[Show abstract][Hide abstract] ABSTRACT: Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. While lacking a nucleus, platelets contain functional mitochondria. Upon activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile non-hemolytic reactions, skin manifestations and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (i.e. lysophospholipids, fatty acids and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused-animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.
[Show abstract][Hide abstract] ABSTRACT: We report new substrates for quantitative enzyme activity measurements of human palmitoyl protein thioesterase (PPT1) and tripeptidyl peptidase (TPP1) in dried blood spots from newborns using tandem mass spectrometry. Deficiencies in these enzyme activities due to inborn errors of metabolism cause neuronal ceroid lipofuscinoses. The assays use synthetic compounds that were designed to mimic the natural substrates. Incubation produces nanomole quantities of enzymatic products per a blood spot that are quantified by tandem mass spectrometry using synthetic internal standards and selected reaction monitoring. The assays utilize a minimum steps for sample work up and can be run in a duplex format for the detection of neuronal ceroid lipofuscinoses or potentially multiplexed with other mass spectrometry-based assays for newborn screening of lysosomal storage disorders.
[Show abstract][Hide abstract] ABSTRACT: Metabolic disorders, including obesity and insulin resistance, have their basis in dysregulated lipid metabolism and low-grade inflammation. In a microarray search of unique lipase-related genes whose expressions are associated with obesity, we found that two secreted phospholipase A2s (sPLA2s), PLA2G5 and PLA2G2E, were robustly induced in adipocytes of obese mice. Analyses of Pla2g5(-/-) and Pla2g2e(-/-) mice revealed distinct roles of these sPLA2s in diet-induced obesity. PLA2G5 hydrolyzed phosphatidylcholine in fat-overladen low-density lipoprotein to release unsaturated fatty acids, which prevented palmitate-induced M1 macrophage polarization. As such, PLA2G5 tipped the immune balance toward an M2 state, thereby counteracting adipose tissue inflammation, insulin resistance, hyperlipidemia, and obesity. PLA2G2E altered minor lipoprotein phospholipids, phosphatidylserine and phosphatidylethanolamine, and moderately facilitated lipid accumulation in adipose tissue and liver. Collectively, the identification of "metabolic sPLA2s" adds this gene family to a growing list of lipolytic enzymes that act as metabolic coordinators.
[Show abstract][Hide abstract] ABSTRACT: Tandem mass spectrometry for the multiplex and quantitative analysis of enzyme activities in dried blood spots on newborn screening cards has emerged as a powerful technique for early assessment of lysosomal storage diseases. Here we report the design and process-scale synthesis of substrates for the enzymes alpha-L-iduronidase, iduronate-2-sulfatase, and N-acetylgalactosamine-4-sulfatase that are used for newborn screening of mucopolysaccharidosis types I, II and VI. The products contain a bis-amide unit that is hypothesized to readily protonate in the gas phase, which improves detection sensitivity by tandem mass spectrometry. The products contain a benzoyl group, which provides a useful site for inexpensive deuteration, thus facilitating the preparation of internal standards for the accurate quantification of enzymatic products. Finally, the reagents are designed with ease of synthesis in mind, thus permitting scale up preparation to support worldwide newborn screening of lysosomal storage diseases. The new reagents provide the most sensitive assay for the 3 lysosomal enzymes reported to date as shown by their performance in reactions using dried blood spots as the enzyme source. Also, the ratio of assay signal to that measured in the absence of blood (background) is superior to all previously reported mucopolysaccharidosis types I, II an VI assays.
[Show abstract][Hide abstract] ABSTRACT: Sandhoff disease is a rare progressive neurodegenerative genetic disorder with a high incidence among certain isolated communities and ethnic groups around the world. Previous reports have shown a high occurrence of Sandhoff disease in northern Saskatchewan. Newborn screening cards from northern Saskatchewan were retrospectively screened in order to investigate the incidence and determine the carrier frequency of Sandhoff disease in these communities. PCR-based screening was conducted for the c.115delG (p.(Val39fs)) variant in the HEXB gene that was previously found in 4 Sandhoff disease patients from this area. The carrier frequency for this allele was estimated to be ~1:27. MS/MS-based screening of hexosaminidase activity along with genetic sequencing allowed for the identification of additional variants based on low total hexosaminidase activity and high % hexosaminidase A activity relative to c.115delG carriers. In total 4 pathogenic variants were discovered in the population (c.115delG, c.619A>G, c.1601G>T, and c.1652G>A) of which two are previously unreported (c.1601G>T and c.1652G>A). The combined carrier frequency of these alleles in the study area was estimated at ~1:15. Based on the number of cases of Sandhoff disease from this area we estimate the incidence to be ~1:390 corresponding to a child being born with the disease every 1-2years on average. The results from our study were then compared with variants in the HEXB gene from the genomes available from the 1000 Genomes project. A total of 19 HEXB variants were found in the 1092 genomes of which 5 are suspected of having a deleterious effect on hexosaminidase activity. The estimated carrier frequency of Sandhoff disease in Saskatchewan at 1:15 is more than 3 times higher than the carrier frequency in the global sample provided by the 1000 Genomes project at 1:57.
Molecular Genetics and Metabolism 01/2014; · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX−/−) model and found that survival after infection was significantly greater in GX−/− mice than in GX+/+ mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower in GX−/− mice BAL fluid. Lung microarray analysis identified an earlier and more robust induction of T and B cell associated genes in GX−/− mice. Based on the central role of sPLA2 enzymes as key initiators of inflammatory processes, we propose that activation of GX-sPLA2 during H1N1pdm infection is an early step of pulmonary inflammation and its inhibition increases adaptive immunity and improves survival. Our findings suggest that GX-sPLA2 may be a potential therapeutic target during influenza.
Virology 01/2014; s 454–455:78–92. · 3.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Treatments are being developed for metachromatic leukodystrophy (MLD), suggesting the need for eventual newborn screening. Previous studies have shown that sulfatide molecular species are increased in the urine of MLD patients compared to samples from non-MLD individuals, but there is no data using dried blood spots (DBS), the most common sample available for newborn screening laboratories.
We used ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) to quantify sulfatides in DBS and dried urine spots from 14 MLD patients and 50 non-MLD individuals.
Several sulfatide molecular species were increased in dried urine samples from all MLD samples compared to non-MLD samples. Sulfatides, especially low molecular species, were increased in DBS from MLD patients, but the sulfatide levels were relatively low. There was good separation in sulfatide levels between MLD and non-MLD samples when dried urine spots were used, but not with DBS, because DBS from non-MLD individuals have measurable levels of sulfatides.
Sulfatide accumulation studies in urine, but not in DBS, emerges as the method of choice if newborn screening is to be proposed for MLD.
Clinica chimica acta; international journal of clinical chemistry 12/2013; · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of human African trypanosomiasis, led to the identification of substituted 2-(3-aminophenyl)oxazolopyridines as a starting point for hit-to-lead medicinal chemistry. A total of 110 analogs were prepared, which led to the identification of 87, a substituted 2-(3-aminophenyl)imidazopyridine. This compound showed antiparasitic activity of parasites in vitro with an EC50 of 2 nM and displayed reasonable drug-like properties when tested in a number of in vitro assays. The compound was orally bioavailable and displayed good plasma and brain exposure in mice. Compound 87 cured mice infected with Trypanosoma brucei when dosed orally down to 2.5 mg/kg. Given its potent anti-parasitic properties and its ease of synthesis, compound 87 represents a new lead for the development of drugs to treat human African trypanosomiasis.
Journal of Medicinal Chemistry 12/2013; · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, LDH release and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2α (cPLA2α) or calcium independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2α-deficient fibroblasts was inhibited by the cPLA2α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calmodulin-dependent protein kinase II, a mitochondrial Ca2+ uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and by H2O2. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and OAG but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation and necrotic cell death.
Journal of Biological Chemistry 12/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mammalian epidermis provides both an interface and a protective barrier between the organism and its environment. Lipid, processed into water-impermeable bilayers between the outermost layers of the epidermal cells, forms the major barrier that prevents water from exiting the organism, and also prevents toxins and infectious agents from entering. The secretory phospholipase 2 (sPLA2) enzymes control important processes in skin and other organs, including inflammation and differentiation. sPLA2 activity contributes to epidermal barrier formation and homeostasis by generating free fatty acids, which are required both for formation of lamellar membranes and also for acidification of the stratum corneum (SC). sPLA2 is especially important in controlling SC acidification and establishment of an optimum epidermal barrier during the first postnatal week. Several sPLA2 isoforms are present in the epidermis. We find that two of these isoforms, sPLA2 IIA and sPLA2 IIF, localize to the upper stratum granulosum and increase in response to experimental barrier perturbation. sPLA2F (-/-) mice also demonstrate a more neutral SC pH than do their normal littermates, and their initial recovery from barrier perturbation is delayed. These findings confirm that sPLA2 enzymes perform important roles in epidermal development, and suggest that the sPLA2IIF isoform may be central to SC acidification and barrier function.
Biochimica et Biophysica Acta 11/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exercise-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.
We 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.
Endobronchial 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.
Tryptase 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.
Mast 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.
The Journal of allergy and clinical immunology 11/2013; · 12.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Little is known about the physiological role of the phospholipase A2 receptor (PLA2R1). PLA2R1 has been described to regulate the replicative senescence, a telomerase-dependant proliferation arrest. The downstream PLA2R1 signaling and its role on cancer are currently unknown. Senescence induction in response to activated oncogenes is a failsafe program of tumor suppression that must be bypassed for tumorigenesis. We now present evidence that PLA2R1 functions in vitro as a tumor suppressor the depletion of which is sufficient to escape oncogene-induced senescence (OIS), thereby facilitating oncogenic cell transformation. Further, mice that are genetically deficient in PLA2R1 display increased sensitivity to RAS-induced tumorigenesis by facilitating OIS escape, highlighting its physiological role as a tumor suppressor. Unexpectedly, PLA2R1 activated JAK2 and its effector signaling, with PLA2R1-mediated inhibition of cell transformation largely reverted in JAK2-depleted cells. This result inding was unexpected as the JAK2 pathway has been associated mainly with pro-tumoral functions and several inhibitors are currently in clinical trials. Taken together, our findings uncover an unanticipated tumor suppressive role for PLA2R1 that is mediated by targeting downstream JAK2 effector signaling.
[Show abstract][Hide abstract] ABSTRACT: Quantitative analysis of fatty acids (FAs) is an important area of analytical biochemistry. Ultra high sensitivity FA analysis usually is done with gas chromatography of pentafluorobenzyl esters coupled to an electron capture detector. With the popularity of electrospray ionization mass spectrometers coupled to liquid chromagraphy, it would be convenient to develop a method for ultra high sensitivity FA detection using this equipment. Although fatty acids can be analyzed by electrospray ionization in negative ion mode, this method is not very sensitive. In this study, we demonstrate a new method of FA analysis based on conversion of the carboxylic acid to an amide bearing a permanent positive charge (N-(4-aminomethylphenyl)pyridinium) (AMPP) combined with analysis on a reverse phase liquid chromatography column coupled to an electrospray ionization mass spectrometer operating in positive ion mode. This leads to a ~60,000-fold increase in sensitivity compared to the same method carried out with underivatized FAs. The new method is about 10-fold more sensitive than the existing method of gas chromatography/electron-capture mass spectrometry of fatty acid pentafluorobenzyl esters. Furthermore, significant fragmentation of the precursor ions in the non-tag portion improves analytical specificity. We show that a large number of FA molecular species can be analyzed with this method in complex biological samples such as mouse serum.
The Journal of Lipid Research 08/2013; · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: New dialkylimidazole based sterol 14α-demethylase inhibitors were prepared and tested as potential anti-Trypanosoma cruzi agents. Previous studies had identified compound 2 as the most potent and selective inhibitor against parasite cultures. In addition, animal studies had demonstrated that compound 2 is highly efficacious in the acute model of the disease. However, compound 2 has a high molecular weight and high hydrophobicity, issues addressed here. Systematic modifications were carried out at four positions on the scaffold and several inhibitors were identified which are highly potent (EC50 <1nM) against T. cruzi in culture. The halogenated derivatives 36j, 36k, and 36p, display excellent activity against T. cruzi amastigotes, with reduced molecular weight and lipophilicity, and exhibit suitable physicochemical properties for an oral drug candidate.
[Show abstract][Hide abstract] ABSTRACT: Resolution of inflammation is an active process that is mediated in part by antiinflammatory lipid mediators. Although phospholipase A2 (PLA2) enzymes have been implicated in the promotion of inflammation through mobilizing lipid mediators, the molecular entity of PLA2 subtypes acting upstream of antiinflammatory lipid mediators remains unknown. Herein, we show that secreted PLA2 group IID (PLA2G2D) is preferentially expressed in CD11c(+) dendritic cells (DCs) and macrophages and displays a pro-resolving function. In hapten-induced contact dermatitis, resolution, not propagation, of inflammation was compromised in skin and LNs of PLA2G2D-deficient mice (Pla2g2d(-/-)), in which the immune balance was shifted toward a proinflammatory state over an antiinflammatory state. Bone marrow-derived DCs from Pla2g2d(-/-) mice were hyperactivated and elicited skin inflammation after intravenous transfer into mice. Lipidomics analysis revealed that PLA2G2D in the LNs contributed to mobilization of a pool of polyunsaturated fatty acids that could serve as precursors for antiinflammatory/pro-resolving lipid mediators such as resolvin D1 and 15-deoxy-Δ(12,14)-prostaglandin J2, which reduced Th1 cytokine production and surface MHC class II expression in LN cells or DCs. Altogether, our results highlight PLA2G2D as a "resolving sPLA2" that ameliorates inflammation through mobilizing pro-resolving lipid mediators and points to a potential use of this enzyme for treatment of inflammatory disorders.
Journal of Experimental Medicine 05/2013; · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.
[Show abstract][Hide abstract] ABSTRACT: Rationale: Indirect airway hyperresponsiveness (AHR) is a fundamental feature of asthma that is manifest as exercise-induced bronchoconstriction (EIB). Secreted phospholipase A2 group X (sPLA2-X) plays a key role in regulating eicosanoid formation and the development of inflammation and AHR in murine models. Objectives: We sought to examine sPLA2-X in the airway epithelium and airway wall of patients with asthma, the relationship to AHR in humans, and the regulation and function of sPLA2-X within the epithelium. Methods: We precisely phenotyped 34 asthmatics (19 with and 15 without EIB), and 10 normal controls to examine in vivo differences in epithelial gene expression, quantitative morphometry of endobronchial biopsies, and levels of secreted protein. The regulation of sPLA2-X gene (PLA2G10) expression was examined in primary airway epithelial cell cultures. The function of epithelial sPLA2-X in eicosanoid formation was examined using PLA2 inhibitors and murine tracheal epithelial cells with Pla2g10 deletion. Measurements and Main Results: We found that sPLA2-X protein is increased in the airways of patients with asthma, and that epithelial-derived sPLA2-X may be increased in association with indirect AHR. The expression of sPLA2-X increases during in vitro epithelial differentiation, is regulated by inflammatory signals including TNF, IL-13, and IL-17, and is both secreted from the epithelium and directly participates in the release of arachidonic acid by epithelial cells. Conclusions: These data reveal a relationship between epithelial-derived sPLA2-X and indirect AHR in asthma, and that sPLA2-X serves as an epithelial regulator of inflammatory eicosanoid formation. Therapies targeting epithelial sPLA2-X may be useful in asthma.
American Journal of Respiratory and Critical Care Medicine 04/2013; · 11.04 Impact Factor