a key molecule in lymphedema pathogenesis. Surgical, nonsurgical, and pharmacological treatments have been proposed; however, they do not cure the disease and only ameliorate the symptoms. Nutrition and nutritional status are extremely important in lymphedema physiopathology. Obesity is a comorbidity that exacerbates the risk for secondary lymphedema and constitutes a negative prognostic factor. Indeed, anti-inflammatory foods and their effects on the inflammatory state and on oxidative stress are now being investigated for their possible therapeutic role in lymphedema. Although no special diet has so far been proven to be very effective, specific dietary tips could help in alleviating the edematous state of patients with lymphedema. A few supplements have been tested for lymphedema treatment. Among them, GARLIVE® containing hydroxytyrosol, hesperidin, spermidine and vitamin A, exhibited promising effects in the animal model. Hydroxytyrosol, a polyphenol from olives, showed anti-inflammatory effects and reduced leukotriene B4 synthesis, thus holding promise as a potential natural candidate for lymphedema treatment.

Validating methods for testing natural molecules on molecular pathways of interest in silico and in vitro.

Differentially expressed genes can serve as drug targets and are used to predict drug response and disease progression. In silico drug analysis based on the expression of these genetic biomarkers allows the detection of putative therapeutic agents, which could be used to reverse a pathological gene expression signature. Indeed, a set of bioinformatics tools can increase the accuracy of drug discovery, helping in biomarker identification. Once a drug target is identified, in vitro cell line models of disease are used to evaluate and validate the therapeutic potential of putative drugs and novel natural molecules. This study describes the development of efficacious PCR primers that can be used to identify gene expression of specific genetic pathways, which can lead to the identification of natural molecules as therapeutic agents in specific molecular pathways. For this study, genes involved in health conditions and processes were considered. In particular, the expression of genes involved in obesity, xenobiotics metabolism, endocannabinoid pathway, leukotriene B4 metabolism and signaling, inflammation, endocytosis, hypoxia, lifespan, and neurotrophins were evaluated. Exploiting the expression of specific genes in different cell lines can be useful in in vitro to evaluate the therapeutic effects of small natural molecules.

Respiratory Syncytial Virus Nonstructural Protein 1 Promotes 5-Lipoxygenase via miR-19a-3p.

Background Respiratory syncytial virus (RSV) infection can regulate the expression of a wide range of noncoding microRNAs (miRNAs), in which mir-19a-3p can participate in airway inflammatory response by regulating 5-lipoxygenase (5-LO) pathway. RSV nonstructural protein (NS) 1 is involved in the airway hyperresponsiveness during RSV infection. Methods The expression levels of miR-19a-3p and inflammatory signaling-related indicators were detected using quantitative real-time PCR and western blot analyses on the A549 cells transfected with NS1 expression plasmids (pNS1). The 5-LO-mediated inflammatory signaling pathway was assessed when the miR-19a-3p or 5-LO was inhibited. Results The immunofluorescence analysis showed that the plasmid-mediated NS1 protein was observed in both the cytoplasm and nucleus. The expression level of miR-19a-3p was significantly upregulated in the pNS1 or RSV-treated cells, which was reversed by the NS1 small interfering RNA. In addition, pNS1 also upregulated the expression of 5-LO, interleukin-5 (IL-5), and leukotriene B4 (LTB4), which was also significantly inhibited by the miR-19a-3p antagonists. The 5-LO inhibitor MK886 prevented the increase in the expression level of IL-5 induced by pNS1. Conclusions These results suggested that the RSV NS1 might play an important role in the pathogenesis of RSV by activating the 5-LO and subsequent inflammatory cytokines through miR-19a-3p.

Baseline Elevations of Leukotriene Metabolites and Altered Plasmalogens Are Prognostic Biomarkers of Plaque Progression in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is associated with an increased incidence of acute and chronic cardiovascular disease as compared to the general population. This study uses a comprehensive metabolomic screen of baseline sera from lupus patients to identify metabolites that predict future carotid plaque progression, following 8–9 years of follow-up. Nine patients had SLE without plaque progression, 8 had SLE and went on to develop atherosclerotic plaques (SLEPP), and 8 patients were controls who did not have SLE. The arachidonic acid pathway metabolites, leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE), and the oxidized lipids 9/13-hydroxyoctodecadienoic acid (HODE) were found to be significantly altered (p < 0.05 and fold-change >2) in SLEPP patients compared to SLE patients without plaque progression. SLEPP patients also exhibited significantly altered levels of branched chain amino acid (BCAA) metabolites and plasmalogens compared to the non-SLE controls. Taken together with the rich literature on these metabolites, these findings suggest that the identified metabolites may not only be prognostic of cardiovascular disease development in SLE patients, but they may also be active drivers of atheroma formation. Early identification of these high risk SLE patients may help institute preventive measures early in the disease course.

α-lipoic acid ameliorates inflammation state and oxidative stress by reducing the content of bioactive lipid derivatives in the left ventricle of rats fed a high-fat diet

Lipid mediators derived from arachidonic acid (AA) are implicated with the occurrence of inflammation and oxidative stress. The current knowledge of AA metabolism focuses on searching for the therapeutic strategy to subvert affected AA metabolism. The aim of our study was to evaluate the potential protective effect of chronic α-lipoic acid (α-LA) supplementation on myocardial inflammation state and oxidative stress in obesity-related cardiovascular dysfunction. The experiment was carried out on male Wistar rats receiving a standard or a high-fat diets with intragastric α-LA administration for 8 weeks. Plasma and myocardial AA concentrations were determined using the gas-liquid chromatography (GLC). The Western blot technique was used to examine the expression of proteins from the inflammatory pathway. The content of selected cytokines, inflammatory mediators, and oxidative stress indicators was detected by the ELISA, colorimetric, and multiplex assay kits. Our results revealed that α-LA caused a notable reduction in AA content, mainly in the phospholipid fraction with a simultaneous diminishment in the synthesis of pro-inflammatory mediators, i.e., prostaglandin E2, leukotrienes B4 and C4 by decreasing the expression of COX-2 and 5-LOX. α-LA also augmented the level of antioxidative SOD2 and GSH and decreased the level of lipid peroxidation products, which improved oxidative system impairment in the left ventricle tissue. The data clearly showed that α-lipoic acid has a significant role in inflammation and oxidative stress development ameliorating the risk of cardiac obesity induced by high-fat feeding.

Modeling of cancer-related body-wide effects identifies LTB4 as a diagnostic biomarker for pancreatic cancer.

SummaryBackgroundCancer elicits a complex adaptive response in an organism. Limited information is available for the body-wide effects induced by cancer. Here, we evaluated multiorgan changes in mouse models of pancreatic ductal adenocarcinoma (PDAC) and its precursor lesions (pancreatic intraepithelial neoplasia, PanIN) to decipher changes that occur during PDAC development.MethodsRNA-sequencing was employed in the brain, colon, stomach, kidney, heart, liver, and lung tissues of mice with PanIN and PDAC. A combination of differential expression analysis and functional-category enrichment was applied for an in-depth understanding of the multiorgan transcriptome. Differentially expressed genes were verified by quantitative real-time polymerase chain reaction. Neutrophil and macrophage infiltration in multiple organs was analyzed by immunohistochemical staining. Leukotriene B4 (LTB4) levels in mouse and human serum samples were determined by enzyme-linked immunosorbent assay.FindingsTranscriptional changes within diverse organs during PanIN and PDAC stages were identified. Using Gene Ontology enrichment analysis, increased neutrophil infiltration was discovered as a central and prominent affected feature, which occurred in the liver, lung, and stomach at the PanIN stage. The brain appeared to be well protected from the sequels of PanIN or PDAC. Importantly, serum LTB4 was able to discriminate PDAC from normal controls, chronic pancreatitis, and intraductal papillary mucinous neoplasms with high performance.InterpretationOur study provides a high-resolution cartographic view of the dynamic multiorgan transcriptomic landscape of mice with PDAC and its precursor lesions. Our findings suggest that LTB4 could serve as a biomarker for the early detection of PDAC.FundingThe complete list of funders can be found in the Acknowledgement section.

Evaluation of Nomacopan for Treatment of Bullous Pemphigoid

Bullous pemphigoid is a difficult-to-treat autoimmune blistering skin disease that predominantly affects older adults and is associated with an increased mortality rate. To examine the safety and therapeutic potential of nomacopan, an inhibitor of leukotriene B4 and complement C5, in patients with bullous pemphigoid. This multicenter, single-group, phase 2a nonrandomized controlled trial was conducted in the dermatology departments of universities in the Netherlands and Germany. Participants were enrolled between September 2018 and April 2020. Older adult patients (aged ≥55 years) with mild to moderate, new-onset or relapsing bullous pemphigoid were recruited into the study. Patients received nomacopan, 90 mg, subcutaneously on day 1 and 30 mg subcutaneously daily until day 42. The primary end point was the proportion of patients with grade 3 to 5 (severe) adverse events associated or possibly associated with nomacopan. Secondary end points included mean absolute and percentage changes in the Bullous Pemphigoid Disease Area Index (BPDAI) activity score, the BPDAI pruritus score, and the patient-reported outcome measures Dermatology Life Quality Index (DLQI) and Treatment of Autoimmune Bullous Disease Quality of Life (TABQOL). A total of 9 patients (median [range] age, 75 [55-85] years) with bullous pemphigoid were included in the trial, of whom 5 were women (55.6%). No serious adverse events associated with nomacopan were found. The mean (90% CI) BPDAI activity score decreased from 32.0 (8.7) points on day 1 to 19.6 (9.0) points on day 42. Seven of 9 patients (77.8%) responded to nomacopan with a reduction in the BPDAI activity score of at least 8 points between days 1 and 42; in 3 responders, the reduction was 80% or greater. On day 42, the mean (90% CI) BPDAI pruritus score had decreased by 6.8 (4.6) points from 17.6 (4.0) points on day 1. The mean (90% CI) DLQI score decreased from 11.3 (4.2) points at baseline to 6.4 (3.8) points by day 42, and the mean (90% CI) TABQOL score decreased from 14.6 (5.4) points at baseline to 10.3 (5.0) points on day 42. Results of this nonrandomized controlled trial suggest that nomacopan can be well tolerated in older patients with bullous pemphigoid and may have therapeutic benefits for suppressing acute flares of this disease. A larger, placebo-controlled randomized clinical trial is warranted to confirm this safety profile and to establish nomacopan as a new therapeutic option for bullous pemphigoid. ClinicalTrials.gov Identifier: NCT04035733.

Exosomes as Key Regulators of Neutrophil Chemotaxis

Neutrophils, the most abundant leukocytes in human blood, are the first line of defense at sites of injury or infection. Upon activation by primary chemoattractants such as the formyl peptide N‐Formyl‐methionine‐leucyl‐phenylalanine (fMLF), neutrophils secrete the secondary chemoattractant leukotriene B4 (LTB4) which serves to relay primary chemotactic signals and increase the range and persistence of migration. LTB4 is synthesized from arachidonic acid (AA) through the sequential action of the 5‐lipoxygenase (5‐LO), its associated activating protein (FLAP) and leukotriene A4 hydrolase (LTA4H). We previously demonstrated that LTB4 and its synthesizing enzymes are packaged and released in extracellular vesicles called exosomes. Conventional exosome biogenesis originates from the plasma membrane. However, the LTB4 synthesizing machinery resides on the nuclear envelope (NE). We found that the localized activation of membrane curvature inducing proteins facilitates the selective sorting of the LTB4‐synthesis machinery on the NE and its packaging within NE‐derived exosomes. We show that chemoattractant stimulation leads to the generation of 5‐LO‐ and FLAP‐positive NE buds and cytosolic vesicles, which are also positive for the inner nuclear membrane protein lamin B receptor (LBR). Furthermore, we found that the generation of ceramide from sphingomyelin through the action of the neutral sphingomyelinase 1 (nSMase1) is essential for the generation of these NE buds and cytosolic vesicles by generating ordered lipid microdomains on the NE. Together, our findings show that the spatiotemporal orchestration of chemoattractant‐initiated events leads to the recruitment of the LTB4 biogenesis machinery on the NE followed by NE‐budding and subsequent release of cytosolic vesicles containing exosomes. We envision that this novel mechanism will represent an important means of exosome biogenesis for the release of NE‐associated components in other cells, including immune cells and invasive cancer cells.

Isoflurane targets BLT1 to attenuate lung injury

Abstract Sepsis-associated lung injury is one of major comorbidities in sepsis, largely responsible for its mortality. Lack of specific therapeutic for lung injury is an urgent issue to be addressed. We found that volatile anesthetic isoflurane attenuated lung injury associated with experimental polymicrobial abdominal sepsis. This was associated with a reduction in the number of recruited neutrophils in the lung and bronchoalveolar space. Furthermore, isoflurane attenuated neutrophil chemotaxis induced by major chemoattractant leukotriene B4 (LTB4), not by C5a. Although LTB4 level did not differ by isoflurane exposure, isoflurane was shown to interact with LTB4’s neutrophil receptor BLT1 in silico. The interaction site was predicted to overlap with BLT1 antagonist BIIL260 binding site, suggesting isoflurane as a BLT1 antagonist. Five biotinylated compounds with a range of spacer lengths were developed and subjected to calcium mobilization test using CHO-BLT1 cells to determine their property as BLT1 antagonist. Two compounds showed adequate inhibition and we performed the competition assay for BLT1 between the compound and isoflurane. Isoflurane significantly competed with the BIIL260 biotinylated compound, indicating that isofluane bound to the BIIL260 binding site on BLT1. Isoflurane is considered as an alternative sedative in Europe and Canada, and this result could support the potential use of isoflurane as an ICU sedative in lung injury setting. Supported by GM118277

Induction of ALOX5 during Polarization of M1 Macrophages by Multi‐walled Carbon Nanotubes

Fibrogenic carbon nanotubes (CNTs) induce the polarization of M1 and M2 macrophages in mouse lungs. Polarization of the macrophages differentiates the production of proinflammatory and pro‐resolving lipid mediators (LMs) to mediate acute inflammation and its resolution in a time‐dependent manner. In the present study, we examined the molecular mechanism by which multi‐walled CNTs (MWCNTs) induce M1 polarization in vitro, with focus on induction of arachidonate 5‐lipoxygenase (ALOX5), which is required to produce proinflammatory LMs, such as leukotriene B4 (LTB4). Treatment of J774A.1 macrophages with MWCNTs at a concentration of 2.5 µg/mL increased the expression of ALOX5 mRNA by 2.0‐fold at 1‐day and 2.5‐fold at 3‐day post‐exposure. At a concentration of 10 µg/mL, MWCNTs increased the ALOX5 mRNA expression by 3.5‐fold at 1‐day and 5.0‐fold at 3‐day post‐exposure. Immunoblotting shows that MWCNTs significantly increased the ALOX5 protein expression in macrophages. Luciferase reporter assays with the mouse ALOX5 promoter of 2 kilobase upstream of translation start codon demonstrate that the ALOX5 promoter activity increased more than 5‐fold over background. This activity was significantly elevated following 5’‐deletion analyses of ALOX5 promoter and further enhanced by MWCNT treatment, implicating a transcriptional mechanism in the induction of ALOX5. MWCNTs preferentially induced the expression of CD68, a cell surface marker of M1, by 2.5‐fold and induction was persistently high to 3‐day post‐exposure. Moreover, both the expression and activity of the inducible nitric oxide synthase, an intracellular marker of M1 macrophages, were increased by MWCNTs, indicating induction of M1 polarization. Consistent with this notion, MWCNTs increased the capacity of the macrophages to produce proinflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β), and proinflammatory LMs, such as LTB4 and prostaglandin E2 (PGE2), as measured by enzyme‐linked immunosorbent assays. Taken together, these results support that persistent exposure to MWCNTs polarizes macrophages to M1 cells with induction of ALOX5 and elevated production of proinflammatory LMs to boost the acute inflammatory response to fibrogenic nanoparticles.

Pathological metabolic adaptation of neutrophils recruited to cystic fibrosis airway microenvironment can not be reversed by modulator therapy

Abstract Background Neutrophilic inflammation is a hallmark of cystic fibrosis (CF) lung disease. Prior studies from our group show that blood neutrophils undergo metabolic and functional adaptations upon entry into the CF lung lumen that causes them to become anabolic, while repressing bacterial killing function and enhancing degranulation. The recent advent of highly effective modulator therapy (HEMT) for the CFTR channel (mutated in CF) has improved patient outcomes, but it is unclear how HEMT impact CF lung neutrophilic inflammation. Method We used an organotypic model in which blood neutrophils are transmigrated through differentiated airway epithelial cells towards a chemoattractant control (leukotriene B4, LTB4), or airway supernatant from CF patients not on HEMT (CFASN) or on HEMT (CFMOD). We conducted phenotypic (FACS, CFU-assay) and metabolomic (metabolomics, 13C6-glucose tracing) analysis of the transmigrated neutrophils. Results CFMOD-recruited neutrophils contained ivacaftor (a HEMT drug), demonstrating exposure to the therapy. Compared to LTB4-recruited neutrophils, CFASN/CFMOD-recruited neutrophils showed increased AMP, GMP and intracellular and secreted metabolites related to citric acid cycle activity. 13C6-glucose flux indicated similar rates of extracellular glucose utilization in all transmigrated neutrophils, but higher total glycolysis to lactate in CFASN/CFMOD-transmigrated neutrophils. Conclusion Pathological metabolic adaptations of neutrophils following transmigration into CF airway fluid are not substantially altered by HEMT. Further studies are needed to assess the potential impact on functional adaptations by these cells. Supported by NIH (R56 HL150658), Cystic Fibrosis Foundation (CAMMAR21F0, TIROUV19G0), I3 Teams Research Award (Emory), and CF@LANTA, a component of Emory University and Children’s Healthcare of Atlanta.

12/15-lipoxygenase is required for inflammation resolution in a murine model of Lyme arthritis

Abstract Lyme arthritis is a major sequela of disseminated Lyme disease that can persist despite antibiotic clearance of the etiological agent Borrelia burgdorferi. In the murine model of Lyme arthritis (mLA), infected mice develop an inflammatory arthritis characterized by robust innate cell infiltrate and synovial hyperplasia in the tibiotarsal joint, followed by spontaneous resolution around 4–5 weeks post-infection. Eicosanoids are bioactive lipid mediators that play a critical role in both the development and resolution of inflammation. As such, temporal regulation of eicosanoid class-switching from proinflammatory to proresolving is vital. In other inflammation models, late-phase cyclooxygenase (COX)-2 metabolite prostaglandin (PG) E2 skews the 5-lipoxygenase (LO) eicosanoid biosynthesis pathway away from leukotriene B4 production and towards proresolving lipoxin (LX) A4 production via 12/15-LO activity. Indeed, we have previously described a defect in mLA resolution in COX-2−/− and 5-LO−/− mice. Therefore, we hypothesize that 12/15-LO production of LXA4 is required to facilitate mLA resolution. To investigate this, we characterized mLA in 12/15-LO−/− mice and observed a pronounced resolution defect, as evidenced by significant ankle swelling and persistent innate immune infiltrate compared to wild-type mice after 4–5 weeks, despite efficient control of Borrelia burdens. We are currently investigating the mLA eicosanoid profile in 12/15LO−/− mice as well as whether exogenous treatment with LXA4 promotes mLA resolution. These experiments will determine how eicosanoids regulate inflammation resolution and may identify therapeutic targets for inflammatory diseases, including human Lyme arthritis. Supported by a grant from the NIH (R21 AR175160) and the G. Ellsworth Huggins Graduate Fellowship.

Cysteinyl–leukotriene and prostaglandin pathways in bronchial versus alveolar lavage in allergic asthmatics

Cysteinyl–leukotriene and prostaglandin pathways in bronchial versus alveolar lavage in allergic asthmatics

Association between the clinical-functional parameters and lipid markers of systemic inflammation in mild asthma complicated with obesity

Aim. To identify the features of the association between the clinical-functional conditions and lipid markers of systemic inflammation (fatty acids, eicosanoids, plasmalogens) of patients with mild asthma and obesity. Materials and methods. Obese (n=80) and nonobese (n = 55) adults with mild, partially controlled asthma aged 28 to 65 years, and nonobese (n = 30) healthy controls had venous blood samples analysed. The ACQ-5 test, spirometry were evaluated. Serum thromboxane B2 (ТХВ2) and leukotriene B4 (LTB4) were analyzed by the enzyme immunoassay method. Fatty acidsas methyl esters (FAMEs) and plasmalogens in the form of dimethyl acetals (DMA) were estimated in blood plasma by gas chromatography-mass spectrometry. Results. In obese patients with asthma, the ACQ-5 test was lower in comparison to nonobese patients. FEV1 decreased by 8 %, FVC by 12% (p &lt; 0.05). A decrease in the level of DMA16: 0 by 17.5 % and DMA18: 0 by 25 % was established in obese asthma patients. The ratio of DMA16:0/FAME 16: 0 declined by 21 %, DMA18:0/FAME18: 0 − by 28 %. A fall in eicosapentaenoic acid (20: 5n3) and a change in the 20:4n6/20:5n3 ratio were found. The level of ТХВ2 and LTB4 was increased regardless of body weight. There was a positive correlation between body mass index and levels of LTB4 (r = 0.46), ТХВ2 (r = 0.49); and a negative correlation between ACQ and ТХВ2 (r = - 0.58). Indicators of lung function (FEV1, FVC, VC) had correlative links with DMA18:0, DMA20:0, eicosapentaenoic acid. Conclusion. We showed the change in the inflammatory fatty acids and eicosanoids synthesis and plasmalogen deficiency in obese-asthma. Established relationships between clinical and functional parameters and lipid markers of systemic inflammation, plasmalogen deficiency in patients with asthma and obesity reveal new pathogenetic mechanisms of the systemic inflammatory process in chronic respiratory diseases.

Influence of N-acyl-ethanolamine of arachidonic acid on the synthesis of cytokines and oxylipins by the blood leukocytes of patients with asthma under &lt;i&gt;in vitro&lt;/i&gt; conditions

Introduction. The search for hihly effective methods for the treatment and control of asthma remains an urgent task of modern pathophysiology and pharmacology. N-acyl-ethanolamines of arachidonic acid (NAE 20: 4) are promising biomolecules for the regulation of the immune processes. Aim. To investigate the in vitro influence of NAE 20: 4 on the immune cells' ability from patients with asthma to synthesize pro-inflammatory cytokines and oxylipins. Materials and methods. The objective of the study was the blood of 11 patients with mild controlled asthma and 10 healthy volunteers. The in vitro experiment was carried out in stimulated and not stimulated by lipopolysaccharide (LPS) blood for 6 hours. NAE 20: 4 was added at 1.0 µM, 3.0 µM or 10.0 µM doses. The levels of cytokines (TNFα, IL-8), thromboxaneB2 (TXB2) and leukotriene B4 (LTB4) were analyzed by the ELISA method. Results. We found an increase in TNFα, IL-8, TXB2, LTB4 in the blood of persons with asthma before in vitro NAE 20: 4 exposure, which indicates systemic chronic inflammation. Incubation of blood cells without LPS did not affect the change in the spectrum of mediators studied. Whereas the treatment of NAE 20: 4 at a 10 µM reduced the synthesis of TNFα, IL-8, LTB4, TXB2. In vitro blood cells stimulation by LPS led to hyperproduction of cytokines and oxylipins. NAE 20: 4 at a dose of 1 µM in LPS-stimulated blood had no effect on the production of signaling molecules, while NAE 20:4 at a concentration of 3 µM reduced the level of TNFα, IL-8. NAE 20:4 at a dose of 10.0 µM inhibited the cytokines and LTB4 synthesis. The level of TXB2 underthe influence of NAE 20: 4 at 10.0 µM did not change. Conclusion. The results of the study revealed a dose-dependent anti-inflammatory effect of NAE 20:4, characterized by its ability to influence the synthesis of pro-inflammatory cytokines and oxylipins by the immune cells of individuals with asthma in vitro. Further study of NAE 20: 4 opens up new prospects for the development of targeted methods for regulating immune processes in asthma.

Plasma lipidome acts as diagnostic marker and predictor for cyclosporin response in patients with aplastic anemia.

The lipid metabolomic profile has been well defined in the pathogenesis and differential diagnosis in patients with different myeloid diseases. We assumed that the serum lipid metabolites could also help the diagnosis and prognostic prediction of aplastic anemia (AA). In this study, serum lipid profiles were explored in AA patients before and after cyclosporin (CsA) treatment. Meanwhile, hypocellular myelodysplastic syndrome (h-MDS) patients and the healthy volunteers were compared as controls. 15 AA patients, 11h-MDS patients and 20 age and sex matched health controls were enrolled. All the AA patients were diagnosed to be non-severe aplastic anemia with transfusion dependency and were treated by CsA 3-5mg/kg/d for at least 6months. AA patients had decreased arachidonic acid pathway metabolites and retinol metabolism-related metabolites as compared with h-MDS and the health (P < 0.05), whereas h-MDS patients had increased metabolism of proline and threonine and abnormal sphingolipid metabolism compared with AA patients and the normal controls. After 6month of CsA treatment, serum arachidonic acid, PGE2, PGJ2, 15(S)-HETE, leukotriene B4 and Protectin D1 decreased significantly. Patients who had response to CsA had higher levels of baseline protectin D1 (P = 0.011), leukotriene B4 (P = 0.011), 15(S)-HETE (P = 0.004) and all-trans-retinal (P = 0.000) than those who had no response.

Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy

To successfully feed on blood, hematophagous arthropods must combat the host's natural hemostatic and inflammatory responses. Salivary proteins of blood-feeding insects such as mosquitoes contain compounds that inhibit these common host defenses against blood loss, including vasoconstriction, platelet aggregation, blood clotting, pain, and itching. The D7 proteins are some of the most abundantly expressed proteins in female mosquito salivary glands and have been implicated in inhibiting host hemostatic and inflammatory responses. Anopheles gambiae, the primary vector of malaria, expresses three D7 long-form and five D7 short-form proteins. Previous studies have characterized the AngaD7 short-forms, but the D7 long-form proteins have not yet been characterized in detail. Here, we characterized the A. gambiae D7 long-forms by first determining their binding kinetics to hemostatic agonists such as leukotrienes and serotonin, which are potent activators of vasoconstriction, edema formation, and postcapillary venule leakage, followed by ex vivo functional assays. We found that AngaD7L1 binds leukotriene C4 and thromboxane A2 analog U-46619; AngaD7L2 weakly binds leukotrienes B4 and D4; and AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. It is therefore possible that AngaD7L proteins counteract host hemostasis by scavenging these mediators. Finally, we demonstrate that AngaD7L2 had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI. The uncovering of these interactions in the present study will be essential for comprehensive understanding of the vector-host biochemical interface.