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Cecil R Paceasciak - One of the best experts on this subject based on the ideXlab platform.

  • a lipid mediator Hepoxilin a3 is a natural inducer of neutrophil extracellular traps in human neutrophils
    Mediators of Inflammation, 2015
    Co-Authors: David N Douda, Cecil R Paceasciak, Hartmut Grasemann, Nades Palaniyar
    Abstract:

    Pulmonary exacerbations in cystic fibrosis airways are accompanied by inflammation, neutrophilia, and mucous thickening. Cystic fibrosis sputum contains a large amount of uncleared DNA contributed by neutrophil extracellular trap (NET) formation from neutrophils. The exact mechanisms of the induction of NETosis in cystic fibrosis airways remain unclear, especially in uninfected lungs of patients with early cystic fibrosis lung disease. Here we show that Hepoxilin A3, a proinflammatory eicosanoid, and the synthetic analog of Hepoxilin B3, PBT-3, directly induce NETosis in human neutrophils. Furthermore, we show that Hepoxilin A3-mediated NETosis is NADPH-oxidase-dependent at lower doses of Hepoxilin A3, while it is NADPH-oxidase-independent at higher doses. Together, these results demonstrate that Hepoxilin A3 is a previously unrecognized inducer of NETosis in cystic fibrosis lungs and may represent a new therapeutic target for treating cystic fibrosis and other inflammatory lung diseases.

  • Hepoxilins in cancer and inflammation use of Hepoxilin antagonists
    Cancer and Metastasis Reviews, 2011
    Co-Authors: Cecil R Paceasciak
    Abstract:

    Cancer is often accompanied with inflammatory, thrombotic, and diabetic complications. Alternatively, chronic inflammation is believed to be a causative factor in several cancers. This review article brings together reported biological actions in these areas of the unstable naturally derived Hepoxilins (HX), metabolites of arachidonic acid formed through the 12-LO pathway, and those of their synthetically derived stable HX antagonists (PBT; proprietary bioactive therapeutics). Although the HX pathway has been known for some three decades since its discovery by the author with much data originating from the author's laboratory, studies by others over the past few years have confirmed early findings of the actions of HX as potent pro-inflammatory chemoattractant mediators and further showed HX to be involved in bacterial infection (Salmonella-induced intestinal inflammation and in bone inflammation caused by infection with the Lyme bacterium). The HX pathway appears to be an important early signal leading to inflammation. This provides important therapeutic potential for the PBTs as the only available selective antagonists of this pathway. The PBTs have shown benefit and efficacy in animal models of cancer and inflammation, which together with their known actions as anti-thrombotic (thromboxane (TPα) receptor antagonists) and hypoglycemic agents in vivo appears to make the PBTs suitable as therapeutics to control these disorders. The PBT structure is both stable in vivo and is essentially devoid of side effects in the animal models tested. The PBT structure serves as an important platform for selective HX and TX antagonists.

  • formation metabolism and action of Hepoxilin a3 in the rat pineal gland
    Journal of Neurochemistry, 2008
    Co-Authors: Denis Reynaud, Isabelle Delton, Abdallah Gharib, Nicole Sarda, Michel Lagarde, Cecil R Paceasciak
    Abstract:

    The present study was undertaken to investigate the possible formation of Hepoxilin A3 in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 microM) led to the appearance of Hepoxilin A3 (HxA3) analyzed as its stable trihydroxy derivative, trioxilin A3 by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA3 is estimated to be 1.43 +/- 0.66 ng/micrograms of protein. This amount is not modified when the tissue is boiled (2.07 +/- 0.66 ng/micrograms of protein). However, the formation of this compound was stimulated to 21.26 +/- 5.82 ng/micrograms of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 micrograms) resulted in a partial blockade of Hepoxilin formation. Using [1-14C]HxA3, we demonstrated that the pineal gland contained Hepoxilin epoxide hydrolase, which hydrolyzed HxA3 into trioxilin A3. This hydrolysis was inhibited by 1 mumol/L of 3,3,3-trichloropropene-1,2-oxide. In a separate study, HxA3 in the presence of 3,3,3-trichloropropene-1,2-oxide to block the hydrolysis of HxA3 decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5'-N-ethylcarboxamidoadenosine, an A1/A2 adenosine receptor agonist. This effect is stereospecific because the (8S)-enantiomer is more active in decreasing cyclic AMP production (-88.7%) than the (8R)-enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA3 in the rat pineal gland.

  • Hepoxilin analogs pbt 3 and pbt 4 cause apoptosis of gleevec resistant k562 cells in vitro
    in Vivo, 2007
    Co-Authors: Na Qiao, Denis Reynaud, Mohamed Abdelhaleem, Cecil R Paceasciak
    Abstract:

    The use of Gleevec ® in the treatment of leukemia has been widely accepted, although resistance to Gleevec is commonly observed. Gleevec represents a new direction in the development of target-focused chemotherapeutic agents in cancer. Gleevec inhibits the tyrosine kinase activity of Bcr-Abl, which is responsible for leukemic cell survival. We have previously shown that PBT-3 (racemic anti-10(R/S)-hydroxy- 11,12-cyclopropyl-eicosa-5Z, 8Z,14Z-trienoic acid methyl ester) and PBT-4 (racemic syn-10(R/S)-hydroxy-11,12-cyclopropyl- eicosa-5Z 8Z,14Z-trienoic acid methyl ester), stable analogs of the Hepoxilins, caused apoptosis of the human leukemic K562 cell line in vitro and in vivo. We also showed that PBTs inhibited the growth of tumours derived from the inoculation of immunodeficient mice with K562 cells and that the effect of PBTs was synergistic with that of Gleevec. We now show that the effect of PBT-3 and of PBT-4 is independent of that of Gleevec, demonstrating that Gleevec-resistant K562 cells retain their responsiveness to PBT treatment, resulting in apoptosis. These findings provide important information suggesting that the two compounds, PBT and Gleevec, can be used together in the treatment of leukemia. The PBTs may provide a new platform for the development of apoptotic drugs in cancer. Leukemia is a heterogeneous disease characterized by malignant proliferation of cells of the hematopoietic system. Leukemic cells contain a Bcr-Abl chimeric fusion protein,

  • Hepoxilin analogs potential new therapeutics in disease
    Current Pharmaceutical Design, 2006
    Co-Authors: Cecil R Paceasciak, Denis Reynaud, Peter Demin, N Qiao, Mohamed Abdelhaleem
    Abstract:

    We have chemically synthesized several stable analogs of the naturally occurring Hepoxilins, 12-LO products derived from arachidonic acid, which we found to have promising actions in a variety of test systems of disease. The analogs, PBTs, afford chemical and biological stability to the Hepoxilin molecule. This article reviews some of our latest observations with the PBTs in the areas of inflammation (inhibition of the bleomycin-evoked lung fibrosis in mice in vivo), platelet aggregation (antagonism of the thromboxane receptor in human platelets in vitro) and thrombosis (inhibitors in vivo), and cancer (apoptosis of the human leukemia cell line, K562 in vitro and in vivo). The demonstration that the PBTs are active in vivo suggests that they can serve as a platform for their further development as novel therapeutics in disease.

Santosh Nigam - One of the best experts on this subject based on the ideXlab platform.

  • Hepoxilin a3 protects β cells from apoptosis in contrast to its precursor 12 hydroperoxyeicosatetraenoic acid
    Biochimica et Biophysica Acta, 2011
    Co-Authors: Mariapatapia Zafiriou, Santosh Nigam, Laura C Zelarayan, Claudia Noack, Anke Renger, Athanassia Siafakakapadai
    Abstract:

    Abstract Pancreatic β-cells have a deficit of scavenging enzymes such as catalase (Cat) and glutathione peroxidase (GPx) and therefore are susceptible to oxidative stress and apoptosis. Our previous work showed that, in the absence of cytosolic GPx in insulinoma RINm5F cells, an intrinsic activity of 12 lipoxygenase (12(S)-LOX) converts 12S-hydroperoxyeicosatetraenoic acid (12(S)-HpETE) to the bioactive epoxide Hepoxilin A3 (HXA3). The aim of the present study was to investigate the effect of HXA3 on apoptosis as compared to its precursor 12(S)-HpETE and shed light upon the underlying pathways. In contrast to 12(S)-HpETE, which induced apoptosis via the extrinsic pathway, we found HXA3 not only to prevent it but also to promote cell proliferation. In particular, HXA3 suppressed the pro-apoptotic BAX and upregulated the anti-apoptotic Bcl-2. Moreover, HXA3 induced the anti-apoptotic 12(S)-LOX by recruiting heat shock protein 90 (HSP90), another anti-apoptotic protein. Finally, a co-chaperone protein of HSP90, protein phosphatase 5 (PP5), was upregulated by HXA3, which counteracted oxidative stress-induced apoptosis by dephosphorylating and thus inactivating apoptosis signal-regulating kinase 1 (ASK1). Taken together, these findings suggest that HXA3 protects insulinoma cells from oxidative stress and, via multiple signaling pathways, prevents them from undergoing apoptosis.

  • Hepoxilin a3 hxa3 synthase deficiency is causative of a novel ichthyosis form
    FEBS Letters, 2008
    Co-Authors: Santosh Nigam, Roberto Ciccoli, Mariapatapia Zafiriou, Rupal Deva, Nadja Kerstin, Christoph C Geilen, Marco Sczepanski, Maren Lohse
    Abstract:

    Non-bullous congenital ichthyosis erythroderma (NCIE) and lamellar ichthyosis (LI) are characterized by mutations in 12R-lipoxygenase (12R-LOX) and/or epidermal lipoxygenase 3 (eLOX3) enzymes. The eLOX3 lacks oxygenase activity, but is capable of forming Hepoxilin-type products from arachidonic acid-derived hydroperoxide from 12R-LOX, termed 12R-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12R-HpETE). Mutations in either of two enzymes lead to NCIE or LI. Moreover, 12R-LOX-deficient mice exhibit severe phenotypic water barrier dysfunctions. Here, we demonstrate that 12R-HpETE can also be transformed to 8R-HXA3 by Hepoxilin A3 (HXA3) synthase (12-lipoxygenase), which exhibits oxygenase activity. We also presented a novel form of ichthyosis in a patient, termed Hepoxilin A3 synthase-linked ichthyosis (HXALI), whose scales expressed high levels of 12R-LOX, but were deficient of HXA3 synthase.

  • Hepoxilin a3 synthase
    Biochemical and Biophysical Research Communications, 2005
    Co-Authors: Santosh Nigam, Mariapatapia Zafiriou
    Abstract:

    Hepoxilins constitute a group of 12S-hydroperoxyeicosatetraenoic acid (12S-HpETE)-derived epoxy-hydroxy fatty acids that have been detected in various cell types and tissues. Although Hepoxilin A3 (HXA3) exhibits a myriad of biological activities, its biosynthetic mechanism was not investigated in detail. Here we review the isolation, cloning, and characterization of a leukocyte-type 12S-lipoxygenase (12S-LOX) from rat insulinoma cells RINm5F, which exhibits an intrinsic Hepoxilin A3 synthase activity. Confirmation for this observation was achieved by coimmunoprecipitation of HXA3 synthase activity with an anti-leukocyte 12S-LOX antibody, preparation of recombinant rat 12S-LOX enzyme from RINm5F cells, and assay of HXA3 synthase activity therein. Site-directed mutagenesis studies performed on rat 12S-LOX showed that 12-lipoxygenating enzyme species exhibit a strong HXA3 synthase activity that is impaired when the positional specificity of arachidonic acid is altered in favor of 15-lipoxygenation. Inasmuch as cellular glutathione peroxidases (cGPx and PHGPx) and HXA3 synthase compete for the same substrate 12S-HpETE, it can be proposed that the overall activity of glutathione peroxidases, representing the overall peroxide tone, finely tunes the rate of HXA3 formation.

  • the rat leukocyte type 12 lipoxygenase exhibits an intrinsic Hepoxilin a3 synthase activity
    Journal of Biological Chemistry, 2004
    Co-Authors: Santosh Nigam, Shankarnarayanan Patabhiraman, Roberto Ciccoli, Ganchimeg Ishdorj, Kristin Schwarz, Branka Petrucev, Hartmut Kuhn, Jesper Z Haeggstrom
    Abstract:

    Hepoxilins are biologically relevant eicosanoids formed via the 12-lipoxygenase pathway of the arachidonic acid cascade. Although these eicosanoids exhibit a myriad of biological activities, their biosynthetic mechanism has not been investigated in detail. We examined the arachidonic acid metabolism of RINm5F rat insulinoma cells and found that they constitutively express a leukocyte-type 12S-lipoxygenase. Moreover, we observed that RINm5F cells exhibit an active Hepoxilin A3 synthase that converts exogenous 12S-HpETE (12S-5Z,8-Z,10E,14Z-12-hydro(pero)xy-eicosa-5,8,10,14-tetraenoic acid) or arachidonic acid predominantly to Hepoxilin A3. 12S-lipoxygenase and Hepoxilin A3 synthase activities were co-localized in the cytosol; immunoprecipitation with an anti-12S-lipoxygenase antibody co-precipitated the two catalytic activities. These data suggested that Hepoxilin A3 synthase activity may be considered an intrinsic catalytic property of the leukocyte-type 12S-lipoxygenase. To test this hypothesis we cloned the leukocyte-type 12S-LOX from RINm5F cells, expressed it in Pichia pastoris, and found that the recombinant enzyme exhibited both 12S-lipoxygenase and Hepoxilin A3 synthase activities. The recombinant human platelet-type 12S-lipoxygenase and the porcine leukocyte-type 12S-lipoxygenase also exhibited Hepoxilin A3 synthase activity. In contrast, the native rabbit reticulocyte-type 15S-lipoxygenase did not convert 12S-HpETE to Hepoxilin isomers. These data suggest that the positional specificity of lipoxygenases may be crucial for this catalytic function. This hypothesis was confirmed by site-directed mutagenesis studies that altered the positional specificity of the rat leukocyte-type 12S- and the rabbit reticulocyte-type 15-lipoxygenase. In summary, it may be concluded that naturally occurring 12S-lipoxygenases exhibit an intrinsic Hepoxilin A3 synthase activity that is minimal in lipoxygenase isoforms with different positional specificity.

  • biosynthesis of Hepoxilins evidence for the presence of a Hepoxilin synthase activity in rat insulinoma cells
    FEBS Letters, 2003
    Co-Authors: Pattabhiraman Shankaranarayanan, Roberto Ciccoli, Santosh Nigam
    Abstract:

    The 12(S)-lipoxygenase (12-LOX) pathway of arachidonic acid (AA) metabolism after dioxygenation to 12(S)-hydroperoxy-eicosatetraenoic acid is bifurcated in a reduction route to formation of 12(S)-hydroxy-eicosatetraenoic acid (12-HpETE) and an isomerization route to formation of Hepoxilins. Interestingly, we found that the rat insulinoma RINm5F cells, which are devoid of cytoplasmic glutathione peroxidase (cGPx)/phospholipid hydroperoxide glutathione peroxidase (PHGPx), produce solely Hepoxilin A(3) (HXA(3)). Since HXA(3) synthesis was abolished in heat-denatured or cGPx- or PHGPx-transfected cells, it was tempting to speculate that a HXA(3) synthase activity regulated by cGPx/PHGPx is present. To confirm this assumption we incubated AA with HeLa cells overexpressing the rat leukocyte-type 12-LOX. Neither HXA(3) nor 12(S)-HETE were detected due to abundance of cGPx/PHGPx. But, pretreatment of transfected cells with diethyl maleate, an inhibitor of glutathione and PHGPx, restored HXA(3) synthase and 12-LOX activities. Thus, we conclude, that cells containing rat leukocyte-type 12-LOX also possess an intrinsic HXA(3) synthase activity, which is activated by inhibition of cGPx/PHGPx. In normal cells HXA(3) is down-regulated by cGPx/PHGPx, but, it is persistently activated in oxidatively stressed cells deficient in cGPx/PHGPx, such as RINm5F.

Denis Reynaud - One of the best experts on this subject based on the ideXlab platform.

  • formation metabolism and action of Hepoxilin a3 in the rat pineal gland
    Journal of Neurochemistry, 2008
    Co-Authors: Denis Reynaud, Isabelle Delton, Abdallah Gharib, Nicole Sarda, Michel Lagarde, Cecil R Paceasciak
    Abstract:

    The present study was undertaken to investigate the possible formation of Hepoxilin A3 in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 microM) led to the appearance of Hepoxilin A3 (HxA3) analyzed as its stable trihydroxy derivative, trioxilin A3 by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA3 is estimated to be 1.43 +/- 0.66 ng/micrograms of protein. This amount is not modified when the tissue is boiled (2.07 +/- 0.66 ng/micrograms of protein). However, the formation of this compound was stimulated to 21.26 +/- 5.82 ng/micrograms of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 micrograms) resulted in a partial blockade of Hepoxilin formation. Using [1-14C]HxA3, we demonstrated that the pineal gland contained Hepoxilin epoxide hydrolase, which hydrolyzed HxA3 into trioxilin A3. This hydrolysis was inhibited by 1 mumol/L of 3,3,3-trichloropropene-1,2-oxide. In a separate study, HxA3 in the presence of 3,3,3-trichloropropene-1,2-oxide to block the hydrolysis of HxA3 decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5'-N-ethylcarboxamidoadenosine, an A1/A2 adenosine receptor agonist. This effect is stereospecific because the (8S)-enantiomer is more active in decreasing cyclic AMP production (-88.7%) than the (8R)-enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA3 in the rat pineal gland.

  • Hepoxilin analogs pbt 3 and pbt 4 cause apoptosis of gleevec resistant k562 cells in vitro
    in Vivo, 2007
    Co-Authors: Na Qiao, Denis Reynaud, Mohamed Abdelhaleem, Cecil R Paceasciak
    Abstract:

    The use of Gleevec ® in the treatment of leukemia has been widely accepted, although resistance to Gleevec is commonly observed. Gleevec represents a new direction in the development of target-focused chemotherapeutic agents in cancer. Gleevec inhibits the tyrosine kinase activity of Bcr-Abl, which is responsible for leukemic cell survival. We have previously shown that PBT-3 (racemic anti-10(R/S)-hydroxy- 11,12-cyclopropyl-eicosa-5Z, 8Z,14Z-trienoic acid methyl ester) and PBT-4 (racemic syn-10(R/S)-hydroxy-11,12-cyclopropyl- eicosa-5Z 8Z,14Z-trienoic acid methyl ester), stable analogs of the Hepoxilins, caused apoptosis of the human leukemic K562 cell line in vitro and in vivo. We also showed that PBTs inhibited the growth of tumours derived from the inoculation of immunodeficient mice with K562 cells and that the effect of PBTs was synergistic with that of Gleevec. We now show that the effect of PBT-3 and of PBT-4 is independent of that of Gleevec, demonstrating that Gleevec-resistant K562 cells retain their responsiveness to PBT treatment, resulting in apoptosis. These findings provide important information suggesting that the two compounds, PBT and Gleevec, can be used together in the treatment of leukemia. The PBTs may provide a new platform for the development of apoptotic drugs in cancer. Leukemia is a heterogeneous disease characterized by malignant proliferation of cells of the hematopoietic system. Leukemic cells contain a Bcr-Abl chimeric fusion protein,

  • Hepoxilin analogs potential new therapeutics in disease
    Current Pharmaceutical Design, 2006
    Co-Authors: Cecil R Paceasciak, Denis Reynaud, Peter Demin, N Qiao, Mohamed Abdelhaleem
    Abstract:

    We have chemically synthesized several stable analogs of the naturally occurring Hepoxilins, 12-LO products derived from arachidonic acid, which we found to have promising actions in a variety of test systems of disease. The analogs, PBTs, afford chemical and biological stability to the Hepoxilin molecule. This article reviews some of our latest observations with the PBTs in the areas of inflammation (inhibition of the bleomycin-evoked lung fibrosis in mice in vivo), platelet aggregation (antagonism of the thromboxane receptor in human platelets in vitro) and thrombosis (inhibitors in vivo), and cancer (apoptosis of the human leukemia cell line, K562 in vitro and in vivo). The demonstration that the PBTs are active in vivo suggests that they can serve as a platform for their further development as novel therapeutics in disease.

  • the Hepoxilin analog pbt 3 induces apoptosis in bcr abl positive k562 leukemia cells
    Anticancer Research, 2003
    Co-Authors: Na Qiao, Denis Reynaud, Mohamed Abdelhaleem, Janice Lam, Cecil R Paceasciak
    Abstract:

    Background: Leukemia is a heterogeneous disease characterized by malignant proliferation of cells of the hematopoietic system. The use of chemotherapeutic agents is still the mainstay of anti-leukemia therapy. Despite this, significant morbidity and mortality still occurs. We describe herein novel apoptotic effects of PBT-3, one of a family of stable analogs of the Hepoxilins, natural products derived from arachidonic acid. Materials and Methods: Inhibition of [ 3 H]-thymidine incorporation, nuclear fragmentation, DNA laddering, FACS analysis as well as Annexin V binding were assessed. Results: PBT-3 dose-dependently causes apoptosis of the CML cell line, K562, in vitro. PBT-3 acts by increasing cytochrome c release into the cytoplasm and by activation of caspase-3 degradation. The effects of PBT-3 compare favorably with those of STI571 (Gleevec), while thromboxane agonists and antagonists are without effect. Conclusion: These results suggest that PBT analogs may provide a new platform for the development of apoptotic drugs in leukemia.

  • the Hepoxilin stable analogue pbt 3 inhibits primary platelet related hemostasis in whole blood measured in vitro with the pfa 100
    Thrombosis Research, 2003
    Co-Authors: Denis Reynaud, Dewi Clark, Na Qiao, Margaret L Rand, Cecil R Paceasciak
    Abstract:

    Hepoxilins are hydroxy epoxide metabolites of arachidonic acid formed through a coupled reaction involving 12(S)-lipoxygenase and the putative enzyme, Hepoxilin synthase, that transforms 12S-HPETE into the Hepoxilins (Fig. 1) (see Refs. [1–3] for reviews). Hepoxilins possess a variety of biological actions in vitro as well as in vivo including insulin release from pancreatic islets, potentiation of vascular contraction, blockade of neurotransmitter release, regulation of cell volume and they provoke skin vascular permeability. Because the native Hepoxilins are unstable chemically, a new family of stable analogues, PBTs, designed to have chemical and biological stability suitable for in vivo studies, were prepared by total chem-

Beth A Mccormick - One of the best experts on this subject based on the ideXlab platform.

  • pneumolysin induces 12 lipoxygenase dependent neutrophil migration during streptococcus pneumoniae infection
    Journal of Immunology, 2020
    Co-Authors: Walter Adams, Rodney K Tweten, Beth A Mccormick, Kristin R Wade, Rudra Bhowmick, Elsa Bou N Ghanem, Mikhail Shchepetov, Jeffrey N Weiser, John M Leong
    Abstract:

    Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator Hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting Hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.

  • systemic disease during streptococcus pneumoniae acute lung infection requires 12 lipoxygenase dependent inflammation
    Journal of Immunology, 2013
    Co-Authors: Rudra Bhowmick, Bryan P Hurley, Beth A Mccormick, Elsa Bou N Ghanem, Karsten Gronert, Nang Tin H Maung, John M Leong
    Abstract:

    Acute pulmonary infection by Streptococcus pneumoniae is characterized by high bacterial numbers in the lung, a robust alveolar influx of polymorphonuclear cells (PMNs), and a risk of systemic spread of the bacterium. We investigated host mediators of S. pneumoniae-induced PMN migration and the role of inflammation in septicemia following pneumococcal lung infection. Hepoxilin A3 (HXA3) is a PMN chemoattractant and a metabolite of the 12-lipoxygenase (12-LOX) pathway. We observed that S. pneumoniae infection induced the production of 12-LOX in cultured pulmonary epithelium and in the lungs of infected mice. Inhibition of the 12-LOX pathway prevented pathogen-induced PMN transepithelial migration in vitro and dramatically reduced lung inflammation upon high-dose pulmonary challenge with S. pneumoniae in vivo, thus implicating HXA3 in pneumococcus-induced pulmonary inflammation. PMN basolateral-to-apical transmigration in vitro significantly increased apical-to-basolateral transepithelial migration of bacteria. Mice suppressed in the expression of 12-LOX exhibited little or no bacteremia and survived an otherwise lethal pulmonary challenge. Our data suggest that pneumococcal pulmonary inflammation is required for high-level bacteremia and systemic infection, partly by disrupting lung epithelium through 12-LOX–dependent HXA3 production and subsequent PMN transepithelial migration.

  • Hepoxilin a3 facilitates neutrophilic breach of lipoxygenase expressing airway epithelial barriers
    Journal of Immunology, 2012
    Co-Authors: David L Tamang, Bruce D. Hammock, John R Falck, Christophe Morisseau, Beth A Mccormick, Waheed Pirzai, Gregory P Priebe, David C Traficante, Gerald B Pier, Karsten Gronert
    Abstract:

    A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A(3) is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of Hepoxilin A(3) in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate Hepoxilin A(3), including phospholipase A(2) and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate Hepoxilin A(3) precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing Hepoxilin A(3) are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased Hepoxilin A(3) and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with Hepoxilin A(3) serving a chemotactic role. Stable inhibitory structural analogs of Hepoxilin A(3) are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites Hepoxilin A(3) and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that Hepoxilin A(3) is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.

  • of lipoxygenase expressing airway facilitates neutrophilic breach 3 Hepoxilin a
    2012
    Co-Authors: Karsten Gronert, Bruce D. Hammock, John R Falck, Bryan P Hurley, Beth A Mccormick, Gerald B Pier, C Traficante, L Tamang, Gregory P Priebe
    Abstract:

    Karsten Gronert and Bryan P. HurleyMorisseau, Bruce D. Hammock, Beth A. McCormick,C. Traficante, Gerald B. Pier, John R. Falck, Christophe David L. Tamang, Waheed Pirzai, Gregory P. Priebe, Davidhttp://www.jimmunol.org/content/189/10/4960doi: 10.4049/jimmunol.1201922October 2012;J Immunol€2012; 189:4960-4969; Prepublished online 8Referenceshttp://www.jimmunol.org/content/189/10/4960.full#ref-list-1This article cites 63 articles, 31 of which you can access for free at: Subscriptionshttp://jimmunol.org/subscriptionsInformation about subscribing to The Journal of Immunology is online at: Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at: Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

  • the erm protein ezrin regulates neutrophil transmigration by modulating the apical localization of mrp2 in response to the sipa effector protein during salmonella typhimurium infection
    Cellular Microbiology, 2011
    Co-Authors: Terence A Agbor, Jeffrey D Bien, Karen L Mumy, Zachary Demma, Beth A Mccormick
    Abstract:

    In human disease induced by Salmonella enterica serovar Typhimurium (S. Typhimurium), transepithelial migration of neutrophils rapidly follows attachment of the bacteria to the epithelial apical membrane. We have previously shown that during S. Typhimurium infection the multidrug resistance-associated protein 2 (MRP2) is highly expressed at the apical surface of the intestinal epithelia, and that it functions as an efflux pump for the potent neutrophil chemoattractant Hepoxilin A(3) . However, the molecular mechanisms regulating its apical localization during active states of inflammation remain unknown. Thus, our objective was to determine the mechanistic basis for the translocation of MRP2 to the apical surface of intestinal epithelial cells during S. Typhimurium infection. We show that suppression of ezrin, through either RNAi or truncation of the C-terminus, results not only in a decrease in S. Typhimurium-induced neutrophil transmigration but also significantly attenuates the apical membrane expression of MRP2 during Salmonella infection. In addition, we determined that S. Typhimurium induces the activation of ezrin via a PKC-α-dependent pathway and that ezrin activation is coupled to apical localization of MRP2. Based on these results we propose that activation of ezrin is required for the apical localization of MRP2 during S. Typhimurium infection.

Bryan P Hurley - One of the best experts on this subject based on the ideXlab platform.

  • pseudomonas aeruginosa exou augments neutrophil transepithelial migration
    PLOS Pathogens, 2017
    Co-Authors: Michael A Pazos, Waheed Pirzai, Karsten Gronert, Lael M Yonker, Bernard B Lanter, Joseph V Bonventre, Alex D Eaton, Bryan P Hurley
    Abstract:

    Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce Hepoxilin A3 (HXA3), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.

  • distinct cellular sources of Hepoxilin a3 and leukotriene b4 are used to coordinate bacterial induced neutrophil transepithelial migration
    Journal of Immunology, 2015
    Co-Authors: Michael A Pazos, Christophe Morisseau, Waheed Pirzai, Karsten Gronert, Lael M Yonker, Bryan P Hurley
    Abstract:

    Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states, including cystic fibrosis. Hepoxilin A 3 (HXA 3 ) is a chemotactic eicosanoid shown to mediate the transepithelial passage of neutrophils in response to infection in several model systems and at multiple mucosal surfaces. Another well-known eicosanoid mediating general neutrophil chemotaxis is leukotriene B 4 (LTB 4 ). We sought to distinguish the roles of each eicosanoid in the context of infection of lung epithelial monolayers by Pseudomonas aeruginosa . Using human and mouse in vitro transwell model systems, we used a combination of biosynthetic inhibitors, receptor antagonists, as well as mutant sources of neutrophils to assess the contribution of each chemoattractant in driving neutrophil transepithelial migration. We found that following chemotaxis to epithelial-derived HXA 3 signals, neutrophil-derived LTB 4 is required to amplify the magnitude of neutrophil migration. LTB 4 signaling is not required for migration to HXA 3 signals, but LTB 4 generation by migrated neutrophils plays a significant role in augmenting the initial HXA 3 -mediated migration. We conclude that HXA 3 and LTB 4 serve independent roles to collectively coordinate an effective neutrophilic transepithelial migratory response.

  • pathogen induced chemo attractant Hepoxilin a3 drives neutrophils but not eosinophils across epithelial barriers
    Prostaglandins & Other Lipid Mediators, 2014
    Co-Authors: S A Kubala, Sarita U Patil, Wayne G Shreffler, Bryan P Hurley
    Abstract:

    Pathogen induced migration of neutrophils across mucosal epithelial barriers requires epithelial production of the chemotactic lipid mediator, Hepoxilin A3 (HXA3). HXA3 is an eicosanoid derived from arachidonic acid. Although eosinophils are also capable of penetrating mucosal surfaces, eosinophilic infiltration occurs mainly during allergic processes whereas neutrophils dominate mucosal infection. Both neutrophils and eosinophils can respond to chemotactic gradients of certain eicosanoids, however, it is not known whether eosinophils respond to pathogen induced lipid mediators such as HXA3. In this study, neutrophils and eosinophils were isolated from human blood and placed on the basolateral side of polarized epithelial monolayers grown on permeable Transwell filters and challenged by various chemotactic gradients of distinct lipid mediators. We observed that both cell populations migrated across epithelial monolayers in response to a leukotriene B4 (LTB4) gradient, whereas only eosinophils migrated toward a prostaglandin D2 (PGD2) gradient. Interestingly, while pathogen induced neutrophil trans-epithelial migration was substantial, pathogen induced eosinophil trans-epithelial migration was not observed. Further, gradients of chemotactic lipids derived from pathogen infected epithelial cells known to be enriched for HXA3 as well as purified HXA3 drove significant numbers of neutrophils across epithelial barriers, whereas eosinophils failed to respond to these gradients. These data suggest that although the eicosanoid HXA3 serves as an important neutrophil chemo-attractant at mucosal surfaces during pathogenic infection, HXA3 does not appear to exhibit chemotactic activity toward eosinophils.

  • systemic disease during streptococcus pneumoniae acute lung infection requires 12 lipoxygenase dependent inflammation
    Journal of Immunology, 2013
    Co-Authors: Rudra Bhowmick, Bryan P Hurley, Beth A Mccormick, Elsa Bou N Ghanem, Karsten Gronert, Nang Tin H Maung, John M Leong
    Abstract:

    Acute pulmonary infection by Streptococcus pneumoniae is characterized by high bacterial numbers in the lung, a robust alveolar influx of polymorphonuclear cells (PMNs), and a risk of systemic spread of the bacterium. We investigated host mediators of S. pneumoniae-induced PMN migration and the role of inflammation in septicemia following pneumococcal lung infection. Hepoxilin A3 (HXA3) is a PMN chemoattractant and a metabolite of the 12-lipoxygenase (12-LOX) pathway. We observed that S. pneumoniae infection induced the production of 12-LOX in cultured pulmonary epithelium and in the lungs of infected mice. Inhibition of the 12-LOX pathway prevented pathogen-induced PMN transepithelial migration in vitro and dramatically reduced lung inflammation upon high-dose pulmonary challenge with S. pneumoniae in vivo, thus implicating HXA3 in pneumococcus-induced pulmonary inflammation. PMN basolateral-to-apical transmigration in vitro significantly increased apical-to-basolateral transepithelial migration of bacteria. Mice suppressed in the expression of 12-LOX exhibited little or no bacteremia and survived an otherwise lethal pulmonary challenge. Our data suggest that pneumococcal pulmonary inflammation is required for high-level bacteremia and systemic infection, partly by disrupting lung epithelium through 12-LOX–dependent HXA3 production and subsequent PMN transepithelial migration.

  • of lipoxygenase expressing airway facilitates neutrophilic breach 3 Hepoxilin a
    2012
    Co-Authors: Karsten Gronert, Bruce D. Hammock, John R Falck, Bryan P Hurley, Beth A Mccormick, Gerald B Pier, C Traficante, L Tamang, Gregory P Priebe
    Abstract:

    Karsten Gronert and Bryan P. HurleyMorisseau, Bruce D. Hammock, Beth A. McCormick,C. Traficante, Gerald B. Pier, John R. Falck, Christophe David L. Tamang, Waheed Pirzai, Gregory P. Priebe, Davidhttp://www.jimmunol.org/content/189/10/4960doi: 10.4049/jimmunol.1201922October 2012;J Immunol€2012; 189:4960-4969; Prepublished online 8Referenceshttp://www.jimmunol.org/content/189/10/4960.full#ref-list-1This article cites 63 articles, 31 of which you can access for free at: Subscriptionshttp://jimmunol.org/subscriptionsInformation about subscribing to The Journal of Immunology is online at: Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at: Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at: