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Background Unexplained heterogeneity in clinical trials has resulted in questions regarding the effectiveness of ɣ-linolenic acid (GLA)-containing botanical oil supplements. This heterogeneity may be explained by genetic variation within the fatty acid desaturase (FADS) gene cluster that is associated with circulating and tissue concentrations of arachidonic acid (ARA) and dihomo-ɣ-linolenic acid (DGLA), both of which may be synthesized from GLA and result in proinflammatory and anti-inflammatory metabolites, respectively. Objectives The objective of this study was to prospectively compare the capacity of a non-Hispanic white cohort, stratified by FADS genotype at the key single-nucleotide polymorphism (SNP) rs174537, to metabolize 18-carbon omega-6 (n-6) PUFAs in borage oil (BO) and soybean oil (SO) to GLA, DGLA, and ARA. Methods Healthy adults (n = 64) participated in a randomized, double-blind, crossover intervention. Individuals received encapsulated BO (Borago officinalis L.; 37% LA and 23% GLA) or SO [Glycine max (L.) Merr.; 50% LA and 0% GLA] for 4 wk, followed by an 8-wk washout period, before consuming the opposite oil for 4 wk. Serum lipids and markers of inflammation (C-reactive protein) were assessed for both oil types at baseline and during weeks 2 and 4 of the intervention. Results SO supplementation failed to alter circulating concentrations of any n-6 long-chain PUFAs. In contrast, a modest daily dose of BO elevated serum concentrations of GLA and DGLA in an rs174537 genotype-dependent manner. In particular, DGLA increased by 57% (95% CI: 0.38, 0.79) in GG genotype individuals, but by 141% (95% CI: 1.03, 2.85) in TT individuals. For ARA, baseline concentrations varied substantially by genotype and increased modestly with BO supplementation, suggesting a key role for FADS variation in the balance of DGLA and ARA. Conclusions The results of this study clearly suggest that personalized and population-based approaches considering FADS genetic variation may be necessary to optimize the design of future clinical studies with GLA-containing oils. This trial was registered at clinicaltrials.gov as NCT02337231.

There is an urgent need to identify the cellular and molecular mechanisms responsible for severe COVID-19 that results in death. We initially performed both untargeted and targeted lipidomics as well as focused biochemical analyses of 127 plasma samples and found elevated metabolites associated with secreted phospholipase A(2) (sPLA(2)) activity and mitochondrial dysfunction in patients with severe COVID-19. Deceased COVID-19 patients had higher levels of circulating, catalytically active sPLA(2) group IIA (sPLA(2)-IIA), with a median value that was 9.6-fold higher than that for patients with mild disease and 5.0-fold higher than the median value for survivors of severe COVID-19. Elevated sPLA(2)-IIA levels paralleled several indices of COVID-19 disease severity (e.g., kidney dysfunction, hypoxia, multiple organ dysfunction). A decision tree generated by machine learning identified sPLA(2)-IIA levels as a central node in the stratification of patients who died from COVID-19. Random forest analysis and least absolute shrinkage and selection operator–based (LASSO-based) regression analysis additionally identified sPLA(2)-IIA and blood urea nitrogen (BUN) as the key variables among 80 clinical indices in predicting COVID-19 mortality. The combined PLA-BUN index performed significantly better than did either one alone. An independent cohort (n = 154) confirmed higher plasma sPLA(2)-IIA levels in deceased patients compared with levels in plasma from patients with severe or mild COVID-19, with the PLA-BUN index–based decision tree satisfactorily stratifying patients with mild, severe, or fatal COVID-19. With clinically tested inhibitors available, this study identifies sPLA(2)-IIA as a therapeutic target to reduce COVID-19 mortality.

Long chain polyunsaturated fatty acids (LC-PUFAs) have critical signaling roles that regulate dyslipidemia and inflammation. Genetic variation in the FADS gene cluster accounts for a large portion of interindividual differences in circulating and tissue levels of LC-PUFAs, with the genotypes most strongly predictive of low LC-PUFA levels at strikingly higher frequencies in Amerind ancestry populations. In this study, we examined relationships between genetic ancestry and FADS variation in 1102 Hispanic American participants from the Multi-Ethnic Study of Atherosclerosis. We demonstrate strong negative associations between Amerind genetic ancestry and LC-PUFA levels. The FADS rs174537 single nucleotide polymorphism (SNP) accounted for much of the AI ancestry effect on LC-PUFAs, especially for low levels of n-3 LC-PUFAs. Rs174537 was also strongly associated with several metabolic, inflammatory and anthropomorphic traits including circulating triglycerides (TGs) and E-selectin in MESA Hispanics. Our study demonstrates that Amerind ancestry provides a useful and readily available tool to identify individuals most likely to have FADS-related n-3 LC-PUFA deficiencies and associated cardiovascular risk., Yang et al. examine whether genetic ancestry is associated with genetic variation in fatty acid desaturases and plasma phospholipid levels of long chain polyunsaturated fatty acids (LC-PUFAs) in Hispanic Americans. They find strong associations between Amerind genetic ancestry and LC-PUFA levels; and report that the well-known FADS rs174537 variant was associated with several metabolic, inflammatory and anthropomorphic traits including circulating triglycerides.

Hispanic populations have higher rates of obesity, elevated triglycerides, and a greater prevalence of diabetes. Long chain polyunsaturated fatty acids (LC-PUFAs) and LC-PUFA metabolites have critical signaling roles that regulate dyslipidemia and inflammation. Genetic variation in the FADS cluster accounts for a large part of the interindividual differences in circulating and tissue levels of LC-PUFAs, with the genotypes most strongly predictive of low LC-PUFA levels at strikingly higher frequencies in Amerind (AI) ancestry populations. In this study, we examined relationships between genetic ancestry and FADS variation, plasma phospholipid levels of LC-PUFAs, anthropometric measures, and circulating metabolic and inflammatory biomarkers in 1,102 Hispanic American participants, representing six distinct ancestry populations from the Multi-Ethnic Study of Atherosclerosis. We demonstrate strong negative associations between AI genetic ancestry and LC-PUFA levels. The FADS rs174537 single nucleotide polymorphism (SNP) accounted for much of the AI ancestry effect on LC-PUFAs, especially for low levels of n-3 LC-PUFAs. Rs174537 was also strongly associated with several metabolic, inflammatory and anthropomorphic traits including circulating triglycerides (TGs) and E-selectin in MESA Hispanics. We further replicated the association with circulating TGs in two additional Hispanic cohorts: the Hispanic Community Health Study/Study of Latinos and the Arizona Insulin Resistance Registry. Our study demonstrates that Amerind ancestry provides a useful and readily available tool to identify individuals most likely to have FADS-related n-3 LC-PUFA deficiencies and associated cardiovascular risk.

There is an urgent need to identify cellular and molecular mechanisms responsible for severe COVID-19 disease accompanied by multiple organ failure and high mortality rates. Here, we performed untargeted/targeted lipidomics and focused biochemistry on 127 patient plasma samples, and showed high levels of circulating, enzymatically active secreted phospholipase A2 Group IIA (sPLA2-IIA) in severe and fatal COVID-19 disease compared with uninfected patients or mild illness. Machine learning demonstrated that sPLA2-IIA effectively stratifies severe from fatal COVID-19 disease. We further introduce a PLA-BUN index that combines sPLA2-IIA and blood urea nitrogen (BUN) threshold levels as a critical risk factor for mitochondrial dysfunction, sustained inflammatory injury and lethal COVID-19. With the availability of clinically tested inhibitors of sPLA2-IIA, our study opens the door to a precision intervention using indices discovered here to reduce COVID-19 mortality.

The mechanisms responsible for driving endogenous airway hyperresponsiveness (AHR) in the form of exercise-induced bronchoconstriction (EIB) are not fully understood. We examined alterations in airway phospholipid hydrolysis, surfactant degradation, and lipid mediator release in relation to AHR severity and changes induced by exercise challenge. Paired induced sputum ( n = 18) and bronchoalveolar lavage (BAL) fluid ( n = 11) were obtained before and after exercise challenge in asthmatic subjects. Samples were analyzed for phospholipid structure, surfactant function, and levels of eicosanoids and secreted phospholipase A2group 10 (sPLA2-X). A primary epithelial cell culture model was used to model effects of osmotic stress on sPLA2-X. Exercise challenge resulted in increased surfactant degradation, phospholipase activity, and eicosanoid production in sputum samples of all patients. Subjects with EIB had higher levels of surfactant degradation and phospholipase activity in BAL fluid. Higher basal sputum levels of cysteinyl leukotrienes (CysLTs) and prostaglandin D2(PGD2) were associated with direct AHR, and both the postexercise and absolute change in CysLTs and PGD2levels were associated with EIB severity. Surfactant function either was abnormal at baseline or became abnormal after exercise challenge. Baseline levels of sPLA2-X in sputum and the absolute change in amount of sPLA2-X with exercise were positively correlated with EIB severity. Osmotic stress ex vivo resulted in movement of water and release of sPLA2-X to the apical surface. In summary, exercise challenge promotes changes in phospholipid structure and eicosanoid release in asthma, providing two mechanisms that promote bronchoconstriction, particularly in individuals with EIB who have higher basal levels of phospholipid turnover.

Prostate cancer (PCa) is the most common male cancer and the second leading cause of cancer death in United States men. Controversy continues over the effectiveness of prostate-specific antigen (PSA) for distinguishing aggressive from indolent PCa. There is a critical need for more specific and sensitive biomarkers to detect and distinguish low- versus high-risk PCa cases. Discovery metabolomics were performed utilizing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) on plasma samples from 159 men with treatment na&iuml, ve prostate cancer participating in the North Carolina-Louisiana PCa Project to determine if there were metabolites associated with aggressive PCa. Thirty-five identifiable plasma small molecules were associated with PCa aggressiveness, 15 of which were sphingolipids, nine common molecules were present in both African-American and European-American men. The molecules most associated with PCa aggressiveness were glycosphingolipids, levels of trihexosylceramide and tetrahexosylceramide were most closely associated with high-aggressive PCa. The Cancer Genome Atlas was queried to determine gene alterations within glycosphingolipid metabolism that are associated with PCa and other cancers. Genes that encode enzymes associated with the metabolism of glycosphingolipids were altered in 12% of PCa and &gt, 30% of lung, uterine, and ovarian cancers. These data suggest that the identified plasma (glyco)sphingolipids should be further validated for their association with aggressive PCa, suggesting that specific sphingolipids may be included in a diagnostic signature for PCa.

The “modern western” diet (MWD) has increased the onset and progression of chronic human diseases as qualitatively and quantitatively maladaptive dietary components give rise to obesity and destructive gene-diet interactions. There has been a three-fold increase in dietary levels of the omega-6 (n-6) 18 carbon (C18), polyunsaturated fatty acid (PUFA) linoleic acid (LA; 18:2n-6), with the addition of cooking oils and processed foods to the MWD. Intense debate has emerged regarding the impact of this increase on human health. Recent studies have uncovered population-related genetic variation in the LCPUFA biosynthetic pathway (especially within the fatty acid desaturase gene (FADS) cluster) that is associated with levels of circulating and tissue PUFAs and several biomarkers and clinical endpoints of cardiovascular disease (CVD). Importantly, populations of African descent have higher frequencies of variants associated with elevated levels of arachidonic acid (ARA), CVD biomarkers and disease endpoints. Additionally, nutrigenomic interactions between dietary n-6 PUFAs and variants in genes that encode for enzymes that mobilize and metabolize ARA to eicosanoids have been identified. These observations raise important questions of whether gene-PUFA interactions are differentially driving the risk of cardiovascular and other diseases in diverse populations, and contributing to health disparities, especially in African American populations.

Secretory phospholipases A2 (sPLA2) hydrolyze phospholipids in cell membranes and extracellular structures such as pulmonary surfactant. This study tests the hypothesis that sPLA2 are elevated in human lungs during acute respiratory distress syndrome (ARDS) and that sPLA2 levels are associated with surfactant injury by hydrolysis of surfactant phospholipids.Bronchoalveolar lavage (BAL) fluid was obtained from 18 patients with early ARDS (72 hours) and compared with samples from 10 healthy volunteers. Secreted phospholipase A2 levels were measured (enzyme activity and enzyme immunoassay) in conjunction with ARDS subjects' surfactant abnormalities including surfactant phospholipid composition, large and small aggregates distribution and surface tension function.BAL sPLA2 enzyme activity was markedly elevated in ARDS samples relative to healthy subjects when measured by ex vivo hydrolysis of both phosphatidylglycerol (PG) and phosphatidylcholine (PC). Enzyme immunoassay identified increased PLA2G2A protein in the ARDS BAL fluid, which was strongly correlated with the sPLA2 enzyme activity against PG. Of particular interest, the authors demonstrated an average depletion of 69% of the PG in the ARDS sample large aggregates relative to the normal controls. Furthermore, the sPLA2 enzyme activity against PG and PC ex vivo correlated with the BAL recovery of in vivo PG and PC, respectively, and also correlated with the altered distribution of the large and small surfactant aggregates.These results support the hypothesis that sPLA2-mediated hydrolysis of surfactant phospholipid, especially PG by PLA2G2A, contributes to surfactant injury during early ARDS.

SummaryCalgranulins are a family of powerful chemoattractants, which have been implicated as biomarkers in inflammatory diseases. To determine how different respiratory diseases affect the expression of calgranulins, we measured the expression of S100A8/A9 and S100A12 in bronchoalveolar lavage fluid (BALF) of acute respiratory distress syndrome (ARDS) patients and healthy volunteers by ELISA. Analysis of calgranulin expression revealed a high level of S100A12 in the lavages of patients suffering from ARDS compared to controls (p

BACKGROUND In vitro and experimental animal studies have demonstrated that high levels of omega-6 (n-6) polyunsaturated fatty acids (PUFAs) and high ratios of n-6 to omega-3 (n-3) PUFAs are strongly associated with the development and progression of prostate cancer (PCA). However, epidemiological studies in humans have demonstrated inconsistent findings linking dietary PUFAs and PCA risk. We hypothesize that genetic and epigenetic variations within the fatty acid desaturase (FADS) gene cluster produce gene-diet interactions that may explain these disparate findings. This study tested the relationship of the genotype of a single nucleotide polymorphism, rs174537, and the methylation status of a CpG site, cg27386326, with PUFA composition, and markers of PUFA biosynthesis in PCA tissue. METHODS Sixty PCA specimens from patients undergoing radical prostatectomy were genotyped, pyrosequenced and quantitated for fatty acids (FAs). RESULTS Long-chain (LC)-PUFAs, such as arachidonic acid (ARA), were abundant in these specimens, with ARA accounting for 15.8% of total FAs. In addition, there was a positive association of the G allele at rs174537 with concentrations of ARA and adrenic acid and ratios of products to precursors within the n-6 PUFA pathway such that specimens from homozygous G individuals exhibited increasingly higher values as compared to specimens from heterozygous individuals and homozygous T individuals. Finally, the methylation status of cg27386326 was inversely correlated with tissue concentrations of LC-PUFAs and markers of LC-PUFA biosynthesis. CONCLUSIONS These data reveal that genetic and epigenetic variations within the FADS cluster are highly associated with LC-PUFA concentrations and LC-PUFA biosynthetic capacity in PCA tissue. They also raise the potential that gene-PUFA interactions play an important role in PCA risk and severity. Prostate 76:1182-1191, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.

Stressed cells undergoing necrosis release molecules that acts as endogenous danger signals to alert and activate innate immune cells. Both HMGB1 and HSP70 are induced in activated monocytes/macrophages and also are released from stressed or injured cells. We investigated whether HMGB1 and HSP70 released from necrotic monocytes/macrophages, can act as danger signals to mediate proinflammatory cytokine responses to bacterial endotoxin or lipopolysaccharide (LPS). We show that cell lysate, obtained from necrotic cells directly stimulates the proinflammatory cytokine and chemokine responses in human monocyte/macrophage cell line, THP-1, as revealed by the induction of TNF-alpha, IL-6 and IL-8 mRNA expression and protein production. In the presence of LPS, necrotic cell lysate induced a more robust increase in all three proteins. We found that HMGB1 and HSP70 were indeed present in the necrotic cell lysate and were responsible for the significant induction of the proinflammatory cytokine expression, as neutralization with antibodies against both proteins blocked the increase in the cytokine production seen after incubating LPS-stimulated cells with the necrotic cell lysate. We also found that the newly identified triggering receptor expressed on myeloid cells-1 (TREM-1) was involved in mediating the HMGB1- and HSP70-induced cytokine production. Blocking TREM-1 on THP-1 cells with a recombinant chimera prevented the increase in cytokine production, while simultaneous blocking of TLR4 and TREM-1 completely abolished the proinflammatory response, suggesting that TREM-1 synergizes with TLR4 to mediate the effects of such signals from necrotic cells. In addition, blocking HMGB1 or HSP70 simultaneously with TREM-1 did not decrease the cytokine level further, confirming the involvement of TREM-1 in mediating the effect of HMGB1 and HSP70. Although the interaction of HMGB1 and HSP70 with TREM-1 induced I kappa B alpha and p38 expression, both of which are required for the inflammatory cytokine expression, blockade of TREM-1 did not affect I kappa B alpha expression but markedly reduced p38 activation, as revealed by Western blot analysis. Together, these results demonstrate that HMGB1 and HSP70 released from necrotic cells function as endogenous danger signals to augment the proinflammatory responses in monocytes/macrophage and that TREM-1 relays such signals to the cytokine expression cascade. This mechanism may contribute to the amplification and persistence of the inflammatory response to bacterial infection.

In the absence of activation signals, circulating human neutrophils and eosinophils undergo spontaneous apoptosis. The glucocorticoid Dexamethasone (Dex) accelerates apoptosis in inflammatory cells such as eosinophils, but uniquely delays neutrophil apoptosis. Corresponding to the opposite effects of Dex on granulocyte apoptosis, we demonstrate that in neutrophils and eosinophils Dex oppositely affects expression of the anti-apoptotic Bcl-2 family protein Mcl-1L. Mcl-1L expression declines over time in vitro; however, Dex maintains Mcl-1L expression in neutrophils. In contrast, Dex accelerates Mcl-1L protein loss in eosinophils. Neither Mcl-1S, a pro-apoptotic splice variant, nor Bax were affected. Dex treatment in the presence of a translation inhibitor stabilized existing Mcl-1L protein in neutrophils, while Mcl-1L stability in eosinophils was unaffected. Accordingly, delay of neutrophil apoptosis by Dex was prevented by antisense Mcl-1L siRNA. Our findings suggest that regulation of Mcl-1L degradation plays an important role in the opposite effects of Dex on granulocyte apoptosis.

In asthma, inflammation-mediated surfactant dysfunction contributes to increased airway resistance, but the mechanisms for dysfunction are not understood. To test mechanisms that alter surfactant function, atopic asthmatics underwent endobronchial antigen challenge and bronchoalveolar lavage (BAL). BAL fluids were sequentially separated into cells, surfactant, and supernatant, and multiple end points were analyzed. Each end point's unique relationship to surfactant dysfunction was determined. Our results demonstrate that minimum surface tension (γmin) of surfactant after antigen challenge was significantly increased with a spectrum of responses that included dysfunction in 6 of 13 asthmatics. Antigen challenge significantly altered the partitioning of surfactant phospholipid measured as a decreased ratio of large surfactant aggregates (LA) to small surfactant aggregates (SA), LA/SA ratio. Phosphatidylglycerol (PG) was significantly reduced in the LA of the dysfunctional asthmatic BALs. There was a corresponding significant increase in the ratio of phosphatidylcholine to PG, which strongly correlated with both increased γminand decreased LA/SA. Altered surfactant phospholipid properties correlated with surfactant dysfunction as well or better than either increased eosinophils or protein. Secretory phospholipase activity, measured in vitro, increased after antigen challenge and may explain the decrease in surfactant PG. In summary, alteration of phospholipids, particularly depletion of PG, in the LA of surfactant may be an important mechanism in asthma-associated surfactant dysfunction.

Early mobilization of critically ill patients with the acute respiratory distress syndrome (ARDS) has emerged as a therapeutic strategy that improves patient outcomes, such as the duration of mechanical ventilation and muscle strength. Despite the apparent efficacy of early mobility programs, their use in clinical practice is limited outside of specialized centers and clinical trials. To evaluate the mechanisms underlying mobility therapy, we exercised acute lung injury (ALI) mice for 2 days after the instillation of lipopolysaccharides into their lungs. We found that a short duration of moderate intensity exercise in ALI mice attenuated muscle ring finger 1 (MuRF1)–mediated atrophy of the limb and respiratory muscles and improved limb muscle force generation. Exercise also limited the influx of neutrophils into the alveolar space through modulation of a coordinated systemic neutrophil chemokine response. Granulocyte colony-stimulating factor (G-CSF) concentrations were systemically reduced by exercise in ALI mice, and in vivo blockade of the G-CSF receptor recapitulated the lung exercise phenotype in ALI mice. Additionally, plasma G-CSF concentrations in humans with acute respiratory failure (ARF) undergoing early mobility therapy showed greater decrements over time compared to control ARF patients. Together, these data provide a mechanism whereby early mobility therapy attenuates muscle wasting and limits ongoing alveolar neutrophilia through modulation of systemic neutrophil chemokines in lung-injured mice and humans., Facultad de Ciencias Médicas

Levels and ratios of omega-6 (n-6) and omega-3 (n-3) long chain polyunsaturated fatty acids (LcPUFAs) impact a wide range of biological activities and human diseases. We have shown marked differences in the synthesis of the n-6 LcPUFA, arachidonic acid (20:4, n-6), between African (AfAm) and European (EuAm) ancestry populations in the US. N-3 LcPUFA biosynthesis is more complex, including up to 4 additional biochemical steps to make docosahexaenoic acid (DHA; 22:6, n-3). Given the critical role of the balance of n-6 and n-3 LcPUFAs on biological responses, the objective of the current study was to compare the efficiency of biosynthesis of both n-6 and n-3 LcPUFA in AfAm and EuAm subjects. Serum levels of PUFAs were examined in two cohorts, and AfAm contained higher DHA levels in both cohorts (p= 4 x 10-6 and 1.7 x 10-9) and higher DHA to precursor n-3 PUFA ratios (p=1.7x10-8 and 6.8x10-20) than EuAm. Analysis of 77 candidate SNPs in the six genes proposed to participate in LcPUFA biosynthesis revealed str...

Over the past 100 years, changes in the food supply in Western nations have resulted in alterations in dietary fatty acid consumption, leading to a dramatic increase in the ratio of omega-6 (omega6) to omega3 polyunsaturated fatty acids (PUFA) in circulation and in tissues. Increased omega6/omega3 ratios are hypothesized to increase inflammatory mediator production, leading to higher incidence of inflammatory diseases, and may impact inflammatory gene expression. To determine the effect of reducing the omega6/omega3 ratio on expression of inflammatory pathway genes in mononuclear cells, healthy humans were placed on a controlled diet for 1 week, then given fish oil and borage oil for an additional 4 weeks. Serum and neutrophil fatty acid composition and ex vivo leukotriene B(4) production from stimulated neutrophils were measured at the start and end of the supplementation period and after a 2-week washout. RNA was isolated from mononuclear cells and expression of PI3K, Akt, NFkappaB, and inflammatory cytokines was measured by real-time PCR. A marked increase was seen in serum and neutrophil levels of long-chain omega3 PUFA concomitant with a reduction in the omega6/omega3 PUFA ratio (40%). The ex vivo capacity of stimulated neutrophils to produce leukotriene B(4) was decreased by 31%. Expression of PI3Kalpha and PI3Kgamma and the quantity of PI3Kalpha protein in mononuclear cells was reduced after supplementation, as was the expression of several proinflammatory cytokines. These data reveal that PUFA may exert their clinical effects via their capacity to regulate the expression of signal transduction genes and genes for proinflammatory cytokines.

Although the role of arachidonic acid (AA) metabolism to eicosanoids has been well established in allergy and asthma, recent studies in neoplastic cells have revealed that AA remodeling through phospholipids impacts cell survival. This study tests the hypothesis that regulation of AA/phospholipid-remodeling enzymes, cytosolic phospholipase A(2) alpha(cPLA(2)-alpha, gIValphaPLA(2)) and CoA-independent transacylase (CoA-IT), provides a mechanism for altered eosinophil survival during allergic asthma. In vitro incubation of human eosinophils (from donors without asthma) with IL-5 markedly increased cell survival, induced gIValphaPLA(2) phosphorylation, and increased both gIValphaPLA(2) and CoA-IT activity. Furthermore, treatment of eosinophils with nonselective (ET18-O-CH(3)) and selective (SKF 98625) inhibitors of CoA-IT triggered apoptosis, measured by changes in morphology, membrane phosphatidylserine exposure, and caspase activation, completely reversing IL-5-induced eosinophil survival. To determine if similar activation occurs in vivo, human blood eosinophils were isolated from either normal individuals at baseline or from subjects with mild asthma, at both baseline and 24 hours after inhaled allergen challenge. Allergen challenge of subjects with allergic asthma induced a marked increase in cPLA(2) phosphorylation, augmented gIValphaPLA(2) activity, and increased CoA-IT activity. These findings indicate that both in vitro and in vivo challenge of eosinophils activated gIValphaPLA(2) and CoA-IT, which may play a key role in enhanced eosinophil survival.

Changes in diet over the past century have markedly altered the consumption of fatty acids. The dramatic increase in the ingestion of saturated and n-6 fatty acids and concomitant decrease in n-3 fatty acids are thought to be a major driver of the increase in the incidence of inflammatory diseases such as asthma, allergy, and atherosclerosis. The central objective of the Center for Botanical Lipids at Wake Forest University School of Medicine and the Brigham and Women's Hospital is to delineate the mechanisms by which fatty acid-based dietary supplements inhibit inflammation leading to chronic human diseases such as cardiovascular disease and asthma. The key question that this center addresses is whether botanical n-6 and n-3 fatty acids directly block recognized biochemical pathways or the expression of critical genes that lead to asthma and atherosclerosis. Dietary supplementation with flaxseed oil, borage oil, and echium oil affects the biochemistry of fatty acid metabolism and thus the balance of proinflammatory mediators and atherogenic lipids. Supplementation studies have begun to identify key molecular and genetic mechanisms that regulate the production of lipid mediators involved in inflammatory and hyperlipidemic diseases. Echium oil and other oils containing stearidonic acid as well as botanical oil combinations (such as echium and borage oils) hold great promise for modulating inflammatory diseases.

Influenza A virus (IAV) has previously been shown to alter chemotactic, oxidative, and secretory functions of polymorphonuclear leukocytes (PMNL). Because of the role of cytoskeletal proteins in these processes, studies were carried out to determine if IAV altered the PMNL cytoskeleton. PMNL were incubated with buffer or IAV, stimulated with f-met-leu-phe (FMLP), fixed and stained with NBD-phallacidin (NBD-Ph) and studied by flow cytometry. Mean F-actin fluorescence was increased 18% in virus treated cells pre-FMLP stimulation and 13% 5 and 10 min post-FMLP (P

Exposure of airway smooth muscle (ASM) cells to the cytokine IL-1β results in an induction of PGE2 synthesis that affects numerous cell functions. Current dogma posits induction of COX-2 protein as the critical, obligatory event in cytokine-induced PGE2 production, although PGE2 induction can be inhibited without a concomitant inhibition of COX-2. To explore other putative regulatory features we examined the role of phospholipase A2 (PLA2) and PGE synthase (PGES) enzymes in IL-1β-induced PGE2 production. Treatment of human ASM cultures with IL-1β caused a time-dependent induction of both cytosolic PLA2 (cPLA2) and microsomal PGES (mPGES) similar to that observed for COX-2. Regulation of COX-2 and mPGES induction was similar, being significantly reduced by inhibition of p42/p44 or p38, whereas cPLA2 induction was only minimally reduced by inhibition of p38 or PKC. COX-2 and mPGES induction was subject to feed-forward regulation by PKA, whereas cPLA2 induction was not. SB-202474, an SB-203580 analog lacking the ability to inhibit p38 but capable of inhibiting IL-1β-induced PGE2 production, was effective in inhibiting mPGES but not COX-2 or cPLA2 induction. These data suggest that although COX-2, cPLA2, and mPGES are all induced by IL-β in human ASM cells, regulatory features of cPLA2 are dissociated, whereas those of COX-2 and mPGES are primarily associated, with regulation of PGE2 production. mPGES induction and, possibly, cPLA2 induction appear to cooperate with COX-2 to determine IL-1β-mediated PGE2 production in human ASM cells.

Cystic fibrosis (CF) is characterized by progressive airway obstruction. Although it has been postulated that this is due in part to mucus hypersecretion, there are no published data showing an increase in the gel-forming mucins MUC5AC or MUC5B in CF secretions. We used confocal microscopy to assess the amount of mucin-like glycoprotein and DNA in CF sputum and found more mucin in bronchitis sputum and a much greater amount of DNA in CF sputum. We then used antibodies to MUC5AC and MUC5B with Western gels and dot-blot to quantify mucin in sputum from 12 patients with CF and 11 subjects without lung disease. There was a 70% decrease in MUC5B and a 93% decrease in MUC5AC in CF sputum (P < 0.005 for both). We conclude that the vol/vol concentration of MUC5AC and MUC5B are decreased in the CF airways relative to normal mucus. This may be due to a relative increase in other components of sputum in the CF airway or to a primary defect in mucin secretion in CF.

Secretory phospholipase A(2) (sPLA(2)) enzymes contribute to inflammatory injury in human lungs by several mechanisms, including eicosanoid production and hydrolytic damage to surfactant phospholipids. Several distinct sPLA(2) genes have been described in human tissue but little is known regarding their presence, localization, or function(s) within lungs. We hypothesized that sPLA(2)s would have cell-specific distributions within lung. We used reverse transcriptase/polymerase chain reaction to identify sPLA(2) messenger RNAs (mRNAs) in adult human lung tissue. Resulting complementary DNA (cDNA) sequences indicated that total lung extracts contained mRNA for Groups IB, IIA, V, and X sPLA(2). An epithelial cell line, BEAS cells, expressed only Groups IIA, V, and X. We used these cDNAs to clone these enzymes, especially the recently described Group X and Group V enzymes. Digoxigenin-labeled complementary RNA probes were used to determine localization of each sPLA(2) by in situ hybridization of human lung. Hybridization was strongly positive for Group X and Group V in airway epithelial cells, which failed to hybridize Group IB or IIA probes. Although four known mammalian sPLA(2) isotypes were expressed in lung, only Group X and Group V sPLA(2) mRNAs appear uniquely expressed in airway epithelium, suggesting they could provide a mechanism of pulmonary surfactant hydrolysis during lung injury.

Cytokines alter neutrophil (PMN) function during inflammation, and Tumor Necrosis Factor (TNF) in vitro primes PMN such that receptor-mediated stimulation causes markedly enhanced release of arachidonic acid. We hypothesized that two Ca 2+ -dependent PLA 2 's in PMN might be activated during priming of the cell, thus affecting arachidonate release. A low molecular weight, secretory PLA 2 was identified by enzymatic activity in the cell free supernates of primed or stimulated PMN, and in PMN disrupted by nitrogen cavitation. The enzymatic activity was calcium-dependent, acid stable, destroyed by dithiothreitol, and blocked by anti-sPLA 2 antibodies. TNF caused secretion of sPLA 2 and also caused an increase in cell-associated sPLA 2 enzymatic activity. Activation and release were maximal with fMLP stimulation of TNF-primed PMN. Neutrophils also contained a cytosolic PLA 2 (cPLA 2 ) characterized by enzymatic activity which was calcium dependent, enhanced by dithiothreitol, and blocked by anti-cPLA 2 antibody. TNF caused a doubling of cPLA 2 enzymatic activity which was associated with phosphorylation of the enzyme as judged by a migration shift on Western blots. Thus, TNF priming of human PMN caused marked increase in fMLP stimulated AA release in parallel to enhanced activity of two different PLA 2 's.

Phospholipases A2 (PLA2) hydrolyze phospholipids resulting in the release of fatty acids including arachidonic acid (AA) and lysophospholipids. AA, in turn, serves as a substrate for the synthesis of leukotrienes which can cause bronchoconstriction and airways edema and appear to be important mediators of clinical asthma. Further, lysophospholipids may be cytotoxic and/or impair the function of surfactant. We examined the release of secretory PLA2 (sPLA2) and AA into the airways after antigen challenge in 16 subjects with allergic asthma. Asthmatic subjects underwent bronchoscopy with bronchoalveolar lavage (BAL) before and after inhaled antigen challenge; in addition, a single BAL, without inhaled antigen, was performed in 10 control subjects. BAL was obtained at 4 h (n = 7), the time of the late asthmatic response (LAR) (n = 5), or 24 h (n = 4) after challenge. There was no difference between normal and asthmatic subjects in either BAL fluid (BALF) sPLA2 activity or AA concentration at baseline. Both sPLA2 and AA increased after antigen challenge (p0.01 and 0.05, respectively). These changes were most marked 4 h after challenge (p0.03 for both). sPLA2 may play an important role in the generation of AA in patients with asthma.

Increasing evidence suggests that the metabolism of arachidonic acid (AA) may be different in inflammatory cells isolated from blood or migrating into tissues. To explore the possibility that changes in AA metabolism between blood and tissue inflammatory cells could be due in part to a different content or distribution of AA in glycerolipid classes, we studied these parameters in six human inflammatory cells isolated from blood (eosinophils, monocytes, neutrophils, and platelets) or from the lung tissue (mast cells and macrophages). Lung cells generally had a higher total cellular content of AA than that found in the blood cells. In addition, both mast cells and macrophages had a large endogenous pool of AA associated with triglycerides (TG), containing 45 and 22% of their total cellular AA, respectively. To address the hypothesis that cells migrating into the lung had a higher cellular level of AA and a larger AA pool in TG, we studied neutrophils isolated from the bronchoalveolar lavage (BAL) of patients with adult respiratory distress syndrome. BAL neutrophils had a fourfold increase in cellular AA as compared with blood neutrophils and contained 25% of their AA in TG versus 3% in blood neutrophils. BAL neutrophils also had a higher number of cytoplasmic lipid bodies (8 +/- 3/cell) relative to blood neutrophils (2 +/- 1/cell). High concentrations of free AA were also found in the cell-free BAL fluid of adult respiratory distress syndrome patients. To explore whether changes in BAL neutrophils may be due to the exposure of the cells to high concentrations of exogenous AA found in BAL, we incubated blood neutrophils in culture with AA (10-100 microM) for 24 h. Neutrophils supplemented with AA had a 10-fold increase in the amount of AA associated with TG and a sixfold increase in the number of lipid bodies. In addition, supplementation with AA induced a dose-dependent formation of hypodense cells. Taken together, these data indicate that human inflammatory cells undergo a fundamental and consistent remodeling of AA pools as they mature or enter the lung from the blood. These biochemical and morphological changes can be mimicked in vitro by exposing the cells to high levels of AA. This mechanism may be responsible for the changes in AA mobilization and eicosanoid metabolism observed in tissue inflammatory cells.

2003;123;372S Chest David L. Bowton, Michael C. Seeds and David A. Bass * Cationic Protein Concentration in Sputum Saliva Does Not Correlate With Eosinophil Eosinophil Cationic Protein Concentration in http://chestjournal.chestpubs.org/content/123/3_suppl/372S.1.full.html services can be found online on the World Wide Web at: The online version of this article, along with updated information and ISSN:0012-3692 ) http://chestjournal.chestpubs.org/site/misc/reprints.xhtml ( written permission of the copyright holder. this article or PDF may be reproduced or distributed without the prior Dundee Road, Northbrook, IL 60062. All rights reserved. No part of Copyright2003by the American College of Chest Physicians, 3300 Physicians. It has been published monthly since 1935. is the official journal of the American College of Chest Chest

Early events of stimulus-response coupling in polymorphonuclear leukocytes (PMNL) reportedly include membrane depolarization as a necessary antecedent to oxidative responses. However, depolarization by nonspecific means (ionophores) is insufficient to elicit an oxidative burst. This apparent conflict might be related to whether depolarization is due to membrane receptor-mediated stimulation of PMNL. We used two fluorescent probes and dual laser flow cytometry to monitor both membrane potential and oxidative product formation in individual PMNL, following stimulation by phorbol myristate acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP). Dipentylindocarbocyanine [di-I-C5(3)] is one of a family of dyes that partition between cells and aqueous media as a function of transmembrane potential. The dye appeared stable in the presence of PMNL oxidative products. Oxidation of intracellularly trapped, nonfluorescent dichlorofluorescin (DCFH) to fluorescent dichlorofluorescein (DCF) provided a quantitative assessment of oxidative metabolism (H2O2 production) of stimulated PMNL. Incubating PMNL with both fluorochromes resulted in stable red [di-l-C5(3)] and green (DCF) fluorescence when examined on a Cytofluorograph. Upon stimulation by 0.03 to 0.1 nmol/L PMA, PMNL showed a unimodal apparent depolarization (decrease in di-l-C5(3) fluorescence). Oxidative activity (increased DCF fluorescence) was first seen at a concentration of PMA of 0.17 nmol/L, higher than that required to elicit depolarization. This oxidative burst appeared as a dose-dependent, graded response. Thus, receptor-mediated membrane depolarization, although antecedent to the onset of the oxidative burst, was not in itself sufficient to trigger the oxidative metabolic response. When PMNL were isolated by centrifugation through Ficoll-metrizoate, fMLP caused an apparent depolarization of a variable subpopulation of the cells. However, such purified PMNL appeared relatively unstable and often depolarized spontaneously. PMNL studied without centrifugation through Ficoll-metrizoate were stable. Moreover, fMLP stimulation of such cells did not cause membrane depolarization but did stimulate a two- to six-fold increase in DCFH oxidation. Apparently, membrane depolarization may antecede oxidative responses in PMNL, but appears to depend upon the method of cell preparation and the nature of the stimulus.

The effect of influenza A virus on the endocytic pathway in polymorphonuclear leukocytes (PMNLs) and the relationship of altered endocytic activity to virus-induced inhibition of other PMNL functions were examined with virus that caused decreased phagosomelysosome fusion and bacterial killing (depressing virus [DV]) and virus that did not (non-DV). Binding of both viruses to PMNL surface receptors was similar, but uptake of DV into PMNLs was decreased compared with that of non-DV. Both viruses were associated with the PMNL plasma membrane and were in endosomes. DV caused less stimulation of pinocytosis than did non-DV. The rate of exocytosis of fluoresceinated-dextran (FL-dextran) from cells stimulated with DV was significantly less than for non-DV. When PMNLs were pretreated with buffer, DV, or non-DV and then exposed to FL-dextran and N-formylmethionylleucylphenylalanine, the pinocytosis of FL-dextran was significantly less in cells pretreated with DV as compared with non-DV or buffer.

Recombinant human interleukin-2 (IL-2) was studied to determine effects on acid production by individual osteoclasts in situ on mouse calvarial bones. This analysis was performed using a microspectrofluorimetric technique to quantify acid production in individual cells. Radioactive calcium release was determined using calvarial bones in a standard tissue culture system. This allowed us to correlate changes in acid production with a measure of bone resorption. IL-2 stimulated acid production and bone resorbing activity. Both effects were inhibited by calcitonin. No stimulation of bone resorption occurred when IL-2-containing test media was incubated with a specific anti-IL-2 antibody and ultrafiltered. Our data demonstrated a correlation between acid production and bone resorbing activity in mouse calvaria exposed to parathyroid hormone (PTH). The data obtained from cultured mouse calvaria exposed to IL-2 demonstrated similar stimulatory effects to those seen during PTH exposure. These data suggest that calvaria exposed to IL-2 in vitro have increased osteoclastic acid production corresponding with increased bone resorption.