WorldWideScience

Sample records for human neutrophil granulocytes

  1. Toxicity of thienopyridines on human neutrophil granulocytes and lymphocytes.

    Science.gov (United States)

    Maseneni, Swarna; Donzelli, Massimiliano; Brecht, Karin; Krähenbühl, Stephan

    2013-06-07

    Thienopyridines can cause neutropenia and agranulocytosis. The aim of the current investigations was to compare cytotoxicity of ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel for human neutrophil granulocytes with the toxicity for lymphocytes and to investigate underlying mechanisms. For granulocytes, clopidogrel, ticlopidine, clopidogrel carboxylate and prasugrel were concentration-dependently toxic starting at 10μM. Cytotoxicity could be prevented by the myeloperoxidase inhibitor rutin, but not by the cytochrome P450 inhibitor ketoconazole. All compounds were also toxic for lymphocytes, but cytotoxicity started at 100μM and could not be prevented by rutin or ketoconazole. Granulocytes metabolized ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel, and metabolization was inhibited by rutin, but not by ketoconazole. Metabolism of these compounds by lymphocytes was much slower and could not be inhibited by ketoconazole or rutin. In neutrophils, all compounds investigated decreased the electrical potential across the inner mitochondrial membrane, were associated with cellular accumulation of ROS, mitochondrial loss of cytochrome c and induction of apoptosis starting at 10μM. All of these effects could be inhibited by rutin, but not by ketoconazole. Similar findings were obtained in lymphocytes; but compared to neutrophils, the effects were detectable only at higher concentrations and were not inhibited by rutin. In conclusion, ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel are toxic for both granulocytes and lymphocytes. In granulocytes, cytotoxicity is more accentuated than in lymphocytes and depends on metabolization by myeloperoxidase. These findings suggest a mitochondrial mechanism for cytotoxicity for both myeloperoxidase-associated metabolites and, at higher concentrations, also for the parent compounds. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  2. Alpha-1-antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis

    DEFF Research Database (Denmark)

    Clemmensen, Stine N; Jacobsen, Lars C; Rørvig, Sara

    2011-01-01

    Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associate...

  3. Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma

    DEFF Research Database (Denmark)

    Kharazmi, A; Nielsen, H; Hovgaard, D

    1991-01-01

    Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit the chemotaxis and enhance the oxidative burst response of human neutrophils in vitro. The present study describes the effect of recombinant GM-CSF on the neutrophil and monocyte function in patients with lymphoma...

  4. Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells

    Science.gov (United States)

    Hsing, Chung-Hsi; Chen, Chia-Ling; Lin, Wei-Chieh; Lin, Chiou-Feng

    2015-01-01

    Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells. PMID:26061531

  5. Effect of salivary agglutination on oral streptococcal clearance by human polymorphonuclear neutrophil granulocytes.

    Science.gov (United States)

    Itzek, A; Chen, Z; Merritt, J; Kreth, J

    2017-06-01

    Salivary agglutination is an important host defense mechanism to aggregate oral commensal bacteria as well as invading pathogens. Saliva flow and subsequent swallowing more easily clear aggregated bacteria compared with single cells. Phagocytic clearance of bacteria through polymorphonuclear neutrophil granulocytes also seems to increase to a certain extent with the size of bacterial aggregates. To determine a connection between salivary agglutination and the host innate immune response by phagocytosis, an in vitro agglutination assay was developed reproducing the average size of salivary bacterial aggregates. Using the oral commensal Streptococcus gordonii as a model organism, the effect of salivary agglutination on phagocytic clearance through polymorphonuclear neutrophil granulocytes was investigated. Here we describe how salivary aggregates of S. gordonii are readily cleared through phagocytosis, whereas single bacterial cells showed a significant delay in being phagocytosed and killed. Furthermore, before phagocytosis the polymorphonuclear neutrophil granulocytes were able to induce a specific de-aggregation, which was dependent on serine protease activity. The data presented suggest that salivary agglutination of bacterial cells leads to an ideal size for recognition by polymorphonuclear neutrophil granulocytes. As a first line of defense, these phagocytic cells are able to recognize the aggregates and de-aggregate them via serine proteases to a more manageable size for efficient phagocytosis and subsequent killing in the phagolysosome. This observed mechanism not only prevents the rapid spreading of oral bacterial cells while entering the bloodstream but would also avoid degranulation of involved polymorphonuclear neutrophil granulocytes, so preventing collateral damage to nearby tissue. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Granulocyte

    Science.gov (United States)

    Granulocytes are a type of white blood cell that has small granules, which contain proteins. The specific types of granulocytes are neutrophils, eosinophils, and basophils. Granulocytes, specifically neutrophils, ...

  7. Irradiation causes biphasic neutrophilic granulocyte phagocytic function

    Energy Technology Data Exchange (ETDEWEB)

    Micke, O. [Dept. of Radiotherapy, Muenster Univ. Hospital, Muenster (Germany); Haidenberger, A.; Auer, T.; Egger, S.; DeVries, A.F. [Dept. of Radiotherapy, Innsbruck Univ. Medical Hospital, Innsbruck (Austria); Seegenschmiedt, M.H. [Dept. of Radiotherapy, Alfried Krupp Hospital, Essen (Germany); Hengster, P. [Dept. of General and Transplant Surgery, Daniel Swarovski Research Lab., Innsbruck Univ. Medical Hospital, Innsbruck (Austria)

    2005-05-01

    Background and purpose: the anti-inflammatory effect of low-dose radiotherapy is clinically well described. Nevertheless, until now neither the optimal dose nor the background of tissue reactions have been defined. The current study examines the influence of low radiation doses on neutrophilic granulocyte function, which could be helpful in finding the optimal dose for either stimulation or suppression of anti-inflammatory activity. Material and methods: Lymphoprep registered density gradient-purified neutrophilic granulocytes of three voluntary, healthy donors were used for all experiments. Granulocytes were incubated 48 h in RPMI 1640 and irradiated with single doses of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, and 12 Gy using a {sup 137}Cs IBL 437L irradiator. Their function was assessed by measuring granulocytic release of reactive oxygen species (ROS) with luminol-enhanced chemiluminescence after stimulation with phorbol myristate acetate (PMA). Results: relative changes of ROS release (ROS release before stimulation was set to 100%) increased after stimulation with PMA (mean {+-} standard deviation [SD]): 0 Gy: 147.6% {+-} 60%; 0.5 Gy: 153.6% {+-} 70%; 1.0 Gy: 164.9% {+-} 63%; 1.5 Gy: 177.8% {+-} 66%; 2.0 Gy: 162.5% {+-} 57%; 2.5 Gy: 156.2% {+-} 60%; 3.0 Gy: 159.2% {+-} 60%; 3.5 Gy: 126.9% {+-} 55%; 4.0 Gy: 137.9% {+-} 71%; 6.0 Gy: 148.3% {+-} 65%; 12.0 Gy: 156.1% {+-} 52%. The relative ROS release showed a significant increase at 1.5 Gy (p < 0.001) after PMA stimulation and a significant decrease of ROS release at 3.5 Gy (p < 0.005) and less markedly at 4.0 Gy (p < 0.05). 6.0 and 12.0 Gy showed a significant (p < 0.05) increase again. Conclusion: this ex vivo in vitro study on native human neutrophilic granulocytes shows an increase at 1.5 Gy and a significant decrease of granulocyte function at 3.5 and 4.0 Gy, as it was described for different other phenomena in low-dose radiotherapy. These results may provide a further explanation for the local anti

  8. Effects of gadolinium oxide nanoparticles on the oxidative burst from human neutrophil granulocytes

    Science.gov (United States)

    Abrikossova, Natalia; Skoglund, Caroline; Ahrén, Maria; Bengtsson, Torbjörn; Uvdal, Kajsa

    2012-07-01

    We have previously shown that gadolinium oxide (Gd2O3) nanoparticles are promising candidates to be used as contrast agents in magnetic resonance (MR) imaging applications. In this study, these nanoparticles were investigated in a cellular system, as possible probes for visualization and targeting intended for bioimaging applications. We evaluated the impact of the presence of Gd2O3 nanoparticles on the production of reactive oxygen species (ROS) from human neutrophils, by means of luminol-dependent chemiluminescence. Three sets of Gd2O3 nanoparticles were studied, i.e. as synthesized, dialyzed and both PEG-functionalized and dialyzed Gd2O3 nanoparticles. In addition, neutrophil morphology was evaluated by fluorescent staining of the actin cytoskeleton and fluorescence microscopy. We show that surface modification of these nanoparticles with polyethylene glycol (PEG) is essential in order to increase their biocompatibility. We observed that the as synthesized nanoparticles markedly decreased the ROS production from neutrophils challenged with prey (opsonized yeast particles) compared to controls without nanoparticles. After functionalization and dialysis, more moderate inhibitory effects were observed at a corresponding concentration of gadolinium. At lower gadolinium concentration the response was similar to that of the control cells. We suggest that the diethylene glycol (DEG) present in the as synthesized nanoparticle preparation is responsible for the inhibitory effects on the neutrophil oxidative burst. Indeed, in the present study we also show that even a low concentration of DEG, 0.3%, severely inhibits neutrophil function. In summary, the low cellular response upon PEG-functionalized Gd2O3 nanoparticle exposure indicates that these nanoparticles are promising candidates for MR-imaging purposes.

  9. Neutrophil granulocytes promote the migratory activity of MDA-MB-468 human breast carcinoma cells via ICAM-1.

    Science.gov (United States)

    Strell, Carina; Lang, Kerstin; Niggemann, Bernd; Zaenker, Kurt S; Entschladen, Frank

    2010-01-01

    Tumor infiltrating neutrophil granulocytes do not only exhibit tumor eliminating functions but also promote tumor progression. We have recently shown that neutrophil granulocytes can serve as linking cells for the adhesion of MDA-MB-468 breast carcinoma cells to pulmonary endothelium. Neutrophil granulocytes but not MDA-MB-468 cells express beta(2)-integrins, the ligands of the intercellular adhesion molecule (ICAM)-1, whereas ICAM-1 is strongly expressed on MDA-MB-468 cells. Consequently, the herein presented study was performed to investigate if this interaction has also an influence on the migratory activity of the tumor cells and whether ICAM-1 signaling plays a role in this process, too. We found that the continuous release of interleukin-8 (IL-8) and GRO-alpha by MDA-MB-468 cells increases the migratory activity of neutrophil granulocytes and attracts these cells towards the tumor cells which enables direct cell-cell interactions. These interactions in turn increase the migratory activity of the tumor cells in an ICAM-1 clustering-dependent mechanism since transfection of the tumor cells with specific siRNA against ICAM-1 abolished the effect. Moreover, ICAM-1 cross-linking on tumor cells induces the phosphorylation of focal adhesion components such as focal adhesion kinase and paxillin via src kinase as well as the activation of the p38 MAPK pathway via Rho kinase in a time-dependent manner. Our results provide evidence that ICAM-1 is coupled to intracellular signaling pathways involved in tumor cell migration. Thus, neutrophil granulocytes can act as modulators of the metastatic capability of tumor cells by ligation of ICAM-1.

  10. Effects of interferon-γ and granulocyte colony-stimulating factor on antifungal activity of human polymorphonuclear neutrophils against Candida albicans grown as biofilms or planktonic cells.

    Science.gov (United States)

    Katragkou, Alphaspasia; Simitsopoulou, Maria; Chatzimoschou, Athanasios; Georgiadou, Elpiniki; Walsh, Thomas J; Roilides, Emmanuel

    2011-09-01

    Candida albicans is a leading cause of biofilm-related infections. As Candida biofilms are recalcitrant to host defenses, we sought to determine the effects of interferon-γ and granulocyte colony-stimulating factor, two pro-inflammatory cytokines, on the antifungal activities of human polymorphonuclear neutrophils (PMNs) against C. albicans biofilms, using an in vitro biofilm model. Priming of PMNs by these cytokines augmented fungal damage of planktonic cells; however, priming of PMNs did not have the same effect against Candida biofilms. Biofilm phenotype appears to play an important role in protecting C. albicans from the innate immune system. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Granulocytic spongiotic papulovesiculosis (neutrophilic spongiosis: A rare entity

    Directory of Open Access Journals (Sweden)

    Vibhu Mendiratta

    2017-01-01

    Full Text Available Neutrophilic spongiosis also known as granulocytic spongiotic papulovesiculosis (GSPV is an uncommon disorder of uncertain classification. We report the case of a 45-year-old woman suffering from recurrent episodes of itchy, grouped papulovesicles over her body, histologically showing granulocytic spongiosis. The eruptions showed complete response to dapsone.

  12. Human Granulocytic Anaplasmosis, Japan

    Science.gov (United States)

    Gaowa; Wuritu; Kawamori, Fumihiko; Wu, Dongxing; Yoshikawa, Yuko; Chiya, Seizou; Fukunaga, Kazutoshi; Funato, Toyohiko; Shiojiri, Masaaki; Nakajima, Hideki; Hamauzu, Yoshiji; Takano, Ai; Kawabata, Hiroki; Ando, Shuji; Kishimoto, Toshio

    2013-01-01

    We retrospectively confirmed 2 cases of human Anaplasma phagocytophilum infection. Patient blood samples contained unique p44/msp2 for the pathogen, and antibodies bound to A. phagocytophilum antigens propagated in THP-1 rather than HL60 cells. Unless both cell lines are used for serodiagnosis of rickettsiosis-like infections, cases of human granulocytic anaplasmosis could go undetected. PMID:23460988

  13. In vitro effects of bisphosphonates on chemotaxis, phagocytosis, and oxidative burst of neutrophil granulocytes.

    Science.gov (United States)

    Hagelauer, Nadine; Pabst, Andreas Max; Ziebart, Thomas; Ulbrich, Holger; Walter, Christian

    2015-01-01

    Bisphosphonate-associated osteonecrosis of the jaws is a serious side effect that mainly occurs in patients receiving highly potent, nitrogen-containing bisphosphonates. Usually the diagnosis is made due to exposed bone and a nonhealing wound. Neutrophil granulocytes are essential for sufficient wound healing; therefore, the influence of different bisphosphonates on neutrophil granulocytes was the focus of this study. The effect of nitrogen-containing bisphosphonates (ibandronate, pamidronate, and zoledronate) and one non-nitrogen-containing bisphosphonate (clodronate) on chemotaxis, phagocytosis, and oxidative burst of neutrophil granulocytes in human whole blood was analyzed using standard cytometric flow assays. Chemotaxis of neutrophils was reduced by almost 50 % when cells were treated with ibandronate and zoledronate. All tested nitrogen-containing bisphosphonates moderately increased the percentage of phagocytizing neutrophils, whereas the percentage of oxidizing cells was extremely affected. Zoledronate increased the oxidative burst activity even at low concentrations. Treatment with ibandronate and pamidronate reached the same level, but only in at least 10 times the higher concentrations. The maximal burst activity of a single cell reached nearly 150 % compared to control. In this case, zoledronate also caused maximal effects even at low concentrations. Clodronate did not show any effects. The results show a proinflammatory effect of the nitrogen-containing effect on neutrophil granulocytes which might contribute to the development of osteonecrosis. The altered neutrophil defense might play a key role in the pathogenesis of bisphosphonate-associated osteonecrosis of the jaws, although the underlying causation between inflammatory reaction and the development of necrosis is yet unknown.

  14. Cortisol inhibits apoptosis in carp neutrophilic granulocytes.

    NARCIS (Netherlands)

    Weyts, F.A.A.; Flik, G.; Verburg-van Kemenade, B.M.L.

    1998-01-01

    The direct effect of cortisol treatment on carp neutrophil viability was examined in vitro. Cortisol treatment caused an inhibition of neutrophil apoptosis. The effect was blocked by glucocorticoid receptor blocker RU486, showing that rescue from apoptosis was receptor mediated. Using binding

  15. The human granulocyte nucleus: Unusual nuclear envelope and heterochromatin composition.

    Science.gov (United States)

    Olins, Ada L; Zwerger, Monika; Herrmann, Harald; Zentgraf, Hanswalter; Simon, Amos J; Monestier, Marc; Olins, Donald E

    2008-05-01

    The human blood granulocyte (neutrophil) is adapted to find and destroy infectious agents. The nucleus of the human neutrophil has a segmented appearance, consisting of a linear or branched array of three or four lobes. Adequate levels of lamin B receptor (LBR) are necessary for differentiation of the lobulated nucleus. The levels of other components of the nuclear envelope may also be important for nuclear shape determination. In the present study, immunostaining and immunoblotting procedures explored the levels of various components of the nuclear envelope and heterochromatin, comparing freshly isolated human neutrophils with granulocytic forms of HL-60 cells, a tissue culture model system. In comparison to granulocytic HL-60 cells, blood neutrophil nuclear envelopes contain low-to-negligible amounts of LBR, lamins A/C, B1 and B2, LAP2beta and emerin. Surprisingly, a "mitotic" chromosome marker, H3(S10)phos, is elevated in neutrophil nuclei, compared to granulocytic HL-60 cells. Furthermore, neutrophil nuclei appear to be more fragile to methanol fixation, than observed with granulocytic HL-60 cells. Thus, the human neutrophil nucleus appears to be highly specialized, possessing a paucity of nuclear envelope-stabilizing proteins. In consequence, the neutrophil nucleus appears to be very malleable, supporting rapid migration through tight tissue spaces.

  16. Neutrophil Evolution and Their Diseases in Humans

    Directory of Open Access Journals (Sweden)

    Jennifer W. Leiding

    2017-08-01

    Full Text Available Granulocytes have been preserved and have evolved across species, developing into cells that provide one of the first lines of host defense against pathogens. In humans, neutrophils are involved in early recognition and killing of infectious pathogens. Disruption in neutrophil production, emigration, chemotaxis, and function cause a spectrum of primary immune defects characterized by host susceptibility to invasive infections.

  17. Influence of fractionated irradiation on neutrophilic granulocyte function

    Energy Technology Data Exchange (ETDEWEB)

    Haidenberger, A.; Wolfgruber, T.; Auer, T.; Lukas, P.; DeVries, A. [Dept. of Radiotherapy, Leopold Franzens Univ. Innsbruck (Austria); Hengster, P. [Dept. of Transplant Surgery, Leopold Franzens Univ. Innsbruck (Austria); Kunc, M. [Daniel Swarovski Research Lab., Leopold Franzens Univ. Innsbruck (Austria); Micke, O. [Dept. of Radiotherapy, Univ. of Muenster (Germany)

    2003-01-01

    Background: A recent study has demonstrated that radiation therapy with single doses of up to 32 Gy has only a minor effect on neutrophilic granulocyte function. In clinical practice, by contrast, fractionated irradiation is applied. Therefore, the aim of the current study was to verify the influence of fractionated radiation therapy on granulocyte function. Material and Methods: Density gradient-purified granulocytes of voluntary healthy donors were used for all experiments. Granulocytes were kept in RPMI 1640 without fetal calf serum, incubated for 48 h and irradiated. Their function was assessed by measuring luminol-enhanced chemiluminescence after stimulation with phorbol myristate acid (PMA). All tests were performed at least five times. Results: Relative changes (any reactive oxygen species [ROS] release before stimulation was defined as being equal to 100%) in ROS release increased after stimulation with PMA (mean {+-} SD): 0 Gy: 785 {+-} 462.6%; 2 Gy: 704.3 {+-} 388.1%; 6 Gy: 1,360.3 {+-} 710.5%; 12 Gy: 1,119.4 {+-} 581.1%; 18 Gy: 1,087.4 {+-} 622.4; 6 Gy (3 x 2 Gy): 279.4 {+-} 201.1%; 12 Gy (6 x 2 Gy): 278.8 {+-} 175.3%; 18 Gy (9 x 2 Gy): 84.2 {+-} 41.5%. Comparing relative changes in ROS release after PMA stimulation, the differences between 0, 2, 6, 12, 18 Gy, and 6 Gy (3 x 2 Gy), 12 Gy (6 x 2 Gy), 18 Gy (9 x 2 Gy), and between 6 Gy (3 x 2 Gy), 12 Gy (6 x 2 Gy) and 18 Gy (9 x 2 Gy) proved to be significant (all p < 0.005). Conclusion: The study shows, that clinically used fractionated irradiation has an impact on granulocyte function, but contrary to common assumption, it is not the total dose itself but rather the fractionation which influences granulocyte function. This could have a major clinical impact on radiation treatment schemes especially for benign diseases or anti-inflammatory treatment. (orig.)

  18. Granulocyte colony-stimulating factor delays neutrophil apoptosis by inhibition of calpains upstream of caspase-3

    NARCIS (Netherlands)

    van Raam, Bram J.; Drewniak, Agata; Groenewold, Vincent; van den Berg, Timo K.; Kuijpers, Taco W.

    2008-01-01

    Neutrophils have a very short life span and undergo apoptosis within 24 hours after leaving the bone marrow. Granulocyte colony-stimulating factor (G-CSF) is essential for the recruitment of fresh neutrophils from the bone marrow but also delays apoptosis of mature neutrophils. To determine the

  19. Raman microspectroscopic approach to the study of human granulocytes

    NARCIS (Netherlands)

    Puppels, G.J.; Garritsen, H.S.; Segers-Nolten, Gezina M.J.; de Mul, F.F.M.; Greve, Jan

    1991-01-01

    A sensitive confocal Raman microspectrometer was employed to record spectra of nuclei and cytoplasmic regions of single living human granulocytes. Conditions were used that ensured cell viability and reproducibility of the spectra. Identical spectra were obtained from the nuclei of neutrophilic,

  20. Human neutrophil alloantigens systems

    Directory of Open Access Journals (Sweden)

    Elyse Moritz

    2009-09-01

    Full Text Available Neutrophil alloantigens are involved in a variety of clinical conditions including immune neutropenias, transfusion-related acute lung injury (TRALI, refractoriness to granulocyte transfusions and febrile transfusion reactions. In the last decade, considerable progress has been made in the characterization of the implicated antigens. Currently, seven antigens are assigned to five human neutrophil antigen (HNA systems. The HNA-1a, HNA-1b and HNA-1c antigens have been identified as polymorphic forms of the neutrophil Fcγ receptor IIIb (CD16b, encoded by three alleles. Recently, the primary structure of the HNA-2a antigen was elucidated and the HNA-2a-bearing glycoprotein was identified as a member of the Ly-6/uPAR superfamily, which has been clustered as CD177. The HNA-3a antigen is located on a 70-95 kDa glycoprotein; however, its molecular basis is still unknown. Finally, the HNA-4a and HNA-5a antigens were found to be caused by single nucleotide mutations in the αM (CD11b and αL (CD11a subunits of the leucocyte adhesion molecules (β2 integrins. Molecular and biochemical characterization of neutrophil antigenshave expanded our diagnostic tools by the introduction of genotyping techniques and immunoassays for antibody identification. Further studies in the field of neutrophil immunology will facilitate the prevention and management of transfusion reactions and immune diseases caused by neutrophil antibodies.Os aloantígenos de neutrófilos estão associados a várias condições clínicas como neutropenias imunes, insuficiência pulmonar relacionada à transfusão (TRALI, refratariedade à transfusão de granulócitos, e reações transfusionais febris. Na última década, foi observado considerável progresso na caracterização dos aloantígenos envolvidos nestas condições clínicas. Atualmente sete antígenos estão incluídos em cinco sistemas de antígenos de neutrófilo humano (HNA. Os antígenos HNA-1a, HNA-1b e HNA-1c foram

  1. Granulocytes as models for human protein marker identification following nicotine exposure.

    Science.gov (United States)

    Mulcahy, Matthew J; Lester, Henry A

    2017-08-01

    Nicotinic acetylcholine receptors (nAChRs) are pentameric cation channels expressed in the mammalian CNS, in the peripheral nervous system, and in skeletal muscle. Neuronal-type nAChRs are also found in several non-neuronal cell types, including leukocytes. Granulocytes are a subtype of leukocytes that include basophils, eosinophils, and neutrophils. Granulocytes, also known as polymorphonuclear leukocytes, are characterized by their ability to produce, store, and release compounds from intracellular granules. Granulocytes are the most abundant type of leukocyte circulating in the peripheral blood. Granulocyte abundance, nAChR expression, and nAChR upregulation following chronic nicotine administration makes granulocytes interesting models for identifying protein markers of nicotine exposure. Nicotinic receptor subunits and several non-nAChR proteins have been identified as protein markers of granulocyte nicotine exposure. We review methods to isolate granulocytes from human tissue, summarize present data about the expression of nAChRs in the three granulocyte cell types (basophils, eosinophils, and neutrophils), describe current knowledge of the effects of nicotine exposure on human granulocyte protein expression, and highlight areas of interest for future investigation. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

  2. Human Granulocytic Ehrlichiosis Complicating Early Pregnancy

    Directory of Open Access Journals (Sweden)

    Tyler Muffly

    2008-01-01

    Full Text Available Background. The goal of this case is to review the zoonotic infection, human granulocytic ehrlichiosis, presenting with pyrexia. Case. A 22-year-old multigravid female presented to the emergency department with a painful skin rash, high fever, and severe myalgias. The patient underwent a diagnostic evaluation for zoonotic infections due to her geographical and seasonal risk factors. Treatment of human granulocytic ehrlichiosis was successful though the patient spontaneously aborted presumably due to the severity of the acute illness. Conclusion. Treatment of human granulocytic ehrlichiosis in pregnancy presents unique challenges. Management of pyrexia during pregnancy is limited to external cooling in the setting of thrombocytopenia and elevated aminotransferases. Extensive counseling regarding teratogenic potential of medications allows the patient to weigh the pros and cons of treatment.

  3. Behavior of Neutrophil Granulocytes during Toxoplasma gondii Infection in the Central Nervous System

    Science.gov (United States)

    Biswas, Aindrila; French, Timothy; Düsedau, Henning P.; Mueller, Nancy; Riek-Burchardt, Monika; Dudeck, Anne; Bank, Ute; Schüler, Thomas; Dunay, Ildiko Rita

    2017-01-01

    Cerebral toxoplasmosis is characterized by activation of brain resident cells and recruitment of specific immune cell subsets from the periphery to the central nervous system (CNS). Our studies revealed that the rapidly invaded Ly6G+ neutrophil granulocytes are an early non-lymphoid source of interferon-gamma (IFN-γ), the cytokine known to be the major mediator of host resistance to Toxoplasma gondii (T. gondii). Upon selective depletion of Ly6G+ neutrophils, we detected reduced IFN-γ production and increased parasite burden in the CNS. Ablation of Ly6G+ cells resulted in diminished recruitment of Ly6Chi monocytes into the CNS, indicating a pronounced interplay. Additionally, we identified infiltrated Ly6G+ neutrophils to be a heterogeneous population. The Ly6G+CD62-LhiCXCR4+ subset released cathelicidin-related antimicrobial peptide (CRAMP), which can promote monocyte dynamics. On the other hand, the Ly6G+CD62-LloCXCR4+ subset produced IFN-γ to establish early inflammatory response. Collectively, our findings revealed that the recruited Ly6G+CXCR4+ neutrophil granulocytes display a heterogeneity in the CNS with a repertoire of effector functions crucial in parasite control and immune regulation upon experimental cerebral toxoplasmosis. PMID:28680853

  4. Granulocyte transfusions for preventing infections in people with neutropenia or neutrophil dysfunction

    Science.gov (United States)

    Estcourt, Lise J; Stanworth, Simon; Doree, Carolyn; Blanco, Patricia; Hopewell, Sally; Trivella, Marialena; Massey, Edwin

    2015-01-01

    Background Despite modern antimicrobials and supportive therapy, bacterial and fungal infections are still major complications in people with prolonged disease-related or therapy-related neutropenia. Since the late 1990s there has been increasing demand for donated granulocyte transfusions to treat or prevent severe infections in people who lack their own functional granulocytes. This is an update of a Cochrane review first published in 2009. Objectives To determine the effectiveness and safety of prophylactic granulocyte transfusions compared with a control population not receiving this intervention for preventing all-cause mortality, mortality due to infection, and evidence of infection due to infection or due to any other cause in people with neutropenia or disorders of neutrophil function. Search methods We searched for randomised controlled trials (RCTs) and quasi-RCTs in the Cochrane Central Register of Controlled Trials (Cochrane Library 2015, Issue 3), MEDLINE (from 1946), EMBASE (from 1974), CINAHL (from 1937), the Transfusion Evidence Library (from 1980) and ongoing trial databases to April 20 2015. Selection criteria Randomised controlled trials (RCTs) and quasi-RCTs comparing people receiving granulocyte transfusions to prevent the development of infection with a control group receiving no granulocyte transfusions. Neonates are the subject of another Cochrane review and were excluded from this review. There was no restriction by outcomes examined, but this review focuses on mortality, mortality due to infection and adverse events. Data collection and analysis We used standard methodological procedures expected by The Cochrane Collaboration. Main results Twelve trials met the inclusion criteria. One trial is still ongoing, leaving a total of 11 trials eligible involving 653 participants. These trials were conducted between 1978 and 2006 and enrolled participants from fairly comparable patient populations. None of the studies included people with

  5. Swell activated chloride channel function in human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Salmon, Michael D. [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom); Ahluwalia, Jatinder, E-mail: j.ahluwalia@uel.ac.uk [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom)

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  6. Low density granulocytes: a distinct class of neutrophils in systemic autoimmunity

    Science.gov (United States)

    Carmona-Rivera, Carmelo; Kaplan, Mariana J.

    2013-01-01

    Recent studies have renewed the interest on the potential role that neutrophils play in the development of systemic lupus erythematosus (SLE) and other autoimmune conditions. A distinct subset of proinflammatory, low-density granulocytes (LDGs) isolated from the peripheral blood mononuclear cell fractions of patients with SLE has been described. While the origin and role of LDGs needs to be fully characterized, there is evidence that these cells may contribute to lupus pathogenesis and to the development of end-organ damage through heightened proinflammatory responses, altered phagocytic capacity, enhanced ability to synthesize type I Interferons and to kill endothelial cells. Furthermore, these cells readily form neutrophil extracellular traps (NETs), a phenomenon that may promote autoantigen externalization and organ damage. This review examines the biology and potential origin of LDGs, describes the ultrastructural characteristics of these cells, and discusses their putative pathogenic role in systemic autoimmune diseases. PMID:23553215

  7. [Prognostic value of the leukogram and the myeloperoxidase activity of neutrophilic granulocytes in infectious complications in children with severe burns].

    Science.gov (United States)

    Kozinets, G P; Kiriliuk, A N; Tsygankov, V P; Osadchaia, O I; Boiarskaia, A M

    1992-01-01

    Clinical course of the burn disease of a severe, or extremely severe degree was studied in 56 children aging from 1 to 5 years. It was established that intensity of a leukocytic reaction and the redistribution reactions of neutrophil granulocytes with different activity of myeloperoxidase objectively reflected the course of a pathologic process, permitted to predict the development of local and general infective complications, and to make up a rational program for treatment with differentiated use of the methods of detoxication and immunotherapy. Activity of acid phosphatase and content of PAS-positive substances in neutrophil granulocytes didn't correlated with severity of the course of the burn disease.

  8. Flow cytometry evidence of human granulocytes interaction with polyhedral oligomeric silsesquioxanes: effect of nanoparticle charge

    Science.gov (United States)

    Renò, Filippo; Carniato, Fabio; Rizzi, Manuela; Olivero, Francesco; Pittarella, Pamela; Marchese, Leonardo

    2013-05-01

    Nanoparticles (NPs) entering the human body are immediately confronted with the innate part of human immune system. In particular, monocyte and neutrophil granulocytes readily clear particles by phagocytosis, even if in the case of NPs the uptake mechanism may be classified as macropinocytosis. Among engineered nanoparticles, in the last years, siliceous materials have emerged as promising materials for several applications ranging from catalysis to biomedical. The polyhedral oligomeric silsesquioxanes (POSS) are nanodimensional, easily synthesizable molecular compounds and POSS-based systems are promising carriers for biological molecules. In this work, the ability of human granulocytes to uptake positively and negatively charged POSS was measured using a simple flow cytometry analysis based on cell size modifications. The data obtained showed that after a 30 min exposure only positive NPs were uptaken by human granulocyte using both macropinocytosis and clathrin-mediated mechanisms as demonstrated by uptake inhibition mediated by amiloride and chlorpromazine.

  9. Type I Interferon Transcriptional Signature in Neutrophils and Low-Density Granulocytes Are Associated with Tissue Damage in Malaria

    Directory of Open Access Journals (Sweden)

    Bruno Coelho Rocha

    2015-12-01

    Full Text Available Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of Plasmodium sp. infection. However, the role of these polymorphonuclear cells in mediating either the resistance or the pathogenesis of malaria is poorly understood. We report that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, enhanced release of reactive oxygen species and myeloperoxidase, and a high frequency of low-density granulocytes. The activation of neutrophils was associated with increased levels of serum alanine and aspartate aminotransferases, indicating liver damage. In a rodent malaria model, we observed intense recruitment of neutrophils to liver sinusoids. Neutrophil migration and IL-1β and chemokine expression as well as liver damage were all dependent on type I interferon signaling. The data suggest that type I interferon signaling has a central role in neutrophil activation and malaria pathogenesis.

  10. Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease.

    Directory of Open Access Journals (Sweden)

    Cindy Franklin

    Full Text Available Chronic graft-versus-host disease (cGVHD is a common side effect of allogeneic stem cell transplantation and a major cause of morbidity and mortality in affected patients. Especially skin, eyes and oral mucosa are affected. This can lead to pain and functional impairment. Extracorporeal photopheresis (ECP is an effective immunomodulatory therapy with minimal side effects but its mode of action is still largely unknown. The objective of the present study was to examine the effects of ECP on neutrophil granulocytes in patients with cGVHD. Analysis of leukocytes from cGVHD patients obtained from the ECP device during treatment showed that neutrophil granulocytes account for the majority of cells treated during ECP. Neutrophils from healthy donors treated in vitro with 8-methoxypsoralen and UVA light as well as neutrophils from buffy coats of patients with cGVHD treated by ECP showed increased apoptosis and decreased half-life. In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions. This was accompanied by an increase in extracellular arginase-1 activity. Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo. These observations strongly suggest that ECP induces both apoptosis and physiological changes in neutrophils and that these changes also take place in vivo. This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

  11. Granulocyte and monocyte adsorption apheresis for refractory skin diseases due to activated neutrophils, psoriasis, and associated arthropathy.

    Science.gov (United States)

    Sakanoue, Masanao; Takeda, Koichiro; Kawai, Kazuhiro; Kanekura, Takuro

    2013-10-01

    Granulocyte and monocyte adsorption apheresis (GMA), an extracorporeal apheresis instrument whose column contains cellulose acetate (CA) beads, is designed to remove activated granulocytes and monocytes. We previously demonstrated that GMA was useful for treating neutrophilic dermatoses and associated arthropathy as it adsorbs Mac-1 (CD11b/CD18)-expressing neutrophils to the CA beads by the binding of complement component (iC3b) and CD11b expressed on activated neutrophils. The objective of this study is to further assess the clinical effectiveness of GMA in the treatment of neutrophilic dermatoses and associated arthropathy. The effect of GMA for skin lesions and joint lesions was assessed in 44 and 23 patients, respectively. Mac-1 expression on peripheral neutrophils was measured by flow cytometry. Skin lesions and arthropathy improved in 39 of 44 patients (88.6%) and 22 of 23 (95.6%), respectively. Mac-1 (CD11b/CD18) expression on the peripheral neutrophils, 27.1 ± 6.66 MFI (mean fluorescence intensity) before treatment, was reduced to 17.9 ± 3.02 MFI by GMA (P < 0.05). Clinical effectiveness of GMA for the treatment of intractable neutrophilic dermatoses and associated arthropathy was further confirmed. © 2013 The Authors. Therapeutic Apheresis and Dialysis © 2013 International Society for Apheresis.

  12. Innate Recognition by Neutrophil Granulocytes Differs between Neisseria gonorrhoeae Strains Causing Local or Disseminating Infections

    Science.gov (United States)

    Roth, Alexandra; Mattheis, Corinna; Muenzner, Petra; Unemo, Magnus

    2013-01-01

    Members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family serve as cellular receptors for Neisseria gonorrhoeae. More specifically, neisserial colony opacity (OpaCEA) proteins bind to epithelial CEACAMs (CEACAM1, CEA, CEACAM6) to promote bacterial colonization of the mucosa. In contrast, recognition by CEACAM3, expressed by human granulocytes, results in uptake and destruction of OpaCEA-expressing bacteria. Therefore, CEACAM3-mediated uptake might limit the spread of gonococci. However, some strains can cause disseminating gonococcal infections (DGIs), and it is currently unknown how these strains escape detection by granulocyte CEACAM3. Therefore, the opa gene loci from N. gonorrhoeae strain VP1, which was derived from a patient with disseminated gonococcal disease, were cloned and constitutively expressed in Escherichia coli. Similar to Opa proteins of the nondisseminating strain MS11, the majority of Opa proteins from strain VP1 bound epithelial CEACAMs and promoted CEACAM-initiated responses by epithelial cells. In sharp contrast to the Opa proteins of strain MS11, the Opa proteins of strain VP1 failed to interact with the human granulocyte receptor CEACAM3. Accordingly, bacteria expressing VP1 Opa proteins were not taken up by primary human granulocytes and did not trigger a strong oxidative burst. Analysis of Opa variants from four additional clinical DGI isolates again demonstrated a lack of CEACAM3 binding. In summary, our results reveal that particular N. gonorrhoeae strains express an Opa protein repertoire allowing engagement of epithelial CEACAMs for successful mucosal colonization, while avoiding recognition and elimination via CEACAM3-mediated phagocytosis. A failure of CEACAM3-mediated innate immune detection might be linked to the ability of gonococci to cause disseminated infections. PMID:23630956

  13. Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage

    DEFF Research Database (Denmark)

    Porse, Bo T; Bryder, David; Theilgaard-Mönch, Kim

    2005-01-01

    CCAAT/enhancer binding protein (C/EBP)alpha is a myeloid-specific transcription factor that couples lineage commitment to terminal differentiation and cell cycle arrest, and is found mutated in 9% of patients who have acute myeloid leukemia (AML). We previously showed that mutations which...... dissociate the ability of C/EBP alpha to block cell cycle progression through E2F inhibition from its function as a transcriptional activator impair the in vivo development of the neutrophil granulocyte and adipose lineages. We now show that such mutations increase the capacity of bone marrow (BM) myeloid...... progenitors to proliferate, and predispose mice to a granulocytic myeloproliferative disorder and transformation of the myeloid compartment of the BM. Both of these phenotypes were transplantable into lethally irradiated recipients. BM transformation was characterized by a block in granulocyte differentiation...

  14. Molecular and functional characterization of Hv1 proton channel in human granulocytes.

    Science.gov (United States)

    Petheo, Gábor L; Orient, Anna; Baráth, Mónika; Kovács, István; Réthi, Bence; Lányi, Arpád; Rajki, Anikó; Rajnavölgyi, Eva; Geiszt, Miklós

    2010-11-23

    Voltage-gated proton current (I(Hv)) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of I(Hv) in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently H(v)1, a voltage-gated proton channel, was cloned, and leukocytes from H(v)1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about H(v)1 in human granulocytes. Using qPCR and a self made antibody, we detected a significant amount of H(v)1 in human eosinophil and neutrophil granulocytes and in PLB-985 leukemia cells. Using different crosslinking agents and detergents in reducing and non-reducing PAGE, significant expression of H(v)1 homodimers, but not that of higher-order multimers, could be detected in granulocytes. Results of subcellular fractionation and confocal imaging indicate that H(v)1 is resident in both plasmalemmal and granular membrane compartments of resting neutrophils. Furthermore, it is also demonstrated that H(v)1 accumulates in phagosome wall during zymosan engulfment together with, but independently of Nox2. During granulocytic differentiation early and parallel upregulation of H(v)1 and Nox2 expression was observed in PLB-985 cells. The upregulation of H(v)1 or Nox2 expression did not require the normal expression of the other molecule. Using RNA interference, we obtained strong correlation between H(v)1 expression and I(Hv) density in PLB-985 cells. It is also demonstrated that a massive reduction in H(v)1 expression can limit the Nox2 mediated superoxide production of PLB-985 granulocytes. In summary, beside monomers native H(v)1 forms stable proton channel dimer in resting and activated human granulocytes. The expression pattern of H(v)1 in granulocytes is optimized to support intense NADPH

  15. β-lactam antibiotic-induced release of lipoteichoic acid from Staphylococcus aureus leads to activation of neutrophil granulocytes

    Directory of Open Access Journals (Sweden)

    Hartung Thomas

    2006-06-01

    Full Text Available Abstract Background Polymorphonuclear neutrophil granulocytes (PMN are phagocytes of the first line of antimicrobial defense. Previously we demonstrated that lipoteichoic acid (LTA from Staphylococcus aureus (S. aureus directly activates neutrophil granulocytes. Others have reported that exposure of S. aureus to β-lactam antibiotics leads to LTA release. In the present study we addressed the question whether exposure of S. aureus to β-lactam antibiotics or antibiotics of other groups results in the generation of PMN-stimulating activity and whether this activity can be attributed to LTA. Methods S. aureus were exposed to flucloxacillin, a β-lactam antibiotic or to the protein synthesis-inhibitors erythromycin and gentamicin, or to ciprofloxacin, a gyrase inhibitor. Supernatants of the antibiotic-treated bacteria were assayed for their LTA content and for their effect on PMN functions. Results We observed that exposure of S. aureus to flucloxacillin and, to a lesser degree to ciprofloxacin, but not to erythromycin or gentamicin led to LTA release. Co-incubation of neutrophil granulocytes with LTA-containing supernatants led to PMN activation as assed by morphological changes, release of IL-8, delay of spontaneous apoptosis and enhanced phagocytic activity. Depletion of LTA from the supernatants markedly reduced their PMN-activating capacity. Conclusion The findings suggest that, via the activation of PMN, antibiotic-induced LTA release from S. aureus leads to enhanced antimicrobial activity of the innate immune defense mechanisms.

  16. CHEMILUMINESCENT ACTIVITY OF NEUTROPHILIC GRANULOCYTES IN PROGRESSION OF OBSTRUCTIVE JAUNDICE DEPENDING ON ITS ORIGIN AND BILIRUBIN LEVELS

    Directory of Open Access Journals (Sweden)

    O. V. Smirnova

    2016-01-01

    Full Text Available Mechanical jaundice (MJ is a severe pathological condition, caused by obstruction of the bile ducts, requiring immediate surgical intervention. Etiologically, MJ can be of benign (60-80% of the cases, or malignant origin. MJ progression depends on the underlying pathology, and, moreover, on bilirubin levels. Focal inflammation in affected area represents a significant mechanism of the MJ progression. Neutrophilic granulocytes, are primarily involved into the immune response, i.e., pathogen elimination. Hence, the MJ progression may depend on their functional activity. In this context, the aim of our study was to investigate chemiluminescent activity of neutrophil granulocytes in progression of MJ, depending on the bilirubin levels and origin of the jaundice. All the MJ patients showed altered chemiluminescent activity of granulocytes. Both spontaneous and induced chemiluminescence (CL intensity was decreased in the patients with gallstoneassociated MJ. Meanwhile, the CL intensity did not change in MJ caused by benign tumors (BTP. An increased activation index reflected higher induced activity of phagocytes. In patients with MJ of malignant origin, the largest number of changes was found, i.e., an increase in the induced luminescence intensity and higher activation indexes were revealed. The MJ progression depends on blood bilirubin levels. All the patients with gallstone-related MJ exhibited a decrease in spontaneous and induced CL activity of the neutrophils. In cases of BTP-caused MJ, the indexes of spontaneous and induced CL decreased at the bilirubin levels of 60 to 200 mmol/L, with increased activation index suggesting elevated induced activity over its spontaneous levels. In patients with MJ and bilirubin levels >200 mmol/L, distinct unidirectional changes in the granulocyte activation were observed, with increased spontaneous and induced CL intensity. In patients with MJ caused by malignancies with bilirubin levels <60

  17. Effects of Granulocyte Colony-Stimulating Factor on Opsonin Receptor Expression and Neutrophil Antibacterial Activity in a Mouse Model of Severe Acute Pancreatitis

    Directory of Open Access Journals (Sweden)

    Tuo Hong-Fang

    2017-05-01

    Full Text Available The antimicrobial function of neutrophils, which is dependent on opsonin receptors, deteriorates in severe acute pancreatitis (SAP. Granulocyte colony-stimulating factor (G-CSF putatively enhanced levels of the opsonin receptors CD11b and CD32/16 in healthy human subjects, and provided protection against infection in animal models of SAP. A statistically convincing study of the effect of G-CSF on CD32/16 expression in an SAP model is lacking. We used a mouse model of SAP to investigate the association between G-CSF administration and CD32/16 levels on neutrophils and bacterial translocation. G-CSF or saline was subcutaneously injected into SAP-induced mice. The pancreases were histologically examined, and leukocytes were stained to count neutrophils. The expression of CD11b and CD32/16 on neutrophils was measured by flow cytometry, and bacterial translocation was observed by bacterial culture.The numbers of CD11b and CD32/16-positive neutrophils were significantly elevated in the SAP mice treated with G-CSF, and the mean fluorescence intensities of these receptors on neutrophils were significantly elevated. Bacterial translocations to cavity organs were suppressed from 17% to 6% by G-CSF treatment. Our results indicated that the number of neutrophils significantly increased with increasing expression of CD11b and CD32/16 and their mean fluorescence intensities (MFIs. This inhibited bacterial translocation to other organs. These results are in accord with other studies in SAP dogs and SAP mice. Our findings suggest that G-CSF was effective in protecting against bacterial infection in SAP mice.

  18. Demonstration of the neutrophil granulocyte functional capacity in silicosis, using the NBT test.

    Science.gov (United States)

    Niculescu, T; Cojocaru, M

    1995-01-01

    The functional capacity of neutrophils was studied using the NBT test in 68 patients with silicosis in various evolutive stages, comparatively with a group of 35 controls. The NBT test showed an initial increase of nonspecific cellular reactivity which decreased as the disease evolved. The results in the control group showed 9 +/- 5% NBT positive neutrophils. The highest proportion of NBT positive neutrophils (17.6 +/- 2.3, p NBT positive neutrophils may be considered as a biochemical marker of neutrophil functionality.

  19. Effect of sevoflurane on human neutrophil apoptosis.

    LENUS (Irish Health Repository)

    Tyther, R

    2012-02-03

    BACKGROUND AND OBJECTIVE: Both chronic occupational exposure to volatile anaesthetic agents and acute in vitro exposure of neutrophils to isoflurane have been shown to inhibit the rate of apoptosis of human neutrophils. It is possible that inhibition of neutrophil apoptosis arises through delaying mitochondrial membrane potential collapse. We assessed mitochondrial depolarization and apoptosis in unexposed neutrophils and neutrophils exposed to sevoflurane in vivo. METHODS: A total of 20 mL venous blood was withdrawn pre- and postinduction of anaesthesia, the neutrophils isolated and maintained in culture. At 1, 12 and 24 h in culture, the percentage of neutrophil apoptosis was assessed by dual staining with annexin V-FITC and propidium iodide. Mitochondrial depolarization was measured using the dual emission styryl dye JC-1. RESULTS: Apoptosis was significantly inhibited in neutrophils exposed to sevoflurane in vivo at 24 (exposed: 38 (12)% versus control: 28 (11)%, P = 0.001), but not at 1 or 12 h, in culture. Mitochondrial depolarization was not delayed in neutrophils exposed to sevoflurane. CONCLUSIONS: The most important findings are that sevoflurane inhibits neutrophil apoptosis in vivo and that inhibition is not mediated primarily by an effect on mitochondrial depolarization.

  20. Radiation-induced muscositis and neutrophil granulocytes in oral mucosa; Strahleninduzierte Mukositis und neutrophile Granulozyten in der Mundschleimhaut

    Energy Technology Data Exchange (ETDEWEB)

    Schmidberger, H.; Rave-Fraenk, M.; Kim, S.; Hille, A.; Pradier, O.; Hess, C.F. [Klinik fuer Strahlentherapie und Radioonkologie, Univ. Goettingen (Germany)

    2003-10-01

    Background: Chemotherapy-induced mucositis can be related to a decrease in oral neutrophils. We tested the relationship between radiation-induced mucositis and oral neutrophil counts. Patients and Methods: Oral neutrophil counts were obtained for ten patients with head and neck cancer who received radiotherapy of the pharynx and oral cavity. Four patients received additional chemotherapy (5-FU, Mitomycin). Counts were obtained before and during treatment; four healthy volunteers were included in the study as well. For evaluation, a quantitative mouth rinse assay, including neutrophil-staining with acridin-orange, was applied. Results: We observed large inter-individual variations with respect to neutrophil counts for patients and control persons (Table 1). During treatment (irradiation or chemoirradiation), large intra-individual variations were seen additionally (Figure 1). We found a correlation between neutrophil counts and clinical reaction grade. Neutrophil counts increased with increasing mucositis (Figure 2). This increase was more pronounced for patients treated with chemoirradiation compared to radiation alone. Treatment breaks at weekends had no clear influence on neutrophil counts. Conclusions: We observed a weak correlation between neutrophil counts and clinical reaction grade. However, the variations in neutrophil counts are too large to utilize this parameter as a surrogate for clinical mucositis grading. The assumption that a decrease in oral neutrophils is associated with radiation-induced mucositis was clearly negated. (orig.) [German] Hintergrund: Die chemotherapieinduzierte Mukositis kann mit einer Verarmung der Mundschleimhaut an neutrophilen Granulozyten vergesellschaftet sein. Wir ueberprueften den Zusammenhang zwischen der radiogenen Mukositis und der Anzahl neutrophiler Granulozyten. Patienten und Methoden: Bei zehn Patienten mit Tumoren der Kopf-Hals-Region, die sich einer Strahlentherapie unterzogen, wurde die Anzahl enoraler neutrophiler

  1. Moraxella catarrhalis induces CEACAM3-Syk-CARD9-dependent activation of human granulocytes.

    Science.gov (United States)

    Heinrich, A; Heyl, K A; Klaile, E; Müller, M M; Klassert, T E; Wiessner, A; Fischer, K; Schumann, R R; Seifert, U; Riesbeck, K; Moter, A; Singer, B B; Bachmann, S; Slevogt, H

    2016-11-01

    The human restricted pathogen Moraxella catarrhalis is an important causal agent for exacerbations in chronic obstructive lung disease in adults. In such patients, increased numbers of granulocytes are present in the airways, which correlate with bacteria-induced exacerbations and severity of the disease. Our study investigated whether the interaction of M. catarrhalis with the human granulocyte-specific carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-3 is linked to NF-κB activation, resulting in chemokine production. Granulocytes from healthy donors and NB4 cells were infected with M. catarrhalis in the presence of different inhibitors, blocking antibodies and siRNA. The supernatants were analysed by enzyme-linked immunosorbent assay for chemokines. NF-κB activation was determined using a luciferase reporter gene assay and chromatin-immunoprecipitation. We found evidence that the specific engagement of CEACAM3 by M. catarrhalis ubiquitous surface protein A1 (UspA1) results in the activation of pro-inflammatory events, such as degranulation of neutrophils, ROS production and chemokine secretion. The interaction of UspA1 with CEACAM3 induced the activation of the NF-κB pathway via Syk and the CARD9 pathway and was dependent on the phosphorylation of the CEACAM3 ITAM-like motif. These findings suggest that the CEACAM3 signalling in neutrophils is able to specifically modulate airway inflammation caused by infection with M. catarrhalis. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

  2. Neutrophil-induced transmigration of tumour cells treated with tumour-conditioned medium is facilitated by granulocyte-macrophage colony-stimulating factor.

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

    OBJECTIVE: To investigate the effect of different cytokines that are present in tumour-conditioned medium on human neutrophil (PMN)-induced tumour cell transmigration. DESIGN: Laboratory study. SETTING: University hospital, Ireland. MATERIAL: Isolated human PMN and cultured human breast tumour cell line, MDA-MB-231. Interventions: Human PMN treated with either tumour-conditioned medium or different media neutralised with monoclonal antibodies (MoAb), and MDA-MB-231 cells were plated on macrovascular and microvascular endothelial monolayers in collagen-coated transwells to assess migration of tumour cells. MAIN OUTCOME MEASURES: Cytokines present in tumour-conditioned medium, PMN cytocidal function and receptor expression, and tumour cell transmigration. RESULTS: tumour-conditioned medium contained high concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and interleukin 8 (IL-8), but not granulocyte colony-stimulating factor (G-CSF) and interleukin 3 (IL-3). Anti-GM-CSF MoAb significantly reduced PMN-induced transmigration of tumour cells treated with tumour-conditioned medium (p < 0.05), whereas anti-VEGF and anti-IL-8 MoAbs did not affect their migration. In addition, anti-GM-CSF MoAb, but not anti-VEGF or anti-IL-8 MoAb, reduced PMN CD11b and CD18 overexpression induced by tumour-conditioned medium (p < 0.05). CONCLUSION: These results indicate that the GM-CSF that is present in tumour-conditioned medium may be involved, at least in part, in alterations in PMN function mediated by the medium and subsequently PMN-induced transmigration of tumour cells.

  3. Radioassay of granulocyte chemotaxis. Studies of human granulocytes and chemotactic factors. [/sup 51/Cr tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Gallin, J.I.

    1974-01-01

    The above studies demonstrate that the /sup 51/Cr radiolabel chemotactic assay is a relatively simple and objective means for studying leukocyte chemotaxis in both normal and pathological conditions. Application of this method to studies of normal human chemotaxis revealed a relatively narrow range of normal and little day-to-day variability. Analysis of this variability revealed that there is more variability among the response of different granulocytes to a constant chemotactic stimulus than among the chemotactic activity of different sera to a single cell source. Utilizing the /sup 51/Cr radioassay, the abnormal granulocyte chemotactic behavior reported in Chediak-Higashi syndrome and a patient with recurrent pyogenic infections and mucocutaneous candidiasis has been confirmed. The /sup 51/Cr chemotactic assay has also been used to assess the generation of chemotactic activity from human serum and plasma. The in vitro generation of two distinct chemotactic factors were examined; the complement product (C5a) and kallikrein, an enzyme of the kinin-generating pathway. Kinetic analysis of complement-related chemotactic factor formation, utilizing immune complexes or endotoxin to activate normal sera in the presence or absence of EGTA as well as kinetic analysis of activation of C2-deficient human serum, provided an easy means of distinguishing the classical (antibody-mediated) complement pathway from the alternate pathway. Such kinetic analysis is necessary to detect clinically important abnormalities since, after 60 min of generation time, normal chemotactic activity may be present despite complete absence or inhibition of one complement pathway. The chemotactic factor generated by either pathway of complement activation appears to be predominately attributable to C5a.

  4. Approaches to the preservation of human granulocytes by freezing

    Energy Technology Data Exchange (ETDEWEB)

    Frim, J; Mazur, P

    1979-01-01

    Because of its simplicity, the FDA assay can be used effectively as a screening test to eliminate procedures and treatments that are damaging to cells. In this context, a number of conclusions can be drawn from the data presented: (1) Exposure to 1 and 2 M glycerol at room temperature damages human granulocytes in a few minutes. Reducing the exposure temperature to 0/sup 0/C reduces the amount of injury substantially. (2) Human granulocytes respond to freezing and thawing in a manner typical of many mammalian cells in that they exhibit a maximum in survival at an optimum cooling rate slightly above 1/sup 0/C/min when combined with rapid warming. The use of rapid warming and a high (2 M) concentration of glycerol reduces the dependence of survival on cooling rate by broadening the range of rates over which survival is relatively high. (3) Human granulocytes show some sensitivity to dilution stresses since survival depends somewhat on the concentration of glycerol used and the severity of the dilution procedure. The reasons for the sharp decrease in cell viability following incubation of frozen-thawed granulocytes at 37/sup 0/C are not known. One possibility is that the phosphate buffered saline suspending medium used is not suitable for incubation at 37/sup 0/C. A second possibility is that some cell injury is not expressed at 0/sup 0/C and remains undetected by the FDA assay until the cells are incubated at 37/sup 0/C. There is also the possibility that lysosomal enzymes released by a few damaged cells in a sample will cause additional damage in other cells at 37/sup 0/C.

  5. Human anti-neutrophil cytoplasm autoantibodies to proteinase 3 (PR3-ANCA) bind to neutrophils

    NARCIS (Netherlands)

    Van Rossum, AP; van der Geld, YM; Limburg, PC; Kallenberg, CGM

    Human anti-neutrophil cytoplasm autoantibodies to proteinase 3 (PR3-ANCA) bind to neutrophils. Background. Recently, the in vivo pathogenic role of anti-neutrophil cytoplasm autoantibodies (ANCA) in ANCA-associated vasculitis has been challenged by Abdel-Salam et al. In their report, they observed

  6. Selective and direct activation of human neutrophils but not eosinophils by Toll-like receptor 8.

    Science.gov (United States)

    Janke, Markus; Poth, Jens; Wimmenauer, Vera; Giese, Thomas; Coch, Christoph; Barchet, Winfried; Schlee, Martin; Hartmann, Gunther

    2009-05-01

    Granulocytes represent the largest fraction of immune cells in peripheral blood and are directly exposed to circulating Toll-like receptor (TLR) ligands. Although highly relevant for TLR-based therapies, because of the technical challenge, activation of the granulocyte subsets of neutrophils and eosinophils by TLR ligands is less well studied than activation of other immune cell subsets. The aim of this work was to study direct versus indirect neutrophil and eosinophil activation by TLR7 and TLR8 ligands. We used a new whole-blood assay, single cell-based cytokine detection, and highly purified primary human neutrophils and eosinophils to separate direct and indirect effects on these blood cell subsets. We found indirect but not direct activation of neutrophils but not eosinophils in whole blood by using unmodified immunostimulatory RNA (isRNA; TLR7/8 ligand). In contrast, direct activation and stimulation of the respiratory burst and degranulation was seen with nuclease-stable isRNA and with the small-molecule TLR8 agonist 3M002 but not 3M001 (TLR7). Neutrophils expressed TLR8 but none of the other 2 RNA-detecting TLRs (TLR3 and TLR7). Together, these results demonstrate that neutrophils are directly and fully activated through TLR8 but not TLR7. Furthermore, the results predict that the clinical utility of small-molecule TLR8 ligands or nuclease-stable RNA ligands for TLR8 might be limited because of neutrophil-mediated toxicity and that no such limitation applies for unmodified isRNA, which is known to induce desired T(H)1 activities in other immune cell subsets.

  7. Thalidomide inhibits granulocyte responses in healthy humans after ex vivo stimulation with bacterial antigens

    NARCIS (Netherlands)

    Juffermans, N. P.; Verbon, A.; Schultz, M. J.; Hack, C. E.; van Deventer, S. J.; Speelman, P.; van der Poll, T.

    2001-01-01

    Ingestion of thalidomide was associated with a reduction in the upregulation of the granulocyte activation marker CD11b and a reduced capacity to release elastase and lactoferrin after stimulation with lipopolysaccharide or lipoteichoic acid. A single oral dose of thalidomide attenuates neutrophil

  8. Splenic Marginal Zone Granulocytes Acquire an Accentuated Neutrophil B-Cell Helper Phenotype in Chronic Lymphocytic Leukemia.

    Science.gov (United States)

    Gätjen, Marcel; Brand, Franziska; Grau, Michael; Gerlach, Kerstin; Kettritz, Ralph; Westermann, Jörg; Anagnostopoulos, Ioannis; Lenz, Peter; Lenz, Georg; Höpken, Uta E; Rehm, Armin

    2016-09-15

    Recruitment of tumor-associated macrophages and neutrophils (TAM and TAN) to solid tumors contributes to immunosuppression in the tumor microenvironment; however, their contributions to lymphoid neoplasms are less clear. In human chronic lymphocytic leukemia (CLL), tumor B cells lodge in lymph nodes where interactions with the microenvironment occur. Tumor cell homing stimulates proliferation, such that engagement of the B-cell receptor is important for malignant progression. In the Eμ-Tcl1 murine model of CLL, we identified gene expression signatures indicative of a skewed polarization in the phenotype of monocytes and neutrophils. Selective ablation of either of these cell populations in mice delayed leukemia growth. Despite tumor infiltration of these immune cells, a systemic inflammation was not detected. Notably, in progressive CLL, splenic neutrophils were observed to differentiate toward a B-cell helper phenotype, a process promoted by the induction of leukemia-associated IL10 and TGFβ. Our results suggest that targeting aberrant neutrophil differentiation and restoring myeloid cell homeostasis could limit the formation of survival niches for CLL cells. Cancer Res; 76(18); 5253-65. ©2016 AACR. ©2016 American Association for Cancer Research.

  9. Myeloid Engraftment in Humanized Mice: Impact of Granulocyte-Colony Stimulating Factor Treatment and Transgenic Mouse Strain.

    Science.gov (United States)

    Coughlan, Alice M; Harmon, Cathal; Whelan, Sarah; O'Brien, Eóin C; O'Reilly, Vincent P; Crotty, Paul; Kelly, Pamela; Ryan, Michelle; Hickey, Fionnuala B; O'Farrelly, Cliona; Little, Mark A

    2016-04-01

    Poor myeloid engraftment remains a barrier to experimental use of humanized mice. Focusing primarily on peripheral blood cells, we compared the engraftment profile of NOD-scid-IL2Rγc(-/-) (NSG) mice with that of NSG mice transgenic for human membrane stem cell factor (hu-mSCF mice), NSG mice transgenic for human interleukin (IL)-3, granulocyte-macrophage-colony stimulating factor (GM-CSF), and stem cell factor (SGM3 mice). hu-mSCF and SGM3 mice showed enhanced engraftment of human leukocytes compared to NSG mice, and this was reflected in the number of human neutrophils and monocytes present in these strains. Importantly, discrete classical, intermediate, and nonclassical monocyte populations were identifiable in the blood of NSG and hu-mSCF mice, while the nonclassical population was absent in the blood of SGM3 mice. Granulocyte-colony stimulating factor (GCSF) treatment increased the number of blood monocytes in NSG and hu-mSCF mice, and neutrophils in NSG and SGM3 mice; however, this effect appeared to be at least partially dependent on the stem cell donor used to engraft the mice. Furthermore, GCSF treatment resulted in a preferential expansion of nonclassical monocytes in both NSG and hu-mSCF mice. Human tubulointerstitial CD11c(+) cells were present in the kidneys of hu-mSCF mice, while monocytes and neutrophils were identified in the liver of all strains. Bone marrow-derived macrophages prepared from NSG mice were most effective at phagocytosing polystyrene beads. In conclusion, hu-mSCF mice provide the best environment for the generation of human myeloid cells, with GCSF treatment further enhancing peripheral blood human monocyte cell numbers in this strain.

  10. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    Science.gov (United States)

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

  11. Moraxella catarrhalis induces CEACAM3‐Syk‐CARD9‐dependent activation of human granulocytes

    Science.gov (United States)

    Heinrich, A.; Heyl, K.A.; Klaile, E.; Müller, M.M.; Klassert, T.E.; Wiessner, A.; Fischer, K.; Schumann, R.R.; Seifert, U.; Riesbeck, K.; Moter, A.; Singer, B.B.; Bachmann, S.

    2016-01-01

    Summary The human restricted pathogen Moraxella catarrhalis is an important causal agent for exacerbations in chronic obstructive lung disease in adults. In such patients, increased numbers of granulocytes are present in the airways, which correlate with bacteria‐induced exacerbations and severity of the disease. Our study investigated whether the interaction of M. catarrhalis with the human granulocyte‐specific carcinoembryonic antigen‐related cell adhesion molecule (CEACAM)‐3 is linked to NF‐κB activation, resulting in chemokine production. Granulocytes from healthy donors and NB4 cells were infected with M. catarrhalis in the presence of different inhibitors, blocking antibodies and siRNA. The supernatants were analysed by enzyme‐linked immunosorbent assay for chemokines. NF‐κB activation was determined using a luciferase reporter gene assay and chromatin‐immunoprecipitation. We found evidence that the specific engagement of CEACAM3 by M. catarrhalis ubiquitous surface protein A1 (UspA1) results in the activation of pro‐inflammatory events, such as degranulation of neutrophils, ROS production and chemokine secretion. The interaction of UspA1 with CEACAM3 induced the activation of the NF‐κB pathway via Syk and the CARD9 pathway and was dependent on the phosphorylation of the CEACAM3 ITAM‐like motif. These findings suggest that the CEACAM3 signalling in neutrophils is able to specifically modulate airway inflammation caused by infection with M. catarrhalis. PMID:27038042

  12. Yops of Yersinia enterocolitica Inhibit Receptor-Dependent Superoxide Anion Production by Human Granulocytes

    Science.gov (United States)

    Visser, L. G.; Seijmonsbergen, E.; Nibbering, P. H.; van den Broek, P. J.; van Furth, R.

    1999-01-01

    The virulence plasmid-borne genes encoding Yersinia adhesin A (YadA) and several Yersinia secreted proteins (Yops) are involved in the inhibition of phagocytosis and killing of Yersinia enterocolitica by human granulocytes. One of these Yops, YopH, dephosphorylates multiple tyrosine-phosphorylated proteins in eukaryotic cells and is involved in the inhibition of phagocytosis of Y. enterocolitica by human granulocytes. We investigated whether antibody- and complement-opsonized plasmid-bearing (pYV+) Y. enterocolitica inhibits O2− production by human granulocytes in response to various stimuli and whether YopH is involved. Granulocytes were preincubated with mutant strains unable to express YadA or to secrete Yops or YopH. O2− production by granulocytes during stimulation was assessed by measuring the reduction of ferricytochrome c. PYV+ Y. enterocolitica inhibited O2− production by granulocytes incubated with opsonized Y. enterocolitica or N-formyl-Met-Leu-Phe (f-MLP). This inhibitory effect mediated by pYV did not affect receptor-independent O2− production by granulocytes in response to phorbol myristate acetate, indicating that NADPH activity remained unaffected after activation of protein kinase C. The inhibition of f-MLP-induced O2− production by granulocytes depends on the secretion of Yops and not on the expression of YadA. Insertional inactivation of the yopH gene abrogated the inhibition of phagocytosis of antibody- and complement-opsonized Y. enterocolitica by human granulocytes but not of the f-MLP-induced O2− production by granulocytes or tyrosine phosphorylation of granulocyte proteins. These findings suggest that the specific targets for YopH are not present in f-MLP receptor-linked signal transduction and that other Yop-mediated mechanisms are involved. PMID:10024567

  13. GATA2−/− human ESCs undergo attenuated endothelial to hematopoietic transition and thereafter granulocyte commitment

    Directory of Open Access Journals (Sweden)

    Ke Huang

    2015-01-01

    Conclusion: Our findings revealed the essential roles of GATA2 in EHT and granulocyte development through regulating SPI1, and uncovered a role of Notch signaling in granulocyte generation during hematopoiesis modeled by human ESCs.

  14. Human granulocyte colony stimulating factor (hG-CSF: cloning, overexpression, purification and characterization

    Directory of Open Access Journals (Sweden)

    Vanz Ana LS

    2008-04-01

    Full Text Available Abstract Background Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3 host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4% to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost

  15. Treatment of human granulocytic ehrlichiosis during pregnancy and risk of perinatal transmission.

    Science.gov (United States)

    Casau, Nathalie C; Hewins, Mary Ellen; Zaleznik, Dori F

    2002-01-01

    A pregnant woman from Nantucket Island, MA was diagnosed with human granulocytic ehrlichiosis at 34 weeks gestation. We describe the diagnostic and therapeutic dilemmas involved and discuss the risks of perinatal transmission.

  16. PU.1 regulates the expression of the human neutrophil elastase gene.

    Science.gov (United States)

    Srikanth, S; Rado, T A

    1998-06-16

    PU.1 is a transcription factor present in B-cells and macrophages. Here, we report our studies on the role of PU.1 in myelopoiesis using human neutrophil elastase (HNE) as a model. HNE, a component of the primary granules of mature granulocytes, is a serine protease which is transcriptionally restricted to the late promyelocytic stage of granulocytic maturation. The first 200 bp of the HNE promoter directs myeloid specific expression of a reporter gene and a 30-bp element within this region was been identified as the major determinant of myeloid specific expression [S. Srikanth, T. Rado, A 30-bp element is responsible for the myeloid specific activity of the human neutrophil elastase promoter, J. Biol. Chem. 269 (1994) 32626-32632.]. We now show that the B-cell and macrophage specific transcription factor, PU.1, binds to the PU.1 consensus site within the 30-bp element to activate transcription. Substitution mutations within this recognition sequence results in the loss of PU.1 binding and in a 90% decrease in promoter activity in myeloid cells. Cotransfection of PU.1 and a reporter gene controlled by the HNE promoter into non-myeloid HeLa cells resulted in activation of reporter gene transcription.

  17. Effects Of Gelatine-Coated Vascular Grafts On Human Neutrophils.

    Science.gov (United States)

    Meyer, Frank; Buerger, Thomas; Halloul, Zuhir; Lippert, Hans; König, Brigitte; Tautenhahn, Joerg

    2015-09-01

    The aim of the study was to investigate the immune-modulatory potential of commercially available PTFE and polyester vascular grafts with and without gelatine-coating. The biomaterial-cell-interaction was characterized by changes of established parameters such as PMN-related receptors/mediators, phagocytosis potential and capacity as well as the effect of an additional plasma-dependent modulation. By means of a standardized experimental in vitro model, various vascular graft material (PTFE/polyester/uncoated/gelatine-coated) was used for incubation with or without plasma and co-culturing with human neutrophile granulocytes (PMN) followed by analysis of representative receptors and mediators (CD62L, CD11b, CXCR2, fMLP-R, IL-8, Elastase, LTB4). Oxidative burst assessed phagocytosis capacity. Comparing the vascular grafts, un-coated PTFE induced the lowest magnitude of cell stimulation whereas in case of gelatine-coating, cell response exceeded those of the other vascular grafts. This was also found comparing the polyester-based prosthetic material. Gelatine-coated polyester led to a more pronounced release of elastase than gelatine-coated PTFE and the uncoated materials. The results of oxidative burst indicated a reduced phagocytosis capacity in case of gelatine-coated polyester. Plasma incubation did also provide an impact on the cellular response. While in case of gelatine-coating, PMN-related receptor stimulation became lower, it increased by native polyester. The latter one did also induce more mediators such as IL-8 and LTB4 than gelatine-coated material. There have been no extensive data on cell-cell interactions, cytokines and general histo-/hemocompatibility of human cells by the new generation of vascular grafts. It remains still open whether healing process and infectious resistance can be compromised by material-dependent over-stimulation or reduced phagocytosis potential of the immune cells of the primary unspecific immune response induced by gelatine

  18. Characterization of Yersinia pestis Interactions with Human Neutrophils In vitro

    Directory of Open Access Journals (Sweden)

    Sophia C. Dudte

    2017-08-01

    Full Text Available Yersinia pestis is a gram-negative, zoonotic, bacterial pathogen, and the causative agent of plague. The bubonic form of plague occurs subsequent to deposition of bacteria in the skin by the bite of an infected flea. Neutrophils are recruited to the site of infection within the first few hours and interactions between neutrophils and Y. pestis have been demonstrated in vivo. In contrast to macrophages, neutrophils have been considered non-permissive to Y. pestis intracellular survival. Several studies have shown killing of the vast majority of Y. pestis ingested by human neutrophils. However, survival of 10–15% of Y. pestis after phagocytosis by neutrophils is consistently observed. Furthermore, these surviving bacteria eventually replicate within and escape from the neutrophils. We set out to further characterize the interactions between Y. pestis and human neutrophils by (1 determining the effects of known Y. pestis virulence factors on bacterial survival after uptake by neutrophils, (2 examining the mechanisms employed by the neutrophil to kill the majority of intracellular Y. pestis, (3 determining the activation phenotype of Y. pestis-infected neutrophils, and (4 characterizing the Y. pestis-containing phagosome in neutrophils. We infected human neutrophils in vitro with Y. pestis and assayed bacterial survival and uptake. Deletion of the caf1 gene responsible for F1 capsule production resulted in significantly increased uptake of Y. pestis. Surprisingly, while the two-component regulator PhoPQ system is important for survival of Y. pestis within neutrophils, pre-induction of this system prior to infection did not increase bacterial survival. We used an IPTG-inducible mCherry construct to distinguish viable from non-viable intracellular bacteria and determined the association of the Y. pestis-containing phagosome with neutrophil NADPH-oxidase and markers of primary, secondary and tertiary granules. Additionally, we show that inhibition of

  19. Recombinant human granulocyte colony-stimulating factor (rhG-CSF; filgrastim) treatment of clozapine-induced agranulocytosis

    DEFF Research Database (Denmark)

    Nielsen, H

    1993-01-01

    After 10 weeks of treatment with clozapine, severe agranulocytosis was diagnosed in a 33-year-old female. The patient was treated with filgrastim (granulocyte colony-stimulating factor [G-CSF]) 5 micrograms kg-1 day-1. The neutrophil count was 0.234 x 10(9) l-1 on admission, with a further decrease...... of neutrophil granulocytes (including immature forms) of 33.108 x 10(9) l-1 on day 12 after admission. The patient only had minor infectious complications during the neutropenic period. In conclusion, early treatment with filgrastim seems warranted in severe cases of clozapine-induced agranulocytosis. A dosage...

  20. Transcriptome kinetics of circulating neutrophils during human experimental endotoxemia.

    Directory of Open Access Journals (Sweden)

    Stan de Kleijn

    Full Text Available Polymorphonuclear cells (neutrophils play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n = 4 administered a single dose of endotoxin (LPS, 2 ng/kg iv. In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFα, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFα and IL-1α and IL-1β. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. These changes in neutrophil transcriptome suggest a combination of early activation of circulating neutrophils by TNFα and G-CSF and a mobilization of young neutrophils from the bone marrow.

  1. Evaluation of lactate, white blood cell count, neutrophil count, procalcitonin and immature granulocyte count as biomarkers for sepsis in emergency department patients.

    Science.gov (United States)

    Karon, Brad S; Tolan, Nicole V; Wockenfus, Amy M; Block, Darci R; Baumann, Nikola A; Bryant, Sandra C; Clements, Casey M

    2017-11-01

    Lactate, white blood cell (WBC) and neutrophil count, procalcitonin and immature granulocyte (IG) count were compared for the prediction of sepsis, and severe sepsis or septic shock, in patients presenting to the emergency department (ED). We prospectively enrolled 501 ED patients with a sepsis panel ordered for suspicion of sepsis. WBC, neutrophil, and IG counts were measured on a Sysmex XT-2000i analyzer. Lactate was measured by i-STAT, and procalcitonin by Brahms Kryptor. We classified patients as having sepsis using a simplification of the 1992 consensus conference sepsis definitions. Patients with sepsis were further classified as having severe sepsis or septic shock using established criteria. Univariate receiver operating characteristic (ROC) analysis was performed to determine odds ratio (OR), area under the ROC curve (AUC), and sensitivity/specificity at optimal cut-off for prediction of sepsis (vs. no sepsis), and prediction of severe sepsis or septic shock (vs. no sepsis). There were 267 patients without sepsis; and 234 with sepsis, including 35 patients with severe sepsis or septic shock. Lactate had the highest OR (1.44, 95th% CI 1.20-1.73) for the prediction of sepsis; while WBC, neutrophil count and percent (neutrophil/WBC) had OR>1.00 (psepsis or septic shock, with an odds ratio (95th% CI) of 2.70 (2.02-3.61) and AUC 0.89 (0.82-0.96). Traditional biomarkers (lactate, WBC, neutrophil count, procalcitonin, IG) have limited utility in the prediction of sepsis. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  2. Synchronisation of glycolytic oscillations in a suspension of human neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Poulsen, Allan K.; Olsen, Lars Folke

    Neutrophils are known to be able to synchronize their production of superoxide. We show that glycolysis is also synchronized in human neutrophils being in suspension and suggest that oscillations in glycolysis are driving the pulsatile production of superoxide. The synchronising agent remains so...... far unknown, however, much evident points to that it might be hydrogen peroxide or an intermediate in glycolysis....

  3. Regulation of calcium homeostasis in activated human neutrophils ...

    African Journals Online (AJOL)

    Objectives. The objectives of the current study were to: (i) present an integrated model for the restoration of calcium homeostasis in activated human neutrophils based on current knowledge and recent research; and (ii) identify potential targets for the modulation of calcium fluxes in activated neutrophils based on this model ...

  4. Characterization of neutrophil subsets in healthy human pregnancies.

    Directory of Open Access Journals (Sweden)

    Aloysius Ssemaganda

    Full Text Available We have previously shown that in successful pregnancies increased arginase activity is a mechanism that contributes to the suppression of the maternal immune system. We identified the main type of arginase-expressing cells as a population of activated low-density granulocytes (LDGs in peripheral blood mononuclear cells and in term placentae. In the present study, we analyzed the phenotype of LDGs and compared it to the phenotype of normal density granulocytes (NDGs in maternal peripheral blood, placental biopsies and cord blood. Our data reveal that only LDGs but no NDGs could be detected in placental biopsies. Phenotypically, NDGs and LDGs from both maternal and cord blood expressed different levels of maturation, activation and degranulation markers. NDGs from the maternal and cord blood were phenotypically similar, while maternal, cord and placental LDGs showed different expression levels of CD66b. LDGs present in cord blood expressed higher levels of arginase compared to maternal and placental LDGs. In summary, our results show that in maternal and cord blood, two phenotypically different populations of neutrophils can be identified, whereas in term placentae, only activated neutrophils are present.

  5. Human neutrophils facilitate tumor cell transendothelial migration.

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

    Tumor cell extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain unclear. In this study, using an in vitro transendothelial migration model, we show that human polymorphonuclear neutrophils (PMN) assist the human breast tumor cell line MDA-MB-231 to cross the endothelial barrier. We found that tumor-conditioned medium (TCM) downregulated PMN cytocidal function, delayed PMN apoptosis, and concomitantly upregulated PMN adhesion molecule expression. These PMN treated with TCM attached to tumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did not transmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) but did not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 on PMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells still possessed the ability to proliferate after PMN-assisted transmigration. These results indicate that TCM-treated PMN may serve as a carrier to assist tumor cell transendothelial migration and suggest that tumor cells can exploit PMN and alter their function to facilitate their extravasation.

  6. Human neutrophils facilitate tumor cell transendothelial migration.

    Science.gov (United States)

    Wu, Q D; Wang, J H; Condron, C; Bouchier-Hayes, D; Redmond, H P

    2001-04-01

    Tumor cell extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain unclear. In this study, using an in vitro transendothelial migration model, we show that human polymorphonuclear neutrophils (PMN) assist the human breast tumor cell line MDA-MB-231 to cross the endothelial barrier. We found that tumor-conditioned medium (TCM) downregulated PMN cytocidal function, delayed PMN apoptosis, and concomitantly upregulated PMN adhesion molecule expression. These PMN treated with TCM attached to tumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did not transmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) but did not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 on PMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells still possessed the ability to proliferate after PMN-assisted transmigration. These results indicate that TCM-treated PMN may serve as a carrier to assist tumor cell transendothelial migration and suggest that tumor cells can exploit PMN and alter their function to facilitate their extravasation.

  7. Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes.

    Science.gov (United States)

    Bruzzone, Santina; Basile, Giovanna; Mannino, Elena; Sturla, Laura; Magnone, Mirko; Grozio, Alessia; Salis, Annalisa; Fresia, Chiara; Vigliarolo, Tiziana; Guida, Lucrezia; De Flora, Antonio; Tossi, Vanesa; Cassia, Raul; Lamattina, Lorenzo; Zocchi, Elena

    2012-06-01

    UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation. Copyright © 2011 Wiley Periodicals, Inc.

  8. A comparative study of adverse reactions occurring after administration of glycosylated granulocyte colony stimulating factor and/or dexamethasone for mobilization of neutrophils in healthy donors.

    Science.gov (United States)

    Heuft, H G; Goudeva, L; Blasczyk, R

    2004-05-01

    Both granulocyte colony-stimulating factor (G-CSF) and dexamethasone (DXM) are used for neutrophil (PMN) mobilization and collection. This prospective study was aimed to evaluate and compare the rate, severity and clinical significance of adverse reactions of these drugs alone and in combination in healthy donors. PMN mobilization was carried out using dexamethasone alone (8 mg orally; n=25) or glycosylated G-CSF alone (Lenograstim, 5 microg/kg subcutaneously, n=24) or in combination (n=23) prior to a standard granulocyte apheresis on the Spectra cell separator. The number of PMNs counted in the mobilized peripheral blood of the donors was 7.0 (3.6-20.4) x10(9)/L (DXM), 25.2 (15.5-49.7) x10(9)/L (G-CSF), and 31.6 (20.0-43.0) x10(9)/L (G-CSF+DXM), corresponding to PMN apheresis yields of 13 (8-43) x10(9)/U, 56 (34-118) x10(9)/U, and 83 (33-117) x10(9)/U, respectively. The three groups had comparable percentages of donors with at least one adverse effect (ranging from 75 to 80%), but the G-CSF-containing regimens were generally more toxic, as was reflected by higher percentages of donors with moderate to severe adverse reactions and higher overall severity scores of 2.28 (G-CSF) and 2.08 (G-CSF+DXM) compared with 1.33 in the DXM group ( pdonors; 19%) and unwillingness to give further neutrophil donations (2/47 donors; 4%). The addition of DXM to G-CSF diminished some symptoms, particularly bone pain, headache and the frequency of requests for analgesics. The predominant symptoms in the DXM alone group were mild gastrointestinal complaints. We conclude that G-CSF stimulation improved neutrophil mobilization and apheresis yields at the expense of donor tolerability. Compared with G-CSF alone, the combination G-CSF and DXM did not increase the quantity or the severity of donor symptoms.

  9. Paclitaxel, ifosfamide and cisplatin with granulocyte colony-stimulating factor or recombinant human interleukin 3 and granulocyte colony-stimulating factor in ovarian cancer : A feasibility study

    NARCIS (Netherlands)

    Veldhuis, GJ; Willemse, PHB; Beijnen, JH; Piersma, H; vanderGraaf, WTA; deVries, EGE; Boonstra, J.

    1997-01-01

    The tolerability and efficacy of four courses of paclitaxel and ifosfamide plus cisplatin every 3 weeks was evaluated in patients with residual or refractory ovarian cancer. Additionally, supportive haematological effects of recombinant human interleukin 3 (rhIL-3) and recombinant human granulocyte

  10. Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives.

    Science.gov (United States)

    Lahoz-Beneytez, Julio; Elemans, Marjet; Zhang, Yan; Ahmed, Raya; Salam, Arafa; Block, Michael; Niederalt, Christoph; Asquith, Becca; Macallan, Derek

    2016-06-30

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day. © 2016 by The American Society of Hematology.

  11. Aliphatic alcohol contaminants of illegally produced spirits inhibit phagocytosis by human granulocytes.

    Science.gov (United States)

    Pál, László; Árnyas, Ervin M; Tóth, Béla; Ádám, Balázs; Rácz, Gábor; Ádány, Róza; McKee, Martin; Szűcs, Sándor

    2013-04-01

    Unregulated production of spirits in many countries leads to products containing appreciable levels of aliphatic alcohols (AAs) and is the main source of human exposure to these substances worldwide. Previous studies have confirmed that alcohol abuse can lead to ethanol-induced immunosuppression and thereby increased susceptibility to infectious diseases. Granulocytes, as professional phagocytic cells, play a crucial role in engulfment and killing of pathogenic microorganisms. Thus, a decrease in their phagocytic activity has been invoked as a factor in the impaired antimicrobial defense observed in alcoholics. However, AAs consumed as contaminants of illicit spirits may also influence phagocytosis, thereby contributing to a further decrease in microbicidal activity but, so far, this has not been studied. Therefore, the aim of this study was to measure granulocyte phagocytosis following treatment of granulocytes with those higher alcohols found in illegal spirits. Granulocytes were isolated from human peripheral blood. Then phagocytosis of opsonized zymosan particles by granulocytes treated with AAs individually and in combination was determined. These alcohols inhibited phagocytosis in a concentration-dependent manner and at lower concentrations when combined than when tested individually. Due to their synergistic effects, it is possible that, in combination with ethanol, they may inhibit phagocytosis in a clinically meaningful way in episodic heavy drinkers.

  12. The transcriptional activation program of human neutrophils in skin lesions supports their important role in wound healing

    DEFF Research Database (Denmark)

    Theilgaard-Monch, K.; Knudsen, Steen; Follin, P.

    2004-01-01

    receptors involved in inflammatory responses. These findings indicate a change of responsiveness to chemotactic and immunoregulatory mediators once PMNs have migrated to skin lesions and have been activated. Other effects of the up-regulated cytokines/chemokines/enzymes were critical for wound healing......To investigate the cellular fate and function of polymorphonuclear neutrophilic granulocytes (PMNs) attracted to skin wounds, we used a human skin-wounding model and microarray technology to define differentially expressed genes in PMNs from peripheral blood, and PMNs that had transmigrated to skin...... and function, and promotes wound healing....

  13. Membrane permeability of the human granulocyte to water, dimethyl sulfoxide, glycerol, propylene glycol and ethylene glycol.

    Science.gov (United States)

    Vian, Alex M; Higgins, Adam Z

    2014-02-01

    Granulocytes are currently transfused as soon as possible after collection because they rapidly deteriorate after being removed from the body. This short shelf life complicates the logistics of granulocyte collection, banking, and safety testing. Cryopreservation has the potential to significantly increase shelf life; however, cryopreservation of granulocytes has proven to be difficult. In this study, we investigate the membrane permeability properties of human granulocytes, with the ultimate goal of using membrane transport modeling to facilitate development of improved cryopreservation methods. We first measured the equilibrium volume of human granulocytes in a range of hypo- and hypertonic solutions and fit the resulting data using a Boyle-van't Hoff model. This yielded an isotonic cell volume of 378 μm(3) and an osmotically inactive volume of 165 μm(3). To determine the permeability of the granulocyte membrane to water and cryoprotectant (CPA), cells were injected into well-mixed CPA solution while collecting volume measurements using a Coulter Counter. These experiments were performed at temperatures ranging from 4 to 37°C for exposure to dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol. The best-fit water permeability was similar in the presence of all of the CPAs, with an average value at 21°C of 0.18 μmatm(-1)min(-1). The activation energy for water transport ranged from 41 to 61 kJ/mol. The CPA permeability at 21°C was 6.4, 1.0, 8.4, and 4.0 μm/min for dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol, respectively, and the activation energy for CPA transport ranged between 59 and 68 kJ/mol. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Chemotactic Activity on Human Neutrophils to Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Tetiana Haniastuti

    2013-07-01

    Full Text Available Objective: The aim of this study was to evaluate chemotactic activity o neutrophil to S. mutans. Chemotaxis assay was performed in blind well chambers. Materials and Methods: Hanks balanced salt solution (HBSS containing 106 S. mutans,  108 S. mutans, 10-8 M fMLP, or HBSS alone were placed in the lower wells of the chamber and covered with polycorbonate membrane filter. Neutrophils suspension (2x105 cells was then placed in the upper compartment. After incubation for 60 mins at 37ºC in a humidified atmosphere with 5% CO2, the filters were removed and stained with Giemsa. Result: ANOVA revealed statistically significant differences among groups (p<0.05, indicating that S. mutans induced neutrophils chemotaxis. The number of neutrophils migration in response to 108 S. mutans and 106 S. mutans were signifiantly greater compared to fMLP (p<0.05. Conclusion: S. mutans may activate human neutrophils, resulting in the chemotaxis of the neutrophils.DOI: 10.14693/jdi.v16i2.99

  15. Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives

    OpenAIRE

    Lahoz-Beneytez, J; Elemans, M; Zhang, Y.; R. Ahmed; Salam, A.; Block, M.; Niederalt, C; Asquith, B; Macallan, D

    2016-01-01

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4-18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water which yielded estimates in excess of 3 days. To investigate this disparity we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n=4) and deuterium-labeled glucose (n=9), a compound with more rapid labeling kinetics. To interpret results we developed...

  16. Respiratory syncytial virus fusion protein promotes TLR-4-dependent neutrophil extracellular trap formation by human neutrophils.

    Directory of Open Access Journals (Sweden)

    Giselle A Funchal

    Full Text Available Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.

  17. Cytostasis of tumor cell lines by granulocytes from cancer patients and normal human donors.

    Science.gov (United States)

    Korec, S; Herberman, R B; Cannon, G B; Reid, J; Braatz, J A

    1981-08-15

    Granulocytes of normal human donors were previously shown to have cytostatic activity in vitro against a variety of tumor cell lines. In the present study, we have compared the levels of granulocyte-mediated cytostatic activity in cancer patients and normal donors. In an initial study of 25 tumor-bearing patients and 21 individuals with benign or no disease, decreased cytostatic activity was observed in 84% of the cancer patients. Nine cancer patients with no evidence of disease had reactivity in the normal range. Granulocytes separated by a one-step method on a double Ficoll-Percoll gradient showed decreased reactivity. This procedure eliminated the differences previously detected between tumor-bearing patients and controls. Addition of either pooled normal AB human serum or autologous serum to the assay restored the reactivity. Only with autologous serum and not with allogeneic serum, were the differences between tumor-bearing patients and controls again seen. Therefore, in a subsequent study, we examined the effect of serum on cytostasis by normal granulocytes that were isolated on double gradients. We observed lowered serum restorative activity (SRA) in 41 of the 46 (89%) tumor-bearing patients tested. Fractionation of sera by Sephadex G-200 chromatography indicated that SRA of both cancer patients and normal donors was in the 100,000 molecular weight region.

  18. Recombinant human granulocyte colony-stimulating factor (rhG-CSF; filgrastim) treatment of clozapine-induced agranulocytosis

    DEFF Research Database (Denmark)

    Nielsen, H

    1993-01-01

    of neutrophil granulocytes (including immature forms) of 33.108 x 10(9) l-1 on day 12 after admission. The patient only had minor infectious complications during the neutropenic period. In conclusion, early treatment with filgrastim seems warranted in severe cases of clozapine-induced agranulocytosis. A dosage...... the next day to dosage of filgrastim was increased to 10 micrograms kg-1 day-1 with immediate improvement. A rapid and pronounced leucocytosis developed with maximal value...

  19. Entamoeba histolytica induces human neutrophils to form NETs.

    Science.gov (United States)

    Ventura-Juarez, J; Campos-Esparza, Mr; Pacheco-Yepez, J; López-Blanco, J A; Adabache-Ortíz, A; Silva-Briano, M; Campos-Rodríguez, R

    2016-08-01

    Entamoeba histolytica invades the intestine and other organs during the pathogenesis of amoebiasis. In the early stages, the host organism responds with an inflammatory infiltrate composed mostly of neutrophils. It has been reported that these immune cells, activated by E. histolytica, exert a protective role by releasing proteolytic enzymes and generating reactive oxygen/nitrogen species (ROS/RNS) and antimicrobial peptides. It is now known that neutrophils also produce neutrophil extracellular traps (NETs), which are able to damage and kill pathogens. Studies have shown that intracellular protozoan pathogens, including Toxoplasma gondi, Plasmodium falciparum and Leishmania spp, induce neutrophils to release NETs and are damaged by them. However, the action of this mechanism has not been explored in relation to E. histolytica trophozoites. Through scanning electron, epifluorescence microscopy and viability assays, we show for first time that during in vitro interaction with E. histolytica trophozoites, human neutrophils released NETs that covered amoebas and reduced amoebic viability. These NETs presented histones, myeloperoxidase and decondensed chromatin. The results suggest that NETs participate in the elimination of the parasite. © 2016 John Wiley & Sons Ltd.

  20. [EPIDEMIOLOGIC ASPECTS OF HUMAN GRANULOCYTIC ANAPLASMOSIS IN THE WESTERN REGION OF UKRAINE].

    Science.gov (United States)

    Ben, I I; Biletska, H V

    2015-01-01

    This article presents data of complex study of human granulocytic anaplasmosis the human granulocytic anaplasmosis (HGA) in the western region of Ukraine. Natural HGA foci were identified, where the prevalence of A. phagocytophilum in the main vector (I. ricinus) amount to (12.0 +/- 0.7) %, and seroprevalence of HGA among the healthy population--(28.6 +/- 1.6) %. It's shown that A. phagocytophilum is the etiologic agent of (33.7 +/- 4.9) % of undiagnosed cases of seasonal febrile diseases. Principal characteristics of HGA epidemiology: spring-summer scasonality, the prevalence of disease in the age structure of people of active age (45.30 +/- 1.95 years), a significant level combination with (60.4 +/- 2.2) % Lyme-horreliosis and other tick-borne infections (mixed infections), the different level of activity of HGA epidemic process in forest and steppe geographical landscape zones--were revealed.

  1. Technical advance: immunophenotypical characterization of human neutrophil differentiation

    DEFF Research Database (Denmark)

    Mora-Jensen, Helena Isabel; Jendholm, Johan; Fossum, Anna

    2011-01-01

    The current study reports a flow cytometry-based protocol for the prospective purification of human BM populations representing six successive stages of terminal neutrophil differentiation, including early promyelocytes and late promyelocytes, myelocytes, metamyelocytes, band cells, and PMN...... differentiation in vivo in the human setting and constitutes an important alternative to models that are based on in vitro differentiation of myeloid cell lines and HPCs....

  2. Extracellular NAD+ regulates intracellular calcium levels and induces activation of human granulocytes

    Science.gov (United States)

    2005-01-01

    β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation. PMID:16225456

  3. Recombinant human granulocyte colony-stimulating factor increases circulating CD34-postive cells in patients with AIDS

    DEFF Research Database (Denmark)

    Nielsen, S D; Dam-Larsen, S; Nielsen, C

    1997-01-01

    .e., the circulating hematopoietic progenitor cells (CD34 cells) in patients with AIDS, using the recombinant human granulocyte colony-stimulating factor (G-CSF). Eight patients with AIDS were treated with G-CSF for neutropenia (... G-CSF for 3-5 consecutive days. Within 5 days of initiation of G-CSF therapy, an increase in the absolute neutrophil count (ANC) was seen in all patients. There was a median increase in ANC from 0.4 to 3.4 x 10(9)/l. In addition, G-CSF treatment significantly increased the absolute number of CD34...... cells. The median increase in CD34 cells was from 0.8 to 2.2 x 10(6)/l. Finally, using a highly sensitive HIV-1 RNA PCR, we found that treatment of AIDS patients with G-CSF did not lead to enhanced HIV replication. These observations indicate that G-CSF may be used to mobilize CD34 cells in patients...

  4. Transcellular migration of neutrophil granulocytes through the blood-cerebrospinal fluid barrier after infection with Streptococcus suis

    Directory of Open Access Journals (Sweden)

    Quitsch Ulrike

    2011-05-01

    Full Text Available Abstract Background A critical point during the course of bacterial meningitis is the excessive influx of polymorphnuclear neutrophils (PMNs from the blood into the brain. Both paracellular and transcellular routes of leukocyte transmigration through the blood-brain barrier have been described in CNS diseases so far. Thus, we investigated the mechanism of PMN transmigration through the blood-CSF barrier under inflammatory conditions. Methods In an "inverted" Transwell culture model of the blood-CSF barrier, the zoonotic agent Streptococcus suis (S. suis was used to stimulate porcine choroid plexus epithelial cells (PCPECs specifically from the physiologically relevant basolateral side. Barrier function was analyzed by measuring TEER and TR-dextran-flux, and tight junction morphology was investigated by immunofluorescence. Route and mechanism of PMN transmigration were determined by immunofluorescence, electron microscopy and FACS analysis. Quantitative real time-PCR was used to determine expression levels of ICAM-1 and VCAM-1. Results Here, we show that the transmigration of PMNs through PCPECs was significantly higher after stimulation with TNFα or infection with S. suis strain 10 compared to its non-encapsulated mutant. Barrier function was not significantly affected by PMN migration alone, but in combination with S. suis infection. Tight junction and cytoskeletal actin reorganisation were also observed after stimulation with S. suis or TNFα. Most strikingly, PMNs preferentially migrated across PCPECs via the transcellular route. Extensive sequential analyses of the PMN transmigration process with Apotome®-imaging and electron microscopy revealed that paracellular migrating PMNs stop just before tight junctions. Interestingly, PMNs subsequently appeared to proceed by transcellular migration via funnel-like structures developing from the apical membrane. It is noteworthy that some PMNs contained bacteria during the transmigration process

  5. Congenital Defects in Neutrophil Dynamics

    Directory of Open Access Journals (Sweden)

    Marton Keszei

    2014-01-01

    Full Text Available Neutrophil granulocytes are key effector cells of the vertebrate immune system. They represent 50–70% of the leukocytes in the human blood and their loss by disease or drug side effect causes devastating bacterial infections. Their high turnover rate, their fine-tuned killing machinery, and their arsenal of toxic vesicles leave them particularly vulnerable to various genetic deficiencies. The aim of this review is to highlight those congenital immunodeficiencies which impede the dynamics of neutrophils, such as migration, cytoskeletal rearrangements, vesicular trafficking, and secretion.

  6. Mechanism Underlying Levofloxacin Uptake by Human Polymorphonuclear Neutrophils

    OpenAIRE

    Vazifeh, Doina; Bryskier, André; Labro, Marie-Thérèse

    1999-01-01

    The mechanism of radiolabeled levofloxacin ([3H]levofloxacin) uptake by human polymorphonuclear neutrophils (PMNs) was investigated by a classical velocity centrifugation technique. PMNs were incubated with levofloxacin for 5 to 180 min under various conditions before centrifugation through an oil cushion. Radioactivity was measured in the cell pellet to determine the amount of cell-associated drug. The uptake of levofloxacin was moderate with a cellular concentration/extracellular concentrat...

  7. Tumor Associated Neutrophils in Human Lung Cancer

    Science.gov (United States)

    2016-10-01

    isolated from the samepatientwithNSCLC.Tcellproliferation in responsetoCD3/CD28wasperformedasdescribed inMaterials andMethods. Cell proliferationwas...PMNswere isolated as described inMaterials andMethods and then added to allogeneic MLR in the presence of neutralizing mouse anti-human LOX-1 antibody (10 mg

  8. Mouse neutrophilic granulocytes express mRNA encoding the macrophage colony-stimulating factor receptor (CSF-1R) as well as many other macrophage-specific transcripts and can transdifferentiate into macrophages in vitro in response to CSF-1.

    Science.gov (United States)

    Sasmono, R Tedjo; Ehrnsperger, Achim; Cronau, Stephen L; Ravasi, Timothy; Kandane, Rangi; Hickey, Michael J; Cook, Andrew D; Himes, S Roy; Hamilton, John A; Hume, David A

    2007-07-01

    The differentiation of macrophages from their progenitors is controlled by macrophage colony-stimulating factor (CSF-1), which binds to a receptor (CSF-1R) encoded by the c-fms proto-oncogene. We have previously used the promoter region of the CSF-1R gene to direct expression of an enhanced green fluorescent protein (EGFP) reporter gene to resident macrophage populations in transgenic mice. In this paper, we show that the EGFP reporter is also expressed in all granulocytes detected with the Gr-1 antibody, which binds to Ly-6C and Ly-6G or with a Ly-6G-specific antibody. Transgene expression reflects the presence of CSF-1R mRNA but not CSF-1R protein. The same pattern is observed with the macrophage-specific F4/80 marker. Based on these findings, we performed a comparative array profiling of highly purified granulocytes and macrophages. The patterns of mRNA expression differed predominantly through granulocyte-specific expression of a small subset of transcription factors (Egr1, HoxB7, STAT3), known abundant granulocyte proteins (e.g., S100A8, S100A9, neutrophil elastase), and specific receptors (fMLP, G-CSF). These findings suggested that appropriate stimuli might mediate rapid interconversion of the major myeloid cell types, for example, in inflammation. In keeping with this hypothesis, we showed that purified Ly-6G-positive granulocytes express CSF-1R after overnight culture and can subsequently differentiate to form F4/80-positive macrophages in response to CSF-1.

  9. Effect of probenecid on phagocytosis and intracellular killing of Staphylococcus aureus and Escherichia coli by human monocytes and granulocytes.

    Science.gov (United States)

    Buisman, H P; Buys, L F; Langermans, J A; van den Broek, P J; van Furth, R

    1991-01-01

    The present study concerns the effects of probenecid on the phagocytosis and intracellular killing of Staphylococcus aureus and Escherichia coli by human monocytes and granulocytes. In both monocytes and granulocytes the inhibitory effect on phagocytosis was very small. Inhibition of intracellular killing of S. aureus by monocytes and granulocytes by probenecid was concentration dependent, being half-maximal at about 2 mM probenecid, and near-maximal at about 5 mM probenecid. The intracellular killing could also be inhibited when probenecid was added when this process was already started. Probenecid also inhibited the intracellular killing of E. coli by granulocytes, but not by monocytes. In the concentration range used, probenecid had no toxic effect on phagocytes or bacteria during the 2 hr of the experiments. PMID:1748482

  10. Fibroblast growth factor 23 weakens chemotaxis of human blood neutrophils in microfluidic devices

    OpenAIRE

    Yang, Ke; Peretz-Soroka, Hagit; Wu, Jiandong; Zhu, Ling; Cui, Xueling; Zhang, Michael; Rigatto, Claudio; Liu, Yong; Lin, Francis

    2017-01-01

    Neutrophil trafficking in tissues critically regulates the body?s immune response. Neutrophil migration can either play a protective role in host defense or cause health problems. Fibroblast growth factor 23 (FGF23) is a known biomarker for chronic kidney disease (CKD) and was recently shown to impair neutrophil arrest on endothelium and transendothelial migration. In the present study, we further examined the effect of FGF23 on human blood neutrophil chemotaxis using two new microfluidic dev...

  11. Bone marrow changes associated with recombinant granulocyte-macrophage and granulocyte colony-stimulating factors. Discrimination of granulocytic regeneration.

    Science.gov (United States)

    Harris, A C; Todd, W M; Hackney, M H; Ben-Ezra, J

    1994-06-01

    The hematopoietic growth factors recombinant human granulocyte-macrophage colony-stimulating factor and recombinant human granulocyte colony-stimulating factor are associated with changes of the bone marrow. To evaluate the morphologic features and to differentiate them from leukemia, bone marrow specimens from 12 patients who had been treated with one of these agents were evaluated. The bone marrow displayed marked promyelocytic hyperplasia and a less striking increased percentage of myeloblasts. In each of the 11 patients without leukemia at the time of bone marrow biopsy, the percentage of promyelocytes in the bone marrow was greater than that of myeloblasts. Cytologic features of stimulated regeneration included diffuse cytoplasmic hypergranulation of immature neutrophilic precursors that had prominent perinuclear spherical clear areas representing the Golgi zones. With consideration of bone marrow composition and careful attention to cytologic detail, the distinction of bone marrow regeneration from acute leukemia can be made in most patients who are being treated with recombinant hematopoietic growth factors.

  12. Staphylococcus epidermidis Strategies to Avoid Killing by Human Neutrophils

    Science.gov (United States)

    Cheung, Gordon Y. C.; Rigby, Kevin; Wang, Rong; Queck, Shu Y.; Braughton, Kevin R.; Whitney, Adeline R.; Teintze, Martin; DeLeo, Frank R.; Otto, Michael

    2010-01-01

    Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus. PMID:20949069

  13. Anti-inflammatory sesquiterpene lactones from Tithonia diversifolia trigger different effects on human neutrophils

    Directory of Open Access Journals (Sweden)

    Aneli E. Abe

    Full Text Available Abstract The tagitinins isolated of Tithonia diversifolia (Hemsl. A. Gray, Asteraceae, are the most studied sesquiterpene lactones due to their wide spectrum of pharmacologic activities, especially related with nuclear factor-kappa B inhibition. Nevertheless, detailed studies about the mechanism of action of its active compounds are still lacking. Neutrophils perform a fundamental role in the inflammatory response to several etiologic factors. However, the effect of tagitinins on human neutrophil is not yet clearly known. We investigated the role of tagitinin C (1, tagitinin F (2 and tagitinin A (3 in activation and survival of human neutrophils to establish possible effects in their mechanisms of inflammation. Human neutrophils were purified from the peripheral blood and cultivated with tagitinins C (1, F (2 and A (3 in the presence or not of Escherichia coli lipopolysaccharide. The enzymatic activity, apoptosis and secretion of cytokines rate were determined after 18 h. Lipopolysaccharide-induced myeloperoxidase activity of human neutrophils was significantly inhibited only by tagitinin F (2. Apoptosis of neutrophils was increased in the presence of tagitinin C (1, and it occurred independently of the presence of lipopolysaccharide or dexamethasone. Tagitinins C (1, F (2 and A (3 decrease lipopolysaccharide-induced interleukin-6, interleukin-8 and Tumor necrosis factor alpha production by human neutrophils. Together, these results indicate that tagitinins exhibit anti-inflammatory action on human neutrophils. However, tagitinin F (2 was the only sesquiterpene lactone that decreased secretion of inflammatory products by neutrophils without inducing neutrophil apoptosis.

  14. Culture of human oocytes with granulocyte-macrophage colony-stimulating factor has no effect on embryonic chromosomal constitution

    DEFF Research Database (Denmark)

    Agerholm, Inge; Loft, Anne; Hald, Finn

    2010-01-01

    The effect on ploidy rate in donated human oocytes after in-vitro culture with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; 2 ng/ml) from fertilization until day 3 was examined in a multicentre, prospective placebo-controlled and double-blinded study including 73...

  15. A novel bacterial transport mechanism of Acinetobacter baumannii via activated human neutrophils through interleukin-8.

    Science.gov (United States)

    Kamoshida, Go; Tansho-Nagakawa, Shigeru; Kikuchi-Ueda, Takane; Nakano, Ryuichi; Hikosaka, Kenji; Nishida, Satoshi; Ubagai, Tsuneyuki; Higashi, Shouichi; Ono, Yasuo

    2016-12-01

    Hospital-acquired infections as a result of Acinetobacter baumannii have become problematic because of high rates of drug resistance. Although neutrophils play a critical role in early protection against bacterial infection, their interactions with A. baumannii remain largely unknown. To elucidate the interactions between A. baumannii and human neutrophils, we cocultured these cells and analyzed them by microscopy and flow cytometry. We found that A. baumannii adhered to neutrophils. We next examined neutrophil and A. baumannii infiltration into Matrigel basement membranes by an in vitro transmigration assay. Neutrophils were activated by A. baumannii, and invasion was enhanced. More interestingly, A. baumannii was transported together by infiltrating neutrophils. Furthermore, we observed by live cell imaging that A. baumannii and neutrophils moved together. In addition, A. baumannii-activated neutrophils showed increased IL-8 production. The transport of A. baumannii was suppressed by inhibiting neutrophil infiltration by blocking the effect of IL-8. A. baumannii appears to use neutrophils for transport by activating these cells via IL-8. In this study, we revealed a novel bacterial transport mechanism that A. baumannii exploits human neutrophils by adhering to and inducing IL-8 release for bacterial portage. This mechanism might be a new treatment target. © Society for Leukocyte Biology.

  16. Proteinase-activated receptor-2 up-regulation by Fcγ-receptor activation in human neutrophils

    Science.gov (United States)

    St-Onge, Mireille; Lagarde, Stéphanie; Laflamme, Cynthia; Rollet-Labelle, Emmanuelle; Marois, Louis; Naccache, Paul H.; Pouliot, Marc

    2010-01-01

    We shed new light on the expression and function of the proteinase-activated receptor (PAR) family, associated with inflammation and hyperalgesia, in human granulocytes. Resting cells expressed constitutive levels of PAR-2 and PAR-3 mRNA but not PAR-1 or PAR-4. Based on flow cytometry, stimulation with opsonized bacteria (Bop) specifically up-regulated cell surface expression of PAR-2 in a concentration-dependent and time-dependent manner, independent of transcription or de novo protein synthesis. Primary granules were identified as a source of preformed PAR-2 that can readily be mobilized at the surface on fusion with the plasma membrane. Cellular response to PAR-2 activation, measured as changes in intracellular calcium concentration, was enhanced in PAR-2 up-regulated cells. Increase of cell-surface PAR-2 and of cell responsiveness were dependent specifically on the engagement of immunoglobulin (Ig)-binding receptors. Together, our results reveal that mobilization of intracellular granules, in response to Ig-receptor activation, up-regulates PAR-2 surface expression and makes neutrophils more responsive to proteinase activity. This enhanced response to PAR-2 activation indicates that molecular communication between pain and inflammation may be more important than previously believed.—St-Onge, M., Lagarde, S., Laflamme, C., Rollet-Labelle, E., Marois, L., Naccache, P. H., Pouliot, M. Proteinase-activated receptor-2 up-regulation by Fcγ-receptor activation in human neutrophils. PMID:20154268

  17. Form Matters: Stable Helical Foldamers Preferentially Target Human Monocytes and Granulocytes.

    Science.gov (United States)

    Del Secco, Benedetta; Malachin, Giulia; Milli, Lorenzo; Zanna, Nicola; Papini, Emanuele; Cornia, Andrea; Tavano, Regina; Tomasini, Claudia

    2017-02-20

    Some hybrid foldamers of various length, all containing the (4R,5S)-4-carboxy-5-methyloxazolidin-2-one (d-Oxd) moiety alternating with an l-amino acid (l-Val, l-Lys, or l-Ala), were prepared in order to study their preferred conformations and to evaluate their biological activity. Surprisingly, only the longer oligomers containing l-Ala fold into well-established helices, whereas all the other oligomers give partially unfolded turn structures. Nevertheless, they all show good biocompatibility, with no detrimental effects up to 64 μm. After equipping some selected foldamers with the fluorescent tag rhodamine B, a quantitative analysis was performed by dose- and time-response fluorescence-activated cell sorting (FACS) assays with human HeLa cells and primary blood lymphocytes, granulocytes, and monocytes. Among the cell types analyzed, the oligomers associated with monocytes and granulocytes with greatest efficacy, still visible after 24 h incubation. This effect is even more pronounced for foldamers that are able to form stable helices. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. The granulocyte nucleus and lamin B receptor: avoiding the ovoid.

    Science.gov (United States)

    Hoffmann, Katrin; Sperling, Karl; Olins, Ada L; Olins, Donald E

    2007-06-01

    The major human blood granulocyte, the neutrophil, is an essential component of the innate immunity system, emigrating from blood vessels and migrating through tight tissue spaces to the site of bacterial or fungal infection where they kill and phagocytose invading microbes. Since the late nineteenth century, it has been recognized that the human neutrophil nucleus is distinctly not ovoid as in other cell types, but possesses a lobulated (segmented) shape. This deformable nucleus enhances rapid migration. Recent studies have demonstrated that lamin B receptor (LBR) is necessary for the non-ovoid shape. LBR is an integral membrane protein of the nuclear envelope. A single dominant mutation in humans leads to neutrophils with hypolobulated nuclei (Pelger-Huet anomaly); homozygosity leads to ovoid granulocyte nuclei. Interestingly, LBR is also an enzyme involved in cholesterol metabolism. Homozygosity for null mutations is frequently lethal and associated with severe skeletal deformities. In addition to the necessity for LBR, formation of the mature granulocyte nucleus also depends upon lamin composition and microtubule integrity. These observations are part of a larger question on the relationships between nuclear shape and cellular function.

  19. Effects of budlein A on human neutrophils and lymphocytes

    Directory of Open Access Journals (Sweden)

    Carollinie Dias KNOB

    Full Text Available ABSTRACT Sesquiterpene lactones (SLs are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases and other ailments. Objective In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. Material and Methods Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. Results Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-β, and IFN-γ production, but it did not lead to cell death. Conclusions Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells.

  20. Effects of budlein A on human neutrophils and lymphocytes

    Science.gov (United States)

    KNOB, Carollinie Dias; SILVA, Milena; GASPAROTO, Thaís Helena; OLIVEIRA, Carine Ervolino; AMÔR, Nádia Ghinelli; ARAKAWA, Nilton Syogo; COSTA, Fernando Batista; CAMPANELLI, Ana Paula

    2016-01-01

    ABSTRACT Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases and other ailments. Objective In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. Material and Methods Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. Results Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-β, and IFN-γ production, but it did not lead to cell death. Conclusions Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells. PMID:27383709

  1. Dual effect of neutrophils on secretory component production by human bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    C. Pilette

    2006-12-01

    Full Text Available A decreased bronchial expression of secretory component (SC was demonstrated in severe COPD, and correlated with neutrophils. Mechanisms of epithelial cell/neutrophils interactions remain however poorly understood. Calu-3 (human bronchial epithelial cells were incubated after confluence (in triplicate conditions with various ratios of activated neutrophils (0.5:1 to 15:1, neutrophils: Calu-3 cells. After 48hrs of co-culture supernatants were assayed for SC by ELISA. SC production by Calu-3 cells increased at intermediate neutrophil numbers (316±32 versus 193±19ng·ml–1, ratio of 5:1 versus control, mean±SEM of 3 experiments, p = 0.05. In contrast, a trend for decrease in SC was observed with high neutrophil numbers (111±19 versus 193±19ng·ml–1, ratio of 15:1 versus control, p = 0.06. The addition of secretory leukocyte protease inhibitor further increased SC upregulation at intermediate ratios, and inhibited the SC decrease at high neutrophil numbers. The mechanism of SC up-regulation by neutrophils did not implicate TNF-alpha or IL-1beta. This study provides direct evidence of a dual effect of neutrophils on epithelial SC. Our data suggest that neutrophils could differently affect epithelial immune secretory function according to the extent of neutrophil influx and/or to the reactivity of airway epithelial cells.

  2. Recombinant human granulocyte-macrophage colony-stimulating factor as treatment for chronic leg ulcers.

    Science.gov (United States)

    Borbolla-Escoboza, J R; María-Aceves, R; López-Hernández, M A; Collados-Larumbe, M T

    1997-01-01

    To evaluate the safety and effectiveness of a single subcutaneous perilesional administration of 300 micrograms of recombinant human granulocyte-macrophage colony stimulating factor (rHGM-CSF) for the treatment of chronic leg ulcers. Prospective, descriptive evaluation in an outpatient group. The Centro Médico Nacional 20 de Noviembre, ISSSTE, Mexico City. 10 patients with chronic leg ulcers. Ulcer diameter and side effects. After 4 weeks observation, 8 of the 10 ulcers had healed; the other two had a mean diameter decrease of 21%. The only side effect was found in a 58 year old female who complained of moderate perilesional pain two days after having received treatment: it was successfully treated with paracetamol. We believe that a single perilesional subcutaneous administration of rhGM-CSF is safe and effective for the treatment of chronic leg ulcers.

  3. Human neutrophil cytoskeletal dynamics and contractility actively contribute to trans-endothelial migration.

    Science.gov (United States)

    Stroka, Kimberly M; Hayenga, Heather N; Aranda-Espinoza, Helim

    2013-01-01

    Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of transmigration in our in vitro model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks transmigration, thus establishing a critical role for neutrophil actin dynamics in transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which "opens the door" in the endothelium and facilitates transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the transmigration step of the immune response.

  4. Human neutrophil cytoskeletal dynamics and contractility actively contribute to trans-endothelial migration.

    Directory of Open Access Journals (Sweden)

    Kimberly M Stroka

    Full Text Available Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of transmigration in our in vitro model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks transmigration, thus establishing a critical role for neutrophil actin dynamics in transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which "opens the door" in the endothelium and facilitates transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the transmigration step of the immune response.

  5. Delayed inhibition of agonist-induced granulocyte-platelet aggregation after low-dose sevoflurane inhalation in humans.

    Science.gov (United States)

    Wacker, Johannes; Lucchinetti, Eliana; Jamnicki, Marina; Aguirre, José; Härter, Luc; Keel, Marius; Zaugg, Michael

    2008-06-01

    Sevoflurane can be used as sedative-analgesic drug with endothelial protective properties. We tested whether low-dose sevoflurane inhalation provides sustained inhibition of detrimental granulocyte-platelet aggregation in humans. Ten healthy male volunteers were enrolled in this crossover study. Each subject inhaled sevoflurane for 1 h at 0.5-1 vol % end-tidal concentration in oxygen (50 vol %). Inhaling oxygen (50 vol %) alone served as control. Venous blood samples were collected at baseline before inhalation, immediately after inhalation, and 24 h thereafter, and were used for flow cytometry to determine platelet surface marker (CD41, CD42b, CD62P/P-selectin, and PAC-1) on platelets and granulocytes and for kaolin-induced clot formation, as assessed by thromboelastography. In flow cytometry experiments, platelets were stimulated with arachidonic acid (AA, 30 microM), adenosine diphosphate (ADP, 1 microM), and thrombin receptor agonist peptide-6 (TRAP-6, 6 microM). AA, ADP, and TRAP-6 markedly increased the expression of CD62P on platelets, whereas CD42b (shedding) and PAC-1 (heterotypic conjugates) expression decreased. The amount of granulocyte-platelet aggregates increased upon agonist stimulation. Low-dose sevoflurane inhalation reduced ADP-induced CD62P expression on platelets 24 h after inhalation, and inhibited the formation of granulocyte-platelet aggregates under stimulation with AA and ADP after 1 and 24 h, and with TRAP-6 after 24 h compared with control. Inhibition of granulocyte-platelet aggregates was accompanied by reduced clot firmness 24 h after sevoflurane inhalation compared with control. We demonstrated for the first time that inhaling low-dose sevoflurane (<1 vol % end-tidal) inhibits agonist-induced granulocyte-platelet interactions 24 h after administration and thus counteracts thromboinflammatory processes.

  6. Olfactomedin 4 defines a subset of human neutrophils

    DEFF Research Database (Denmark)

    Clemmensen, Stine N; Bohr, Christina T; Rørvig, Sara

    2012-01-01

    blood neutrophils demonstrated complete colocalization of OLFM4 with the specific granule protein NGAL, and stimulation of neutrophils with PMA resulted in corelease of NGAL and OLFM4, proving that OLFM4 is a genuine constituent of neutrophil-specific granules. In accordance with this, OLFM4 mRNA peaked...... at the MY/MM stage of maturation. OLFM4 was, however, present in only 20-25% of peripheral blood neutrophils, as determined by immunocytochemistry and flow cytometry, whereas mRNA for OLFM4 was present in all MY/MM, indicating post-transcriptional regulation as a basis for the heterogeneous expression...... of OLFM4 protein....

  7. Social networking of human neutrophils within the immune system.

    Science.gov (United States)

    Scapini, Patrizia; Cassatella, Marco A

    2014-07-31

    It is now widely recognized that neutrophils are highly versatile and sophisticated cells that display de novo synthetic capacity and may greatly extend their lifespan. In addition, concepts such as "neutrophil heterogeneity" and "neutrophil plasticity" have started to emerge, implying that, under pathological conditions, neutrophils may differentiate into discrete subsets defined by distinct phenotypic and functional profiles. A number of studies have shown that neutrophils act as effectors in both innate and adaptive immunoregulatory networks. In fact, once recruited into inflamed tissues, neutrophils engage into complex bidirectional interactions with macrophages, natural killer, dendritic and mesenchymal stem cells, B and T lymphocytes, or platelets. As a result of this cross-talk, mediated either by contact-dependent mechanisms or cell-derived soluble factors, neutrophils and target cells reciprocally modulate their survival and activation status. Altogether, these novel aspects of neutrophil biology have shed new light not only on the potential complex roles that neutrophils play during inflammation and immune responses, but also in the pathogenesis of several inflammatory disorders including infection, autoimmunity, and cancer. © 2014 by The American Society of Hematology.

  8. Two lytic transglycosylases in Neisseria gonorrhoeae impart resistance to killing by lysozyme and human neutrophils.

    Science.gov (United States)

    Ragland, Stephanie A; Schaub, Ryan E; Hackett, Kathleen T; Dillard, Joseph P; Criss, Alison K

    2017-03-01

    Symptomatic infection by Neisseria gonorrhoeae (Gc) produces a potent inflammatory response, resulting in a neutrophil-rich exudate. A population of Gc can survive the killing activities of neutrophils for reasons not completely understood. Unlike other Gram-negative bacteria, Gc releases monomeric peptidoglycan (PG) extracellularly, dependent on two nonessential, nonredundant lytic transglycosylases (LTs), LtgA and LtgD. PG released by LtgA and LtgD can stimulate host immune responses. We report that ΔltgAΔltgD Gc were decreased in survival in the presence of primary human neutrophils but otherwise grew equally to wild-type Gc. Adding PG monomer failed to alter ΔltgAΔltgD Gc survival. Thus, LTs protect Gc from neutrophils independently of monomer release. We found two reasons to explain decreased survival of the double LT mutant. First, ΔltgAΔltgD Gc was more sensitive to the neutrophil antimicrobial proteins lysozyme and neutrophil elastase, but not others. Sensitivity to lysozyme correlated with decreased Gc envelope integrity. Second, exposure of neutrophils to ΔltgAΔltgD Gc increased the release of neutrophil granule contents extracellularly and into Gc phagosomes. We conclude that LtgA and LtgD protect Gc from neutrophils by contributing to envelope integrity and limiting bacterial exposure to select granule-localized antimicrobial proteins. These observations are the first to link bacterial degradation by lysozyme to increased neutrophil activation. © 2016 John Wiley & Sons Ltd.

  9. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

    Science.gov (United States)

    Ong, Catherine W M; Elkington, Paul T; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T; Tezera, Liku B; Pabisiak, Przemyslaw J; Moores, Rachel C; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H; Porter, Joanna C; Friedland, Jon S

    2015-05-01

    Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

  10. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

    Directory of Open Access Journals (Sweden)

    Catherine W M Ong

    2015-05-01

    Full Text Available Pulmonary cavities, the hallmark of tuberculosis (TB, are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8 secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

  11. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis

    Science.gov (United States)

    Ong, Catherine W. M.; Elkington, Paul T.; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T.; Tezera, Liku B.; Pabisiak, Przemyslaw J.; Moores, Rachel C.; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H.; Porter, Joanna C.; Friedland, Jon S.

    2015-01-01

    Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease. PMID:25996154

  12. Subcellular Fractionation of Human Neutrophils and Analysis of Subcellular Markers

    DEFF Research Database (Denmark)

    Clemmensen, Stine Novrup; Udby, Lene; Borregaard, Niels

    2014-01-01

    The neutrophil has long been recognized for its impressive number of cytoplasmic granules that harbor proteins indispensable for innate immunity. Analysis of isolated granules has provided important information on how the neutrophil grades its response to match the challenges it meets on its...

  13. Computer-assisted image analysis assay of human neutrophil chemotaxis in vitro

    DEFF Research Database (Denmark)

    Jensen, P; Kharazmi, A

    1991-01-01

    We have developed a computer-based image analysis system to measure in-filter migration of human neutrophils in the Boyden chamber. This method is compared with the conventional manual counting techniques. Neutrophils from healthy individuals and from patients with reduced chemotactic activity we...

  14. Human Neutrophil Peptides 1-3 – Early Markers in Development of Colorectal Adenomas and Carcinomas

    Directory of Open Access Journals (Sweden)

    Henning Mothes

    2008-01-01

    Full Text Available Expression of Human Neutrophil Peptides (HNP 1–3 was recently found to be associated with development of colorectal cancer. Raised defensin-expression in tumours is believed to stem from increased infiltration of neutrophils into tumour environment.

  15. Hematological indices, inflammatory markers and neutrophil CD64 expression: comparative trends during experimental human endotoxemia.

    NARCIS (Netherlands)

    Meer, W. van der; Pickkers, P.; Scott, C.S.; Hoeven, J.G. van der; Gunnewiek, J.K.

    2007-01-01

    CD64 is a high-affinity Fc(gamma)RI receptor expressed by activated neutrophils that has been recently evaluated as a potential sepsis parameter. In the present study, the kinetics of neutrophil membrane CD64 expression were examined during a standardized inflammatory response, using a human

  16. Aliphatic alcohols of illegally produced spirits can act synergistically on superoxide-anion production by human granulocytes.

    Science.gov (United States)

    Arnyas, Ervin M; Pál, László; Kovács, Csilla; Adány, Róza; McKee, Martin; Szűcs, Sándor

    2012-10-01

    Aliphatic alcohols present in illegally produced spirits in a large number of low and middle income countries have been implicated in the etiology of chronic liver disease and cirrhosis. Previous studies have confirmed that chronic alcoholism can lead to increased susceptibility to infectious diseases. Reduced superoxide-anion (O(2)·(-)) production by granulocytes could provide a mechanism by which antimicrobial defense is impaired in alcoholics. In vitro experiments have also demonstrated that ethanol can inhibit granulocyte O(2)·(-) generation. Aliphatic alcohols consumed as contaminants of illicit spirits may also influence O(2)·(-) production thereby contributing to a decrease in microbicidal activity. The aim of this study was to investigate this possibility. It measured the O(2)·(-) production by human granulocytes following treatment of the cells with aliphatic alcohol contaminants found in illicit spirits. Granulocytes were isolated from human buffy coats with centrifugal elutriation and then treated with individual aliphatic alcohols and their mixture. The O(2)·(-) production was stimulated with phorbol-12-13-dibutyrate and N-formyl-methionyl-leucyl-phenylalanine (FMLP) and measured by superoxide dismutase inhibitable reduction of ferricytochrome c. Aliphatic alcohols of illegally produced spirits inhibited the FMLP-induced O(2)·(-) production in a concentration dependent manner. They suppressed O(2)·(-) generation at 2.5-40 times lower concentrations when combined than when tested individually. Aliphatic alcohols found in illegally produced spirits can inhibit FMLP-induced O(2)·(-) production by granulocytes in a concentration-dependent manner. Due to their synergistic effects, it is possible that, in combination with ethanol, they may inhibit O(2)·(-) formation in heavy episodic drinkers.

  17. NO signaling in exercise training-induced anti-apoptotic effects in human neutrophils.

    Science.gov (United States)

    Su, Shu-Hui; Jen, Chauying J; Chen, Hsiun-ing

    2011-02-04

    Short-lived neutrophils play a predominant role in innate immunity, the effects of exercise training on neutrophil survival is unclear. In this study, we investigated the underlying mechanisms of training effects on human neutrophil apoptosis. Healthy male subjects were trained on a cycling ergometer for 8 weeks and followed by 4 weeks of detraining. Blood neutrophils were collected before exercise, after training, and after detraining. Comparing with pre-exercise specimens, neutrophils collected after training showed reduced apoptosis rate, which partially returned after detraining. Various intracellular proteins, including iNOS, Mcl-1, A1, Grp78, and IL-8, were upregulated by training, and they remained high after detraining. Upregulated iNOS was closely correlated with these anti-apoptotic molecules in neutrophils. Furthermore, the possible mechanism by which iNOS suppressed apoptosis was explored. Neutrophil apoptosis was accelerated by blocking and retarded by stimulating the endogenous iNOS activity. As an anti-apoptosis mediator of NO signaling, the Mcl-1 level dropped by depletion of the major NO downstream molecule cGMP and such loss of Mcl-1 was avoidable when supplying exogenous NO. Upon activation of NO-cGMP signaling, neutrophils held increased Mcl-1 expression and delayed apoptosis. Collectively, our results suggested that exercise training may retard neutrophil apoptosis by upregulating the iNOS-NO-cGMP-Mcl-1 pathway. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Granulocyte macrophage colony stimulating factor (GM-CSF biological actions on human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    S Montagnani

    2009-12-01

    Full Text Available Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GMCSFR on human normal skin fibroblasts from healthy subjects (NFPC and on a human normal fibroblast cell line (NHDF and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GMCSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such “differentiation” is an important event induced by such cytokine.

  19. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps.

    Science.gov (United States)

    Ávila, Eva E; Salaiza, Norma; Pulido, Julieta; Rodríguez, Mayra C; Díaz-Godínez, César; Laclette, Juan P; Becker, Ingeborg; Carrero, Julio C

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica.

  20. Pathogenic Neisseria hitchhike on the uropod of human neutrophils.

    Directory of Open Access Journals (Sweden)

    Niklas Söderholm

    Full Text Available Polymorphonuclear neutrophils (PMNs are important components of the human innate immune system and are rapidly recruited at the site of bacterial infection. Despite the effective phagocytic activity of PMNs, Neisseria gonorrhoeae infections are characterized by high survival within PMNs. We reveal a novel type IV pilus-mediated adherence of pathogenic Neisseria to the uropod (the rear of polarized PMNs. The direct pilus-uropod interaction was visualized by scanning electron microscopy and total internal reflection fluorescence (TIRF microscopy. We showed that N. meningitidis adhesion to the PMN uropod depended on both pilus-associated proteins PilC1 and PilC2, while N. gonorrhoeae adhesion did not. Bacterial adhesion elicited accumulation of the complement regulator CD46, but not I-domain-containing integrins, beneath the adherent bacterial microcolony. Electrographs and live-cell imaging of PMNs suggested that bacterial adherence to the uropod is followed by internalization into PMNs via the uropod. We also present data showing that pathogenic Neisseria can hitchhike on PMNs to hide from their phagocytic activity as well as to facilitate the spread of the pathogen through the epithelial cell layer.

  1. Distinct Trypanosoma cruzi isolates induce activation and apoptosis of human neutrophils.

    Directory of Open Access Journals (Sweden)

    Luísa M D Magalhães

    Full Text Available Neutrophils are critical players in the first line of defense against pathogens and in the activation of subsequent cellular responses. We aimed to determine the effects of the interaction of Trypanosoma cruzi with human neutrophils, using isolates of the two major discrete type units (DTUs associated with Chagas' disease in Latin America (clone Col1.7G2 and Y strain, DTU I and II, respectively. Thus, we used CFSE-stained trypomastigotes to measure neutrophil-T. cruzi interaction, neutrophil activation, cytokine expression and death, after infection with Col1.7G2 and Y strain. Our results show that the frequency of CFSE+ neutrophils, indicative of interaction, and CFSE intensity on a cell-per-cell basis were similar when comparing Col1.7G2 and Y strains. Interaction with T. cruzi increased neutrophil activation, as measured by CD282, CD284, TNF and IL-12 expression, although at different levels between the two strains. No change in IL-10 expression was observed after interaction of neutrophils with either strain. We observed that exposure to Y and Col1.7G2 caused marked neutrophil death. This was specific to neutrophils, since interaction of either strain with monocytes did not cause death. Our further analysis showed that neutrophil death was a result of apoptosis, which was associated with an upregulation of TNF-receptor, TNF and FasLigand, but not of Fas. Induction of TNF-associated neutrophil apoptosis by the different T. cruzi isolates may act as an effective common mechanism to decrease the host's immune response and favor parasite survival.

  2. Distinct Trypanosoma cruzi isolates induce activation and apoptosis of human neutrophils

    Science.gov (United States)

    Magalhães, Luísa M. D.; Viana, Agostinho; de Jesus, Augusto C.; Chiari, Egler; Galvão, Lúcia; Gomes, Juliana A.; Gollob, Kenneth J.

    2017-01-01

    Neutrophils are critical players in the first line of defense against pathogens and in the activation of subsequent cellular responses. We aimed to determine the effects of the interaction of Trypanosoma cruzi with human neutrophils, using isolates of the two major discrete type units (DTUs) associated with Chagas’ disease in Latin America (clone Col1.7G2 and Y strain, DTU I and II, respectively). Thus, we used CFSE-stained trypomastigotes to measure neutrophil-T. cruzi interaction, neutrophil activation, cytokine expression and death, after infection with Col1.7G2 and Y strain. Our results show that the frequency of CFSE+ neutrophils, indicative of interaction, and CFSE intensity on a cell-per-cell basis were similar when comparing Col1.7G2 and Y strains. Interaction with T. cruzi increased neutrophil activation, as measured by CD282, CD284, TNF and IL-12 expression, although at different levels between the two strains. No change in IL-10 expression was observed after interaction of neutrophils with either strain. We observed that exposure to Y and Col1.7G2 caused marked neutrophil death. This was specific to neutrophils, since interaction of either strain with monocytes did not cause death. Our further analysis showed that neutrophil death was a result of apoptosis, which was associated with an upregulation of TNF-receptor, TNF and FasLigand, but not of Fas. Induction of TNF-associated neutrophil apoptosis by the different T. cruzi isolates may act as an effective common mechanism to decrease the host’s immune response and favor parasite survival. PMID:29176759

  3. CFTR Expression in Human Neutrophils and the Phagolysosomal Chlorination Defect in Cystic Fibrosis

    Science.gov (United States)

    Painter, Richard G.; Valentine, Vincent G.; Lanson, Nicholas A.; Leidal, Kevin; Zhang, Qiang; Lombard, Gisele; Thompson, Connie; Viswanathan, Anand; Nauseef, William M.; Wang, Guangdi; Wang, Guoshun

    2010-01-01

    Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC/MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically pre-labeled with 13C-L-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides the evidence to suggest that CFTR channel expression in neutrophils and its dysfunction affects neutrophil chlorination of phagocytosed bacteria. PMID:16922501

  4. Safety of recombinant human granulocyte-macrophage colony-stimulating factor in healing pediatric severe burns.

    Science.gov (United States)

    Chi, Y F; Chai, J K; Luo, H M; Zhang, Q X; Feng, R

    2015-03-31

    We explored the safety of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for healing burns in children. Subjects were randomly assigned to two groups: the experimental group received external rhGM-CSF gel, and the control group received rhGM-CSF gel matrix components, applied to the burn surface. Neither group was given any other drugs that promote wound healing. Each day we recorded the pulse, body temperature, and respiration status in the two groups. We detected the blood routine, urine routine, and hepatic and renal function before the patients received drug treatment and after 72 h. The wound scab and healing states in the two groups were recorded every 4 days to evaluate wound healing rate and time taken for complete healing. Adverse reactions and their rate of occurrence were also recorded. The median time of healing was 15 days in the experimental group and 19 days in the control group (log-rank χ(2) = 5.139, P 0.05). Compared with saline treatment of severe burns, rhGM-CSF can effectively shorten the healing time without significant adverse reactions, and is an effective and safe treatment for burns in children.

  5. Human Metapneumovirus Attachment Protein Contributes to Neutrophil Recruitment into the Airways of Infected Mice

    Directory of Open Access Journals (Sweden)

    Nagarjuna R. Cheemarla

    2017-10-01

    Full Text Available Human Metapneumovirus (HMPV is a leading respiratory pathogen that causes lower respiratory tract infections worldwide. Acute HMPV infection induces an exacerbated inflammatory neutrophilic response leading to bronchiolitis and pneumonia. However, the mechanism by which the virus regulates neutrophil infiltration into the airways still remains unexplored. In this work, we used an experimental mouse model of HMPV infection to demonstrate that the attachment (G protein of HMPV contributes to the recruitment of neutrophils into the airways and modulate the production of neutrophil chemoattractants and Type I IFN responses, specifically IFN-α. These findings provide the first evidence that the HMPV G protein contributes to the in vivo neutrophilic response to HMPV infection and furthers our understanding on virus induced inflammatory responses in the airways.

  6. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation

    DEFF Research Database (Denmark)

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G

    2016-01-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages...... and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD m......RNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA...

  7. Activated human neutrophils release hepatocyte growth factor/scatter factor.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Hepatocyte growth factor or scatter factor (HGF\\/SF) is a pleiotropic cytokine that has potent angiogenic properties. We have previously demonstrated that neutrophils (PMN) are directly angiogenic by releasing vascular endothelial growth factor (VEGF). We hypothesized that the acute inflammatory response can stimulate PMN to release HGF. AIMS: To examine the effects of inflammatory mediators on PMN HGF release and the effect of recombinant human HGF (rhHGF) on PMN adhesion receptor expression and PMN VEGF release. METHODS: In the first experiment, PMN were isolated from healthy volunteers and stimulated with tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-8 (IL-8), and formyl methionyl-leucyl-phenylalanine (fMLP). Culture supernatants were assayed for HGF using ELISA. In the second experiment, PMN were lysed to measure total HGF release and HGF expression in the PMN was detected by Western immunoblotting. Finally, PMN were stimulated with rhHGF. PMN CD 11a, CD 11b, and CD 18 receptor expression and VEGF release was measured using flow cytometry and ELISA respectively. RESULTS: TNF-alpha, LPS and fMLP stimulation resulted in significantly increased release of PMN HGF (755+\\/-216, 484+\\/-221 and 565+\\/-278 pg\\/ml, respectively) compared to controls (118+\\/-42 pg\\/ml). IL-8 had no effect. Total HGF release following cell lysis and Western blot suggests that HGF is released from intracellular stores. Recombinant human HGF did not alter PMN adhesion receptor expression and had no effect on PMN VEGF release. CONCLUSIONS: This study demonstrates that pro-inflammatory mediators can stimulate HGF release from a PMN intracellular store and that activated PMN in addition to secreting VEGF have further angiogenic potential by releasing HGF.

  8. Human neutrophil peptide-1 (HNP-1: a new anti-leishmanial drug candidate.

    Directory of Open Access Journals (Sweden)

    Sara Dabirian

    Full Text Available The toxicity of available drugs for treatment of leishmaniasis, coupled with emerging drug resistance, make it urgent to find new therapies. Antimicrobial peptides (AMPs have a strong broad-spectrum antimicrobial activity with distinctive modes of action and are considered as promising therapeutic agents. The defensins, members of the large family of AMPs, are immunomodulatory molecules and important components of innate immune system. Human neutrophil peptide-1 (HNP-1, which is produced by neutrophils, is one of the most potent defensins. In this study, we described anti-parasitic activity of recombinant HNP-1 (rHNP-1 against Leishmania major promastigotes and amastigotes. Furthermore, we evaluated the immunomodulatory effect of rHNP-1 on parasite-infected neutrophils and how neutrophil apoptosis was affected. Our result showed that neutrophils isolated from healthy individuals were significantly delayed in the onset of apoptosis following rHNP-1 treatment. Moreover, there was a noteworthy increase in dying cells in rHNP-1- and/or CpG-treated neutrophils in comparison with untreated cells. There is a considerable increase in TNF-α production from rHNP-1-treated neutrophils and decreased level of TGF-β concentration, a response that should potentiate the immune system against parasite invasion. In addition, by using real-time polymerase chain reaction (real-time PCR, we showed that in vitro infectivity of Leishmania into neutrophils is significantly reduced following rHNP-1 treatment compared to untreated cells.

  9. Effects of areca nut extracts on the functions of human neutrophils in vitro.

    Science.gov (United States)

    Hung, S L; Chen, Y L; Wan, H C; Liu, T Y; Chen, Y T; Ling, L J

    2000-08-01

    Aqueous extracts of ripe areca nut without husk (ripe ANE) and fresh and tender areca nut with husk (tender ANE) were examined for their effects on the defensive functions of human neutrophils. Exposure of peripheral blood neutrophils to ripe ANE and tender ANE inhibited their bactericidal activity against oral pathogens, including Actinobacillus actinomycetemcomitans and Streptococcus mutans, in a dose-dependent manner. At the concentrations tested, ripe and tender ANEs did not significantly affect the viability of neutrophils as verified by their ability to exclude trypan blue dye. However, both ANEs inhibited the production of bactericidal superoxide anion by neutrophils as measured by cytochrome c reduction. Moreover, the ripe ANE inhibited neutrophils more effectively than did tender ANE. Arecoline, a major alkaloid of areca nut, only exhibited an inhibitory effect on the functions of neutrophils when high concentrations were used. Therefore, arecoline could not be used to explain the inhibitory effects observed for ANEs. In conclusion, our results demonstrated that ripe and tender ANEs reduced the antibacterial activity and the superoxide anion production of neutrophils. This effect may contribute to a less efficient elimination of bacteria from the periodontal environment. Inhibition of the antimicrobial functions of neutrophils may alter the microbial ecology of the oral cavity, and this may be one possible mechanism by which areca nut compromises the oral health of users of areca nut products.

  10. [CD15 focus score for diagnostics of periprosthetic joint infections : Neutrophilic granulocytes quantification mode and the development of morphometric software (CD15 quantifier)].

    Science.gov (United States)

    Kölbel, B; Wienert, S; Dimitriadis, J; Kendoff, D; Gehrke, T; Huber, M; Frommelt, L; Tiemann, A; Saeger, K; Krenn, V

    2015-09-01

    The aim of this project was to devise a quantification method for neutrophils within a single focal point through the development of a CD15 focus score which enables bacterial infections in synovial-like interface membranes (SLIM) to be diagnosed. In this study a histopathological classification of 91 SLIM removed during revision surgery from the hips (n = 59) and knees (n = 32) was performed. Neutrophils were identified immunohistochemically by means of a CD15-specific monoclonal antibody. The quantitative evaluation of CD15-positive neutrophils (CD15Ne) used the principle of maximum focal infiltration (focus) together with an assessment of a single focal point (0.3 mm(2)). This immunohistochemical approach made it possible to develop the CD15 quantifier software, which automatically quantifies CD15Ne. The SLIM cases with positive microbiological findings (n = 47) had significantly (p software (n = 31) deviated by an average of 12 cells from the histopathological evaluation findings (CD15 focus score). Above a cell count of 62, the CD15-quantifier needs on average 32 s less than the pathologist. The immunohistochemical CD15 focus score has a high diagnostic value and allowed the development of the CD15 quantifier software. This provides an automated procedure, which shortens the mentally tiring and time-consuming process of microscopic cell counting and thus makes a contribution towards the standardization of tools for diagnosing PJI.

  11. Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

    Energy Technology Data Exchange (ETDEWEB)

    Williams, C. David; Bajt, Mary Lynn [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Sharpe, Matthew R. [Department of Internal Medicine, University of Kansas Hospital, Kansas City, KS (United States); McGill, Mitchell R. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2014-03-01

    Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: > 800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91{sup phox}−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury. - Highlights: • Neutrophil (PMN) function increases during liver repair after acetaminophen overdose. • Liver repair after acetaminophen (APAP)-overdose is not dependent on NADPH oxidase. • Human PMNs do not appear

  12. Doxycycline induced photodamage to human neutrophils and tryptophan

    Energy Technology Data Exchange (ETDEWEB)

    Sandberg, S.; Glette, J.; Hopen, G.; Solberg, C.O. (Haukeland Sykehus, Bergen (Norway))

    1984-01-01

    Neutrophil function were studied following irradiation (340-380 nm) of the cells in the presence of 22 ..mu..M doxycycline. At increasing light fluence the locomotion, chemiluminescence and glucose oxidation (by the hexose monophosphate shunt) of the neutrophils steadily decreased. The photodamage increased with increasing preincubation temperature and time and was enhanced in D/sub 2/O, reduced in azide and abolished in anaerobiosis. Superoxide dismutase, catalase or mannitol did not influence the photodamage. Photooxidation of tryptophan in the presence of doxycycline was increased 9-10-fold in D/sub 2/O and nearly abolished in the presence of 0.25 mM NaN/sub 3/, indicating that singlet oxygen is the most important reactive oxygen species in the doxycycline-induced photodamage. The results may explain some of the features of tetracycline-induced photosensitivity and why other authors have obtained diverging results when studying the influence of tetracyclines on neutrophil functions.

  13. Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages.

    Directory of Open Access Journals (Sweden)

    Henrik Andersson

    Full Text Available Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.

  14. Characterization of the Na+/HCO3- Cotransport in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Miriam S. Giambelluca

    2014-04-01

    Full Text Available Background: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. Methods: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. Results: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na+/H+ exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na+/HCO3- cotransporter (NBC selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. Conclusion: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.

  15. α-1 Antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and IL-8.

    LENUS (Irish Health Repository)

    Bergin, David A

    2010-12-01

    Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor-mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT-IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.

  16. Granulocytes affect double-strand break repair assays in primary human lymphocytes.

    Science.gov (United States)

    Lacoste, Sandrine; Bhatia, Ravi; Bhatia, Smita; O'Connor, Timothy R

    2014-01-01

    Patients who develop therapy-related myelodysplasia/acute myeloid leukemia after autologous-hematopoietic stem cell (aHCT) transplant show lower expression levels of DNA repair genes in their pre-aHCT CD34+ cells. To investigate whether this leads to functional differences in DNA repair abilities measurable in patients, we adapted two plasmid-based host-cell reactivation assays for use in primary lymphocytes. Prior to applying these assays to patients who underwent aHCT, we wanted first to verify whether sample preparation affected repair measurements, as patient samples were simply depleted of erythrocytes (with hetastarch) prior to freezing, which is not the classical way to prepare lymphocytes prior to DNA repair experiments (with a density gradient). We show here that lymphocytes from healthy donors freshly prepared with hetastarch show systematically a higher level of double-strand break repair as compared to when prepared with a density gradient, but that most of this difference disappears after samples were frozen. Several observations points to granulocytes as the source for this effect of sample preparation on repair: 1) removal of granulocytes makes the effect disappear, 2) DSB repair measurements for the same individual correlate to the percentage of granulocytes in the sample and 3) nucleofection in presence of granulocytes increases the level of reactive oxygen species (ROS) in neighboring lymphocytes in a dose-dependent manner (R2 of 0.95). These results indicate that co-purified granulocytes, possibly through the release of ROS at time of transfection, can lead to an enhanced repair in lymphocytes that obfuscates any evaluation of inter individual differences in repair as measured by host-cell reactivation. As a result, hetastarch-prepared samples are likely unsuitable for the assessment of DSB repair in primary cells with that type of assay. Granulocyte contamination that exists after a density gradient preparation, although much more limited, could

  17. Physiological concentrations of leptin do not affect human neutrophils.

    NARCIS (Netherlands)

    Kamp, V.M.; Langereis, J.D.; Aalst, C.W. van; Linden, J. van der; Ulfman, L.H.; Koenderman, L.

    2013-01-01

    Leptin is an adipokine that is thought to be important in many inflammatory diseases, and is known to influence the function of several leukocyte types. However, no clear consensus is present regarding the responsiveness of neutrophils for this adipokine. In this study a 2D DIGE proteomics approach

  18. Nitric oxide regulates the aggregation of stimulated human neutrophils.

    Science.gov (United States)

    Forslund, T; Nilsson, H M; Sundqvist, T

    2000-08-02

    Neutrophil aggregation is mediated by both CD18 integrin and L-selectin. Nitric oxide attenuates the integrin-mediated adhesion of neutrophils to collagen and to endothelium and may therefore affect aggregation as well. FMLP-stimulated neutrophils exposed to l-arginine showed increased and prolonged aggregation, whereas cells pretreated with L-NAME did not differ from FMLP-stimulated controls. Nitric oxide is known to induce ADP ribosylation of G-actin, which inhibits polymerization. We detected equivalent levels of total F-actin in cells pretreated with l-arginine or L-NAME and non-pretreated controls. However, neutrophils pretreated with l-arginine and stimulated by CD18 integrin cross-linking exhibited a more limited increase in total F-actin, compared to control and L-NAME-pretreated cells. Thus at least two signaling pathways may be involved FMLP-stimulated aggregation, mediated by CD18 integrins. More specifically, it is plausible that FMLP-receptor signaling upregulates CD18 integrins and endogenous NO subsequently modulates CD18-mediated signaling to prolong aggregation, possibly through ADP-ribosylation of actin. Copyright 2000 Academic Press.

  19. Effect of Legionella pneumophila cytotoxic protease on human neutrophil and monocyte function

    DEFF Research Database (Denmark)

    Rechnitzer, C; Kharazmi, A

    1992-01-01

    The extracellular metalloprotease of Legionella pneumophila, also called tissue-destructive protease or major secretory protein, has been proposed as one of the virulence factors of this organism. Considering the decisive role played by the phagocytic cells in host defense against Legionella infe....... pneumophila protease on neutrophil chemotaxis and on the listericidal activity of human neutrophils and monocytes demonstrated in this study provides evidence for a role of this enzyme in the pathogenesis of Legionnaires' disease....

  20. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane

    DEFF Research Database (Denmark)

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels Henrik Helweg

    2013-01-01

    granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in...... subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes....

  1. A 30-base pair element is responsible for the myeloid-specific activity of the human neutrophil elastase promoter.

    Science.gov (United States)

    Srikanth, S; Rado, T A

    1994-12-23

    Human neutrophil elastase (HNE), a serine protease, is expressed only in the promyelocytic stages of granulocyte maturation. We examined several regions of the promoter for transcriptional activity and report that a 30-base pair (bp) element located between -76 and -106 in the 5'-flanking region of HNE is sufficient for myeloid-specific expression of HNE. Gel shift assays using nuclear extracts from myeloid and non-myeloid cells reveal several myeloid-specific complexes binding to the 30-bp element. Examination of DNA-protein interactions shows that at least two myeloid-specific proteins of 38 and 55 kDa bind to this element. DNase I protection analysis reveals two distinct footprints between -80 to -91 and -94 to -104 within this element. Transient expression studies using deletion constructs of the HNE 5'-flanking region show that the 30-bp element is active in myeloid cells K 562 and U 937 but not in HeLa cells. Internal deletion of this element results in a 60-85% loss of promoter activity in myeloid cells. Additional functional studies also show that a 19-bp region between -112 and -131 contributes to transcriptional activity of the elastase promoter as well.

  2. Impact of human granulocyte and monocyte isolation procedures on functional studies

    NARCIS (Netherlands)

    L. Zhou (Lili); R. Somasundaram (Rajesh); E. Nederhof (Esther); G. Dijkstra (Gerard); K.N. Faber (Klaas Nico); M.P. Peppelenbosch (Maikel); G.M. Fuhler (Gwenny)

    2012-01-01

    textabstractOne of the first lines of defense against infection is the activation of the innate immune system. It is becoming clear that autoimmune diseases, such as rheumatoid arthritis and Crohn's disease, may be caused by disturbed innate immunity, and relating granulocyte and monocyte functions

  3. Impact of Human Granulocyte and Monocyte Isolation Procedures on Functional Studies

    NARCIS (Netherlands)

    Zhou, Lu; Somasundaram, Rajesh; Nederhof, Rosa F.; Dijkstra, Gerard; Faber, Klaas Nico; Peppelenbosch, Maikel P.; Fuhler, Gwenny M.

    One of the first lines of defense against infection is the activation of the innate immune system. It is becoming clear that autoimmune diseases, such as rheumatoid arthritis and Crohn's disease, may be caused by disturbed innate immunity, and relating granulocyte and monocyte functions to the

  4. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

    Science.gov (United States)

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Praetorius, Jeppe; Borregaard, Niels; Petersen, Steen V

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Anti-neutrophil cytoplasm autoantibodies (ANCA) in autoimmune liver diseases

    NARCIS (Netherlands)

    Roozendaal, C.; Kallenberg, Cees

    1999-01-01

    Anti-neutrophil cytoplasm antibodies (ANCA) are autoantibodies directed against cytoplasmic constituents of neutrophil granulocytes and monocytes. ANCA have been detected in serum from patients with inflammatory bowel diseases (mainly ulcerative colitis) and autoimmune mediated liver diseases

  6. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps

    OpenAIRE

    Eva E Ávila; Norma Salaiza; Julieta Pulido; Mayra C Rodríguez; César Díaz-Godínez; Juan P Laclette; Ingeborg Becker; Carrero, Julio C.

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce th...

  7. Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils.

    Directory of Open Access Journals (Sweden)

    Cheng-Yuk Lee

    Full Text Available Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis as well as upon contact (by serum-dependent adhesion and phagocytosis. This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.

  8. Investigating the potential for cryopreservation of human granulocytes with concentrated glycerol.

    Science.gov (United States)

    Moss, Alden C; Higgins, Adam Z

    2016-06-01

    The purpose of this study was to investigate the potential for cryopreservation of granulocytes using 30% glycerol. Recently reported permeability data was used to design two different methods for addition and removal of glycerol: a fast method that is predicted to keep cell volumes between 80% and 150% of the isotonic volume and a slow method that is predicted to keep cell volumes between 80% and 115% of the isotonic volume. The fast method resulted in cell recoveries of 31% ± 9% and 11% ± 3% before and after freezing, respectively, whereas the slow method resulted in even lower cell recoveries of 5% ± 2% and 4% ± 2%. The reduced cell recovery for the slow method is consistent with an increase in damage as a result of glycerol toxicity. Our results suggest that cryopreservation of granulocytes in concentrated glycerol is not feasible. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.

    1987-05-01

    Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of /sup 51/Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of /sup 51/Cr release from radiolabeled monolayers.

  10. Molecular docking analysis of curcumin analogues as human neutrophil elastase inhibitors

    Directory of Open Access Journals (Sweden)

    Radhakrishnan Narayanaswamy

    2014-03-01

    Full Text Available In the present study, we aimed to dock 17 different ligands of curcumin analogues with that of human neutrophil elastase. Molecular descriptors analysis using Molinspiration online tool was carried out including investigation on human neutrophil elastase putative binding sites using Discovery Studio. The molecular physicochemical analysis revealed that all of the curcumin analogues complied well with the five rules of thumb. With regard to bioact-ivity score, compound 17 has exhibited least score towards nuclear receptor ligand (0.05 and enzyme inhibitor (0.10 compared to all other ligands. Compounds 2, 4 and 13 exhibited the maximum interaction energy (-40 kcal/mol. Interestingly, seven compounds namely 3, 11-14, 16 and 17 interacted well with Arg147 amino acid residue. The present study outcomes therefore might provide new insight in understanding these 17 curcumin analogues as potential candidates for human neutrophil elastase inhibitory agents.

  11. TNF-α potentiates uric acid-induced interleukin-1β (IL-1β) secretion in human neutrophils.

    Science.gov (United States)

    Yokose, Kohei; Sato, Shuzo; Asano, Tomoyuki; Yashiro, Makiko; Kobayashi, Hiroko; Watanabe, Hiroshi; Suzuki, Eiji; Sato, Chikako; Kozuru, Hideko; Yatsuhashi, Hiroshi; Migita, Kiyoshi

    2017-09-14

    Monosodium urate (MSU) has been shown to promote interleukin-1β (IL-1β) secretion in human monocytes, but the priming signals for NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway remains elusive. In this study, we investigated the role of Tumor necrosis factor-alpha (TNF-α) on MSU-mediated IL-1β induction in human neutrophils. Human neutrophils were stimulated with MSU, in the presence or absence of TNF-α priming. The cellular supernatants were analyzed for IL-1β, IL-18, and caspase-1 by enzyme-linked immunosorbent assay (ELISA) methods. Pro-IL-1β mRNA expressions in human neutrophils were analyzed by real-time PCR method. TNF-α stimulation induced pro-IL-1β mRNA expression; however, MSU stimulation did not induce pro-IL-1β mRNA expression in human neutrophils. TNF-α alone or MSU stimulation did not result in efficient IL-1β secretion in human neutrophils, whereas in TNF-α-primed neutrophils, MSU stimulation resulted in a marked IL-1β and IL-18 secretion. TNF-α-primed neutrophils secreted cleaved caspase-1 (p20), in response to MSU stimulation. Our data demonstrate that priming of human neutrophils with TNF-α promotes uric acid-mediated IL-1β secretion in the absence of microbial stimulation. These findings provide insights into the neutrophils-mediated inflammatory processes in gouty arthritis.

  12. Immune Complexes Isolated from Patients with Pulmonary Tuberculosis Modulate the Activation and Function of Normal Granulocytes

    Science.gov (United States)

    Senbagavalli, P.; Hilda, J. Nancy; Ramanathan, V. D.; Kumaraswami, V.; Nutman, Thomas B.

    2012-01-01

    Circulating immune complexes (ICs) are associated with the pathogenesis of several diseases. Very little is known about the effect of ICs on the host immune response in patients with tuberculosis (TB). The effects of ICs isolated from patients with TB in modulating the release of calcium, cytokines, and granular proteins were studied in normal granulocytes, as were their chemotactic, phagocytic, and oxidative burst processes. ICs from TB patients induced decreased production of cytokines and platelet-activating factor (PAF) from normal granulocytes. ICs from TB patients also induced enhanced chemotaxis and phagocytosis but caused diminished oxidative burst. This was accompanied by an increased release in intracellular calcium. On the other hand, ICs from TB patients induced increased release of the granular proteins human neutrophil peptides 1 to 3 (HNP1–3). Thus, ICs from patients with TB exhibit a profound effect on granulocyte function with activation of certain effector mechanisms and dampening of others. PMID:23100480

  13. Activated Human Valvular Interstitial Cells Sustain Interleukin-17 Production To Recruit Neutrophils in Infective Endocarditis

    Science.gov (United States)

    Yeh, Chiou-Yueh; Shun, Chia-Tung; Kuo, Yu-Min; Jung, Chiau-Jing; Hsieh, Song-Chou; Chiu, Yen-Ling; Chen, Jeng-Wei; Hsu, Ron-Bin; Yang, Chia-Ju

    2015-01-01

    The mechanisms that underlie valvular inflammation in streptococcus-induced infective endocarditis (IE) remain unclear. We previously demonstrated that streptococcal glucosyltransferases (GTFs) can activate human heart valvular interstitial cells (VIC) to secrete interleukin-6 (IL-6), a cytokine involved in T helper 17 (Th17) cell differentiation. Here, we tested the hypothesis that activated VIC can enhance neutrophil infiltration through sustained IL-17 production, leading to valvular damage. To monitor cytokine and chemokine production, leukocyte recruitment, and the induction or expansion of CD4+ CD45RA− CD25− CCR6+ Th17 cells, primary human VIC were cultured in vitro and activated by GTFs. Serum cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA), and neutrophils and Th17 cells were detected by immunohistochemistry in infected valves from patients with IE. The expression of IL-21, IL-23, IL-17, and retinoic acid receptor-related orphan receptor C (Rorc) was upregulated in GTF-activated VIC, which may enhance the proliferation of memory Th17 cells in an IL-6-dependent manner. Many chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), were upregulated in GTF-activated VIC, which might recruit neutrophils and CD4+ T cells. Moreover, CXCL1 production in VIC was induced in a dose-dependent manner by IL-17 to enhance neutrophil chemotaxis. CXCL1-expressing VIC and infiltrating neutrophils could be detected in infected valves, and serum concentrations of IL-17, IL-21, and IL-23 were increased in patients with IE compared to healthy donors. Furthermore, elevated serum IL-21 levels have been significantly associated with severe valvular damage, including rupture of chordae tendineae, in IE patients. Our findings suggest that VIC are activated by bacterial modulins to recruit neutrophils and that such activities might be further enhanced by the production of Th17-associated cytokines. Together, these factors can amplify the

  14. Equol Effectively Inhibits Toxic Activity of Human Neutrophils without Influencing Their Viability.

    Science.gov (United States)

    Pažoureková, Silvia; Lucová, Marianna; Nosál, Radomír; Drábiková, Katarína; Harmatha, Juraj; Šmidrkal, Ján; Jančinová, Viera

    2016-01-01

    Equol (7,4'-dihydroxy-isoflavan, or 4',7-isoflavandiol) is a chroman derivative produced by intestinal bacteria in response to soy isoflavone intake in some, but not in all, humans. Equol shows strong anti-oxidant, anti-estrogenic, anti-cancerous and anti-inflammatory properties. The antioxidative capacity of equol has recently received considerable attention, and it has been used for preventing and treating several diseases. We investigated the effect of equol on human neutrophils, extra- and intracellular formation of oxidants, the phosphorylation of protein regulating NADPH oxidase and its effect on apoptosis. Neutrophils, isolated from blood from healthy subjects, were tested upon activation with various stimulants, proper for reactive oxygen species (ROS) production and treated by equol. Equol has the ability to reduce the toxic action of neutrophils. With increasing concentrations, equol decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. The phosphorylation of p40(phox) (a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular membranes) was reduced in the presence of equol. The experiments showed that equol did not change the number of viable, apoptotic or dead neutrophils significantly in all concentrations used. These results indicate the promising effect of equol in the operation of ROS in different mechanisms in the model of inflammation. © 2016 S. Karger AG, Basel.

  15. A bovine whey protein extract can enhance innate immunity by priming normal human blood neutrophils.

    Science.gov (United States)

    Rusu, Daniel; Drouin, Réjean; Pouliot, Yves; Gauthier, Sylvie; Poubelle, Patrice E

    2009-02-01

    Bovine milk-derived products, in particular whey proteins, exhibit beneficial properties for human health, including the acquired immune response. However, their effects on innate immunity have received little attention. Neutrophils are key cells of innate defenses through their primary functions of chemotaxis, phagocytosis, oxidative burst, and degranulation. A whey protein extract (WPE) purified from bovine lactoserum was evaluated for its direct and indirect effects on these primary functions of normal human blood neutrophils in vitro. Although WPE had no direct effects on primary functions, a 24-h pretreatment of neutrophils with WPE was associated with a significant and dose-dependent increase of their chemotaxis, superoxide production, and degranulation in response to N-formyl-methionine-leucine-phenylalanine, as well as of their phagocytosis of bioparticles. The pretreatment increased the surface expression of CD11b, CD16B, and CD32A receptors. The major WPE protein components beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) were the main active fractions having an additive effect on human neutrophils that became more responsive to a subsequent stimulation. This effect on NADPH oxidase activity was associated with translocation of p47(phox) to plasma membrane. Glycomacropeptide, a peptide present in measurable amounts in WPE products, was able to enhance the individual effect of beta-LG or alpha-LA on neutrophils. The present data suggest that WPE, through beta-LG and alpha-LA, has the capacity to enhance or "prime" human neutrophil responses to a subsequent stimulation, an effect that could be associated with increased innate defenses in vivo.

  16. Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

    DEFF Research Database (Denmark)

    Jensen, T; Aliouat, E M; Lundgren, B

    1998-01-01

    The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide...... generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

  17. Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis.

    Directory of Open Access Journals (Sweden)

    Lawrence J Druhan

    Full Text Available Leucine-rich α2 glycoprotein (LRG1, a serum protein produced by hepatocytes, has been implicated in angiogenesis and tumor promotion. Our laboratory previously reported the expression of LRG1 in murine myeloid cell lines undergoing neutrophilic granulocyte differentiation. However, the presence of LRG1 in primary human neutrophils and a role for LRG1 in regulation of hematopoiesis have not been previously described. Here we show that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment. Using immunofluorescence microscopy and direct biochemical measurements, we demonstrate that LRG1 is present in the peroxidase-negative granules of human neutrophils. Exocytosis assays indicate that LRG1 is differentially glycosylated in neutrophils, and co-released with the secondary granule protein lactoferrin. Like LRG1 purified from human serum, LRG1 secreted from activated neutrophils also binds cytochrome c. We also show that LRG1 antagonizes the inhibitory effects of TGFβ1 on colony growth of human CD34+ cells and myeloid progenitors. Collectively, these data invoke an additional role for neutrophils in innate immunity that has not previously been reported, and suggest a novel mechanism whereby neutrophils may modulate the microenvironment via extracellular release of LRG1.

  18. In vitro inhibition of human neutrophil histotoxicity by ambroxol: evidence for a multistep mechanism

    Science.gov (United States)

    Ottonello, Luciano; Arduino, Nicoletta; Bertolotto, Maria; Dapino, Patrizia; Mancini, Marina; Dallegri, Franco

    2003-01-01

    Neutrophils are major culprits for the protease/antiprotease imbalance during various lung diseases, that is, chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis and adult respiratory distress syndrome. Thus, these cells are presently considered an ideal target for the pharmacologic control of tissue injury during these diseases. This study was planned in order to investigate if ambroxol and its precursor bromhexine are actually capable of preventing alpha-1-antitrypsin (A1AT) inactivation by stimulated neutrophils and possibly to look into the mechanisms underlying this event. Ambroxol inhibited the production of superoxide anion by activated neutrophils, whereas bromhexine had no inhibitory effect. Ambroxol decreased the production of hypochlorous acid (HOCl) from activated neutrophils with high efficiency, whereas bromhexine had a modest activity. Ambroxol and bromhexine were capable of limiting the chlorination of monochlorodimedon by HOCl, displaying the capacity of directly scavenging the oxidant. Ambroxol decreased the release of elastase and myeloperoxidase from activated neutrophils, whereas bromhexine was ineffective. Ambroxol prevented the A1AT inactivation by neutrophils, whereas bromhexine was completely ineffective. Among drugs currently available for in vivo use in humans, ambroxol is unique by virtue of its ability to prevent neutrophil-mediated A1AT inactivation via inhibition of HOCl production as well as HOCl scavenging. Also taking into account its capacity for curbing elastase release, the drug displays the potential to lessen the burden of oxidants/proteases and to increase the antiprotease shield at the site of inflammation. Thus, ambroxol appears to be a good candidate for raising attempts to develop new therapeutic histoprotective approaches to inflammatory bronchopulmonary diseases. PMID:14534155

  19. Up-regulation of cluster of differentiation (CD) 11b expression on the surface of canine granulocytes with human granulocyte-macrophage colony-stimulating factor (GM-CSF).

    Science.gov (United States)

    Nakagaki, Kazuhide; Nunomura, Yuka; Uchida, Kanji; Nakata, Koh; Tazawa, Ryushi

    2014-08-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine, sharing a common beta subunit (CDw131) with interleukins 3 and 5. GM-CSF is important for its direct and indirect involvement in host defense. In veterinary medicine, human (h) GM-CSF has been used as a substitute for canine GM-CSF to stimulate canine granulocytes and macrophages. In this study, we compared the effects of three distinct hGM-CSFs produced by bacteria, yeasts and Chinese hamster ovary (CHO) cells with those of Escherichia (E) coli-produced canine GM-CSF on the cluster of differentiation 11b (CD11b) expression in canine granulocytes. The median effective dose (ED50) of hGM-CSFs from bacteria, yeasts and CHO cells was 3.09, 4.09 and 4.27 ng/ml, respectively, with no significant difference among three. In contrast, a significant difference was observed between ED50 of canine GM-CSF (0.56 ng/ml) and three hGM-CSFs according to the paired t-test (Pgranulocytes, but the average activity of the three rhGM-CSFs was approximately 15% of that of canine GM-CSF.

  20. Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils

    Directory of Open Access Journals (Sweden)

    Hytönen Jukka

    2006-10-01

    Full Text Available Abstract Background Species of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry. Results In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain. Conclusion These results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The

  1. Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways.

    NARCIS (Netherlands)

    Aarbiou, J.; Schadewijk, A. van; Stolk, J.; Sont, J.K.; Boer, W.I.; Rabe, K.F.; Krieken, J.H.J.M. van; Mauad, T.; Hiemstra, P.S.

    2004-01-01

    OBJECTIVE: The aim of this study was to analyze a possible contribution of human neutrophil defensins and secretory leukocyte proteinase inhibitor (SLPI) to the induction of airway epithelial changes such as squamous cell metaplasia. MATERIALS AND METHODS: The presence of these molecules and the

  2. Human Neutrophil Peptide 1 Limits Hypercholesterolemia-induced Atherosclerosis by Increasing Hepatic LDL Clearance

    NARCIS (Netherlands)

    Paulin, Nicole; Döring, Yvonne; Kooijman, Sander; Blanchet, Xavier; Viola, Joana R.; de Jong, Renske; Mandl, Manuela; Hendrikse, Jeffrey; Schiener, Maximilian; von Hundelshausen, Philipp; Vogt, Anja; Weber, Christian; Bdeir, Khalil; Hofmann, Susanna M.; Rensen, Patrick C. N.; Drechsler, Maik; Soehnlein, Oliver

    2017-01-01

    Increases in plasma LDL-cholesterol have unequivocally been established as a causal risk factor for atherosclerosis. Hence, strategies for lowering of LDL-cholesterol may have immediate therapeutic relevance. Here we study the role of human neutrophil peptide 1 (HNP1) in a mouse model of

  3. Activated host neutrophils in the larval midgut lumen of the human bot fly Dermatobia hominis.

    Science.gov (United States)

    Leite, Antônio C R; Evangelista, L G

    2002-04-01

    Light microscopy (LM) and transmission electron microscopy (TEM) were used to observe activated polymorphonuclear neutrophils from mammalian hosts as well as invading bacteria in the midgut lumen of larvae of the human bot fly Dermatobia hominis. Other resident or recruited cells associated with dermal myiasis were fed on by larvae and digested more rapidly than neutrophils. The latter were observed moving towards bacteria and particles of food, extending the filopodia and engulfing material to be digested within phagosomes. The larval midgut lumen, thus, appears to be a suitable environment to produce neutrophil activation at least for short periods, as seen in mammalian hosts. Although interactions between phagocytes and bacteria in the midgut lumen may be important in bot fly larval development, further studies are required to confirm this.

  4. Effect of Legionella pneumophila sonicate on killing of Listeria monocytogenes by human polymorphonuclear neutrophils and monocytes

    DEFF Research Database (Denmark)

    Rechnitzer, C; Bangsborg, Jette Marie; Shand, G H

    1993-01-01

    will be exposed to bacterial components, either expressed on the surface of the organisms or released in the environment upon cell lysis. In this study, we have investigated the effect of water-soluble bacterial components present in L. pneumophila sonicate on the phagocytosis and bactericidal activity of human...... polymorphonuclear neutrophils and monocytes. Preincubation of neutrophils with L. pneumophila sonicate did not affect phagocytosis of L. monocytogenes, whereas Listeria killing was significantly inhibited at sonicate concentrations of 1 and 2 mg/ml. The phenol phase of a phenol-water extraction, containing most...... of the lipopolysaccharide (LPS), had no inhibitory effect on the listericidal activity of neutrophils. Killing of Listeria by monocytes was inhibited in a similar manner. The inhibitory activity was mainly recovered in the sonicate fraction above 100 kDa, suggesting that components organized in larger molecular complexes...

  5. Two new monoclonal antibodies to human monocytes and granulocytes: isolation of membrane antigens and lack of effects of antibodies on leukocyte functions in vitro.

    Science.gov (United States)

    Stevenson, H C; Kimball, E; Schroff, R W; Buescher, S; Clarke, G; Gregorio, T; Wilburn, S; Foon, K A

    1984-01-01

    Mice were immunized with purified human monocytes or granulocytes obtained by leukapheresis and isolated on dextran gradients or by countercurrent centrifugation-elutriation. A monoclonal antibody, Mo95, was generated in response to monocytes and was found to react strongly with monocytes, large granular lymphocytes (LGL), granulocytes, eosinophils, and some myelomonocytic leukemia cells, but not with normal T or B lymphocytes, platelets, red cells, or leukemic cell lines. Mo95 is an IgG1 antibody, which precipitated a 95 kD molecular weight antigen. Addition of the Mo95 antibody to monocytes in the absence of complement did not inhibit lysozyme secretion nor did it affect superoxide production, C3b-rosetting, nitrotetrazolium blue reduction, phagocytosis, or chemotactic responses. A second antibody, PMN70, was found to react exclusively with granulocytes and not with monocytes, lymphocytes, LGL, platelets, red cells, or any of the myelomonocytic, T-cell-derived or B-cell-derived leukemic cell lines tested. The PMN70 antibody immunoprecipitated a 70 kD molecular weight antigen found only on mature granulocytes. Mo95 and PMN70 appear to be distinct from five other tested monoclonal antibodies reactive to monocytes and/or granulocytes on the basis of the fluorescent cell sorter and immunoprecipitation studies performed.

  6. Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue.

    Directory of Open Access Journals (Sweden)

    James A Cotton

    Full Text Available Giardia duodenalis (syn. G. intestinalis, G. lamblia is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn's disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time

  7. Giardia duodenalis Infection Reduces Granulocyte Infiltration in an In Vivo Model of Bacterial Toxin-Induced Colitis and Attenuates Inflammation in Human Intestinal Tissue

    Science.gov (United States)

    Cotton, James A.; Motta, Jean-Paul; Schenck, L. Patrick; Hirota, Simon A.; Beck, Paul L.; Buret, Andre G.

    2014-01-01

    Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn’s disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain

  8. Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue.

    Science.gov (United States)

    Cotton, James A; Motta, Jean-Paul; Schenck, L Patrick; Hirota, Simon A; Beck, Paul L; Buret, Andre G

    2014-01-01

    Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn's disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain

  9. Nucleobindin co-localizes and associates with cyclooxygenase (COX-2 in human neutrophils.

    Directory of Open Access Journals (Sweden)

    Patrick Leclerc

    2008-05-01

    Full Text Available The inducible cyclooxygenase isoform (COX-2 is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc, a ubiquitous Ca(2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE(2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE(2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

  10. Neutrophils extracellular traps damage Naegleria fowleri trophozoites opsonized with human IgG.

    Science.gov (United States)

    Contis-Montes de Oca, A; Carrasco-Yépez, M; Campos-Rodríguez, R; Pacheco-Yépez, J; Bonilla-Lemus, P; Pérez-López, J; Rojas-Hernández, S

    2016-08-01

    Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri. © 2016 John Wiley & Sons Ltd.

  11. Nucleobindin Co-Localizes and Associates with Cyclooxygenase (COX)-2 in Human Neutrophils

    Science.gov (United States)

    Leclerc, Patrick; Biarc, Jordane; St-Onge, Mireille; Gilbert, Caroline; Dussault, Andrée-Anne; Laflamme, Cynthia; Pouliot, Marc

    2008-01-01

    The inducible cyclooxygenase isoform (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis. PMID:18493301

  12. Cloning and Characterization of the Human Interleukin-3 (IL-3)/IL-5/ Granulocyte-Macrophage Colony-Stimulating Factor Receptor bc Gene: Regulation by Ets Family Members

    NARCIS (Netherlands)

    Dijk, Thamar B. van; Baltus, Belinda; Caldenhoven, Eric; Handa, Hiroshi; Raaijmakers, J.A.M.; Lammers, J.W.J.; Koenderman, L.; Groot, Rolf P. de

    1998-01-01

    High-affinity receptors for interleukin-3 (IL-3), IL-5, and granu-locyte- macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific a chain and a common ß chain (ßc). Whereas the mouse has two homologous ß subunits (ßc and bIL-3), in humans, only

  13. Novel Human Cytomegalovirus Viral Chemokines, vCXCL-1s, Display Functional Selectivity for Neutrophil Signaling and Function

    NARCIS (Netherlands)

    Heo, Jinho; Dogra, Pranay; Masi, Tom J; Pitt, Elisabeth A; de Kruijf, Petra; Smit, Martine J; Sparer, Tim E

    2015-01-01

    Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable

  14. Innate Defense against Influenza A Virus: Activity of Human Neutrophil Defensins and Interactions of Defensins with Surfactant Protein D

    DEFF Research Database (Denmark)

    Hartshorn, Kevan L.; White, Mitchell R.; Tecle, Tesfaldet

    2006-01-01

    Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study...

  15. High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

    DEFF Research Database (Denmark)

    Shin, Yun-Ji; Hong, Shin-Young; Kwon, Tae-Ho

    2003-01-01

    this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield......Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome...... of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system....

  16. Vitamin E prevents neutrophil accumulation and attenuates tissue damage in ischemic-reperfused human skeletal muscle.

    Science.gov (United States)

    Formigli, L; Ibba Manneschi, L; Tani, A; Gandini, E; Adembri, C; Pratesi, C; Novelli, G P; Zecchi Orlandini, S

    1997-07-01

    Neutrophil accumulation and the consequent production of oxygen-derived free radicals are involved in the pathogenesis of Ischemia-Reperfusion syndrome. In this study we investigated whether a treatment with Vitamin E, which has antioxidant properties, could attenuate the tissue damage by interfering with the influx of neutrophils within the ischemic and reperfused human skeletal muscle. To this purpose, patients undergoing aortic cross-clamping during the surgical repair of aortic abdominal aneurysm were studied as a model of ischemia-reperfusion of the lower limb muscles. Muscle biopsies from the right femoral quadriceps of patients not receiving and receiving Vitamin E pretreatment before surgery were taken: a) after the induction of anaesthesia, as control samples, and b) after a period of ischemia followed by 30 min of reperfusion. The tissue samples were either routinely processed for morphological study and immunohistochemical analysis to detect an altered expression of specific endothelial adhesion proteins, such as E-selectin and ICAM-1. The results obtained showed that Vitamin E administration was able to prevent the accumulation of neutrophils within the ischemic and reperfused muscle. This beneficial effect of Vitamin E was due to its ability to hinder the expression of E-selectin and ICAM-1, molecules known to increase the adhesiveness of endothelium to circulating neutrophils. After treatment with Vitamin E a marked attenuation of the reperfusion injury was also evident. In conclusion, Vitamin E treatment may be considered a valuable tool for protection against the ischemia-reperfusion damage of human skeletal muscle.

  17. High seroprevalence of Borrelia miyamotoi antibodies in forestry workers and individuals suspected of human granulocytic anaplasmosis in the Netherlands

    Directory of Open Access Journals (Sweden)

    S. Jahfari

    2014-09-01

    Full Text Available Substantial exposure to Borrelia miyamotoi occurs through bites from Ixodes ricinus ticks in the Netherlands, which also transmit Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum. Direct evidence for B. miyamotoi infection in European populations is scarce. A flu-like illness with high fever, resembling human granulocytic anaplasmosis, has been attributed to B. miyamotoi infections in relatively small groups. Borrelia miyamotoi infections associated with chronic meningoencephalitis have also been described in case reports. Assuming that an IgG antibody response against B. miyamotoi antigens reflects (endured infection, the seroprevalence in different risk groups was examined. Sera from nine out of ten confirmed B. miyamotoi infections from Russia were found to be positive with the recombinant antigen used, and no significant cross-reactivity was observed in secondary syphilis patients. The seroprevalence in blood donors was set at 2.0% (95% CI 0.4–5.7%. Elevated seroprevalences in individuals with serologically confirmed, 7.4% (2.0–17.9%, or unconfirmed, 8.6% (1.8–23%, Lyme neuroborreliosis were not significantly different from those in blood donors. The prevalence of anti-B. miyamotoi antibodies among forestry workers was 10% (5.3–16.8% and in patients with serologically unconfirmed but suspected human granulocytic anaplasmosis was 14.6% (9.0–21.8%; these were significantly higher compared with the seroprevalence in blood donors. Our findings indicate that infections with B. miyamotoi occur in tick-exposed individuals in the Netherlands. In addition, B. miyamotoi infections should be considered in patients reporting tick bites and febrile illness with unresolved aetiology in the Netherlands, and other countries where I. ricinus ticks are endemic.

  18. Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yang-Chang [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan (China); Sureshbabu, Munisamy; Fang, Yao-Ching; Wu, Yi-Hsiu [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Lan, Yu-Hsuan [School of Pharmacy, China Medical University, Taichung 404, Taiwan (China); Chang, Fang-Rong [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Ya-Wen [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan 333, Taiwan (China)

    2013-02-01

    Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl) -1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O{sub 2}{sup ·−}) production, elastase release, and CD11b expression in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O{sub 2}{sup ·−} production. The peak cytosolic calcium concentration ([Ca{sup 2+}]{sub i}) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca{sup 2+}]{sub i} was significantly shortened. In a calcium-free solution, changes in [Ca{sup 2+}]{sub i} caused by the addition of extracellular Ca{sup 2+} were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca{sup 2+}]{sub i} changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE. Highlights: ► Bractelactone isolated from Fissistigma bracteolatum. ► Bractelactone inhibited FMLP-induced human neutrophil activations. ► Bractelactone had no effect on IP3 formation. ► Bractelactone did not alter MAPKs, AKT, and cAMP pathways. ► Bractelactone inhibited store-operated calcium entry.

  19. Rapid engraftment by peripheral blood progenitor cells mobilized by recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in nonhuman primates.

    Science.gov (United States)

    Andrews, R G; Briddell, R A; Knitter, G H; Rowley, S D; Appelbaum, F R; McNiece, I K

    1995-01-01

    We have previously shown that administration of low-dose recombinant human stem cell factor (rhSCF) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) to baboons mobilizes greater numbers of progenitor cells in the blood than does administration of rhG-CSF alone. The purpose of the present study was to determine whether marrow repopulating cells are present in the blood of nonhuman primates administered low-dose rhSCF plus rhG-CSF, and if present, whether these cells engraft lethally irradiated recipients as rapidly as blood cells mobilized by treatment with rhG-CSF alone. One group of baboons was administered low-dose rhSCF (25 micrograms/kg/d) plus rhG-CSF (100 micrograms/kg/d) while a second group received rhG-CSF alone (100 micrograms/kg/d). Each animal underwent a single 2-hour leukapheresis occurring the day when the number of progenitor cells per volume of blood was maximal. For baboons administered low-dose rhSCF plus rhG-CSF, the leukapheresis products contained 1.8-fold more mononuclear cells and 14.0-fold more progenitor cells compared to the leukapheresis products from animals treated with rhG-CSF alone. All animals successfully engrafted after transplantation of cryopreserved autologous blood cells. In animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells, we observed a time to a platelet count of > 20,000 was 8 days +/- 0, to a white blood cell count (WBC) of > 1,000 was 11 +/- 1 days, and to an absolute neutrophil count (ANC) of > 500 was 12 +/- 1 days. These results compared with 42 +/- 12, 16 +/- 1, and 24 +/- 4 days to achieve platelets > 20,000, WBC > 1,000, and ANC > 500, respectively, for baboons transplanted with rhG-CSF mobilized blood cells. Animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells had blood counts equivalent to pretransplant values within 3 weeks after transplant. The results suggest that the combination of low-dose rhSCF plus rhG-CSF mobilizes greater numbers of

  20. Possible in vivo tolerance of human polymorphonuclear neutrophil to low-grade exercise-induced endotoxaemia

    Directory of Open Access Journals (Sweden)

    G. Camus

    1998-01-01

    Full Text Available To address the question of whether translocation of bacterial lipopolysaccharide (LPS into the blood could be involved in the process of exercise-induced polymorphonuclear neutrophil (PMN activation, 12 healthy male subjects who took part in a sprint triathlon (1.5 km river swim, 40 km bicycle race, 10 km road race were studied. While there was no detectable amount of endotoxin in the blood samples drawn at rest, exercise was followed by the appearance of circulating endotoxin molecules at the end of competition in four subjects, and after one and 24 h recovery in three and seven athletes, respectively. The concentrations of plasma granulocyte myeloperoxidase ([MPO], were significantly higher immediately after exercise and one hour later than baseline values (P<0.001. This variable returned to pre-race levels the day after exercise, despite the presence of detectable amounts of LPS, at that time, in seven athletes. The absence of significant correlation (r=0.26;P=0.383 and temporal association between [MPO]and plasma endotoxin levels led us to conclude that endotoxaemia was not involved in the process of exercise-induced PMN degranulation observed in our subjects.

  1. Granulocytic nuclear differentiation of lamin B receptor-deficient mouse EPRO cells.

    Science.gov (United States)

    Zwerger, Monika; Herrmann, Harald; Gaines, Peter; Olins, Ada L; Olins, Donald E

    2008-08-01

    Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane. Recent studies have demonstrated that genetic deficiency of LBR during granulopoiesis results in hypolobulation of the mature neutrophil nucleus, as observed in human Pelger-Huët anomaly and mouse ichthyosis (ic). In this study, we utilized differentiated early promyelocytes (EPRO cells) that were derived from the bone marrow of homozygous and heterozygous ichthyosis mice to examine changes to the expression of nuclear envelope proteins and heterochromatin structure that result from deficient LBR expression. Wild-type (+/+), heterozygous (+/ic), and homozygous (ic/ic) granulocytic forms of EPRO cells were analyzed for the expression of multiple lamins and inner nuclear envelope proteins by immunostaining and immunoblotting techniques. The heterochromatin architecture was also examined by immunostaining for histone lysine methylation. Wild-type (+/+) and heterozygous (+/ic) granulocytic forms revealed ring-shaped nuclei and contained LBR within the nuclear envelope; ic/ic granulocytes exhibited smaller ovoid nuclei devoid of LBR. The pericentric heterochromatin of undifferentiated and granulocytic ic/ic cells was condensed into larger spots and shifted away from the nuclear envelope, compared to +/+ and +/ic cell forms. Lamin A/C, which is normally not present in mature granulocytes, was significantly elevated in LBR-deficient EPRO cells. Our observations suggest roles for LBR during granulopoiesis, which can involve augmenting nuclear membrane growth, facilitating compartmentalization of heterochromatin, and promoting downregulation of lamin A/C expression.

  2. Low-Density Granulocytes Are Elevated in Mycobacterial Infection and Associated with the Severity of Tuberculosis.

    Science.gov (United States)

    Deng, Yating; Ye, Jianqing; Luo, Qing; Huang, Zhikun; Peng, Yiping; Xiong, Guoliang; Guo, Yang; Jiang, Hong; Li, Junming

    2016-01-01

    Tuberculosis remains a global health problem caused by infection with Mycobacterium tuberculosis. Numerous studies have established a close correlation between the development of tuberculosis and the roles of neutrophils. Recently, a distinct population of CD15+ granulocytes was found to be present in the peripheral blood mononuclear cell (PBMC) fraction in humans. This population of granulocytes, termed low-density granulocytes (LDGs), was reported to be elevated and associated with disease activity or severity in a number of different conditions including SLE, asthma and HIV infection. However, both the frequency and clinical significance of LDGs associated with tuberculosis are unclear. Here we determined LDG levels and made comparisons between subjects with active pulmonary tuberculosis (PTB) and healthy controls, between PTB patients with mild-to-moderate disease and patients with advanced disease, and among PTB patients following anti-tuberculous therapy of varying durations. The direct correlation between M. tuberculosis infection and LDG levels was confirmed by in vitro infection of whole peripheral blood and isolated granulocytes with mycobacteria. Our results demonstrated that PBMCs in PTB patients contained significantly elevated percentages of LDGs compared with control subjects. LDGs in tuberculosis expressed higher levels of activation markers compared to normal-density granulocytes (NDGs). M. tuberculosis induced the generation of LDGs in both whole blood and isolated NDGs from control subjects, which suggests that LDGs associated with M. tuberculosis infection are likely to originate from in situ activation. Furthermore, our results revealed that the frequency of LDGs is associated with the severity of tuberculosis.

  3. Low-Density Granulocytes Are Elevated in Mycobacterial Infection and Associated with the Severity of Tuberculosis.

    Directory of Open Access Journals (Sweden)

    Yating Deng

    Full Text Available Tuberculosis remains a global health problem caused by infection with Mycobacterium tuberculosis. Numerous studies have established a close correlation between the development of tuberculosis and the roles of neutrophils. Recently, a distinct population of CD15+ granulocytes was found to be present in the peripheral blood mononuclear cell (PBMC fraction in humans. This population of granulocytes, termed low-density granulocytes (LDGs, was reported to be elevated and associated with disease activity or severity in a number of different conditions including SLE, asthma and HIV infection. However, both the frequency and clinical significance of LDGs associated with tuberculosis are unclear. Here we determined LDG levels and made comparisons between subjects with active pulmonary tuberculosis (PTB and healthy controls, between PTB patients with mild-to-moderate disease and patients with advanced disease, and among PTB patients following anti-tuberculous therapy of varying durations. The direct correlation between M. tuberculosis infection and LDG levels was confirmed by in vitro infection of whole peripheral blood and isolated granulocytes with mycobacteria. Our results demonstrated that PBMCs in PTB patients contained significantly elevated percentages of LDGs compared with control subjects. LDGs in tuberculosis expressed higher levels of activation markers compared to normal-density granulocytes (NDGs. M. tuberculosis induced the generation of LDGs in both whole blood and isolated NDGs from control subjects, which suggests that LDGs associated with M. tuberculosis infection are likely to originate from in situ activation. Furthermore, our results revealed that the frequency of LDGs is associated with the severity of tuberculosis.

  4. The roles of integrins in function of human neutrophils after their migration through endothelium into interstitial matrix.

    Science.gov (United States)

    Luo, Ding; McGettrick, Helen M; Stone, Phil C; Rainger, George E; Nash, Gerard B

    2015-01-01

    We investigated the changes in neutrophil phenotype and function after transendothelial migration, and the roles played by integrin receptors in their behaviour. Neutrophils were tracked microscopically as they migrated through endothelial cells into collagen gels, and were retrieved at desired times. When endothelial cells were treated with increasing doses of tumour necrosis factor-α, neutrophils not only migrated in greater number, but also to a greater depth in the gel. Apoptosis was barely detectable in neutrophils retrieved after 24h, and many remained viable and motile at 48h. Neutrophils retrieved after 1h had increased oxidative capacity and at 24h had similar capacity as freshly-isolated neutrophils. However, by then they had impaired ability to phagocytose bacteria. Compared to fresh neutrophils, total mRNA was halved by 24h, but while β2-integrin expression decreased, β1- and β3-integrin increased along with ICAM-1. Studies of integrin blockade indicated that while β2-integrins were needed to cross the endothelial barrier, no integrins were required for migration within the gel. β2-integrins also contributed to phagocytosis, but their binding was not required for prolonged survival. These results demonstrate a model for integrated analysis of neutrophil migration and function, and describe development of effector functions and the roles of integrins in human neutrophils for the first time.

  5. The roles of integrins in function of human neutrophils after their migration through endothelium into interstitial matrix.

    Directory of Open Access Journals (Sweden)

    Ding Luo

    Full Text Available We investigated the changes in neutrophil phenotype and function after transendothelial migration, and the roles played by integrin receptors in their behaviour. Neutrophils were tracked microscopically as they migrated through endothelial cells into collagen gels, and were retrieved at desired times. When endothelial cells were treated with increasing doses of tumour necrosis factor-α, neutrophils not only migrated in greater number, but also to a greater depth in the gel. Apoptosis was barely detectable in neutrophils retrieved after 24h, and many remained viable and motile at 48h. Neutrophils retrieved after 1h had increased oxidative capacity and at 24h had similar capacity as freshly-isolated neutrophils. However, by then they had impaired ability to phagocytose bacteria. Compared to fresh neutrophils, total mRNA was halved by 24h, but while β2-integrin expression decreased, β1- and β3-integrin increased along with ICAM-1. Studies of integrin blockade indicated that while β2-integrins were needed to cross the endothelial barrier, no integrins were required for migration within the gel. β2-integrins also contributed to phagocytosis, but their binding was not required for prolonged survival. These results demonstrate a model for integrated analysis of neutrophil migration and function, and describe development of effector functions and the roles of integrins in human neutrophils for the first time.

  6. Docosahexaenoic acid inhibits the adhesion of flowing neutrophils to cytokine stimulated human umbilical vein endothelial cells.

    Science.gov (United States)

    Yates, Clara M; Tull, Samantha P; Madden, Jackie; Calder, Philip C; Grimble, Robert F; Nash, Gerard B; Rainger, G Ed

    2011-07-01

    The (n-3) PUFA, DHA, is widely thought to posses the ability to modulate the inflammatory response. However, its modes of interaction with inflammatory cells are poorly understood. In particular, there are limited data on the interactions of DHA with vascular endothelium, the cells that regulate the traffic of leukocytes from the blood into inflamed tissue. Using human umbilical vein endothelial cells (EC) cultured in a flow-based adhesion assay and activated with TNFα, we tested whether supplementing human umbilical vein EC with physiologically achievable concentrations of DHA would inhibit the recruitment of flowing neutrophils. DHA caused a dose-dependent reduction in neutrophil recruitment to the EC surface, although cells that became adherent were activated and could migrate across the human umbilical vein EC monolayer normally. Using EPA as an alternative supplement had no effect on the levels of neutrophil adhesion in this assay. Analysis of adhesion receptor expression by qPCR demonstrated that DHA did not alter the transcriptional activity of human umbilical vein EC. However, DHA did significantly reduce E-selectin expression at the human umbilical vein EC surface without altering the total cellular pool of this adhesion receptor. Thus, we have identified a novel mechanism by which DHA alters the trafficking of leukocytes during inflammation and demonstrate that this involves disruption of intracellular transport mechanisms used to present adhesion molecules on the surface of cytokine-stimulated EC.

  7. The hederagenin saponin SMG-1 is a natural FMLP receptor inhibitor that suppresses human neutrophil activation.

    Science.gov (United States)

    Hwang, Tsong-Long; Wang, Chien-Chiao; Kuo, Yao-Haur; Huang, Hui-Chi; Wu, Yang-Chang; Kuo, Liang-Mou; Wu, Yi-Hsiu

    2010-10-15

    The pericarp of Sapindus mukorossi Gaertn is traditionally used as an expectorant in Japan, China, and Taiwan. Activated neutrophils produce high concentrations of the superoxide anion (O(2)(-)) and elastase known to be involved in airway mucus hypersecretion. In the present study, the anti-inflammatory functions of hederagenin 3-O-(3,4-O-di-acetyl-alpha-L-arabinopyranoside)-(1-->3)-alpha-l-rhamnopyranosyl-(1-->2)-alpha-l-arabinopyranoside (SMG-1), a saponin isolated from S. mukorossi, and its underlying mechanisms were investigated in human neutrophils. SMG-1 potently and concentration-dependently inhibited O(2)(*-) generation and elastase release in N-Formyl-Met-Leu-Phe (FMLP)-activated human neutrophils. Furthermore, SMG-1 reduced membrane-associated p47(phox) expression in FMLP-induced intact neutrophils, but did not alter subcellular NADPH oxidase activity in reconstituted systems. SMG-1 attenuated FMLP-induced increase of cytosolic calcium concentration and phosphorylation of p38 MAPK, ERK, JNK, and AKT. However, SMG-1 displayed no effect on cellular cAMP levels and activity of adenylate cyclase and phosphodiesterase. Significantly, receptor-binding analysis showed that SMG-1 inhibited FMLP binding to its receptor in a concentration-dependent manner. In contrast, neither phorbol myristate acetate-induced O(2)(*-) generation and MAPKs activation nor thapsigargin-caused calcium mobilization was altered by SMG-1. Taken together, our results demonstrate that SMG-1 is a natural inhibitor of the FMLP receptor, which may have the potential to be developed into a useful new therapeutic agent for treating neutrophilic inflammatory diseases. Copyright 2010 Elsevier Inc. All rights reserved.

  8. Endocytosis is required for exocytosis and priming of respiratory burst activity in human neutrophils.

    Science.gov (United States)

    Creed, T Michael; Tandon, Shweta; Ward, Richard A; McLeish, Kenneth R

    2017-10-01

    Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47 phox phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis. Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47 phox phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey-Kramer multiple-comparison test. Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47 phox phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis. Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.

  9. Induction of retinoic acid receptor-alpha by granulocyte macrophage colony-stimulating factor in human myeloid leukemia cell lines.

    Science.gov (United States)

    Shimizu, T; Takeda, K

    2000-08-15

    We reported previously that treatment with all-trans retinoic acid (ATRA) and granulocyte macrophage colony-stimulating factor (GM-CSF) induces differentiation of human myeloblastic leukemia ML-1 cells to granulocytes, whereas treatment with ATRA alone induces practically no differentiation of these cells. To investigate the mechanism of the synergistic effect of these factors, we examined the effect of GM-CSF on retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in ML-1 cells. We reveal that GM-CSF induces the expression of RAR alpha mRNA and protein and stimulates the binding of nuclear proteins to direct repeat 5, a consensus sequence with high affinity for RAR-RXR heterodimers. Furthermore, expression of CD38 mRNA mediated through RAR alpha is induced synergistically by treatment with ATRA + GM-CSF. These results suggest that GM-CSF stimulates transcriptional activity mediated via RAR alpha in ML-1 cells. The induction of RAR alpha by GM-CSF may therefore be a mechanism for stimulation by GM-CSF. The induction of RAR alpha by GM-CSF was also detected in other myeloid leukemia cell lines (THP-1 and KG-1) that showed a synergistic effect similar to that seen in ML-1 cells in response to ATRA + GM-CSF. We also found that GM-CSF induced the expression of RAR alpha in blood cells obtained from patients with acute myeloid leukemia. This activity of GM-CSF may serve as a useful adjunct to differentiation therapy for retinoic acid-nonresponsive leukemias.

  10. Properdin-Mediated C5a Production Enhances Stable Binding of Platelets to Granulocytes in Human Whole Blood.

    Science.gov (United States)

    Blatt, Adam Z; Saggu, Gurpanna; Kulkarni, Koustubh V; Cortes, Claudio; Thurman, Joshua M; Ricklin, Daniel; Lambris, John D; Valenzuela, Jesus G; Ferreira, Viviana P

    2016-06-01

    Enhanced levels of platelet/granulocyte aggregates (PGAs) are found in patients suffering from many different inflammatory vascular diseases, and their formation in animal models of vascular disease is associated with increased thromboinflammation and worsened outcomes. The complement system, a part of the innate immune system, influences PGA formation, but the mechanisms for its effects are unknown. In this study, we have defined complement-mediated mechanisms that enhance PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP) using ex vivo flow cytometry assays. We demonstrate that physiological properdin, a positive regulator of complement alternative pathway activity, increases PGA formation when added to TRAP-stimulated blood. All physiological properdin forms increase PGA formation, but properdin tetramers are the most efficient at increasing complement activity and PGA formation. Inhibition of endogenous properdin, either circulating in the blood or produced locally by leukocytes, impairs TRAP-mediated PGA formation to the same level as specific inhibition of either the alternative or classical pathway. Additionally, blocking the interaction of C5a with its cellular receptor prevents properdin-mediated increases in PGA formation. Adding either properdin tetramers or C5a to whole blood increases CD11b expression on granulocytes, and this increase is prevented by blockade of the C5a-C5a receptor axis. Finally, we demonstrate that the effects of properdin on PGA formation are tightly regulated by Factor H. Cumulatively, our data indicate that properdin enhances PGA formation via increased production of C5a, and that inhibition of properdin function has therapeutic potential to limit thromboinflammation in diseases characterized by increased PGA formation. Copyright © 2016 by The American Association of Immunologists, Inc.

  11. Neutrophil extracellular traps form predominantly during the organizing stage of human venous thromboembolism development.

    Science.gov (United States)

    Savchenko, A S; Martinod, K; Seidman, M A; Wong, S L; Borissoff, J I; Piazza, G; Libby, P; Goldhaber, S Z; Mitchell, R N; Wagner, D D

    2014-06-01

    A growing health problem, venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), requires refined diagnostic and therapeutic approaches. Neutrophils contribute to thrombus initiation and development in experimental DVT. Recent animal studies recognized neutrophil extracellular traps (NETs) as an important scaffold supporting thrombus stability. However, the hypothesis that human venous thrombi involve NETs has not undergone rigorous testing. To explore the cellular composition and the presence of NETs within human venous thrombi at different stages of development. We examined 16 thrombi obtained from 11 patients during surgery or at autopsy using histomorphological, immunohistochemical and immunofluorescence analyses. We classified thrombus regions as unorganized, organizing and organized according to their morphological characteristics. We then evaluated them, focusing on neutrophil and platelet deposition as well as micro-vascularization of the thrombus body. We observed evidence of NET accumulation, including the presence of citrullinated histone H3 (H3Cit)-positive cells. NETs, defined as extracellular diffuse H3Cit areas associated with myeloperoxidase and DNA, localized predominantly during the phase of organization in human venous thrombi. NETs are present in organizing thrombi in patients with VTE. They are associated with thrombus maturation in humans. Dissolution of NETs might thus facilitate thrombolysis. This finding provides new insights into the clinical development and pathology of thrombosis and provides new perspectives for therapeutic advances. © 2014 International Society on Thrombosis and Haemostasis.

  12. Recombinant human granulocyte-colony stimulating factor administration for treating amyotrophic lateral sclerosis: A pilot study.

    Science.gov (United States)

    Nefussy, Beatrice; Artamonov, Irena; Deutsch, Varda; Naparstek, Ela; Nagler, Arnon; Drory, Vivian E

    2010-01-01

    Granulocyte-colony stimulating factor (G-CSF) is used to mobilize CD34+ haematopoietic stem cells from the bone marrow to the peripheral blood. We proposed to use cell subsets induced by G-CSF to slow down disease progression in patients with amyotrophic lateral sclerosis (ALS). Patients with definite or probable ALS were assigned in a double-blind manner to receive G-CSF or placebo every three months for a year. The primary outcome measure was the functional decline, measured by the revised ALS Functional Rating Scale, Revised (ALSFRS-R) score. Secondary outcome measures included vital capacity, manual muscle strength, compound muscle action potential amplitudes, neurophysiological index, and McGill single item quality of life score (QoL). Thirty-nine patients were enrolled. Seventeen patients who received G-CSF and 18 who received placebo were evaluated. G-CSF was effective in mobilizing CD34+ to blood. The outcome measures used showed no statistically significant benefit, although there was a trend of slowing disease progression following two G-CSF treatments, as shown by lower slopes of ALSFRS-R and QoL in the first six treatment months. The treatment had no major side-effects. G-CSF administration in ALS patients caused successful mobilization of autologous bone marrow cells, but was not effective in slowing down disease deterioration.

  13. Lipoxin A4 modulates transmigration of human neutrophils across intestinal epithelial monolayers.

    OpenAIRE

    Colgan, S P; Serhan, C N; Parkos, C A; Delp-Archer, C; Madara, J L

    1993-01-01

    Neutrophil (PMN) migration across intestinal epithelial barriers, such as occurs in many disease states, results in modifications in epithelial barrier. Here, we investigated the impact of lipoxin A4 (LXA4), an eicosanoid with counterregulatory inflammatory roles, on PMN migration across cultured monolayers of the human intestinal epithelial cell line T84. Transepithelial migration of PMN was assessed in the apical-to-basolateral direction and in the basolateral-to-apical direction. In the ap...

  14. Evidence that endoplasmic reticulum (ER) stress and caspase-4 activation occur in human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Binet, Francois; Chiasson, Sonia [Laboratoire de recherche en inflammation et physiologie des granulocytes, Universite du Quebec, INRS-Institut Armand-Frappier, Laval, QC (Canada); Girard, Denis, E-mail: denis.girard@iaf.inrs.ca [Laboratoire de recherche en inflammation et physiologie des granulocytes, Universite du Quebec, INRS-Institut Armand-Frappier, Laval, QC (Canada)

    2010-01-01

    Apoptosis can result from activation of three major pathways: the extrinsic, the intrinsic, and the most recently identified endoplasmic reticulum (ER) stress-mediated pathway. While the two former pathways are known to be operational in human polymorphonuclear neutrophils (PMNs), the existence of the ER stress-mediated pathway, generally involving caspase-4, has never been reported in these cells. Recently, we have documented that arsenic trioxide (ATO) induced apoptosis in human PMNs by a mechanism that needs to be further investigated. In this study, using immunofluorescence and electron microscopy, we present evidence of ER alterations in PMNs activated by the ER stress inducer arsenic trioxide (ATO). Several key players of the unfolded protein response, including GRP78, GADD153, ATF6, XBP1 and eIF2{alpha} are expressed and activated in PMNs treated with ATO or other ER stress inducers. Although caspase-4 is expressed and activated in neutrophils, treatment with a caspase-4 inhibitor did not attenuate the pro-apoptotic effect of ATO at a concentration that reverses caspase-4 processing and activation. Our results demonstrate for the first time that the ER stress-mediated apoptotic pathway operates in human neutrophils.

  15. The Frequency of Granulocytes with Spontaneous Somatic Mutations: A Wide Distribution in a Normal Human Population

    Science.gov (United States)

    Peruzzi, Benedetta; Boni, Luca; Caporale, Roberto; Dolara, Piero; Notaro, Rosario; Luzzatto, Lucio

    2013-01-01

    Germ-line mutation rate has been regarded classically as a fundamental biological parameter, as it affects the prevalence of genetic disorders and the rate of evolution. Somatic mutation rate is also an important biological parameter, as it may influence the development and/or the course of acquired diseases, particularly of cancer. Estimates of this parameter have been previously obtained in few instances from dermal fibroblasts and lymphoblastoid cells. However, the methodology required has been laborious and did not lend itself to the analysis of large numbers of samples. We have previously shown that the X-linked gene PIG-A, since its product is required for glycosyl-phosphatidylinositol-anchored proteins to become surface bound, is a good sentinel gene for studying somatic mutations. We now show that by this approach we can accurately measure the proportion of PIG-A mutant peripheral blood granulocytes, which we call mutant frequency, ƒ. We found that the results are reproducible, with a variation coefficient (CV) of 45%. Repeat samples from 32 subjects also had a CV of 44%, indicating that ƒ is a relatively stable individual characteristic. From a study of 142 normal subjects we found that log ƒ is a normally distributed variable; ƒ variability spans a 80-fold range, from less than 1×10−6 to 37.5×10−6, with a median of 4.9×10−6. Unlike other techniques commonly employed in population studies, such as comet assay, this method can detect any kind of mutation, including point mutation, as long as it causes functional inactivation of PIG-A gene. Since the test is rapid and requires only a small sample of peripheral blood, this methodology will lend itself to investigating genetic factors that underlie the variation in the somatic mutation rate, as well as environmental factors that may affect it. It will be also possible to test whether ƒ is a determinant of the risk of cancer. PMID:23342069

  16. Expression of sialyltransferase activity on intact human neutrophils

    OpenAIRE

    Rifat, Salahaldin; Kang, Tae Jin; Mann, Dean; Zhang, Lei; Puche, Adam C.; Stamatos, Nicholas M.; Goldblum, Simeon E.; Brossmer, Reinhard; Cross, Alan S.

    2008-01-01

    Endogenous polymorphonuclear leukocyte (PMN)-associated sialidase activity enhances PMN adhesion to and migration across the endothelium through the removal of sialylated cell-surface residues. We tested the hypothesis that PMNs also express sialyltransferase (ST) activity that restores sialyl residues to the PMN surface. We developed a highly sensitive fluorometric assay to demonstrate that intact human PMNs can mediate and accept sialyl residue transfer. This ST activity is inhibited by a S...

  17. Human neutrophil migration and activation by BJcuL, a galactose binding lectin purified from Bothrops jararacussu venom

    Directory of Open Access Journals (Sweden)

    Fernandes Luiz

    2011-01-01

    Full Text Available Abstract Background Neutrophil migration to an inflamed site constitutes the first line of the innate immune response against invading microorganisms. Given the crucial role of endogenous lectins in neutrophil mobilization and activation, lectins from exogenous sources have often been considered as putative modulators of leukocyte function. Lectins purified from snake venom have been described as galactoside ligands that induce erythrocyte agglutination and platelet aggregation. This study evaluated human neutrophil migration and activation by C-type lectin BJcuL purified from Bothrops jararacussu venom. Results Utilizing fluorescence microscopy, we observed that biotinylated-BJcuL was evenly distributed on the neutrophil surface, selectively inhibited by D-galactose. Lectin was able to induce modification in the neutrophil morphology in a spherical shape for a polarized observed by optical microscopy and exposure to BJcuL in a Boyden chamber assay resulted in cell migration. After 30 minutes of incubation with BJcuL we found enhanced neutrophil functions, such as respiratory burst, zymozan phagocytosis and an increase in lissosomal volume. In addition, BJcuL delays late apoptosis neutrophils. Conclusion These results demonstrate that BJcuL can be implicated in a wide variety of immunological functions including first-line defense against pathogens, cell trafficking and induction of the innate immune response since lectin was capable of inducing potent neutrophil activation.

  18. Minocycline affects human neutrophil respiratory burst and transendothelial migration.

    Science.gov (United States)

    Parenti, Astrid; Indorato, Boris; Paccosi, Sara

    2017-02-01

    This study aimed at investigating the in vitro activity of minocycline and doxycycline on human polymorphonuclear (h-PMN) cell function. h-PMNs were isolated from whole venous blood of healthy subjects; PMN oxidative burst was measured by monitoring ROS-induced oxidation of luminol and transendothelial migration was studied by measuring PMN migration through a monolayer of human umbilical vein endothelial cells. Differences between multiple groups were determined by ANOVA followed by Tukey's multiple comparison test; Student's t test for unpaired data for two groups. Minocycline (1-300 µM) concentration dependently and significantly inhibited oxidative burst of h-PMNs stimulated with 100 nM fMLP. Ten micromolar concentrations, which are superimposable to C max following a standard oral dose of minocycline, promoted a 29.8 ± 4 % inhibition of respiratory burst (P minocycline impaired PMN transendothelial migration, with maximal effect at 100 µM (42.5 ± 7 %, inhibition, n = 5, P minocycline exerted on innate immune h-PMN cell function.

  19. Amburanins A and B from Amburana cearensis: daphnodorin-type biflavonoids that modulate human neutrophil degranulation

    Energy Technology Data Exchange (ETDEWEB)

    Canuto, Kirley M.; Silveira, Edilberto R., E-mail: edil@ufc.br [Universidade Federal do Ceara (UFCE), Fortaleza, CE (Brazil). Departamento de Quimica Organica e Inorganica; Leal, Luzia K.A.M.; Lopes, Amanda A. [Universidade Federal do Ceara (CEFAC/UFCE), Fortaleza, CE (Brazil). Centro de Estudos Farmaceuticos e Cosmeticos. Departamento de Farmacia; Coleman, Christina M.; Ferreira, Daneel [Department of Pharmacognosy and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, MS (United States)

    2014-04-15

    Two new biflavonoids 3,5,7,4'-tetrahydroxyflavanone-(2→O→4':3→3')-2',4',6',4- tetrahydroxydihydrochalcone (1) and 3,5,7,4'-tetrahydroxyflavanone-(2→O→7:3→8)-3,4',5,7-tetrahydroxyflavone (2), named as amburanin A and amburanin B, respectively, were isolated from the trunk bark of Amburana cearensis, and their structures elucidated on the basis of spectroscopic analysis and by comparison with literature data. The effects of 1 and 2 on the pro-inflammatory response of human neutrophils were investigated (0.1; 1; 25; 50 e 100 μg mL{sup -1}). At concentration higher than 25 μg mL{sup -1}, both compounds suppressed nearly 92% of the neutrophil degranulation and 53% of myeloperoxidase activity, thus indicating that they are potential anti-inflammatory lead compounds. (author)

  20. Immunomodulatory effects of Santolina chamaecyparissus leaf extracts on human neutrophil functions.

    Science.gov (United States)

    Boudoukha, Chahra; Bouriche, Hamama; Ortega, Eduardo; Senator, Abderrahmane

    2016-01-01

    Santolina chamaecyparissus L. (Asteraceae) is an aromatic plant wide spread in the Mediterranean region. It is used in folk medicine for its anti-inflammatory properties. The effects of S. chamaecyparissus aqueous extract (SCAE) and polyphenolic extract (SCPE) on human polymorphonuclear neutrophil (PMN) degranulation, chemotaxis, phagocytosis, and microbicidal capacity were examined in vitro. Aqueous and polyphenolic extracts were prepared from S. chamaecyparissus leaves. The elastase release was used as a marker for measuring PMN degranulation, while chemotaxis was performed using a 48-microwell chemotaxis chamber. The phagocytosis and the microbicidal capacity were evaluated using fresh cultures of Candida albicans. The treatment of neutrophils with different concentrations (10-200 µg/ml) of SCAE and SCPE caused a significant (p functions support the anti-inflammatory activity and show new mechanisms of action and effectiveness of S. chamaecyparissus leaf extracts. This plant may be considered as an interesting source of anti-inflammatory and immunomodulatory agents.

  1. Phospholipase A2 Inhibitor from Crotalus durissus terrificus rattlesnake: Effects on human peripheral blood mononuclear cells and human neutrophils cells.

    Science.gov (United States)

    Xavier, Caroline V; da S Setúbal, Sulamita; Lacouth-Silva, Fabianne; Pontes, Adriana S; Nery, Neriane M; de Castro, Onassis Boeri; Fernandes, Carla F C; Soares, Andreimar M; Fortes-Dias, Consuelo L; Zuliani, Juliana P

    2017-12-01

    Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A2 (PLA2), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB4 production, cytosolic PLA2s activity, myeloperoxidase (MPO) and superoxide anion (O2-) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB4, but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O2- release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O2-. However, it induces LTB4 and IL-8 production. These data show the influence of CNF on PBMCs' function by inducing TNF-α and LTB4 production, and on neutrophils, by stimulating chemotaxis and LTB4 production, via cytosolic PLA2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Expression of sialyltransferase activity on intact human neutrophils.

    Science.gov (United States)

    Rifat, Salahaldin; Kang, Tae Jin; Mann, Dean; Zhang, Lei; Puche, Adam C; Stamatos, Nicholas M; Goldblum, Simeon E; Brossmer, Reinhard; Cross, Alan S

    2008-10-01

    Endogenous polymorphonuclear leukocyte (PMN)-associated sialidase activity enhances PMN adhesion to and migration across the endothelium through the removal of sialylated cell-surface residues. We tested the hypothesis that PMNs also express sialyltransferase (ST) activity that restores sialyl residues to the PMN surface. We developed a highly sensitive fluorometric assay to demonstrate that intact human PMNs can mediate and accept sialyl residue transfer. This ST activity is inhibited by a ST inhibitor, CMP, which also inhibits the transendothelial migration of PMNs in response to IL-8 in vitro and in vivo. We conclude that intact PMNs express sialidase and ST activities that permit rapid modulation of their surface sialylation and their ability to adhere to and migrate across the endothelium.

  3. Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

    Directory of Open Access Journals (Sweden)

    Zofia Sulowska

    2005-01-01

    in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD but not by catalase (CAT was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.

  4. Aggregation of beta2 integrins activates human neutrophils through the IkappaB/NF-kappaB pathway.

    Science.gov (United States)

    Kim, Cheol Hyeon; Lee, Kyoung-Hee; Lee, Choon-Taek; Kim, Young Whan; Han, Sung Koo; Shim, Young-Soo; Yoo, Chul-Gyu

    2004-02-01

    Neutrophils are now considered central to the pathogenesis of most forms of acute lung injury. Neutrophils do not cause damage while suspended in the bloodstream; however, a release of cytotoxic agents occurs when neutrophils are adherent to endothelium, epithelium, or extracellular matrix proteins in the interstitium. Such neutrophil adherence is mediated predominantly through beta(2) integrins (CD11/CD18) on its surface. This study was undertaken to investigate whether the IkappaB/nuclear factor (NF)-kappaB cascade is involved in this beta(2) integrin-mediated activation of human neutrophils. beta(2) Integrin Mac-1 (CD11b/CD18) aggregation was induced by antibody cross-linking of the integrins on the cell surface. beta(2) Integrin aggregation induced interleukin-1beta and tumor necrosis factor-alpha production, which suggests the activation of neutrophils by beta(2) integrin. IkappaBalpha was markedly degraded at 1 h, and NF-kappaB-DNA-binding activity markedly increased 2 h after beta(2) integrin aggregation, which activated IkappaB kinase activity at 1 h. beta(2) Integrin-induced cytokine production was suppressed by MG132 or SN50 pretreatment, which blocked the activation of NF-kappaB. These findings suggest that the activation of human neutrophils by beta(2) integrin aggregation is mediated through the activation of the IkappaB/NF-kappaB pathway.

  5. Aggregation of β2integrins activates human neutrophils through the IκB/NF-κB pathway.

    Science.gov (United States)

    Kim, Cheol Hyeon; Lee, Kyoung-Hee; Lee, Choon-Taek; Kim, Young Whan; Han, Sung Koo; Shim, Young-Soo; Yoo, Chul-Gyu

    2004-02-01

    Neutrophils are now considered central to the pathogenesis of most forms of acute lung injury. Neutrophils do not cause damage while suspended in the bloodstream; however, a release of cytotoxic agents occurs when neutrophils are adherent to endothelium, epithelium, or extracellular matrix proteins in the interstitium. Such neutrophil adherence is mediated predominantly through β 2 integrins (CD11/CD18) on its surface. This study was undertaken to investigate whether the IκB/nuclear factor (NF)-κB cascade is involved in this β 2 integrin-mediated activation of human neutrophils. β 2 Integrin Mac-1 (CD11b/CD18) aggregation was induced by antibody cross-linking of the integrins on the cell surface. β 2 Integrin aggregation induced interleukin-1β and tumor necrosis factor-α production, which suggests the activation of neutrophils by β 2 integrin. IκBα was markedly degraded at 1 h, and NF-κB-DNA-binding activity markedly increased 2 h after β 2 integrin aggregation, which activated IκB kinase activity at 1 h. β 2 Integrin-induced cytokine production was suppressed by MG132 or SN50 pretreatment, which blocked the activation of NF-κB. These findings suggest that the activation of human neutrophils by β 2 integrin aggregation is mediated through the activation of the IκB/NF-κB pathway. © 2004 Society for Leukocyte Biology.

  6. Human neutrophil elastase degrades SPLUNC1 and impairs airway epithelial defense against bacteria.

    Directory of Open Access Journals (Sweden)

    Di Jiang

    Full Text Available Acute exacerbations of chronic obstructive pulmonary disease (AECOPD are a significant cause of mortality of COPD patients, and pose a huge burden on healthcare. One of the major causes of AECOPD is airway bacterial (e.g. nontypeable Haemophilus influenzae [NTHi] infection. However, the mechanisms underlying bacterial infections during AECOPD remain poorly understood. As neutrophilic inflammation including increased release of human neutrophil elastase (HNE is a salient feature of AECOPD, we hypothesized that HNE impairs airway epithelial defense against NTHi by degrading airway epithelial host defense proteins such as short palate, lung, and nasal epithelium clone 1 (SPLUNC1.Recombinant human SPLUNC1 protein was incubated with HNE to confirm SPLUNC1 degradation by HNE. To determine if HNE-mediated impairment of host defense against NTHi was SPLUNC1-dependent, SPLUNC1 protein was added to HNE-treated primary normal human airway epithelial cells. The in vivo function of SPLUNC1 in NTHi defense was investigated by infecting SPLUNC1 knockout and wild-type mice intranasally with NTHi. We found that: (1 HNE directly increased NTHi load in human airway epithelial cells; (2 HNE degraded human SPLUNC1 protein; (3 Recombinant SPLUNC1 protein reduced NTHi levels in HNE-treated human airway epithelial cells; (4 NTHi levels in lungs of SPLUNC1 knockout mice were increased compared to wild-type mice; and (5 SPLUNC1 was reduced in lungs of COPD patients.Our findings suggest that SPLUNC1 degradation by neutrophil elastase may increase airway susceptibility to bacterial infections. SPLUNC1 therapy likely attenuates bacterial infections during AECOPD.

  7. Antagonism of selectin-dependent adhesion of human eosinophils and neutrophils by glycomimetics and oligosaccharide compounds.

    Science.gov (United States)

    Kim, M K; Brandley, B K; Anderson, M B; Bochner, B S

    1998-11-01

    Early in inflammation, adhesion occurs between leukocytes and endothelium when selectins bind to sialyl Lewis X (sLex) and related oligosaccharides. We tested novel compounds that mimic sLex for their ability to inhibit selectin-mediated adhesion of human eosinophils and neutrophils in vitro. Neutrophils and eosinophils were isolated by density gradient centrifugation, and eosinophils were further purified by immunomagnetic negative selection. Adhesion to unstimulated or interleukin-1beta-stimulated (5 ng/ml, 4-6 h) umbilical vein endothelial monolayers was tested under static or rotating conditions, where adhesion is primarily E- or L-selectin dependent, respectively. P-selectin-dependent adhesion was tested on immobilized platelets treated with or without phorbol myristate acetate (10(-7) M, 10 min). Stimulus-induced adhesion was always at least 4-fold higher than without stimulus, and selectin dependence was confirmed with specific blocking monoclonal antibodies. E-selectin-dependent adhesion of eosinophils and neutrophils was inhibited by compound GM2296 (the concentration producing 50% inhibition of adhesion [IC50] approximately 0.5-1 mM). E-selectin-dependent adhesion of neutrophils, but not eosinophils, was also inhibited by another compound, sLex with a lipid tail (30 +/- 6% inhibition at 3 mM), whereas compound GM1292 slightly inhibited adhesion of both (23 +/- 5 and 20 +/- 6% inhibition, respectively, at 1 mM). L-selectin-dependent adhesion was more effectively inhibited by GM2296 (IC50 approximately 0.2-0.5 mM), although P-selectin-dependent adhesion was also inhibited (IC50 approximately 1 mM). Inhibition was reversible without affecting viability, and no effect was seen with these compounds in assays testing neutrophil adhesion to immobilized intercellular adhesion molecule-1. Thus, compound GM2296, a carbon-fucosylated derivative of glycyrrhetinic acid, inhibits E-, L-, and P-selectin-dependent eosinophil and neutrophil adhesion. The ability of these

  8. Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

    DEFF Research Database (Denmark)

    Sørensen, A L; Hey, A S; Kharazmi, A

    1994-01-01

    In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes...... by heat inactivation of the protease at 70 degrees C for 15 min. Neither neutrophil nor monocyte chemiluminescence was inhibited by Gp63 when cells were stimulated with PMA. Our data suggest that the major surface protease Gp63 might play an important role in the initial stages of Leishmania...

  9. Radical aspects on arthritis : the role of neutrophil generation of nitric oxide and superoxide in inflammatory conditions

    OpenAIRE

    Cedergren, Jan

    2007-01-01

    The polymorphonuclear neutrophil granulocytes (neutrophils) are gaining renewed interest regarding their involvement in chronic inflammatory disorders, including rheumatoid arthritis (RA). Besides phagocytic and destructive capabilities, neutrophils have regulatory roles, e.g. by influencing responses from dendritic cells and lymphocytes. Several animal models have revealed that neutrophils are crucial for the initiation and maintenance of chronic inflammatory diseases. Neutrophil function is...

  10. The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America.

    Science.gov (United States)

    Wormser, Gary P; Dattwyler, Raymond J; Shapiro, Eugene D; Halperin, John J; Steere, Allen C; Klempner, Mark S; Krause, Peter J; Bakken, Johan S; Strle, Franc; Stanek, Gerold; Bockenstedt, Linda; Fish, Durland; Dumler, J Stephen; Nadelman, Robert B

    2006-11-01

    Evidence-based guidelines for the management of patients with Lyme disease, human granulocytic anaplasmosis (formerly known as human granulocytic ehrlichiosis), and babesiosis were prepared by an expert panel of the Infectious Diseases Society of America. These updated guidelines replace the previous treatment guidelines published in 2000 (Clin Infect Dis 2000; 31[Suppl 1]:1-14). The guidelines are intended for use by health care providers who care for patients who either have these infections or may be at risk for them. For each of these Ixodes tickborne infections, information is provided about prevention, epidemiology, clinical manifestations, diagnosis, and treatment. Tables list the doses and durations of antimicrobial therapy recommended for treatment and prevention of Lyme disease and provide a partial list of therapies to be avoided. A definition of post-Lyme disease syndrome is proposed.

  11. The Combination of Fosfomycin, Metronidazole, and Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor is Stable in vitro and Has Maintained Antibacterial Activity

    DEFF Research Database (Denmark)

    Fonnes, Siv; Holzknecht, Barbara Juliane; Gasbjerg, Lærke Smidt

    2017-01-01

    BACKGROUND: Treatment of secondary peritonitis includes surgery and antimicrobial agents. Antimicrobial agents are often administered intravenously, however, the alternative route intraperitoneal administration could be considered. Investigations must be conducted prior to clinical application....... Therefore, we aimed to investigate the combination of fosfomycin, metronidazole, and recombinant human granulocyte-macrophage colony-stimulating factor with regard to its chemical properties and the solution's stability. In addition, the antibacterial effect of the mixed drug solution was compared...

  12. Production of interleukin 27 by human neutrophils regulates their function in response to bacterial infection

    Science.gov (United States)

    Rinchai, Darawan; Khaenam, Prasong; Kewcharoenwong, Chidchamai; Buddhisa, Surachat; Pankla, Rungnapa; Chaussabel, Damien; Bancroft, Gregory J.; Lertmemongkolchai, Ganjana

    2013-01-01

    Septicemia is the most severe form of melioidosis caused by the Gram-negative bacterium, Burkholderia pseudomallei. Here, we showed that levels of IL-27p28 transcript and protein were both significantly elevated in patients with sepsis, particularly melioidosis and in patients with unfavorable disease outcome. Moreover, monocytes/macrophages and neutrophils were the major source of IL-27 during infection. Addition of exogenous IL-27 in vitro resulted in significantly increased bacterial survival, reduced B. pseudomallei-induced oxidative burst and enhanced IL-1beta and TNF-alpha production by purified neutrophils from healthy subjects. Finally, blockade of endogenous IL-27 in neutrophils using soluble IL-27 receptor antagonist prior to infection led to significantly reduced survival of bacteria and decreased IL-1beta, but not TNF-alpha production. These results indicate a potential role of IL-27 in suppression of antibacterial defense mechanisms that might contribute to disease severity in sepsis. The targeting of this cytokine may be beneficial in the management of human sepsis. PMID:22965735

  13. Quantification of heterotypic granule fusion in human neutrophils by imaging flow cytometry

    Directory of Open Access Journals (Sweden)

    Halla Björnsdottir

    2016-03-01

    Full Text Available Human neutrophils are filled with intracellular storage organelles, called granules and secretory vesicles, which differ in their content of soluble matrix proteins and membrane-bound molecules. To date, at least four distinct granule/vesicle subsets have been identified. These organelles may secrete their content extracellularly following mobilization to and fusion with the plasma membrane, but some of them may also fuse with internal membrane-enclosed organelles, typically a plasma membrane-derived phagosome. There are also instances where different granules appear to fuse with one another, a process that would enable mixing of their matrix and membrane components. Such granule fusion enables e.g., myeloperoxidase-processing of intragranular oxygen radicals, a key event in the formation of neutrophil extracellular traps (Björnsdottir et al., 2015 [1]. Described herein are data that show the quantification of such heterotypic granule–granule fusion by the use of imaging flow cytometry, a technique that combines flow cytometry with microscopy. The analysis described is based on immunofluorescent staining of established granule markers (lactoferrin and/or NGAL for one granule subset; the specific granules, and CD63 for another granule subset, the azurophil granules and calculation of a colocalization score for resting and PMA-stimulated neutrophils.

  14. Human neutrophil elastase-mediated goblet cell metaplasia is attenuated in TACE-deficient mice.

    Science.gov (United States)

    Park, Jin-Ah; Sharif, Asma S; Shiomi, Tetsuya; Kobzik, Lester; Kasahara, David I; Tschumperlin, Daniel J; Voynow, Judith; Drazen, Jeffrey M

    2013-05-15

    Neutrophilic inflammation is associated with chronic airway diseases. It has been observed that human neutrophil elastase (HNE), which is secreted by active neutrophils during inflammation, induces both mucin overproduction and goblet cell metaplasia. Several in vitro studies suggest that tumor necrosis factor-α converting enzyme (TACE) regulates the signaling axis that mediates HNE-induced mucin overproduction; however, it is unknown whether TACE performs a similar function in HNE-induced goblet cell metaplasia in vivo. We conducted this study to determine whether the inactivation of Tace gene expression attenuates HNE-induced goblet cell metaplasia in mice. Deletion of Tace is lethal shortly after birth in mice; therefore, we utilized Tace(flox/flox)R26CreER(+/-) mice and induced conditional deletion of Tace using a tamoxifen injection. Wild-type mice were given tamoxifen to control for its effect. Tace conditional deletion mice and wild-type mice were exposed to HNE via nasal instillation three times at 3-day intervals, and the lungs were harvested on day 11 after initial HNE exposure. Using periodic acid-Schiff staining and MUC5AC immunohistochemical staining to visualize goblet cells in the lungs, we found that HNE induced goblet cell metaplasia in the wild-type mice and that HNE-induced goblet cell metaplasia was significantly attenuated in the Tace conditional deletion mice. These findings suggest that TACE could be a potential target in the treatment of goblet cell metaplasia in patients with chronic airway diseases.

  15. Selection of reference genes for gene expression studies in human neutrophils by real-time PCR

    Directory of Open Access Journals (Sweden)

    Sandford Andrew J

    2005-02-01

    Full Text Available Abstract Background Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. However the expression level of these genes may vary among tissues or cells, and may change under certain circumstances. Thus the selection of reference gene(s is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils. Results Initial screening of the expression pattern demonstrated that 3 of the 10 genes were expressed at very low levels in neutrophils and were excluded from further analysis. The range of expression stability of the other 7 genes was (from most stable to least stable: GNB2L1 (Guanine nucleotide binding protein, beta polypeptide 2-like 1, HPRT1 (Hypoxanthine phosphoribosyl transferase 1, RPL32 (ribosomal protein L32, ACTB (beta-actin, B2M (beta-2-microglobulin, GAPD (glyceraldehyde-3-phosphate dehydrogenase and TBP (TATA-binding protein. Relative expression levels of the genes (from high to low were: B2M, ACTB, GAPD, RPL32, GNB2L1, TBP, and HPRT1. Conclusion Our data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils.

  16. Notch signaling mediates granulocyte-macrophage colony-stimulating factor priming-induced transendothelial migration of human eosinophils.

    Science.gov (United States)

    Liu, L Y; Wang, H; Xenakis, J J; Spencer, L A

    2015-07-01

    Priming with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances eosinophil migration and exacerbates the excessive accumulation of eosinophils within the bronchial mucosa of asthmatics. However, mechanisms that drive GM-CSF priming are incompletely understood. Notch signaling is an evolutionarily conserved pathway that regulates cellular processes, including migration, by integrating exogenous and cell-intrinsic cues. This study investigates the hypothesis that the priming-induced enhanced migration of human eosinophils requires the Notch signaling pathway. Using pan Notch inhibitors and newly developed human antibodies that specifically neutralize Notch receptor 1 activation, we investigated a role for Notch signaling in GM-CSF-primed transmigration of human blood eosinophils in vitro and in the airway accumulation of mouse eosinophils in vivo. Notch receptor 1 was constitutively active in freshly isolated human blood eosinophils, and inhibition of Notch signaling or specific blockade of Notch receptor 1 activation during GM-CSF priming impaired priming-enhanced eosinophil transendothelial migration in vitro. Inclusion of Notch signaling inhibitors during priming was associated with diminished ERK phosphorylation, and ERK-MAPK activation was required for GM-CSF priming-induced transmigration. In vivo in mice, eosinophil accumulation within allergic airways was impaired following systemic treatment with Notch inhibitor, or adoptive transfer of eosinophils treated ex vivo with Notch inhibitor. These data identify Notch signaling as an intrinsic pathway central to GM-CSF priming-induced eosinophil tissue migration. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Modulation of Human Neutrophil Responses by the Essential Oils from Ferula akitschkensis and Their Constituents.

    Science.gov (United States)

    Schepetkin, Igor A; Kushnarenko, Svetlana V; Özek, Gulmira; Kirpotina, Liliya N; Sinharoy, Pritam; Utegenova, Gulzhakhan A; Abidkulova, Karime T; Özek, Temel; Başer, Kemal Hüsnü Can; Kovrizhina, Anastasia R; Khlebnikov, Andrei I; Damron, Derek S; Quinn, Mark T

    2016-09-28

    Essential oils were obtained by hydrodistillation of the umbels+seeds and stems of Ferula akitschkensis (FAEOu/s and FAEOstm, respectively) and analyzed by gas chromatography and gas chromatography-mass spectrometry. Fifty-two compounds were identified in FAEOu/s; the primary components were sabinene, α-pinene, β-pinene, terpinen-4-ol, eremophilene, and 2-himachalen-7-ol, whereas the primary components of FAEOstm were myristicin and geranylacetone. FAEOu/s, β-pinene, sabinene, γ-terpinene, geranylacetone, isobornyl acetate, and (E)-2-nonenal stimulated [Ca(2+)]i mobilization in human neutrophils, with the most potent being geranylacetone (EC50 = 7.6 ± 1.9 μM) and isobornyl acetate 6.4 ± 1.7 (EC50 = 7.6 ± 1.9 μM). In addition, treatment of neutrophils with β-pinene, sabinene, γ-terpinene, geranylacetone, and isobornyl acetate desensitized the cells to N-formyl-Met-Leu-Phe (fMLF)- and interleukin-8 (IL-8)-induced [Ca(2+)]i flux and inhibited fMLF-induced chemotaxis. The effects of β-pinene, sabinene, γ-terpinene, geranylacetone, and isobornyl acetate on neutrophil [Ca(2+)]i flux were inhibited by transient receptor potential (TRP) channel blockers. Furthermore, the most potent compound, geranylacetone, activated Ca(2+) influx in TRPV1-transfected HEK293 cells. In contrast, myristicin inhibited neutrophil [Ca(2+)]i flux stimulated by fMLF and IL-8 and inhibited capsaicin-induced Ca(2+) influx in TRPV1-transfected HEK293 cells. These findings, as well as pharmacophore modeling of TRP agonists, suggest that geranylacetone is a TRPV1 agonist, whereas myristicin is a TRPV1 antagonist. Thus, at least part of the medicinal properties of Ferula essential oils may be due to modulatory effects on TRP channels.

  18. Inactivation of human antithrombin by neutrophil elastase. Kinetics of the heparin-dependent reaction.

    Science.gov (United States)

    Jordan, R E; Nelson, R M; Kilpatrick, J; Newgren, J O; Esmon, P C; Fournel, M A

    1989-06-25

    Human neutrophil elastase catalyzes the inactivation of antithrombin by a specific and limited proteinolytic cleavage. This inactivation reaction is greatly accelerated by an active anticoagulant heparin subfraction with high binding affinity for antithrombin. A potentially complex reaction mechanism is suggested by the binding of both neutrophil elastase and antithrombin to heparin. The in vitro kinetic behavior of this system was examined under two different conditions: 1) at a constant antithrombin concentration in which the active anticoagulant heparin was varied from catalytic to saturating levels; and 2) at a fixed, saturating heparin concentration and variable antithrombin levels. Under conditions of excess heparin, the inactivation could be continuously monitored by a decrease in the ultraviolet fluorescence emission of the inhibitor. A Km of approximately 1 microM for the heparin-antithrombin complex and a turnover number of approximately 200/min was estimated from these analyses. Maximum acceleratory effects of heparin on the inactivation of antithrombin occur at heparin concentrations significantly lower than those required to saturate antithrombin. The divergence in acceleratory effect and antithrombin binding contrasts with the anticoagulant functioning of heparin in promoting the formation of covalent antithrombin-enzyme complexes and is likely to derive from the fact that neutrophil elastase is not consumed in the inactivation reaction. A size dependence was observed for the heparin effect since an anticoagulantly active octasaccharide fragment of heparin, with avid antithrombin binding activity, was without effect on the inactivation of antithrombin by neutrophil elastase. Despite the completely nonfunctional nature of elastase-cleaved antithrombin and the altered physical properties of the inhibitor as indicated by fluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the inactivated inhibitor exhibited a circulating half

  19. [Ultrastructure of granulocytes of bony fishes (orders Salmoniformes, Cypriniformes, Perciformes)].

    Science.gov (United States)

    Flerova, E A; Balabanova, L V

    2013-01-01

    Analysis of data on utrastructure of granulocytes of freshwater and marine bony fish of orders Salmoniformes, Cypriniformes, and Perciformes showed that in all studied species there were revealed two types of granulocytes - neutrophils and eosinophils. The exception was the bluefish Pomatomus saltatrix L. whose pronephros hemopoietic tissue was found to contain one type of the granulocytic line - neutrophils. The identification parameters of granular leukocytes are specific granules filling the cytoplasm. The main form of specific granules in neutrophils of bony fish of various phylogenetic groups is an elongated granule with different distribution of fibrils or a granule that has crystalloid formed from fibrils. The main form of eosinophil granules - large, electron-dense, homogenous.

  20. Phenotypic features of first-generation transgenic goats for human granulocyte-colony stimulation factor production in milk.

    Science.gov (United States)

    Batista, Ribrio I T P; Melo, Carlos H S; Souza-Fabjan, Joanna M G; Teixeira, Dárcio I A; Melo, Luciana M; Freitas, Vicente J F

    2014-11-01

    Human granulocyte-colony stimulating factor (hG-CSF) is a hematopoietic growth factor used in neutropenic patients. It is produced in transgenic bacteria or cultured mammalian cells. As an alternative, we now show that hG-CSF can be expressed in the mammary gland of first-generation (F1) transgenic goats during induced lactation. Despite lower milk production, transgenic females presented a similar milk composition (fat, protein and lactose) when compared to non-transgenic (p > 0.05) ones. The mean concentration (±SD) of recombinant hG-CSF in milk during lactation was 360 ± 178 µg ml(-1). All clinical parameters, as well as kidney and liver function, indicated that F1 transgenic goats were healthy. Additionally, no ectopic hG-CSF expression was detected in studied tissues of F1 transgenic males. Thus, F1 hG-CSF-transgenic goats can express the recombinant protein in milk at quantities compatible with their use as bioreactors in a commercial-scale protein-production program.

  1. Analytical characterization of in vitro refolding in the quality by design paradigm: Refolding of recombinant human granulocyte colony stimulating factor.

    Science.gov (United States)

    Pathak, Mili; Dixit, Shruti; Muthukumar, S; Rathore, Anurag S

    2016-07-15

    Protein based therapeutics dominate most pharmaceutical pipelines today. For a therapeutic product to be effective, it is important that it is in its native form as slight modifications have been known to result in significantly different performance in the clinic. When expressed in hosts such as Escherichia coli, formation of inactive insoluble aggregates of proteins popularly known as inclusion bodies occurs in most cases. This necessitates the need for in vitro refolding to generate the native (and active) form of the therapeutic protein. This paper aims to provide an approach to generate a deeper understanding of refolding of a therapeutic protein and then to use it for its optimal production commercially. Recombinant human granulocyte colony stimulating factor has been chosen as the model protein. Seven orthogonal analytical tools have been used to elucidate the refolding process. By strategically using these tools protein refolding has been segregated into a series of well-defined sequence of events, starting from the unfolded random coil and ending with the uniquely folded metastable state. The study also suggests the choice of tools that can be used to monitor each event. We believe that this paper successfully demonstrates an approach to generate deeper understanding of the protein refolding process as per the expectations laid out in the Quality by Design paradigm. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

    Science.gov (United States)

    Vemula, Sandeep; Dedaniya, Akshay; Thunuguntla, Rahul; Mallu, Maheswara Reddy; Parupudi, Pavani; Ronda, Srinivasa Reddy

    2015-01-30

    Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Granulocytes as modulators of dendritic cell function.

    Science.gov (United States)

    Breedveld, Annelot; Groot Kormelink, Tom; van Egmond, Marjolein; de Jong, Esther C

    2017-10-01

    Effector T cell development is directly driven by APCs, in particular, by antigen-primed dendritic cells (DCs). Depending on the pathogenic stimulus and the microenvironment, DCs induce proliferation and polarization of naive CD4+ T cells into different effector subsets, such as Th1, Th2, Th17, or regulatory T cells (Tregs). During inflammation, DCs are found in close proximity to other innate immune cells, including all granulocyte subtypes, which potentially influence the immunomodulatory capacities of DCs. Neutrophils, eosinophils, and basophils are rapidly recruited into infected tissues where their main function is to eliminate invading pathogens. Mast cells are tissue-resident granulocytes that also contribute to host defense against pathogens but have, thus far, primarily been associated with their detrimental roles in allergic diseases. Although granulocytes have always been considered essential in innate immunity, their ability to influence the development of adaptive immunity has long been overlooked. This view is now changing, as multiple studies showed significant modulating effects of granulocytes on key players of adaptive immunity, including DCs and lymphocytes. Neutrophils, eosinophils, basophils, and mast cells regulate recruitment and activation of DCs through the release of mediators or via direct cell-cell contact, thereby influencing antigen-specific T cell responses. In this review, we will summarize the current knowledge on the impact of granulocytes on DC functioning and the subsequent putative consequences of this cross-talk on T cell proliferation and polarization. Together, this overview underscores the importance of granulocyte-DC communication to establish optimal immune responses. © Society for Leukocyte Biology.

  4. Ultraviolet light A irradiation induces immunosuppression associated with the generation of reactive oxygen species in human neutrophils

    Directory of Open Access Journals (Sweden)

    Cunbo Li

    2016-01-01

    Full Text Available Ultraviolet blood irradiation has been used as a physical therapy to treat many nonspecific diseases in clinics; however, the underlying mechanisms remain largely unclear. Neutrophils, the first line of host defense, play a crucial role in a variety of inflammatory responses. In the present work, we investigated the effects of ultraviolet light A (UVA on the immune functions of human neutrophils at the single-cell level by using an inverted fluorescence microscope. N-Formyl-methionyl-leucyl-phenylalanine (FMLP, a classic physiological chemotactic peptide, was used to induce a series of immune responses in neutrophils in vitro. FMLP-induced calcium mobilization, migration, and phagocytosis in human neutrophils was significantly blocked after treatment with 365nm UVA irradiation, demonstrating the immunosuppressive effects of UVA irradiation on neutrophils. Similar responses were also observed when the cells were pretreated with H2O2, a type of reactive oxygen species (ROS. Furthermore, UVA irradiation resulted in an increase in NAD(PH, a member of host oxidative stress in cells. Taken together, our data indicate that UVA irradiation results in immunosuppression associated with the production of ROS in human neutrophils.

  5. Additive antifungal activity of anidulafungin and human neutrophils against Candida parapsilosis biofilms.

    Science.gov (United States)

    Katragkou, Aspasia; Chatzimoschou, Athanasios; Simitsopoulou, Maria; Georgiadou, Elpiniki; Roilides, Emmanuel

    2011-03-01

    To investigate the activities of two newer triazoles and two echinocandins combined with human phagocytes against Candida parapsilosis biofilms. An in vitro model of C. parapsilosis biofilms was used. Biofilms were grown on silicone elastomer discs in 96-well plates at 37°C for 72 h. Biofilms or planktonic cells were incubated with voriconazole, posaconazole, caspofungin or anidulafungin, at clinically relevant concentrations, and human phagocytes (neutrophils or monocytes) alone or in combination with each of the antifungal agents for a further 22 h. Fungal damage induced by antifungal agents and/or phagocytes was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide] metabolic assay. Each of the antifungal agents alone and in combination with human phagocytes induced less damage against C. parapsilosis biofilms compared with planktonic cells. No antagonistic interactions between antifungal agents and phagocytes were found. Furthermore, anidulafungin, but not caspofungin, and neutrophils exerted additive activity against C. parapsilosis biofilms. Besides a lack of antagonistic interactions between newer antifungal agents and phagocytes, anidulafungin exerts additive immunopharmacological activity against C. parapsilosis biofilms.

  6. Porphyromonas gingivalis participates in pathogenesis of human abdominal aortic aneurysm by neutrophil activation. Proof of concept in rats.

    Directory of Open Access Journals (Sweden)

    Sandrine Delbosc

    Full Text Available BACKGROUND: Abdominal Aortic Aneurysms (AAAs represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia. METHODS AND FINDINGS: Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16, and in particular that of Porphyromonas gingivalis (Pg, the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed. CONCLUSIONS: Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression.

  7. Clickable 4-Oxo-β-lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes.

    Science.gov (United States)

    Ruivo, Eduardo F P; Gonçalves, Lídia M; Carvalho, Luís A R; Guedes, Rita C; Hofbauer, Stefan; Brito, José A; Archer, Margarida; Moreira, Rui; Lucas, Susana D

    2016-09-20

    Human neutrophil elastase (HNE) is a serine protease associated with several inflammatory processes such as chronic obstructive pulmonary disease (COPD). The precise involvement of HNE in COPD and other inflammatory disease mechanisms has yet to be clarified. Herein we report a copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC, or 'click' chemistry) approach based on the 4-oxo-β-lactam warhead that yielded potent HNE inhibitors containing a triazole moiety. The resulting structure-activity relationships set the basis to develop fluorescent and biotinylated activity-based probes as tools for molecular functional analysis. Attaching the tags to the 4-oxo-β-lactam scaffold did not affect HNE inhibitory activity, as revealed by the IC50 values in the nanomolar range (56-118 nm) displayed by the probes. The nitrobenzoxadiazole (NBD)-based probe presented the best binding properties (ligand efficiency (LE)=0.31) combined with an excellent lipophilic ligand efficiency (LLE=4.7). Moreover, the probes showed adequate fluorescence properties, internalization in human neutrophils, and suitable detection of HNE in the presence of a large excess of cell lysate proteins. This allows the development of activity-based probes with promising applications in target validation and identification, as well as diagnostic tools. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. TLR4 ligation induces expression of APRIL molecule in human neutrophils — a preliminary study

    Directory of Open Access Journals (Sweden)

    Ewa Jabłońska

    2012-07-01

    Full Text Available In the present study we investigate the consequences of TLR4 activation by LPS for the synthesis of a proliferation-inducing ligand (APRIL by human neutrophils (PMNs, and the possible role of the ERK1/2 kinases signaling pathway. In order to make a comparison, the same examinations were carried out on autologous peripheral blood mononuclear cells (PBMCs. The levels of mRNA for APRIL and TLR4 were measured using the real-time PCR method. Western blot analysis was used to assay the expressions of APRIL and ERK1/2 in cell lysates. We discovered an increased expression of APRIL accompanying the increased expression of TLR4 in the LPS-stimulated PMNs and PBMCs. Furthermore, stimulation with LPS triggered similar changes in phospho-ERK1/2 proteins expression in those cells. The present study suggests that LPS plays a role in TLR4-ligation in APRIL induction through ERK1/2 pathway activation in human neutrophils and mononuclear cells of peripheral blood. The association between TLR4 activation and APRIL expression in examined leukocytes might have important implications for the  mmune response of the host exposed to TLR4 ligands such as LPS.

  9. Impaired killing of Candida albicans by granulocytes mobilized for transfusion purposes: a role for granule components

    NARCIS (Netherlands)

    Gazendam, Roel P.; van de Geer, Annemarie; van Hamme, John L.; Tool, Anton T. J.; van Rees, Dieke J.; Aarts, Cathelijn E. M.; van den Biggelaar, Maartje; van Alphen, Floris; Verkuijlen, Paul; Meijer, Alexander B.; Janssen, Hans; Roos, Dirk; van den Berg, Timo K.; Kuijpers, Taco W.

    2016-01-01

    Granulocyte transfusions are used to treat neutropenic patients with life-threatening bacterial or fungal infections that do not respond to anti-microbial drugs. Donor neutrophils that have been mobilized with granulocyte-colony stimulating factor (G-CSF) and dexamethasone are functional in terms of

  10. [Effect of anti-inflammatory drugs on the motility of human polymorphonuclear granulocytes].

    Science.gov (United States)

    Spisani, S; Vanzini, G; Traniello, S

    1978-11-15

    Polymorphonuclear leucocytes play an important role in the inflammatory process, and their functions are likely to be regulated by pharmacological agents. In this paper we report the "in vitro" effect of non steroidal anti-inflammatory drugs on random and chemotactic motility of human leucocytes, and demonstrate that the two forms of movement, spontaneous and directional, are differently inhibited by low concentrations of these agents. Leucocytes of patients with recurrent inflammation are unaffected by these drugs.

  11. Cytotoxicity towards human endothelial cells, induced by neutrophil myeloperoxidase: protection by ceftazidime

    Directory of Open Access Journals (Sweden)

    M. Mathy-Hartert

    1995-01-01

    Full Text Available We investigated the effects of the antibiotic ceftazidime (CAZ on the cytolytic action of the neutrophil myeloperoxidase–hydrogen peroxide–chloride anion system (MPO/H2O2/Cl−. In this system, myeloperoxidase catalyses the conversion of H2O2 and CI− to the cytotoxic agent HOCl. Stimulated neutrophils can release MPO into the extracellular environment and then may cause tissue injury through direct endothelial cells lysis. We showed that human umbilical vein endothelial cells (HUVEC were capable of taking up active MPO. In presence of H2O2 (10−4 M, this uptake was accompanied by cell lysis. The cytolysis was estimated by the release of 51Cr from HUVEC and expressed as an index of cytotoxicity (IC. Dose dependent protection was obtained for CAZ concentrations ranging from 10−5 to 10−3 M;this can be attributed to inactivation of HOCl by the drug. This protection is comparable to that obtained with methionine and histidine, both of which are known to neutralize HOCl. This protection by CAZ could also be attributed to inactivation of H2O2, but when cytolysis was achieved with H2O2 or O2− generating enzymatic systems, no protection by CAZ was observed. Moreover, the peroxidation activity of MPO (action on H2O2 was not affected by CAZ, while CAZ prevented the chlorination activity of MPO (chlorination of monochlorodimedon. So, we concluded that CAZ acts via HOCl inactivation. These antioxidant properties of CAZ may be clinically useful in pathological situations where excessive activation of neutrophils occurs, such as in sepsis.

  12. Lysosomal-cationic test and NBT reduction test just partially reflect the completeness of phagocytic process in human granulocytes.

    Science.gov (United States)

    Belokrylov, G A; Popova, O Ya

    2002-01-01

    The results of NBT test correlated with the index of phagocytosis completeness in only 43% of 21 clinically healthy volunteers. The level of the lysosomal-cationic test was significantly reduced only if the phagocytosis completeness index was markedly decreased. The latter is an integral value reflecting the bactericidal activity of granulocytes.

  13. Neutrophil beta-2 microglobulin: an inflammatory mediator

    DEFF Research Database (Denmark)

    Bjerrum, O W; Nissen, Mogens Holst; Borregaard, N

    1990-01-01

    vesicles, and plasma membrane. Beta 2m was released in the native form from neutrophils in response to stimulation with chemotactic stimuli and phorbol ester. The results of experiments designed to study the modification of native beta 2m by neutrophils indicated that neutrophils do not participate...... in the proteolysis of beta 2m. However, we demonstrated that native beta 2m following degranulation may be transformed to Des-Lys58-beta 2m by lymphocytes. We suggest that neutrophil beta 2m following exocytosis may be transformed to Des-Lys58-beta 2m, acting as an extracellular messenger between granulocytes...

  14. Severe exercise and exercise training exert opposite effects on human neutrophil apoptosis via altering the redox status.

    Directory of Open Access Journals (Sweden)

    Guan-Da Syu

    Full Text Available Neutrophil spontaneous apoptosis, a process crucial for immune regulation, is mainly controlled by alterations in reactive oxygen species (ROS and mitochondria integrity. Exercise has been proposed to be a physiological way to modulate immunity; while acute severe exercise (ASE usually impedes immunity, chronic moderate exercise (CME improves it. This study aimed to investigate whether and how ASE and CME oppositely regulate human neutrophil apoptosis. Thirteen sedentary young males underwent an initial ASE and were subsequently divided into exercise and control groups. The exercise group (n = 8 underwent 2 months of CME followed by 2 months of detraining. Additional ASE paradigms were performed at the end of each month. Neutrophils were isolated from blood specimens drawn at rest and immediately after each ASE for assaying neutrophil spontaneous apoptosis (annexin-V binding on the outer surface along with redox-related parameters and mitochondria-related parameters. Our results showed that i the initial ASE immediately increased the oxidative stress (cytosolic ROS and glutathione oxidation, and sequentially accelerated the reduction of mitochondrial membrane potential, the surface binding of annexin-V, and the generation of mitochondrial ROS; ii CME upregulated glutathione level, retarded spontaneous apoptosis and delayed mitochondria deterioration; iii most effects of CME were unchanged after detraining; and iv CME blocked ASE effects and this capability remained intact even after detraining. Furthermore, the ASE effects on neutrophil spontaneous apoptosis were mimicked by adding exogenous H(2O(2, but not by suppressing mitochondrial membrane potential. In conclusion, while ASE induced an oxidative state and resulted in acceleration of human neutrophil apoptosis, CME delayed neutrophil apoptosis by maintaining a reduced state for long periods of time even after detraining.

  15. Sulfhydryl modification induces calcium entry through IP₃-sensitive store-operated pathway in activation-dependent human neutrophils.

    Directory of Open Access Journals (Sweden)

    Leiting Pan

    Full Text Available As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO and alkylation by N-ethylmaleimide (NEM, could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT₁MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca²⁺](c without external calcium, followed by a second [Ca²⁺](c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca²⁺](c change of Ca²⁺-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP, a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca²⁺](c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP₃ receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP₃-sensitive store-operated pathway of human neutrophils via sulfhydryl

  16. Thrombin Production and Human Neutrophil Elastase Sequestration by Modified Cellulosic Dressings and Their Electrokinetic Analysis

    Science.gov (United States)

    Edwards, Judson Vincent; Prevost, Nicolette

    2011-01-01

    Wound healing is a complex series of biochemical and cellular events. Optimally, functional material design addresses the overlapping acute and inflammatory stages of wound healing based on molecular, cellular, and bio-compatibility issues. In this paper the issues addressed are uncontrolled hemostasis and inflammation which can interfere with the orderly flow of wound healing. In this regard, we review the serine proteases thrombin and elastase relative to dressing functionality that improves wound healing and examine the effects of charge in cotton/cellulosic dressing design on thrombin production and elastase sequestration (uptake by the wound dressing). Thrombin is central to the initiation and propagation of coagulation, and elastase is released from neutrophils that can function detrimentally in a stalled inflammatory phase characteristic of chronic wounds. Electrokinetic fiber surface properties of the biomaterials of this study were determined to correlate material charge and polarity with function relative to thrombin production and elastase sequestration. Human neutrophil elastase sequestration was assessed with an assay representative of chronic wound concentration with cotton gauze cross-linked with three types of polycarboxylic acids and one phosphorylation finish; thrombin production, which was assessed in a plasma-based assay via a fluorogenic peptide substrate, was determined for cotton, cotton-grafted chitosan, chitosan, rayon/polyester, and two kaolin-treated materials including a commercial hemorrhage control dressing (QuickClot Combat Gauze). A correlation in thrombin production to zeta potential was found. Two polycarboxylic acid cross linked and a phosphorylated cotton dressing gave high elastase sequestration. PMID:24956451

  17. Arbutin and decrease of potentially toxic substances generated in human blood neutrophils

    Science.gov (United States)

    Pečivová, Jana; Nosál', Radomír; Sviteková, Klára

    2014-01-01

    Neutrophils, highly motile phagocytic cells, constitute the first line of host defense and simultaneously they are considered to be central cells of chronic inflammation. In combination with standard therapeutic procedures, natural substances are gaining interest as an option for enhancing the effectiveness of treatment of inflammatory diseases. We investigated the effect of arbutin and carvedilol and of their combination on 4β-phorbol-12β-myristate-13α-acetate- stimulated functions of human isolated neutrophils. Cells were preincubated with the drugs tested and subsequently stimulated. Superoxide (with or without blood platelets, in the rate close to physiological conditions [1:50]) and HOCl generation, elastase and myeloperoxidase release were determined spectrophotometrically and phospholipase D activation spectrofluorometrically. The combined effect of arbutin and carvedilol was found to be more effective than the effect of each compound alone. Our study provided evidence supporting the potential beneficial effect of arbutin alone or in combination with carvedilol in diminishing tissue damage by decreasing phospholipase D, myeloperoxidase and elastase activity and by attenuating the generation of superoxide and the subsequently derived reactive oxygen species. The presented data indicate the ability of arbutin to suppress the onset and progression of inflammation. PMID:26109900

  18. Hematologic improvement in dogs with parvovirus infection treated with recombinant canine granulocyte-colony stimulating factor.

    Science.gov (United States)

    Duffy, A; Dow, S; Ogilvie, G; Rao, S; Hackett, T

    2010-08-01

    Previously, dogs with canine parvovirus-induced neutropenia have not responded to treatment with recombinant human granulocyte-colony stimulating factor (rhG-CSF). However, recombinant canine G-CSF (rcG-CSF) has not been previously evaluated for treatment of parvovirus-induced neutropenia in dogs. We assessed the effectiveness of rcG-CSF in dogs with parvovirus-induced neutropenia with a prospective, open-label, nonrandomized clinical trial. Endpoints of our study were time to recovery of WBC and neutrophil counts, and duration of hospitalization. 28 dogs with parvovirus and neutropenia were treated with rcG-CSF and outcomes were compared to those of 34 dogs with parvovirus and neutropenia not treated with rcG-CSF. We found that mean WBC and neutrophil counts were significantly higher (P parvovirus infection, but indicate the need for additional studies to evaluate overall safety of the treatment.

  19. Granulocytes in Helminth Infection - Who is Calling the Shots?

    Science.gov (United States)

    Makepeace, BL; Martin, C; Turner, JD; Specht, S

    2012-01-01

    Helminths are parasitic organisms that can be broadly described as “worms” due to their elongated body plan, but which otherwise differ in shape, development, migratory routes and the predilection site of the adults and larvae. They are divided into three major groups: trematodes (flukes), which are leaf-shaped, hermaphroditic (except for blood flukes) flatworms with oral and ventral suckers; cestodes (tapeworms), which are segmented, hermaphroditic flatworms that inhabit the intestinal lumen; and nematodes (roundworms), which are dioecious, cylindrical parasites that inhabit intestinal and peripheral tissue sites. Helminths exhibit a sublime co-evolution with the host´s immune system that has enabled them to successfully colonize almost all multicellular species present in every geographical environment, including over two billion humans. In the face of this challenge, the host immune system has evolved to strike a delicate balance between attempts to neutralize the infectious assault versus limitation of damage to host tissues. Among the most important cell types during helminthic invasion are granulocytes: eosinophils, neutrophils and basophils. Depending on the specific context, these leukocytes may have pivotal roles in host protection, immunopathology, or facilitation of helminth establishment. This review provides an overview of the function of granulocytes in helminthic infections. PMID:22360486

  20. Inhibition of monocyte, lymphocyte, and neutrophil adhesion to endothelial cells by human milk oligosaccharides.

    Science.gov (United States)

    Bode, Lars; Kunz, Clemens; Muhly-Reinholz, Marion; Mayer, Konstantin; Seeger, Werner; Rudloff, Silvia

    2004-12-01

    Excessive leukocyte infiltration causes severe tissue damage in a variety of inflammatory diseases. The initial step in leukocyte extravasation is mediated by selectins and oligosaccharides on their glycoconjugate ligands. Human milk is a rich source of lactose-derived oligosaccharides that are partly absorbed in the intestine and excreted with the urine. As these components contain binding determinants for the selectins we investigated whether human milk oligosaccharides are able to affect leukocyte rolling and adhesion to endothelial cells under dynamic conditions. Therefore, monocytes, lymphocytes, or neutrophils isolated from human peripheral blood were passed over TNF-alpha-activated HUVEC under shear stress. The influence of different oligosaccharide fractions was determined by video-microscopy and compared with the effects of various individual oligosaccharides. Within a physiological range (12.5 - 125 microg/ml) the acidic fraction significantly inhibited leukocyte rolling and adhesion (up to 24.0% and 52.8%, respectively) in a concentration-dependent manner. These effects were even more pronounced than those achieved by soluble sialyl-Lewis x, a physiological binding determinant for selectins. Several active components within the oligosaccharide fraction of human milk were identified, e.g. 3'-sialyl-lactose and 3'-sialyl-3-fucosyl-lactose. These results indicate that specific oligosaccharides in human milk may serve as anti-inflammatory components and might therefore contribute to the lower incidence of inflammatory diseases in human milk-fed infants.

  1. Granulocyte-macrophage colony-stimulating factor as an autocrine survival-growth factor in human gliomas.

    Science.gov (United States)

    Revoltella, Roberto P; Menicagli, Michele; Campani, Daniela

    2012-03-01

    We studied the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptors (GM-CSF.R) in 20 human brain gliomas with different tumor gradings and demonstrated constitutive high levels of both mRNA gene expression and protein production exclusively in the highest-grade tumors (WHO, III-IV grade). Five astrocytic cell lines were isolated in vitro from glioma cells, which had selectively adhered to plates pre-coated with rhGM-CSF. These cells were tumorigenic when xenografted to athymic mice, and produced GM-CSF constitutively in culture. Two lines, particularly lines AS1 and PG1, each from a patient with glioblastoma multiforme, constitutively over-expressed both GM-CSF and GM-CSF.R genes and secreted into their culture media biologically active GM-CSF. Different clones of the AS1 line, isolated after subsequent passages in vitro and then transplanted to athymic mice, demonstrated higher tumorigenic capacity with increasing passages in vivo. Cell proliferation was stimulated by rhGM-CSF in late-stage malignant clones, whereas apoptosis occurred at high frequency in the presence of blocking anti-GM-CSF antibodies. In contrast, rhGM-CSF did not induce any apparent effect in early-stage clones expressing neither GM-CSF nor GM-CSF.R. The addition of rhGM-CSF or rhIL-1β, to cultures induced the overproduction of both GM-CSF and its receptors and increased gene activation for several functional proteins (e.g. NGF, VEGF, VEGF.R1, G-CSF, MHC-II), indicating that these cells may undergo dynamic changes in response to environmental stimuli. These findings thus revealed: (1) that the co-expression of both autocrine GM-CSF and GM-CSF.R correlates with the advanced tumor stage; (2) that an important contribution of GM-CSF in malignant glioma cells is the prevention of apoptosis. These results imply that GM-CSF has an effective role in the evolution and pathogenesis of gliomas. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Chemokine- and adhesion-dependent survival of neutrophils after transmigration through cytokine-stimulated endothelium.

    Science.gov (United States)

    McGettrick, Helen M; Lord, Janet M; Wang, Ke-Qing; Rainger, G Ed; Buckley, Christopher D; Nash, Gerard B

    2006-04-01

    We examined the fate of neutrophils following transmigration through an endothelial monolayer cultured on "Transwell" membrane filters. Treatment of human umbilical vein endothelial cells (HUVEC) with increasing doses of tumor necrosis factor-alpha increased the efficiency of transmigration and markedly reduced apoptosis among the transmigrated neutrophils in a dose-dependent manner. Apoptosis was also inhibited after transmigration of neutrophils through HUVEC stimulated with interleukin (IL)-1beta but not so effectively after chemotaxis through unstimulated HUVEC driven by IL-8 added below the filter. Inhibition of beta2-integrin binding after transmigration or coating the lower chamber with a nonadhesive polymer (polyhydroxyl-ethyl-methacrylate) abrogated neutrophil survival. Although integrin engagement during migration itself was not essential to inhibit apoptosis, activation of neutrophils through CXC chemokine receptors was necessary. Quite brief exposure to the HUVEC (30-120 min) was effective in reducing subsequent apoptosis, although if coincubation with the HUVEC were prolonged, neutrophil apoptosis was reduced further. Neutralization of granulocyte macrophage-colony stimulating factor inhibited this additional effect. Thus, a complex interplay between migration- and activation-dependent signals and adhesive interaction in tissue may combine to effectively prolong the survival of neutrophils recruited during inflammation.

  3. Measurement of the metabolic burst in human neutrophils: a comparison between cytochrome c and NBT reduction.

    Science.gov (United States)

    Elferink, J G

    1984-02-01

    Stimulation of human neutrophils with phorbol myristate acetate results in a metabolic burst, which can be measured as an enhanced cytochrome c reduction or NBT reduction. There is more NBT reduction than cytochrome c reduction. When cytochrome c and NBT are simultaneously present the reduction of each is about the same as when either cytochrome c or NBT is present. Whereas cytochrome c reduction is completely annihilated by externally added superoxide dismutase, NBT reduction is diminished to a lesser extent under the same conditions. It is concluded that cytochrome c reduction only measures extracellularly released superoxide, whereas NBT may be reduced by extracellular superoxide or other molecules as well; thus NBT measures another aspect of the metabolic burst.

  4. Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

    Science.gov (United States)

    Sunami, Yoshitaka; Araki, Marito; Hironaka, Yumi; Morishita, Soji; Kobayashi, Masaki; Liew, Ei Leen; Edahiro, Yoko; Tsutsui, Miyuki; Ohsaka, Akimichi; Komatsu, Norio

    2013-01-01

    Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation. PMID:23460888

  5. Inhibition of the NAD-dependent protein deacetylase SIRT2 induces granulocytic differentiation in human leukemia cells.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Sunami

    Full Text Available Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia-retinoic acid receptor α (PML-RAR-α stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.

  6. Staurosporine enhances ATRA-induced granulocytic differentiation in human leukemia U937 cells via the MEK/ERK signaling pathway.

    Science.gov (United States)

    Shi, Lei; Weng, Xiang-Qin; Sheng, Yan; Wu, Jing; Ding, Ming; Cai, Xun

    2016-11-01

    Although all-trans retinoic acid (ATRA) is regarded as a prominent example of differentiation therapy, it is not effective for the treatment of other subtypes of acute myeloid leukemia (AML) beyond acute promyelocytic leukemia (APL). Therefore, new strategies need to be explored to extend the efficacy of ATRA-based therapy to non-APL AML patients. In the present study, staurosporine, a protein kinase C (PKC) pan-inhibitor, exhibited synergism with ATRA to promote granulocytic differentiation in poorly ATRA-sensitive U937 cells but not in ATRA unresponsive K562 and Kasumi cells. Staurosporine or the combined treatment did not affect PKC activity in U937 cells. Moreover, other selective PKC inhibitors, UCN-01, Go6976 or rottlerin failed to enhance ATRA‑induced granulocytic differentiation in U937 cells. Therefore, staurosporine-enhanced ATRA-induced granulocytic differentiation in U937 cells may be independent of PKC. Staurosporine activated mitogen‑activated protein kinase kinase (MEK) and extracellular signal‑regulated kinase (ERK). Meanwhile, staurosporine also enhanced ATRA-promoted upregulation of the protein level of CCAAT/enhancer‑binding protein β (C/EBPβ) and C/EBPε in U937 cells. Furthermore, blockade of MEK activation suppressed staurosporine‑enhanced differentiation as well as the elevated protein level of C/EBPs. Taken together, we concluded that staurosporine enhanced ATRA‑induced granulocytic differentiation in U937 cells via MEK/ERK-mediated modulation of the protein level of C/EBPs.

  7. Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro

    DEFF Research Database (Denmark)

    Jaeger, K E; Kharazmi, A; Høiby, N

    1991-01-01

    concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes. The chemotaxis and chemiluminescence of these cells were then determined. It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect...

  8. Human neutrophil elastase detection with fluorescent peptide sensors conjugated to cellulosic and nanocellulosic materials: part II, structure/function analysis

    Science.gov (United States)

    Human neutrophil elastase (HNE) is one of a number of proteases that is receiving increased attention as a marker for inflammatory diseases and sensor-based point of care diagnostics. Integral to sensor-based detection is the transducer surface which is the platform of the sensor's signal transmitta...

  9. Human neutrophil elastase peptide sensors conjugated to cellulosic and nanocellulosic materials: part I, synthesis and characterization of fluorescent analogs

    Science.gov (United States)

    Here we describe the synthesis and characterization of peptide conjugated cellulose and nanocellulose materials as sensors for fluorescent detection of human neutrophil elastase (HNE). The cellulose sensor surfaces selected are filter paper (FP) and print cloth (PC) fabric, which are composed of pro...

  10. Involvement of toll-like receptor 4 and Fc receptors gamma in human neutrophil priming by endotoxins from escherichia coli.

    Science.gov (United States)

    Kabanov, D S; Prokhorenko, I R

    2013-02-01

    By using the fMLP-induced respiratory burst approach, the involvement of Toll-like receptor 4 (TLR4) in human neutrophil priming by S- or Re-glycoforms of endotoxin from Escherichia coli has been elucidated. The priming effect of Re-glycoform is more pronounced than that of the S-glycoform. Unexpectedly, fMLP-triggered generation of reactive oxygen species (ROS) by endotoxin primed neutrophils was amplified by preincubation of the cells with anti-TLR4 (HTA125) antibodies or with isotype-matched immunoglobulin IgG2a. The most significant finding of our study is that neutrophils exposed to anti-TLR4 antibodies retain their ability to distinguish between S- or Re-glycoforms being primed, respectively. Moreover, differentiated effect of HTA125 antibodies on functional responses of neutrophils during their priming and fMLP stimulation was revealed. Taking these results into consideration, it is reasonable to assume that there is a contribution of Fcγ receptors to fMLP-induced ROS generation by neutrophils preincubated with HTA125 or IgG2a and primed by endotoxins.

  11. CFP-10 from Mycobacterium tuberculosis selectively activates human neutrophils through a pertussis toxin-sensitive chemotactic receptor.

    Science.gov (United States)

    Welin, Amanda; Björnsdottir, Halla; Winther, Malene; Christenson, Karin; Oprea, Tudor; Karlsson, Anna; Forsman, Huamei; Dahlgren, Claes; Bylund, Johan

    2015-01-01

    Upon infection with Mycobacterium tuberculosis, neutrophils are massively recruited to the lungs, but the role of these cells in combating the infection is poorly understood. Through a type VII secretion system, M. tuberculosis releases a heterodimeric protein complex, containing a 6-kDa early secreted antigenic target (ESAT-6) and a 10-kDa culture filtrate protein (CFP-10), that is essential for virulence. Whereas the ESAT-6 component possesses multiple virulence-related activities, no direct biological activity of CFP-10 has been shown, and CFP-10 has been described as a chaperone protein for ESAT-6. We here show that the ESAT-6:CFP-10 complex induces a transient release of Ca(2+) from intracellular stores in human neutrophils. Surprisingly, CFP-10 rather than ESAT-6 was responsible for triggering the Ca(2+) response, in a pertussis toxin-sensitive manner, suggesting the involvement of a G-protein-coupled receptor. In line with this, the response was accompanied by neutrophil chemotaxis and activation of the superoxide-producing NADPH-oxidase. Neutrophils were unique among leukocytes in responding to CFP-10, as monocytes and lymphocytes failed to produce a Ca(2+) signal upon stimulation with the M. tuberculosis protein. Hence, CFP-10 may contribute specifically to neutrophil recruitment and activation during M. tuberculosis infection, representing a novel biological role for CFP-10 in the ESAT-6:CFP-10 complex, beyond the previously described chaperone function. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Major Neutrophilia Observed in Acute Phase of Human Leptospirosis Is Not Associated with Increased Expression of Granulocyte Cell Activation Markers.

    Science.gov (United States)

    Raffray, Loic; Giry, Claude; Vandroux, David; Kuli, Barbara; Randrianjohany, Andry; Pequin, Anne-Marie; Renou, Frédéric; Jaffar-Bandjee, Marie-Christine; Gasque, Philippe

    2016-01-01

    It has long been known that pathogenic Leptospira can mobilize the immune system but the specific contribution of neutrophils to control the infectious challenge remains to be clarified. We herein analyzed the phenotype of circulating neutrophils of patients with leptospirosis and healthy controls for the expression of toll-like receptor (TLR) type 2 (TLR2, to sense the leptospiral LPS) and several activation markers: interleukin 8 chemokine receptor CD182 (CXCR2), CD11b of the integrin/opsonin complement receptor type 3 (CR3) and CD15 (ligand of the selectin). The plasmatic level of the main CD182 ligand, interleukin 8 (CXCL8), was measured by ELISA. Hospitalized leptospirosis cases showed marked neutrophilia, particularly in the most severe cases. Interestingly, TLR2 was significantly increased in leptospirosis but identical levels of CD182 and CD11b were detected when compared to controls. CD15 was significantly decreased on neutrophils in leptospirosis but returned to normal within 1 month. Basal levels of IL-8 were measured in control subjects and were not increased in leptospirosis cases at the initial stage of the disease. In conclusion, we observed that neutrophils failed to regulate the expression of several of the receptors involved in cell activation and recruitment. This study further emphasizes the paradigm that neutrophils may be impaired in their overall capacity to thwart bacterial infection in leptospirosis patients.

  13. Phagocytosis and killing of Candida albicans by human neutrophils after exposure to structurally different lipid emulsions.

    NARCIS (Netherlands)

    Wanten, G.J.A.; Curfs, J.H.A.J.; Meis, J.F.G.M.; Naber, A.H.J.

    2001-01-01

    BACKGROUND: To test the hypothesis that structurally different lipid emulsions have distinct immune-modulating properties, we analyzed the elimination of Candida albicans by neutrophils after exposure to various emulsions. METHODS: Neutrophils from 8 volunteers were incubated in physiologic 5 mmol/L

  14. New Selective Peptidyl Di(chlorophenyl) Phosphonate Esters for Visualizing and Blocking Neutrophil Proteinase 3 in Human Diseases*

    Science.gov (United States)

    Guarino, Carla; Legowska, Monika; Epinette, Christophe; Kellenberger, Christine; Dallet-Choisy, Sandrine; Sieńczyk, Marcin; Gabant, Guillaume; Cadene, Martine; Zoidakis, Jérôme; Vlahou, Antonia; Wysocka, Magdalena; Marchand-Adam, Sylvain; Jenne, Dieter E.; Lesner, Adam; Gauthier, Francis; Korkmaz, Brice

    2014-01-01

    The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidylP(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases. PMID:25288799

  15. The effects of activin A on the migration of human breast cancer cells and neutrophils and their migratory interaction.

    Science.gov (United States)

    Xie, Dongxue; Liu, Zhonghui; Wu, Jiandong; Feng, Wenfang; Yang, Ke; Deng, Jixian; Tian, Ganghong; Santos, Susy; Cui, Xueling; Lin, Francis

    2017-08-01

    Activin A belongs to the superfamily of transforming growth factor beta (TGFβ) and is a critical regulatory cytokine in breast cancer and inflammation. However, the role of activin A in migration of breast cancer cells and immune cells was not well characterized. Here, a microfluidic device was used to examine the effect of activin A on the migration of human breast cancer cell line MDA-MB-231 cells and human blood neutrophils as well as their migratory interaction. We found that activin A promoted the basal migration but impaired epidermal growth factor (EGF)-induced migration of breast cancer cells. By contrast, activin A reduced neutrophil chemotaxis and transendothelial migration to N-Formyl-Met-Leu-Phe (fMLP). Finally, activin A promoted neutrophil chemotaxis to the supernatant from breast cancer cell culture. Collectively, our study revealed the different roles of activin A in regulating the migration of breast cancer cells and neutrophils and their migratory interaction. These findings suggested the potential of activin A as a therapeutic target for inflammation and breast cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. CXC chemokine receptor 3 expression on CD34(+) hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor

    DEFF Research Database (Denmark)

    Jinquan, T; Quan, S; Jacobi, H H

    2000-01-01

    expressed on CD34(+) hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34(+) progenitors. Freshly isolated CD34(+) progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up......-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase-polymerase chain reaction technique. gamma IP-10 and Mig induced chemotaxis of GM-CSF-stimulated CD34(+) progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block gamma IP-10-induced and Mig......-induced CD34(+) progenitor chemotaxis. These chemotactic attracted CD34(+) progenitors are colony-forming units-granulocyte-macrophage. gamma IP-10 and Mig also induced GM-CSF-stimulated CD34(+) progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 m...

  17. Activation of granulocytes by direct interaction with dendritic cells

    Science.gov (United States)

    Tsuda, M; Inaba, M; Sakaguchi, Y; Fukui, J; Ueda, Y; Omae, M; Ando, Y; Mukaide, H; Guo, K; Yabuki, T; Nakatani, T; Ikehara, S

    2007-01-01

    Granulocytes from human peripheral blood were co-cultured with conventional dendritic cells (cDC) or plasmacytoid DCs (pDC) to examine the effects of DCs on the activation or function of granulocytes. After co-culture of granulocytes with DCs, expression of the activation markers of granulocytes (CD63 and CD64) was up-regulated, and increased expression of CD50, the activation marker and ligand for CD209 (DC-SIGN) was also observed. The interaction of granulocytes with DCs was visualized as the cluster where DCs, especially cDCs, were surrounded by granulocytes to form a ‘rosette’. After co-culture of granulocytes with cDCs, the secretion of elastase from granulocytes was enhanced significantly when examined cytohistochemically and by enzyme-linked immunosorbent assay. An increase in myeloperoxidase (another activation index of granulocytes) was also observed after co-culture with DCs. These findings suggest the functional and phenotypical activation of granulocytes by interaction with DCs. Furthermore, we examined the involvement of adhesion molecules in the granulocyte–DC interaction, and found that CD209 participates to some extent in this interaction. PMID:17937679

  18. Neutrophil influx measured in nasal lavages of humans exposed to ozone

    Energy Technology Data Exchange (ETDEWEB)

    Graham, D.; Henderson, F.; House, D.

    1988-05-01

    Neutrophils (PMNs) obtained by nasal lavage were counted to determine if ozone, an oxidant air pollutant, induces an acute inflammatory response in the upper respiratory tract (URT) of humans. Background data were obtained by the nasal lavages from 200 nonexperimentally exposed subjects. Then, using a known inflammatory agent for the URT, rhinovirus-type 39, the induction, peak, and resolution of an acute inflammatory response was shown to be documented by the nasal lavage PMN counts. To determined if ozone induces this response, 41 subjects were exposed to either filtered air or 0.5 ppm ozone for 4 hr, on 2 consecutive days. Nasal lavages were taken pre-, immediately post each exposure, and 22 hr following the last exposure. Lavage PMN counts increased significantly (p = .005) in the ozone-exposed group, with 3.5-, 6.5-, and 3.9-fold increases over the air-exposed group at the post 1, pre 2, and post 2 time points, respectively. Ozone induces an inflammatory response in the URT of humans, and nasal lavage PMN counts are useful to assay the inflammatory properties of air pollutants.

  19. Solonamide B inhibits quorum sensing and reduces Staphylococcus aureus mediated killing of human neutrophils.

    Science.gov (United States)

    Nielsen, Anita; Månsson, Maria; Bojer, Martin S; Gram, Lone; Larsen, Thomas O; Novick, Richard P; Frees, Dorte; Frøkiær, Hanne; Ingmer, Hanne

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a serious human pathogen, and particularly the spread of community associated (CA)-MRSA strains such as USA300 is a concern, as these strains can cause severe infections in otherwise healthy adults. Recently, we reported that a cyclodepsipeptide termed Solonamide B isolated from the marine bacterium, Photobacterium halotolerans strongly reduces expression of RNAIII, the effector molecule of the agr quorum sensing system. Here we show that Solonamide B interferes with the binding of S. aureus autoinducing peptides (AIPs) to sensor histidine kinase, AgrC, of the agr two-component system. The hypervirulence of USA300 has been linked to increased expression of central virulence factors like α-hemolysin and the phenol soluble modulins (PSMs). Importantly, in strain USA300 Solonamide B dramatically reduced the activity of α-hemolysin and the transcription of psma encoding PSMs with an 80% reduction in toxicity of supernatants towards human neutrophils and rabbit erythrocytes. To our knowledge this is the first report of a compound produced naturally by a Gram-negative marine bacterium that interferes with agr and affects both RNAIII and AgrA controlled virulence gene expression in S. aureus.

  20. Solonamide B inhibits quorum sensing and reduces Staphylococcus aureus mediated killing of human neutrophils.

    Directory of Open Access Journals (Sweden)

    Anita Nielsen

    Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA continues to be a serious human pathogen, and particularly the spread of community associated (CA-MRSA strains such as USA300 is a concern, as these strains can cause severe infections in otherwise healthy adults. Recently, we reported that a cyclodepsipeptide termed Solonamide B isolated from the marine bacterium, Photobacterium halotolerans strongly reduces expression of RNAIII, the effector molecule of the agr quorum sensing system. Here we show that Solonamide B interferes with the binding of S. aureus autoinducing peptides (AIPs to sensor histidine kinase, AgrC, of the agr two-component system. The hypervirulence of USA300 has been linked to increased expression of central virulence factors like α-hemolysin and the phenol soluble modulins (PSMs. Importantly, in strain USA300 Solonamide B dramatically reduced the activity of α-hemolysin and the transcription of psma encoding PSMs with an 80% reduction in toxicity of supernatants towards human neutrophils and rabbit erythrocytes. To our knowledge this is the first report of a compound produced naturally by a Gram-negative marine bacterium that interferes with agr and affects both RNAIII and AgrA controlled virulence gene expression in S. aureus.

  1. Blockade of nicotinic and muscarinic receptors facilitates spontaneous migration of human peripheral granulocytes: failure in cystic fibrosis.

    Science.gov (United States)

    Wessler, Ignaz; Neumann, Stefanie; Razen, Michael; Zepp, Fred; Kirkpatrick, Charles James

    2012-11-27

    Circulating leucocytes express muscarinic (m) and nicotinic (n) receptors and synthesize acetylcholine (ACh) regulating various cell functions. Leucocytes from patients with cystic fibrosis contain less ACh; therefore it was tested whether the regulation of cellular functions like migration differed from healthy volunteers. Peripheral blood (10-20 ml) was used, leucocytes were isolated by Ficoll® gradient and the commercial MIGRATEST® combined with flow cytometric analysis was applied (pore size 3 μm). In the absence of test substances 4900±1800 (n=10) leucocytes migrated within a time period of 2 h. In the presence of tubocurarine (TC, 30 μM) the cell number increased to 7500±2700 [n=10] corresponding to an increase of 162±20% (mean of individual experiments; pGranulocytes isolated from patients with cystic fibrosis did not respond (2h migration) to 30 μM TC (control: 5180±1400 cells [n=10]; TC: 5800±1400 [n=10]). Also in the presence of atropine (1 μM) and TC (30 μM) a significant effect was not detected (5800±1300 [n=10]). Auto-paracrine acetylcholine limits the migration of unstimulated peripheral granulocytes. This effect is impaired in cystic fibrosis most likely because of a reduced endogenous cholinergic tone. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Correlations between Lymphocytes, Mid-Cell Fractions and Granulocytes with Human Blood Characteristics Using Low Power He-Ne Laser Radiation

    Science.gov (United States)

    Houssein, Hend A. A.; Jaafar, M. S.; Ramli, R. M.; Ismail, N. E.; Ahmad, A. L.; Bermakai, M. Y.

    2010-07-01

    In this study, the subpopulations of human blood parameters including lymphocytes, the mid-cell fractions (eosinophils, basophils, and monocytes), and granulocytes were determined by electronic sizing in the Health Centre of Universiti Sains Malaysia. These parameters have been correlated with human blood characteristics such as age, gender, ethnicity, and blood types; before and after irradiation with 0.95 mW He-Ne laser (λ = 632.8 nm). The correlations were obtained by finding patterns, paired non-parametric tests, and an independent non-parametric tests using the SPSS version 11.5, centroid and peak positions, and flux variations. The findings show that the centroid and peak positions, flux peak and total flux, were very much correlated and can become a significant indicator for blood analyses. Furthermore, the encircled flux analysis demonstrated a good future prospect in blood research, thus leading the way as a vibrant diagnosis tool to clarify diseases associated with blood.

  3. Treatment with Rutin - A Therapeutic Strategy for Neutrophil-Mediated Inflammatory and Autoimmune Diseases - Anti-inflammatory Effects of Rutin on Neutrophils -

    Directory of Open Access Journals (Sweden)

    Bahareh Abd Nikfarjam

    2017-03-01

    Full Text Available Objectives: Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO and myeloperoxidase (MPO. These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor (TNF-α productions and MPO activity in human neutrophils were investigated. Methods: Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI medium, pre-incubated with or without rutin (25 μM for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA. Then, the TNF-α, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA, Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT, and neutrophils were treated with various concentrations of rutin (1 - 100 μM, after which MTT was appended and incubated at 37ºC for 4 hour. Results: Rutin at concentrations up to 100 μM did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and TNF-α productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001. Also, MPO activity was significantly reduced by rutin (P < 0.001. Conclusion: In this in vitro study, rutin had an anti-inflammatory effect

  4. Toll-like receptor-induced reactivity and strongly potentiated IL-8 production in granulocytes mobilized for transfusion purposes

    NARCIS (Netherlands)

    Drewniak, Agata; Tool, Anton T. J.; Geissler, Judy; van Bruggen, Robin; van den Berg, Timo K.; Kuijpers, Taco W.

    2010-01-01

    Transfusion of granulocytes from granulocyte-colony stimulating factor (G-CSF)/dexamethasone (dexa)-treated donors can be beneficial for neutropenic recipients that are refractory to antimicrobial therapy. G-CSF/dexa treatment not only increases the number of circulating neutrophils but also affects

  5. Neutrophil Extracellular Traps and Fibrin in Otitis Media: Analysis of Human and Chinchilla Temporal Bones.

    Science.gov (United States)

    Schachern, Patricia A; Kwon, Geeyoun; Briles, David E; Ferrieri, Patricia; Juhn, Steven; Cureoglu, Sebahattin; Paparella, Michael M; Tsuprun, Vladimir

    2017-10-01

    Bacterial resistance in acute otitis can result in bacterial persistence and biofilm formation, triggering chronic and recurrent infections. To investigate the middle ear inflammatory response to bacterial infection in human and chinchilla temporal bones. Six chinchillas underwent intrabullar inoculations with 0.5 mL of 106 colony-forming units (CFUs) of Streptococcus pneumoniae, serotype 2. Two days later, we counted bacteria in middle ear effusions postmortem. One ear from each chinchilla was processed in paraffin and sectioned at 5 µm. The opposite ear was embedded in epoxy resin, sectioned at a thickness of 1 µm, and stained with toluidine blue. In addition, we examined human temporal bones from 2 deceased donors with clinical histories of otitis media (1 with acute onset otitis media, 1 with recurrent infection). Temporal bones had been previously removed at autopsy, processed, embedded in celloidin, and cut at a thickness of 20 µm. Sections of temporal bones from both chinchillas and humans were stained with hematoxylin-eosin and immunolabeled with antifibrin and antihistone H4 antibodies. Histopatological and imminohistochemical changes owing to otitis media. Bacterial counts in chinchilla middle ear effusions 2 days after inoculation were approximately 2 logs above initial inoculum counts. Both human and chinchilla middle ear effusions contained bacteria embedded in a fibrous matrix. Some fibers in the matrix showed positive staining with antifibrin antibody, others with antihistone H4 antibody. In acute and recurrent otitis media, fibrin and neutrophil extracellular traps (NETs) are part of the host inflammatory response to bacterial infection. In the early stages of otitis media the host defense system uses fibrin to entrap bacteria, and NETs function to eliminate bacteria. In chronic otitis media, fibrin and NETs appear to persist.

  6. The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages

    Directory of Open Access Journals (Sweden)

    Rest Richard F

    2006-06-01

    Full Text Available Abstract Background Bacillus anthracis is an animal and human pathogen whose virulence is characterized by lethal and edema toxin, as well as a poly-glutamic acid capsule. In addition to these well characterized toxins, B. anthracis secretes several proteases and phospholipases, and a newly described toxin of the cholesterol-dependent cytolysin (CDC family, Anthrolysin O (ALO. Results In the present studies we show that recombinant ALO (rALO or native ALO, secreted by viable B. anthracis, is lethal to human primary polymorphonuclear leukocytes (PMNs, monocytes, monocyte-derived macrophages (MDMs, lymphocytes, THP-1 monocytic human cell line and ME-180, Detroit 562, and A549 epithelial cells by trypan blue exclusion or lactate dehydrogenase (LDH release viability assays. ALO cytotoxicity is dose and time dependent and susceptibility to ALO-mediated lysis differs between cell types. In addition, the viability of monocytes and hMDMs was assayed in the presence of vegetative Sterne strains 7702 (ALO+, UT231 (ALO-, and a complemented strain expressing ALO, UT231 (pUTE544, and was dependent upon the expression of ALO. Cytotoxicity of rALO is seen as low as 0.070 nM in the absence of serum. All direct cytotoxic activity is inhibited by the addition of cholesterol or serum concentration as low as 10%. Conclusion The lethality of rALO and native ALO on human monocytes, neutrophils, macrophages and lymphocytes supports the idea that ALO may represent a previously unidentified virulence factor of B. anthracis. The study of other factors produced by B. anthracis, along with the major anthrax toxins, will lead to a better understanding of this bacterium's pathogenesis, as well as provide information for the development of antitoxin vaccines for treating and preventing anthrax.

  7. The profiles of gamma-H2AX along with ATM/DNA-PKcs activation in the lymphocytes and granulocytes of rat and human blood exposed to gamma rays

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing; Yin, Lina; Zhang, Junxiang; Zhang, Yaping; Zhang, Xuxia; Ding, Defang; Gao, Yun; Li, Qiang; Chen, Honghong [Fudan University, Department of Radiation Biology, Institute of Radiation Medicine, Shanghai (China)

    2016-08-15

    Establishing a rat model suitable for γ-H2AX biodosimeter studies has important implications for dose assessment of internal radionuclide contamination in humans. In this study, γ-H2AX, p-ATM and p-DNA-PKcs foci were enumerated using immunocytofluorescence method, and their protein levels were measured by Western blot in rat blood lymphocytes and granulocytes exposed to γ-rays compared with human blood lymphocytes and granulocytes. It was found that DNA double-strand break repair kinetics and linear dose responses in rat lymphocytes were similar to those observed in the human counterparts. Moreover, radiation induced clear p-ATM and p-DNA-PKcs foci formation and an increase in ratio of co-localization of p-ATM or p-DNA-PKcs with γ-H2AX foci in rat lymphocytes similar to those of human lymphocytes. The level of γ-H2AX protein in irradiated rat and human lymphocytes was significantly reduced by inhibitors of ATM and DNA-PKcs. Surprisingly, unlike human granulocytes, rat granulocytes with DNA-PKcs deficiency displayed a rapid accumulation, but delayed disappearance of γ-H2AX foci with essentially no change from 10 h to 48 h post-irradiation. Furthermore, inhibition of ATM activity in rat granulocytes also decreased radiation-induced γ-H2AX foci formation. In comparison, human granulocytes showed no response to irradiation regarding γ-H2AX, p-ATM or p-DNA-PKcs foci. Importantly, incidence of γ-H2AX foci in lymphocytes after total-body radiation of rats was consistent with that of in vitro irradiation of rat lymphocytes. These findings show that rats are a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans. ATM and DNA-PKcs participate together in DSB repair in rat lymphocytes similar to that of human lymphocytes. Further, rat granulocytes, which have the characteristic of delayed disappearance of γ-H2AX foci in response to radiation, may be a useful experimental system for biodosimetry studies. (orig.)

  8. The profiles of gamma-H2AX along with ATM/DNA-PKcs activation in the lymphocytes and granulocytes of rat and human blood exposed to gamma rays.

    Science.gov (United States)

    Wang, Jing; Yin, Lina; Zhang, Junxiang; Zhang, Yaping; Zhang, Xuxia; Ding, Defang; Gao, Yun; Li, Qiang; Chen, Honghong

    2016-08-01

    Establishing a rat model suitable for γ-H2AX biodosimeter studies has important implications for dose assessment of internal radionuclide contamination in humans. In this study, γ-H2AX, p-ATM and p-DNA-PKcs foci were enumerated using immunocytofluorescence method, and their protein levels were measured by Western blot in rat blood lymphocytes and granulocytes exposed to γ-rays compared with human blood lymphocytes and granulocytes. It was found that DNA double-strand break repair kinetics and linear dose responses in rat lymphocytes were similar to those observed in the human counterparts. Moreover, radiation induced clear p-ATM and p-DNA-PKcs foci formation and an increase in ratio of co-localization of p-ATM or p-DNA-PKcs with γ-H2AX foci in rat lymphocytes similar to those of human lymphocytes. The level of γ-H2AX protein in irradiated rat and human lymphocytes was significantly reduced by inhibitors of ATM and DNA-PKcs. Surprisingly, unlike human granulocytes, rat granulocytes with DNA-PKcs deficiency displayed a rapid accumulation, but delayed disappearance of γ-H2AX foci with essentially no change from 10 h to 48 h post-irradiation. Furthermore, inhibition of ATM activity in rat granulocytes also decreased radiation-induced γ-H2AX foci formation. In comparison, human granulocytes showed no response to irradiation regarding γ-H2AX, p-ATM or p-DNA-PKcs foci. Importantly, incidence of γ-H2AX foci in lymphocytes after total-body radiation of rats was consistent with that of in vitro irradiation of rat lymphocytes. These findings show that rats are a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans. ATM and DNA-PKcs participate together in DSB repair in rat lymphocytes similar to that of human lymphocytes. Further, rat granulocytes, which have the characteristic of delayed disappearance of γ-H2AX foci in response to radiation, may be a useful experimental system for biodosimetry studies.

  9. In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells.

    Science.gov (United States)

    Andrews, R G; Briddell, R A; Knitter, G H; Opie, T; Bronsden, M; Myerson, D; Appelbaum, F R; McNiece, I K

    1994-08-01

    Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU-MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low-dose rhSCF (2.5 micrograms/kg) with

  10. Human Neutrophil Elastase Mediates Fibrinolysis Shutdown Through Competitive Degradation of Plasminogen and Generation of Angiostatin.

    Science.gov (United States)

    Barrett, Christopher D; Moore, Hunter B; Banerjee, Anirban; Silliman, Christopher C; Moore, Ernest E; Yaffe, Michael B

    2017-08-23

    A subset of trauma patients undergo fibrinolysis shutdown rather than pathologic hyperfibrinolysis, contributing to organ failure. The molecular basis for fibrinolysis shutdown in trauma is incompletely understood. Elastase released from primed/activated human neutrophils (HNE) has historically been described as fibrin(ogen)olytic. However, HNE can also degrade plasminogen (PLG) to angiostatin (ANG), retaining the Kringle domains but not the proteolytic function, and could thereby compete for generation of active plasmin by tPA. We hypothesized that HNE can drive fibrinolysis shutdown rather than fibrinolysis. Turbidometry was performed using light scatter (λ=620nm) in a purified fibrinogen + PLG system and in healthy citrate plasma clotted with Ca/thrombin -/+tPA, -/+HNE, and -/+ANG to evaluate HNE effects on fibrinolysis, quantified by time to transition midpoint (Tm). ΔTm from control is reported as percent of control ±95%CI. Purified HNE coincubated with PLG or tPA was analysed by western blot to identify cleavage products. Exogenous HNE was mixed ex-vivo with healthy volunteer blood (n=7) and used in TEG -/+tPA to evaluate effects on fibrinolysis. HNE did not cause measurable fibrinolysis on fibrin clots, clotted plasma, or whole blood as assessed by turbidometry or TEG in the absence of tPA. Upon tPA treatment, all 3 methods of evaluating fibrinolysis showed delays and decreases in fibrinolysis due to HNE relative to control: fibrin clot turbidometry ΔTm =110.7% (CI 105.0%-116.5%), clotted citrate plasma (n=6 healthy volunteers) ΔTm =126.1% (CI 110.4%-141.8%), and whole blood native TEG (n=7 healthy volunteers) with ΔLY30=28% (p=0.043). Western blot analysis of HNE-PLG co-incubation confirmed that HNE generates angiostatin K1-3, and plasma turbidity assays treated with angiostatin K1-3 delayed fibrinolysis. HNE degrades PLG and generates angiostatin K1-3, which predominates over HNE cleavage of fibrin(ogen). These findings suggest that neutrophil

  11. IL-4 increases human endothelial cell adhesiveness for T cells but not for neutrophils.

    Science.gov (United States)

    Thornhill, M H; Kyan-Aung, U; Haskard, D O

    1990-04-15

    The adhesion of leukocytes to vascular endothelium is the first step in their passage from the blood into inflammatory tissues. By modulating endothelial cell (EC) adhesiveness for leukocytes, cytokines may regulate leukocyte accumulation and hence the nature and progression of inflammatory responses. We have found that the T cell cytokine IL-4 increases the adhesion of T cells, but not neutrophils, to human umbilical vein EC monolayers. The increase in T cell adhesion induced by IL-4 was dose dependent (ED50 = 5 U/ml) and peaked around 33 U/ml. No increase in adhesion of neutrophils was observed at concentrations of IL-4 up to 1000 U/ml. The kinetic of the increase in T cell adhesion exhibited a steady rise peaking between 18 and 24 h before returning to basal levels by 72 h. The IL-4 specificity of the effect was confirmed by the ability of neutralizing anti-IL-4, but not anti-TNF, antibodies to abolish the effect. The increase in T cell-EC adhesion was due to an effect of IL-4 on EC inasmuch as preincubation of the T cells with IL-4 did not increase T cell binding. Furthermore, preincubation of A549 epithelial cell line monolayers with IL-4 caused no increase in T cell binding whereas A549 cells and EC showed a similarly enhanced adhesiveness for T cells after preincubation with IL-1, TNF, or IFN-gamma. EC treated with IL-4 retained their increased adhesiveness for T cells after light fixation, suggesting that IL-4 up-regulates binding by increasing the expression or accessibility of EC surface receptors for lymphocytes. Although antibodies to intercellular adhesion molecule-1 (CD54) and the beta-chain (CD18) of lymphocyte function-associated Ag-1 (CD11a/CD18) partially inhibited T cell adhesion to unstimulated EC, they did not affect the increase in adhesion due to IL-4 stimulation, indicating that the increased binding resulted from the generation of an alternative binding receptor(s) on the EC membrane. These findings suggest that IL-4 may play a role in the

  12. Expression of genes involved in initiation, regulation, and execution of apoptosis in human neutrophils and during neutrophil differentiation of HL-60 cells.

    Science.gov (United States)

    Santos-Beneit, A M; Mollinedo, F

    2000-05-01

    Neutrophils possess a very short lifespan, dying by apoptosis. HL-60 cells undergo apoptosis after neutrophil differentiation with dimethyl sulfoxide (DMSO). We have found that the onset of apoptosis in neutrophil-differentiating HL-60 cells correlates with the achievement of an apoptosis-related gene expression pattern similar to that of peripheral blood mature neutrophils. Using reverse transcriptase-polymerase chain reaction, cloning, and sequencing techniques, we have found that HL-60 cells express bak, bik, bax, bad, bcl-2, bcl-xL, bcl-w, bfl-1, fas, and caspases 1-4 and 7-10. After DMSO treatment, bak, bcl-w, bfl-1, fas, and caspases 1 and 9 were up-regulated, whereas bik, bcl-2, and caspases 2, 3, and 10 were down-regulated at different degrees, achieving mRNA expression levels that correlated with those detected in peripheral blood neutrophils. Caspase-2 mRNA and protein expression was drastically reduced after HL-60 cell differentiation, being absent in both HL-60-differentiated neutrophils and mature neutrophils, whereas caspase-3 and -10 mRNA and protein expression were diminished upon HL-60 cell differentiation until achieving the respective levels found in mature neutrophils. Bak and bfl-1 mRNA levels were largely increased during DMSO-induced differentiation of HL-60 cells, and these genes were the bcl-2 family members that were expressed most abundantly in mature neutrophils. Bcl-2 overexpression or caspase inhibition prevented differentiation-induced apoptosis in HL-60 cells, but not their differentiation capability. Neutrophil spontaneous apoptosis was also blocked by the caspase inhibitor z-Asp-2,6-dichlorobenzoyloxymethylketone. Peripheral blood neutrophils expressed bak, bad, bcl-w, bfl-1, fas, and caspases 1, 3, 4, and 7-10, but hardly expressed bcl-2, bcl-xL, bik, bax, and caspase-2. These results suggest that the above gene expression changes in neutrophil-differentiating HL-60 cells may play a role in the acquisition of the neutrophil

  13. Activation of Triggering Receptor Expressed on Myeloid Cells-1 on Human Neutrophils by Marburg and Ebola Viruses

    Science.gov (United States)

    2006-04-21

    and adaptive immunity by Ebola and Lassa viruses . J. Immunol. 170:2797–2801. 30. Martini, G. A., and R. Siegert. 1971. Marburg virus disease...Immunol. 6:1191–1197. 41. Slenczka, W. G. 1999. The Marburg virus outbreak of 1967 and subsequent episodes. Curr. Top. Microbiol. Immunol. 235:49–75...Microbiology. All Rights Reserved. Activation of Triggering Receptor Expressed on Myeloid Cells-1 on Human Neutrophils by Marburg and Ebola Viruses

  14. Activation of TAK1 by Chemotactic and Growth Factors, and Its Impact on Human Neutrophil Signaling and Functional Responses.

    Science.gov (United States)

    Sylvain-Prévost, Stéphanie; Ear, Thornin; Simard, François A; Fortin, Carl F; Dubois, Claire M; Flamand, Nicolas; McDonald, Patrick P

    2015-12-01

    The MAP3 kinase, TAK1, is known to act upstream of IKK and MAPK cascades in several cell types, and is typically activated in response to cytokines (e.g., TNF, IL-1) and TLR ligands. In this article, we report that in human neutrophils, TAK1 can also be activated by different classes of inflammatory stimuli, namely, chemoattractants and growth factors. After stimulation with such agents, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the inducible phosphorylation of ERK occurring in response to these stimuli but had little or no effect on that of p38 MAPK or PI3K. Inhibition of TAK1 also impaired MEKK3 (but not MEKK1) activation by fMLF. Moreover, both TAK1 and the MEK/ERK module were found to influence inflammatory cytokine expression and release in fMLF- and GM-CSF-activated neutrophils, whereas the PI3K pathway influenced this response independently of TAK1. Besides cytokine production, other responses were found to be under TAK1 control in neutrophils stimulated with chemoattractants and/or GM-CSF, namely, delayed apoptosis and leukotriene biosynthesis. Our data further emphasize the central role of TAK1 in controlling signaling cascades and functional responses in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions. Copyright © 2015 by The American Association of Immunologists, Inc.

  15. EFFECTIVENESS AND SAFETY OF RECOMBINANT HUMAN GRANULOCYTIC COLONY-STIMULATING FACTOR IN TREATMENT OF GRANULOCYTOPENIA DEVELOPED DURING IMMUNOSUPPRESSIVE THERAPY IN PATIENTS WITH JUVENILE RHEUMATOID ARTHRITIS

    Directory of Open Access Journals (Sweden)

    E.I. Alexeeva

    2010-01-01

    Full Text Available Treatment of patients with severe clinical course of juvenile rheumatoid arthritis (JRA is difficult problem. During the last years genetically engineered biological drugs are used equally with traditional immunosuppressive agents in treatment of severe forms of juvenile arthritis. High effectiveness of these drugs can be accompanied with development of unfavorable effects, for example, febrile neutropenia. The article presents results of a study of effectiveness and safety of recombinant human granulocytic colony-stimulating factor — filgrastim (Leucostim — in treatment of granulocytopenia developed during immunosuppressive therapy in 16 patients with JRA. It was shown that administration of filgrastim arrests leucopenia in 100% of patients and granulocytopenia — in 93% of patients in 24 hours after first injection. High effectiveness of drug was combined with good tolerability and safety.Key words: children, treatment, granulocytopenia, filgrastim, juvenile rheumatoid arthritis.(Voprosy sovremennoi pediatrii — Current Pediatrics. – 2010;9(4:94-100

  16. Immunomodulatory activity of methanolic extracts of fruits and bark of Ficus glomerata Roxb. in mice and on human neutrophils.

    Science.gov (United States)

    Heroor, Sanjeev; Beknal, Arun Kumar; Mahurkar, Nitin

    2013-01-01

    To evaluate the immunomodulatory activity of methanolic extracts of fruit and bark of Ficus glomerata Roxb. on cyclophosphamide-induced myelosuppression in mice and the phagocytic effect on human neutrophils. Methanolic extracts of fruits and bark of Ficus glomerata Roxb. at two dose levels of 250 and 500 mg/kg p.o. were administered for 13 days to albino mice and cyclophosphamide (30 mg/kg i.p.) was administered on 11th, 12th, and 13th days, 1 hour after the administration of the respective treatment. On 14th day blood was collected and the hematological parameters were evaluated. The two extracts in the concentration range 100, 50, 25, 12 and 6.25 μg were also tested for phagocytic effect on human neutrophils using the in vitro models-nitroblue tetrazolium (NBT) dye test, phagocytosis of Candida albicans, and chemotaxis assay. Methanolic extracts of fruit and bark of Ficus glomerata Roxb. showed significant counteracting effect (P plant in the concentration range 100, 50, 25, 12, and 6.25 μg also showed significant (P effect on human neutrophils in the parameters studied. Methanolic extracts of fruits and bark of Ficus glomerata Roxb. exhibited immunomodulatory property in both in vivo and in vitro models.

  17. Applying label-free dynamic mass redistribution assay for studying endogenous FPR1 receptor signalling in human neutrophils

    DEFF Research Database (Denmark)

    Christensen, Hanna B; Gloriam, David E; Pedersen, Daniel Sejer

    2017-01-01

    INTRODUCTION: The label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors...... (FPRs) in human neutrophils. METHODS: Neutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist...... potencies. RESULTS: The potencies (pEC50) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H...

  18. Design of an Activity-Based Probe for Human Neutrophil Elastase: Implementation of the Lossen Rearrangement To Induce Förster Resonance Energy Transfers.

    Science.gov (United States)

    Schulz-Fincke, Anna-Christina; Tikhomirov, Alexander S; Braune, Annett; Girbl, Tamara; Gilberg, Erik; Bajorath, Jürgen; Blaut, Michael; Nourshargh, Sussan; Gütschow, Michael

    2018-01-16

    Human neutrophil elastase is an important regulator of the immune response and plays a role in host defense mechanisms and further physiological processes. The uncontrolled activity of this serine protease may cause severe tissue alterations and impair inflammatory states. The design of an activity-based probe for human neutrophil elastase reported herein relies on a sulfonyloxyphthalimide moiety as a new type of warhead that is linker-connected to a coumarin fluorophore. The inhibitory potency of the activity-based probe was assessed against several serine and cysteine proteases, and the selectivity for human neutrophil elastase (Ki = 6.85 nM) was determined. The adequate fluorescent tag of the probe allowed for the in-gel fluorescence detection of human neutrophil elastase in the low nanomolar range. The coumarin moiety and the anthranilic acid function of the probe, produced in the course of a Lossen rearrangement, were part of two different Förster resonance energy transfers.

  19. Factor H C-Terminal Domains Are Critical for Regulation of Platelet/Granulocyte Aggregate Formation

    Directory of Open Access Journals (Sweden)

    Adam Z. Blatt

    2017-11-01

    Full Text Available Platelet/granulocyte aggregates (PGAs increase thromboinflammation in the vasculature, and PGA formation is tightly controlled by the complement alternative pathway (AP negative regulator, Factor H (FH. Mutations in FH are associated with the prothrombotic disease atypical hemolytic uremic syndrome (aHUS, yet it is unknown whether increased PGA formation contributes to the thrombosis seen in patients with aHUS. Here, flow cytometry assays were used to evaluate the effects of aHUS-related mutations on FH regulation of PGA formation and characterize the mechanism. Utilizing recombinant fragments of FH spanning the entire length of the protein, we mapped the regions of FH most critical for limiting AP activity on the surface of isolated human platelets and neutrophils, as well as the regions most critical for regulating PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP. FH domains 19–20 were the most critical for limiting AP activity on platelets, neutrophils, and at the platelet/granulocyte interface. The role of FH in PGA formation was attributed to its ability to regulate AP-mediated C5a generation. AHUS-related mutations in domains 19–20 caused differential effects on control of PGA formation and AP activity on platelets and neutrophils. Our data indicate FH C-terminal domains are key for regulating PGA formation, thus increased FH protection may have a beneficial impact on diseases characterized by increased PGA formation, such as cardiovascular disease. Additionally, aHUS-related mutations in domains 19–20 have varying effects on control of TRAP-mediated PGA formation, suggesting that some, but not all, aHUS-related mutations may cause increased PGA formation that contributes to excessive thrombosis in patients with aHUS.

  20. Production of recombinant human granulocyte macrophage-colony stimulating factor in rice cell suspension culture with a human-like N-glycan structure.

    Science.gov (United States)

    Shin, Yun-Ji; Chong, Yun-Jo; Yang, Moon-Sik; Kwon, Tae-Ho

    2011-12-01

    The rice α-amylase 3D promoter system, which is activated under sucrose-starved conditions, has emerged as a useful system for producing recombinant proteins. However, using rice as the production system for therapeutic proteins requires modifications of the N-glycosylation pattern because of the potential immunogenicity of plant-specific sugar residues. In this study, glyco-engineered rice were generated as a production host for therapeutic glycoproteins, using RNA interference (RNAi) technology to down-regulate the endogenous α-1,3-fucosyltransferase (α-1,3-FucT) and β-1,2-xylosyltransferase (β-1,2-XylT) genes. N-linked glycans from the RNAi lines were identified, and their structures were compared with those isolated from a wild-type cell suspension. The inverted-repeat chimeric RNA silencing construct of α-1,3-fucosyltransferase and β-1,2-xylosyltransferase (Δ3FT/XT)-9 glyco-engineered line with significantly reduced core α-1,3-fucosylated and/or β-1,2-xylosylated glycan structures was established. Moreover, levels of plant-specific α-1,3-fucose and/or β-1,2-xylose residues incorporated into recombinant human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced from the N44 + Δ3FT/XT-4 glyco-engineered line co-expressing ihpRNA of Δ3FT/XT and hGM-CSF were significantly decreased compared with those in the previously reported N44-08 transgenic line expressing hGM-CSF. None of the glyco-engineered lines differed from the wild type with respect to cell division, proliferation or ability to secrete proteins into the culture medium. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  1. Dexamethasone promotes granulocyte mobilization by prolonging the half-life of granulocyte-colony-stimulating factor in healthy donors for granulocyte transfusions.

    Science.gov (United States)

    Hiemstra, Ida H; van Hamme, John L; Janssen, Machiel H; van den Berg, Timo K; Kuijpers, Taco W

    2017-03-01

    Granulocyte transfusion (GTX) is a potential approach to correcting neutropenia and relieving the increased risk of infection in patients who are refractory to antibiotics. To mobilize enough granulocytes for transfusion, healthy donors are premedicated with granulocyte-colony-stimulating factor (G-CSF) and dexamethasone. Granulocytes have a short circulatory half-life. Consequently, patients need to receive GTX every other day to keep circulating granulocyte counts at an acceptable level. We investigated whether plasma from premedicated donors was capable of prolonging neutrophil survival and, if so, which factor could be held responsible. The effects of plasma from G-CSF/dexamethasone-treated donors on neutrophil survival were assessed by annexin-V, CD16. and CXCR4 staining and nuclear morphology. We isolated an albumin-bound protein using α-chymotrypsin and albumin-depletion and further characterized it using protein analysis. The effects of dexamethasone and G-CSF were assessed using mifepristone and G-CSF-neutralizing antibody. G-CSF plasma concentrations were determined by Western blot and Luminex analyses. G-CSF/dexamethasone plasma contained a survival-promoting factor for at least 2 days. This factor was recognized as an albumin-associated protein and was identified as G-CSF itself, which was surprising considering its reported half-life of only 4.5 hours. Compared with coadministration of dexamethasone, administration of G-CSF alone to the same GTX donors led to a faster decline in circulating G-CSF levels, whereas dexamethasone itself did not induce any G-CSF, demonstrating a role for dexamethasone in increasing G-CSF half-life. Dexamethasone increases granulocyte yield upon coadministration with G-CSF by extending G-CSF half-life. This observation might also be exploited in the coadministration of dexamethasone with other recombinant proteins to modulate their half-life. © 2016 AABB.

  2. Neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, interleukin-1alpha and interferon-alpha but not other cytokines in human immunoglobulin preparations.

    Science.gov (United States)

    Wadhwa, M; Meager, A; Dilger, P; Bird, C; Dolman, C; Das, R G; Thorpe, R

    2000-01-01

    Human immunoglobulin preparations are used therapeutically for various disorders. Such therapy is generally safe but adverse effects occasionally occur in recipients. It has been suggested that antibodies to cytokines present in clinical immunoglobulin products may contribute to undesirable effects in recipients. Therefore, we investigated intravenous and intramuscular immunoglobulin products for the presence of cytokine-specific neutralizing antibodies. Using validated bioassays, we detected neutralizing activity against human granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-alpha2a (IFN-alpha2a) and interleukin-1alpha (IL-1alpha) in immunoglobulin products. We found no neutralization of granulocyte colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-12, tumour necrosis factor-alpha, oncostatin M (OSM) and IFN-gamma. Most batches which neutralized IFN-alpha2a activity also neutralized other IFN-alpha subtypes, IFN-omega and IFN-beta. Most products (94%) neutralized the biological activity of GM-CSF. No correlation between batches and their ability to neutralize bioactivities of GM-CSF, IFN-alpha2a and IL-1alpha was found. This neutralizing activity could be traced to plasma pools used for manufacture of immunoglobulins. The neutralization was mediated by specific cytokine antibodies contained within immunoglobulin products as it was present in specific immunoglobulin G (IgG) fractions eluted from cytokine affinity chromatography columns. Specific binding of such IgG fractions to cytokines in immunoblots and in enzyme-linked immunosorbent assays (ELISAs) was observed. This contrasts with the broad non-specific recognition of cytokine proteins observed using unfractionated immunoglobulins in ELISAs. This is the first comprehensive study showing the presence of neutralizing antibodies against GM-CSF, IL-1alpha, or IFN-alpha2a in immunoglobulin products.

  3. Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro.

    LENUS (Irish Health Repository)

    Power, C

    2012-02-03

    Bacterial lipoprotein (BLP) is the most abundant protein in gram-negative bacterial cell walls, heavily outweighing lipopolysaccharide (LPS). Herein we present findings demonstrating the potent in vitro effects of BLP on neutrophil (PMN) activation status, function, and capacity to transmigrate an endothelial monolayer. PMNs are the principal effectors of the initial host response to injury or infection and constitute a significant threat to invading bacterial pathogens. The systemic inflammatory response syndrome (SIRS) is characterised by significant host tissue injury mediated, in part, by uncontrolled regulation of PMN cytotoxic activity. We found that BLP-activated human PMN as evidenced by increased CD11b\\/CD18 (Mac-1) expression. Up-regulation of PMN Mac-1 in response to BLP occurred independently of membrane-bound CD14 (mCD14). A similar up-regulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was observed whilst E-Selectin expression was unaffected. PMN transmigration across a human umbilical vein endothelial cell (HUVEC) monolayer was markedly increased after treating either PMN\\'s or HUVEC independently with BLP. This increased transmigration did not occur as a result of any direct effect of BLP on HUVEC monolayer permeability, assessed objectively using the passage of FITC-labeled Dextran-70. BLP primed PMN for enhanced respiratory burst and superoxide anion production in response to PMA, but did not influence phagocytosis of opsonized Escherichia coli. BLP far exceeds LPS as a gram-negative bacterial wall component, these findings therefore implicate BLP as an additional putative mediator of SIRS arising from gram-negative infection.

  4. Granulocytes as effective anticancer agent in experimental solid tumor models.

    Science.gov (United States)

    Jaganjac, Morana; Poljak-Blazi, Marija; Kirac, Iva; Borovic, Suzana; Joerg Schaur, Rudolf; Zarkovic, Neven

    2010-12-01

    The aim of the study was to elucidate the effects of murine granulocytes on the growth of solid murine tumors when administrated in the vicinity of W256 carcinoma growing in Sprague Dawley rats, and in the vicinity of Ehrlich ascites tumor (EAT) growing in BALBc mice. The administration of granulocytes significantly improved the survival of W256-bearing rats, and increased the tumor regression incidence from 17% up to 75%. Rats with regressing tumors had 2.5 times increased levels of granulocytes in peripheral blood, which were also cytotoxic in vitro for W256 carcinoma cells. However, blood levels of cytokine-induced neutrophil chemoattractant-2, tumor necrosis factor α and interleukin 6 were similar between rats with regressing tumors and control healthy rats, suggesting that the observed regression of W256 carcinoma was caused by specific anticancer effects of the applied granulocytes. Anticancer effects of granulocytes were also found in BALBc mice bearing solid form of EAT, resulting in a 20% increase of survival in EAT-bearing mice. Therefore, the administration of granulocytes, isolated from healthy animals and applied at the site of solid tumors in rats and in mice, reduced experimental tumor growth, and extended the survival of tumor-bearing animals, while in some rats it even caused a W256 regression. Copyright © 2010 Elsevier GmbH. All rights reserved.

  5. Bacterial Siderophores Hijack Neutrophil Functions.

    Science.gov (United States)

    Saha, Piu; Yeoh, Beng San; Olvera, Rodrigo A; Xiao, Xia; Singh, Vishal; Awasthi, Deepika; Subramanian, Bhagawat C; Chen, Qiuyan; Dikshit, Madhu; Wang, Yanming; Parent, Carole A; Vijay-Kumar, Matam

    2017-06-01

    Neutrophils are the primary immune cells that respond to inflammation and combat microbial transgression. To thrive, the bacteria residing in their mammalian host have to withstand the antibactericidal responses of neutrophils. We report that enterobactin (Ent), a catecholate siderophore expressed by Escherichia coli, inhibited PMA-induced generation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) in mouse and human neutrophils. Ent also impaired the degranulation of primary granules and inhibited phagocytosis and bactericidal activity of neutrophils, without affecting their migration and chemotaxis. Molecular analysis revealed that Ent can chelate intracellular labile iron that is required for neutrophil oxidative responses. Other siderophores (pyoverdine, ferrichrome, deferoxamine) likewise inhibited ROS and NETs in neutrophils, thus indicating that the chelation of iron may largely explain their inhibitory effects. To counter iron theft by Ent, neutrophils rely on the siderophore-binding protein lipocalin 2 (Lcn2) in a "tug-of-war" for iron. The inhibition of neutrophil ROS and NETs by Ent was augmented in Lcn2-deficient neutrophils compared with wild-type neutrophils but was rescued by the exogenous addition of recombinant Lcn2. Taken together, our findings illustrate the novel concept that microbial siderophore's iron-scavenging property may serve as an antiradical defense system that neutralizes the immune functions of neutrophils. Copyright © 2017 by The American Association of Immunologists, Inc.

  6. Human neutrophil elastase induces endothelial cell apoptosis by activating the PERK-CHOP branch of the unfolded protein response.

    Science.gov (United States)

    Grechowa, Irina; Horke, Sven; Wallrath, Anja; Vahl, Christian-Friedrich; Dorweiler, Bernhard

    2017-09-01

    Human neutrophil elastase impacts on atherosclerotic plaque stability by inducing apoptosis in endothelial cells. Our aim was to investigate the proapoptotic mechanism of elastase on endothelial cells and to evaluate the presence of elastase in human plaque material. Human endothelial cells were treated with purified human neutrophil elastase. Apoptosis was assayed by capsase-3/7 activation, TUNEL, and sub-G 1 assay. Activation of unfolded protein response (UPR) effector molecules binding Ig protein, soluble X-binding protein-1, protein kinase RNA-like ER kinase (PERK), and C/EBP-homologous protein (CHOP) was analyzed by RT-PCR, immunocytochemistry, and Western blot. Genetic silencing of CHOP was achieved by small interfering RNA. Elastase induces autophagic-apoptotic forms of endothelial cell death in a time- and dose-dependent manner, in conjunction with a significant increase in phosphorylation/expression of the canonical UPR-activation markers PERK and CHOP. By using CHOP knockdown, we identified CHOP as a key mediator of elastase-induced endothelial cell death. Immunohistochemical analysis of human rupture-prone plaque specimens confirmed the presence of elastase and colocalization with apoptosis. We have demonstrated for the first time that the PERK-CHOP branch of the UPR is causally involved in elastase-induced apoptosis of endothelial cells. Ex vivo analysis of human rupture-prone plaques confirmed the presence of elastase and its colocalization with markers of apoptosis. This novel role of elastase underlines the potential of combined targeting of elastase and endoplasmic reticulum stress in the prevention of plaque progression and cardiovascular events.-Grechowa, I., Horke, S., Wallrath, A., Vahl, C.-F., Dorweiler, B. Human neutrophil elastase induces endothelial cell apoptosis by activating the PERK-CHOP branch of the unfolded protein response. © FASEB.

  7. Regulation of Discrete Functional Responses by Syk and Src Family Tyrosine Kinases in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Thornin Ear

    2017-01-01

    Full Text Available Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs, as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1 involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.

  8. Low-Frequency Electromagnetic Field Exposure Enhances Extracellular Trap Formation by Human Neutrophils through the NADPH Pathway.

    Science.gov (United States)

    Golbach, Lieke A; Scheer, Marleen H; Cuppen, Jan J M; Savelkoul, Huub; Verburg-van Kemenade, B M Lidy

    2015-01-01

    Low-frequency (LF) electromagnetic fields (EMFs) are abundantly present in modern society, and the potential biological consequences of exposure to these fields are under intense debate. Immune cells are suggested as possible target cells, though a clear mechanism is lacking. Considering their crucial role in innate immune activation, we selected an ex vivo exposure set-up with human neutrophils to investigate a possible correlation between neutrophil extracellular trap (NET) formation and LF EMF exposure. Our study shows that formation of NETs is enhanced by LF EMF exposure. Enhanced NET formation leads to increased antimicrobial properties as well as damage to surrounding cells. We found that LF-EMF-induced NET formation is dependent on the NADPH oxidase pathway and production of reactive oxygen species. Additionally, LF EMF exposure does not influence autophagy and PAD4 activity. Our study provides a mechanism by which exposure to LF EMFs could influence the innate immune system. © 2015 S. Karger AG, Basel.

  9. Data on human neutrophil activation induced by pepducins with amino acid sequences derived from β2AR and CXCR4

    Directory of Open Access Journals (Sweden)

    André Holdfeldt

    2016-09-01

    Full Text Available The data described here is related to the research article titled (Gabl et al., 2016 [1]. Pepducins with peptide sequence derived from one of the intracellular domains of a given G-protein coupled receptor (GPCR can either activate or inhibit cell functions. Here we include data on human neutrophil function induced by pepducins derived from β2AR (ICL3-8 and CXCR4 (ATI-2341, respectively. ICL3-8 exerts neither direct activating effect on the NADPH-oxidase as measured by superoxide release nor inhibitory effect on FPR signaling. ATI-2341 dose-dependently triggers neutrophil activation and these cells were subsequently desensitized in their response to FPR2 specific agonists F2Pal10 and WKYMVM. Moreover, the ATI-2341 response is inhibited by PBP10 and the peptidomimetic Pam-(Lys-betaNSpe6-NH2 (both are FPR2 specific inhibitors, but not to the FPR1 specific inhibitor cyclosporine H.

  10. Amantadine inhibits platelet-activating factor induced clathrin-mediated endocytosis in human neutrophils

    Science.gov (United States)

    Eckels, Phillip C.; Banerjee, Anirban; Moore, Ernest E.; McLaughlin, Nathan J. D.; Gries, Lynn M.; Kelher, Marguerite R.; England, Kelly M.; Gamboni-Robertson, Fabia; Khan, Samina Y.

    2009-01-01

    Receptor signaling is integral for adhesion, emigration, phagocytosis, and reactive oxygen species production in polymorphonuclear neutrophils (PMNs). Priming is an important part of PMN emigration, but it can also lead to PMN-mediated organ injury in the host. Platelet-activating factor (PAF) primes PMNs through activation of a specific G protein-coupled receptor. We hypothesize that PAF priming of PMNs requires clathrin-mediated endocytosis (CME) of the PAF receptor (PAFr), and, therefore, amantadine, known to inhibit CME, significantly antagonizes PAF signaling. PMNs were isolated by standard techniques to >98% purity and tested for viability. Amantadine (1 mM) significantly inhibited the PAF-mediated changes in the cellular distribution of clathrin and the physical colocalization [fluorescence resonance energy transfer positive (FRET+)] of early endosome antigen-1 and Rab5a, known components of CME and similar to hypertonic saline, a known inhibitor of CME. Furthermore, amantadine had no effect on the PAF-induced cytosolic calcium flux; however, phosphorylation of p38 MAPK was significantly decreased. Amantadine inhibited PAF-mediated changes in PMN physiology, including priming of the NADPH oxidase and shape change with lesser inhibition of increases in CD11b surface expression and elastase release. Furthermore, rimantadine, an amantadine analog, was a more potent inhibitor of PAF priming of the N-formyl-methionyl-leucyl-phenylalanine-activated oxidase. PAF priming of PMNs requires clathrin-mediated endocytosis that is inhibited when PMNs are pretreated with either amantadine or rimantadine. Thus, amantadine and rimantadine have the potential to ameliorate PMN-mediated tissue damage in humans. PMID:19295175

  11. Human neutrophil antigen profiles in Banjar, Bugis, Champa, Jawa and Kelantan Malays in Peninsular Malaysia.

    Science.gov (United States)

    Manaf, Siti M; NurWaliyuddin, Hanis Z A; Panneerchelvam, Sundararajulu; Zafarina, Zainuddin; Norazmi, Mohd N; Chambers, Geoffrey K; Edinur, Hisham A

    2015-10-01

    Human neutrophil antigens (HNA) are polymorphic and immunogenic proteins involved in the pathogenesis of neonatal alloimmune neutropenia, transfusion-related acute lung injury (TRALI) and transfusion-related alloimmune neutropenia. The characterisation of HNA at a population level is important for predicting the risk of alloimmunisation associated with blood transfusion and gestation and for anthropological studies. Blood samples from 192 healthy, unrelated Malays were collected and genotyped using polymerase chain reaction-sequence specific primers (HNA-1, -3, -4) and polymerase chain reaction-restriction fragment length polymorphisms (HNA-5). The group comprised 30 Banjar, 37 Bugis, 51 Champa, 39 Jawa and 35 Kelantan Malays. The most common HNA alleles in the Malays studied were HNA-1a (0.641-0.765), -3a (0.676-0.867), -4a (0.943-1.000) and -5a (0.529-0.910). According to principal coordinate plots constructed using HNA allele frequencies, the Malay sub-ethnic groups are closely related and grouped together with other Asian populations. The risks of TRALI or neonatal neutropenia were not increased for subjects with HNA-1, -3 and -4 loci even for donor and recipient or pairs from different Malay sub-ethnic groups. Nonetheless, our estimates showed significantly higher risks of HNA alloimmunisation during pregnancy and transfusion between Malays and other genetically differentiated populations such as Africans and Europeans. This study reports HNA allele and genotype frequencies for the five Malay sub-ethnic groups living in Peninsular Malaysia for the first time. These Malay sub-ethnic groups show closer genetic relationships with other Asian populations than with Europeans and Africans. The distributions of HNA alleles in other lineages of people living in Malaysia (e.g. Chinese, Indian and Orang Asli) would be an interesting subject for future study.

  12. Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils.

    Science.gov (United States)

    Patel, K D; Zimmerman, G A; Prescott, S M; McEver, R P; McIntyre, T M

    1991-02-01

    The initial step in extravasation of neutrophils (polymorphonuclear leukocytes [PMNs]) to the extravascular space is adherence to the endothelium. We examined the effect of oxidants on this process by treating human endothelial cells with H2O2, t-butylhydroperoxide, or menadione. This resulted in a surface adhesive for PMN between 1 and 4 h after exposure. The oxidants needed to be present only for a brief period at the initiation of the assay. Adhesion was an endothelial cell-dependent process that did not require an active response from the PMN. The adhesive molecule was not platelet-activating factor, which mediates PMN adherence when endothelial cells are briefly exposed to higher concentrations of H2O2 (Lewis, M. S., R. E. Whatley, P. Cain, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 1988. J. Clin. Invest. 82:2045-2055), nor was it ELAM-1, an adhesive glycoprotein induced by cytokines. Oxidant-induced adhesion did not require protein synthesis, was inhibited by antioxidants, and, when peroxides were the oxidants, was inhibited by intracellular iron chelators. Granule membrane protein-140 (GMP-140) is a membrane-associated glycoprotein that can be translocated from its intracellular storage pool to the surface of endothelial cells where it acts as a ligand for PMN adhesion (Geng, J.-G., M. P. Bevilacqua, K. L. Moore, T. M. McIntyre, S. M. Prescott, J. M. Kim, G. A. Bliss, G. A. Zimmerman, and R. P. McEver. 1990. Nature (Lond). 343:757-760). We found that endothelial cells exposed to oxidants expressed GMP-140 on their surface, and that an mAb against GMP-140 or solubilized GMP-140 completely blocked PMN adherence to oxidant-treated endothelial cells. Thus, exposure of endothelial cells to oxygen radicals induces the prolonged expression of GMP-140 on the cell surface, which results in enhanced PMN adherence.

  13. Human Neutrophil Clearance of Bacterial Pathogens Triggers Anti-Microbial γδ T Cell Responses in Early Infection

    Science.gov (United States)

    Roberts, Gareth W.; Heuston, Sinéad; Brown, Amanda C.; Chess, James A.; Toleman, Mark A.; Gahan, Cormac G. M.; Hill, Colin; Parish, Tanya; Williams, John D.; Davies, Simon J.; Johnson, David W.; Topley, Nicholas; Moser, Bernhard; Eberl, Matthias

    2011-01-01

    Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early

  14. Eosinophil Granulocytes Account for Indoleamine 2,3-Dioxygenase-Mediated Immune Escape in Human Non Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Simonetta Astigiano

    2005-04-01

    Full Text Available Indoleamine 2,3-dioxygenase (IDO, a catabolizing enzyme of tryptophan, is supposed to play a role in tumor immune escape. Its expression in solid tumors has not yet been well elucidated: IDO can be expressed by the tumor cells themselves, or by ill-defined infiltrating cells, possibly depending on tumor type. We have investigated IDO expression in 25 cases of non small cell lung cancer (NSCLC. Using histochemistry and immunohistochemistry, we found that IDO was expressed not by tumor cells, but by normal cells infiltrating the peritumoral stroma. These cells were neither macrophages nor dendritic cells, and were identified as eosinophil granulocytes. The amount of IDO-positive eosinophils varied in different cases, ranging from a few cells to more than 50 per field at x200 magnification. IDO protein in NSCLC was enzymatically active. Therefore, at least in NSCLC cases displaying a large amount of these cells in the inflammatory infiltrate, IDO-positive eosinophils could exert an effective immunosuppressive action. On analyzing the 17 patients with adequate follow-up, a significant relationship was found between the amount of IDO-positive infiltrate and overall survival. This finding suggests that the degree of IDO-positive infiltrate could be a prognostic marker in NSCLC.

  15. ANNEXIN A1 N-TERMINAL DERIVED PEPTIDE AC2-26 EXERTS CHEMOKINETIC EFFECTS ON HUMAN NEUTROPHILS

    Directory of Open Access Journals (Sweden)

    Jesmond eDalli

    2012-02-01

    Full Text Available It is postulated that peptides derived from the N-terminal region of Annexin A1, a glucocorticoid-regulated 37-kDa protein, could act as biomimetics of the parent protein. However, recent evidence, amongst which the ability to interact with distinct receptors other then that described for Annexin A1, suggest that these peptides might fulfil other functions at variance to those reported for the parent protein. Here we tested the ability of peptide Ac2-26 to induce chemotaxis of human neutrophils, showing that this peptide can elicit responses comparable to those produced by the canonical activator formyl-Met-Leu-Phe (or FMLP. However, whilst disruption of the chemical gradient abolished the FMLP response, addition of peptide Ac2-26 in the top well of the chemotaxis chamber did not affect (10 µM or augmented (at 30 µM the neutrophil locomotion to the bottom well, as elicited by 10 µM peptide Ac2-26. Intriguingly, the sole addition of peptide Ac2-26 in the top wells produced a marked migration of neutrophils. A similar behaviour was observed when human primary monocytes were used. Thus, peptide Ac2-26 is a genuine chemokinetic agent towards human blood leukocytes.Neutralization strategies indicated that engagement of either the GPCR termed FPR1 or its cognate receptor FPR2/ALX was sufficient to sustain peptide Ac2-26 induced neutrophil migration. Similarly, application of pharmacological inhibitors showed that cell locomotion to peptide Ac2-26 was mediated primarily by the ERK, but not the JNK and p38 pathways.In conclusion, we report here novel in vitro properties for peptide Ac2-26, promoting neutrophil and monocyte chemokinesis, a process that may contribute to accelerate the resolution phase of inflammation. Here we postulate that the generation Annexin A1 N-terminal peptides at the site of inflammation may expedite the egress of migrated leukocytes thus promoting the return to homeostasis.

  16. [Neuro-neutrophilic disease suspected by human leukocyte antigen (HLA) typing and brain biopsy: a case report].

    Science.gov (United States)

    Nakanishi, Etsuro; Sawamura, Masanori; Maruhama, Shinichiro; Yamada, Hiroshi; Kim, Gan; Harada, Kiyoshi

    2015-01-01

    In a 72-year-old female, subacute right hemiplegia and aphasia appeared in late May 2011. The results of hematology, a cerebrospinal fluid test, (13)F-FDG-PET, and cephalic MRI suggested intravascular/malignant lymphoma. Brain biopsy was performed. Pathological findings did not suggest a malignant tumor. In the perivascular space, the infiltration of neutrophils or histiocytes was observed. The patient was referred to the Department of Neurology. Based on the results of various examinations, infection was ruled out, and steroid therapy was conducted. Marked improvement was achieved. Subsequently, the results of human leukocyte antigen (HLA) typing showed B54/Cw1. As dermal findings were absent, it was impossible to make a definitive diagnosis of neuro-Sweet disease, but the disorder was regarded as a neuro-neutrophilic disease, which is a more comprehensive entity. Few studies have reported brain tissue findings of active neuro-neutrophilic disease. We report the present case, which will contribute to future research.

  17. Impact of Anti-Inflammatory Agents on the Gene Expression Profile of Stimulated Human Neutrophils: Unraveling Endogenous Resolution Pathways

    Science.gov (United States)

    St-Onge, Mireille; Dumas, Aline; Michaud, Annick; Laflamme, Cynthia; Dussault, Andrée-Anne; Pouliot, Marc

    2009-01-01

    Adenosine, prostaglandin E2, or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A2A receptor agonist CGS 21680, prostaglandin E2, cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution. PMID:19295914

  18. Impact of anti-inflammatory agents on the gene expression profile of stimulated human neutrophils: unraveling endogenous resolution pathways.

    Directory of Open Access Journals (Sweden)

    Mireille St-Onge

    Full Text Available Adenosine, prostaglandin E(2, or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A(2A receptor agonist CGS 21680, prostaglandin E(2, cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution.

  19. Subcutaneous administration of interleukin-2 triggers Fcgamma receptor I expression on human peripheral blood neutrophils in solid and hematologic malignancies.

    Science.gov (United States)

    Sconocchia, G; Cococcetta, N Y; Campagnano, L; Amadori, S; Iorio, B; Boffo, V; Ferdinandi, V; Del Principe, I; Adorno, D; Casciani, C U

    2001-01-01

    Freshly isolated human polymorphonuclear cells (PMNCs) constitutively express Fcgamma receptor (Fc-gammaR) II and FcgammaRIII on the cell surface but not FcgammaRI. Cytokines such as interferon-gamma (IFNgamma), granulocyte-macrophage colony-stimulating factor (CSF), and granulocyte-CSF trigger FcgammaRI expression on (PMNCs). Because PMNCs express interleukin (IL)-2 receptor, we investigated whether IL-2 can induce FcgammaRI expression on PMNCs isolated from IL-2-treated metastatic renal cell carcinoma (MRCC) and low-grade non-Hodgkin lymphoma (LGNHL) patients. Pretherapy flow cytometry analysis of Fcgamma receptors on PMNCs did not show FcgammaRI expression. Interestingly, 3 days after therapy, PMNCs displayed a detectable amount of FcgammaRI on the cell surface. Kinetic studies on the in vivo effects of IL-2 on MRCC patients showed that FcgammaRI was transiently expressed, starting within 3-6 days of therapy, remaining expressed for 10-15 days, and rapidly declining, whereas such expression remained stable for months in LGNHL patients. In contrast, Fc-gammaRII was not affected. In addition, FcgammaRI+ PMNCs coated in vitro with a bispecific antibody Fab anti-FcgammaRI x anti-HER-2/neu formed intercellular conjugates with a human HER-2/neu-transfected 3T3 cell line (HER-2/neu-3T3). Interleukin-2 treatment increased the number of FcgammaRIII low eosinophils, leading to a change in FcgammaRIII distribution among granulocyte cell subsets. In vitro IL-2 treatment of purified PMNCs failed to generate Fc-gammaRI expression, suggesting that IL-2 indirectly causes FcgammaRI expression. During the IL-2 administration, we did not observe significant changes in IFNgamma serum level. In conclusion, our observation may be used to potentiate the antitumor effects of IL-2 in novel immunotherapy regimens, perhaps by redirecting FcgammaRI+ PMNCs against cancer cells by heteroconjugate antibodies and monitoring the biologic activity of subcutaneous IL-2 in cancer patients.

  20. Frontline Science: Tumor necrosis factor-α stimulation and priming of human neutrophil granule exocytosis.

    Science.gov (United States)

    McLeish, Kenneth R; Merchant, Michael L; Creed, T Michael; Tandon, Shweta; Barati, Michelle T; Uriarte, Silvia M; Ward, Richard A

    2017-07-01

    Neutrophil granule exocytosis plays an important role in innate and adaptive immune responses. The present study examined TNF-α stimulation or priming of exocytosis of the 4 neutrophil granule subsets. TNF-α stimulated exocytosis of secretory vesicles and gelatinase granules and primed specific and azurophilic granule exocytosis to fMLF stimulation. Both stimulation and priming of exocytosis by TNF-α were dependent on p38 MAPK activity. Bioinformatic analysis of 1115 neutrophil proteins identified by mass spectrometry as being phosphorylated by TNF-α exposure found that actin cytoskeleton regulation was a major biologic function. A role for p38 MAPK regulation of the actin cytoskeleton was confirmed experimentally. Thirteen phosphoproteins regulated secretory vesicle quantity, formation, or release, 4 of which-Raf1, myristoylated alanine-rich protein kinase C (PKC) substrate (MARCKS), Abelson murine leukemia interactor 1 (ABI1), and myosin VI-were targets of the p38 MAPK pathway. Pharmacologic inhibition of Raf1 reduced stimulated exocytosis of gelatinase granules and priming of specific granule exocytosis. We conclude that differential regulation of exocytosis by TNF-α involves the actin cytoskeleton and is a necessary component for priming of the 2 major neutrophil antimicrobial defense mechanisms: oxygen radical generation and release of toxic granule contents. © Society for Leukocyte Biology.

  1. Kinetics of Neutrophil-Releasing Activity of Post-Leukapheresis Plasma.

    Science.gov (United States)

    1981-11-01

    Auckland, New Zealand Wellington General Hospital, Wellington, New Zealand New Zealand Defense Ministry, Wellington, New Zeland 52 Arthur D bttle Inc V...GRANULOCYTE COUNT WITH GRANULOCYTE YIELDS FROM NYLON FILTERS Postpheresis Granulocyte Count ( New Experiments) ɝ,000 5,000-15,000 15,100-25,000 >25,000 Number...Boggs, D. Transfusion of neutrophils to prevent or treat infection in neutropenic patients. New Eng. J. Med. 290:1055, 1974. I 12. Lehmann, E

  2. fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    María A. Hidalgo

    2015-01-01

    Full Text Available N-Formyl-methionyl-leucyl-phenylalanine (fMLP and platelet-activating factor (PAF induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8 release and nicotinamide adenine dinucleotide phosphate reduced (NADPH oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP, diphenyleneiodonium (DPI, and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

  3. Effects of intranasal TNFα on granulocyte recruitment and activity in healthy subjects and patients with allergic rhinitis

    Science.gov (United States)

    Widegren, Henrik; Erjefält, Jonas; Korsgren, Magnus; Andersson, Morgan; Greiff, Lennart

    2008-01-01

    Background TNFα may contribute to the pathophysiology of airway inflammation. For example, we have recently shown that nasal administration of TNFα produces late phase co-appearance of granulocyte and plasma exudation markers on the mucosal surface. The objective of the present study was to examine indices of granulocyte presence and activity in response to intranasal TNFα challenge. Methods Healthy subjects and patients with allergic rhinitis (examined out of season) were subjected to nasal challenge with TNFα (10 μg) in a sham-controlled and crossover design. Nasal lavages were carried out prior to and 24 hours post challenge. Nasal biopsies were obtained post challenge. Nasal lavage fluid levels of myeloperoxidase (MPO) and eosinophil cationic protein (ECP) were analyzed as indices of neutrophil and eosinophil activity. Moreover, IL-8 and α2-macroglobulin were analyzed as markers of pro-inflammatory cytokine production and plasma exudation. Nasal biopsy numbers of neutrophils and eosinophils were monitored. Results Nasal lavage fluid levels of MPO recorded 24 hours post TNFα challenge were increased in healthy subjects (p = 0.0081) and in patients with allergic rhinitis (p = 0.0081) (c.f. sham challenge). Similarly, α2-macroglobulin was increased in healthy subjects (p = 0.014) and in patients with allergic rhinitis (p = 0.0034). Lavage fluid levels of ECP and IL-8 were not affected by TNFα challenge. TNFα increased the numbers of subepithelial neutrophils (p = 0.0021), but not the numbers of eosinophils. Conclusion TNFα produces a nasal inflammatory response in humans that is characterised by late phase (i.e., 24 hours post challenge) neutrophil activity and plasma exudation. PMID:18234086

  4. Effects of intranasal TNFα on granulocyte recruitment and activity in healthy subjects and patients with allergic rhinitis

    Directory of Open Access Journals (Sweden)

    Andersson Morgan

    2008-01-01

    Full Text Available Abstract Background TNFα may contribute to the pathophysiology of airway inflammation. For example, we have recently shown that nasal administration of TNFα produces late phase co-appearance of granulocyte and plasma exudation markers on the mucosal surface. The objective of the present study was to examine indices of granulocyte presence and activity in response to intranasal TNFα challenge. Methods Healthy subjects and patients with allergic rhinitis (examined out of season were subjected to nasal challenge with TNFα (10 μg in a sham-controlled and crossover design. Nasal lavages were carried out prior to and 24 hours post challenge. Nasal biopsies were obtained post challenge. Nasal lavage fluid levels of myeloperoxidase (MPO and eosinophil cationic protein (ECP were analyzed as indices of neutrophil and eosinophil activity. Moreover, IL-8 and α2-macroglobulin were analyzed as markers of pro-inflammatory cytokine production and plasma exudation. Nasal biopsy numbers of neutrophils and eosinophils were monitored. Results Nasal lavage fluid levels of MPO recorded 24 hours post TNFα challenge were increased in healthy subjects (p = 0.0081 and in patients with allergic rhinitis (p = 0.0081 (c.f. sham challenge. Similarly, α2-macroglobulin was increased in healthy subjects (p = 0.014 and in patients with allergic rhinitis (p = 0.0034. Lavage fluid levels of ECP and IL-8 were not affected by TNFα challenge. TNFα increased the numbers of subepithelial neutrophils (p = 0.0021, but not the numbers of eosinophils. Conclusion TNFα produces a nasal inflammatory response in humans that is characterised by late phase (i.e., 24 hours post challenge neutrophil activity and plasma exudation.

  5. Capsule influences the deposition of critical complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils.

    Directory of Open Access Journals (Sweden)

    Michael E Woodman

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD(50 via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.

  6. [The effect of continuous low-intensity laser irradiation of the red spectrum on the changes in the functional activity and speed of NADPH-oxidase response of human peripheral blood neutrophils].

    Science.gov (United States)

    Gizinger, O A; Moskvin, S V; Ziganshin, O R; Shemetova, М А

    Registration of the changes in the functional metabolic status of neutrophils under the influence of low-intensity laser radiation (LILR) can be useful for the choice of the exposure parameters in the studies designed to evaluate immunotropic effects. Such investigations, in their turn, are very promising in clinical terms since their results can be used to optimize the laser therapy techniques. The objective of the present study was to evaluate the degranulation potential of neutrophils and the intensity of their NADPH-oxidase reaction in response to the exposure to laser radiation as exemplified by the release of lysosomal granules in vitro. The source of radiation was the LASMIK device operated in the continuous mode at the following parameters: wavelength 635 nm, power density 0.12 mW/cm2, exposure time 10, 30, 90, 120, 150 s in the case of the automatic timer control and 100 s in the case of manual shutdown. It was shown that the maximum lysosomal activity and release of lysosomal granules took place at a 90-10 s exposure and a wavelength of 635 nm with the appearance of a plateau within 120 and 150 s after the onset of irradiation. In the case of a shorter exposure of neutrophil granulocytes to LILR (10 and 30 s) no pronounced effect was observed. It means that low-intensity laser radiation of the red spectrum with a wavelength of 635 nm is a physical stimulus reinforcing exocytosis of lysosomal granules by neutrophils in vitro. The reliable changes of NADPH-oxidase activity of neutrophil granulocytes isolated from donor peripheral blood are recorded at an optimum exposure time of 90-100 s. It is concluded that the laser therapy techniques, at least those designed to regulate neutrophils should be applied with the optimum exposure time of 90-100 s.

  7. YKL-40, a mammalian member of the chitinase family, is a matrix protein of specific granules in human neutrophils

    DEFF Research Database (Denmark)

    Volck, B; Price, P A; Johansen, J S

    1998-01-01

    YKL-40, also called human cartilage glycoprotein-39 (HC gp-39), is a member of family 18 glycosyl hydrolases. YKL-40 is secreted by chondrocytes, synovial cells, and macrophages, and recently it has been reported that YKL-40 has a role as an autoantigen in rheumatoid arthritis (RA). The function...... YKL-40 at the myelocyte-metamyelocyte stage, the stage of maturation at which other specific granule proteins are formed. Assuming that YKL-40 has a role as an autoantigen in RA by inducing T cell-mediated autoimmune response, YKL-40 released from neutrophils in the inflamed joint could be essential...

  8. Prevalence and first molecular characterization of Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, in Rhipicephalus sanguineus ticks attached to dogs from Egypt

    Directory of Open Access Journals (Sweden)

    Mohamed W. Ghafar

    2012-04-01

    Full Text Available PCR targeting 16S rRNA gene integrated with sequence analysis were performed to investigate the prevalence and the molecular identity of Anaplasma phagocytophilum in Egyptian Rhipicephalus sanguineus ticks attached to dogs. A total of 413 adult and nymphal R. sanguineus ticks were collected while attached to 72 free-roaming dogs from four locations (Imbaba, Boulaq, Haram, Monib in Giza Governorate, Egypt. DNA was successfully extracted from 401 specimens (133 nymphs and 268 adults. The overall prevalence rate was 13.7% and adult ticks showed a significantly higher infection rate (16.4% compared to nymphs (8.3%. Sequence comparisons of 218-bp showed that detected organism belongs to A. phagocytophilum. The sequence showed 99.1% similarity (2 nucleotide differences with some strains described as human pathogens and with that detected in the established tick vectors. Phylogenetic analysis placed the bacteria on a separate branch with that found in R. annulatus from Egypt (DQ379972 (99.5% similarity. Our variant strain was designated as A. phagocytophilum-Ghafar-EGY (AB608266. This report is the first molecular characterization of A. phagocytophilum in R. sanguineus in Egypt, suggesting that this tick species may act as a competent vector for a variant strain of human granulocytic anaplasmosis agent.

  9. High pH solubilization and chromatography-based renaturation and purification of recombinant human granulocyte colony-stimulating factor from inclusion bodies.

    Science.gov (United States)

    Li, Ming; Fan, Hua; Liu, Jiahua; Wang, Minhong; Wang, Lili; Wang, Chaozhan

    2012-03-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.

  10. Neonatal Sepsis and Neutrophil Insufficiencies

    Science.gov (United States)

    Melvan, John Nicholas; Bagby, Gregory J.; Welsh, David A.; Nelson, Steve; Zhang, Ping

    2011-01-01

    Sepsis has continuously been a leading cause of neonatal morbidity and mortality despite current advances in chemotherapy and patient intensive care facilities. Neonates are at high risk for developing bacterial infections due to quantitative and qualitative insufficiencies of innate immunity, particularly granulocyte lineage development and response to infection. Although antibiotics remain the mainstay of treatment, adjuvant therapies enhancing immune function have shown promise in treating sepsis in neonates. This chapter reviews current strategies for the clinical management of neonatal sepsis and analyzes mechanisms underlying insufficiencies of neutrophil defense in neonates with emphasis on new directions for adjuvant therapy development. PMID:20521927

  11. [Comparative study on the efficacy and safety between pegfilgrastim (PEG-rhG-CSF) and recombinant human granulocyte colony-stimulating factor in promoting hematopoietic recovery after allogeneic hematopoietic stem cell transplantation after hematological malignancy].

    Science.gov (United States)

    Yang, F; Sun, X D; Yuan, L; Zhang, J C; Hu, J W; Liu, N; Lou, X; Su, Y F; Yu, Z Y; Chen, J L; Li, Y H; Hu, L D; Chen, H; Jiang, M

    2017-10-14

    Objective: To observe the efficacy and safety between Pegfilgrastim (PEG-rhG-CSF) and Recombinant human granulocyte colony stimulating factor (rhG-CSF) in hematological malignancy after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: 157 patients after allo-HSCT were enrolled in this study from June 2015 to November 2016. Two agents of G-CSF were used to stimulate hematopoietic recovery after transplantation. There were 65 cases in PEG-rhG-CSF and 92 cases in rhG-CSF groups. Patients in PEG-rhG-CSF group were given a single subcutaneous dose of 6 mg on the first day and +8 d, while cases in rhG-CSF group were given in dose of 5 μg·kg(-1)·d(-1) by subcutaneous injection from +1 d continuing to neutrophils more than 1.5×10(9)/L, and then the indicators and survival rates in two groups after transplantation were compared. Results: ①There were no significant differences of the neutrophil implantation time[13.5 (8-12) d vs 13 (9-24) d, P =0.393] and platelet implantation time [14 (9-160) d vs 14 (9-92) d, P =0.094] between PEG-rhG-CSF and rhG-CSF groups respectively. There were no significant differences in terms of neutropenia period ( P =0.435) , number of cases who got fever during neutropenia ( P =0.622) , and the median time of fever in neutropenia period ( P =0.460) , respectively between the two groups. There were no significant differences of erythrocyte and platelet transfusions ( P =0.074, P =0.059) within 1 month after transplantation. ②There were no significant differences with regard to the incidences of acute GVHD[23.1% (15/65) vs 34.8% (32/92) , P =0.115], chronic GVHD[20.0% (13/65) vs 32.6% (32/92) , P =0.081], Ⅱ-Ⅳdegree of acute GVHD[30.0% (13/65) vs 30.4% (30/92) , P =0.287] and extensive chronic GVHD[9.2% (6/65) vs 20.7% (19/92) , P =0.135] between PEG-rhG-CSF and rhG-CSF groups. ③There were no significant differences in terms of disease free survival (DFS) (62.5% vs 61.4%, P =0.478) and overall survival (OS

  12. [Kinetic and organization of granulocytes transfusion: a case report].

    Science.gov (United States)

    Lamure, S; Latry, P; Kanouni, T; Hicheri, Y; Sirvent, A; Lenglet, A; Mathieu-Daude, D; Cazal, P; Cartron, G

    2014-12-01

    Therapeutic granulocyte transfusion remains an indication for neutropenic sepsis associated with prolonged neutropenia. However, harvest complexity and lack of proved efficacy mark the limits of its development. A 58-year old man received allogeneic stem cell transplantation for osteomyelofibrosis. Six months later, after a transplant rejection, he presented with perineal cellulitis from hemorrhoid origin, without any microbiological documentation. The evolution was unfavorable despite antibiotic and antifungal therapy. A set of seven granulocytes transfusions was initiated. Re-circulation of granulocytes analysis showed an initial increase (H2) followed by a decrease (H8) reaching the basal rate at H16. No toxicity has been reported during or following the transfusions. Clinical improvement has been reported five days after the first transfusion, scaring over at D15, without any neutrophil recovery. In 2014, granulocyte transfusion therapy is indicated for severe infection associated with long-term neutropenia. Minimal circulation of transfused cells in our observation and fast clinical improvement suggest the concentration of granulocytes on the infected area. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  13. Granulocytes: New Members of the Antigen-Presenting Cell Family

    Directory of Open Access Journals (Sweden)

    Ang Lin

    2017-12-01

    Full Text Available Granulocytes, the most abundant types of leukocytes, are the first line of defense against pathogen invasion. However, the plasticity and diversity of granulocytes have been increasingly revealed, especially with regard to their versatile functions in orchestrating adaptive immune responses. A substantial body of recent evidence demonstrates that granulocytes can acquire the function as antigen-presenting cells under pathological or inflammatory conditions. In addition, they can acquire surface expression of MHC class II and costimulatory molecules as well as T cell stimulatory behavior when cultured with selected cytokines. The classic view of granulocytes as terminally differentiated, short-lived phagocytes is therefore changing to phenotypically and functionally heterogeneous cells that are engaged in cross-talk with other leukocyte populations and provide an additional link between innate and adaptive immunity. In this brief review, we summarize the current knowledge on the antigen-presenting capacity of granulocyte subsets (neutrophils, eosinophils, and basophils. Underlying mechanisms, relevant physiological significance and potential controversies are also discussed.

  14. Suppression of blood monocyte and neutrophil chemotaxis in acute human malaria

    DEFF Research Database (Denmark)

    Nielsen, H; Kharazmi, A; Theander, T G

    1986-01-01

    tested monocyte chemotactic responsiveness in 19 patients with acute primary attack malaria. In addition, the neutrophil chemotaxis was measured in 12 patients. Before the initiation of antimalarial treatment a significant depression of monocyte chemotaxis was observed in approximately half...... suppressed. The monocyte chemotaxis was followed in 14 of the patients, during treatment and after complete recovery. After 3 days of treatment the response had improved in most of the patients, and after 7 days all patients had a normal monocyte chemotaxis, which remained normal after one month....... No significant differences between P. falciparum and P. vivax/ovale malaria was observed with respect to blood monocyte chemotactic responsiveness. Neutrophil chemotaxis in patients with P. falciparum infections was similarly suppressed before treatment (54% of controls), was still defective after 3 days...

  15. In vitro inhibition of human neutrophil histotoxicity by ambroxol: evidence for a multistep mechanism

    OpenAIRE

    Ottonello, Luciano; Arduino, Nicoletta; Bertolotto, Maria; Dapino, Patrizia; Mancini, Marina; Dallegri, Franco

    2003-01-01

    Neutrophils are major culprits for the protease/antiprotease imbalance during various lung diseases, that is, chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis and adult respiratory distress syndrome. Thus, these cells are presently considered an ideal target for the pharmacologic control of tissue injury during these diseases.This study was planned in order to investigate if ambroxol and its precursor bromhexine are actually capable of preventing alpha-1-a...

  16. Chemiluminescence response of human neutrophils to He-Ne laser irradiation (in vivo and in vitro)

    OpenAIRE

    Schepetkin, I.; Udut, V.; Karpov, A.

    1994-01-01

    He-Ne laser irradiation (0.01-6 J/cm3) of the blood and neutrophile suspension in vitrowas shown to modulate reactive oxygen species (ROS) production in healthy donors. Intravascular laser irradiation of the blood (5 mW, 30 minutes, daily) of the patients with chronic gastric ulcer during first 5 days resulted in increasing stimul-induced ROS production in patients with the low initial chemiluminescence response and its decreasing in patients with the high initial chemiluminescence response.

  17. alpha isoforms of soluble and membrane-linked folate-binding protein in human blood

    DEFF Research Database (Denmark)

    Hoier-Madsen, M.; Holm, J.; Hansen, S.I.

    2008-01-01

    supported the hypothesis that serum FBP (29 kDa) mainly originates from neutrophils. The presence of FBP/FR alpha isoforms were established for the first time in human blood using antibodies specifically directed against human milk FBP alpha. The alpha isoforms identified on erythrocyte membranes......, and in granulocytes and serum, only constituted an almost undetectable fraction of the functional FBP The FBP alpha in neutrophil granulocytes was identified as a cytoplasmic component by indirect immunofluorescence. Gel filtration of serum revealed a peak of FBP alpha (>120 kDa), which could represent receptor...... fragments from decomposed erythrocytes and granulocytes. The soluble FBPs may exert bacteriostatic effects and protect folates in plasma from biological degradation, whereas FRs on the surface of blood cells could be involved in intracellular folate uptake or serve as signal proteins. The latter receptors...

  18. Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

    Directory of Open Access Journals (Sweden)

    Elodie Parzy

    Full Text Available Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research.We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4 cells per milliliter, well under the physiological neutrophils concentrations.These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

  19. Protection of Candida parapsilosis from neutrophil killing through internalization by human endothelial cells.

    Science.gov (United States)

    Glass, Kyle A; Longley, Sarah J; Bliss, Joseph M; Shaw, Sunil K

    2015-01-01

    Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence.

  20. Effects of Rhodomyrtus tomentosa extract on virulence factors of Candida albicans and human neutrophil function.

    Science.gov (United States)

    Hmoteh, Jutharat; Musthafa, Khadar Syed; Voravuthikunchai, Supayang Piyawan

    2017-12-10

    Candida albicans has become a major problem of oral candidiasis due to increase in antibiotic resistance. Rhodomyrtus tomentosa, a medicinal plant possessing several phytochemical constituents, has been considered as a potential source of antimicrobial and immunomodulatory agents. The aim of this study was to investigate anti-virulence and immunostimulatory activity of R. tomentosa ethanolic leaf extract against C. albicans. The effects of the extract on C. albicans were assessed on germ tube production, adherence of the organisms to surfaces, and biofilm. In addition, the effects of the extract on phagocytosis and killing activity of neutrophils against the pathogen were investigated. Suppression of germ tube production following 30 min exposure to the extract at 256 μg/mL was significantly increased in comparison with that of the unexposed cells (p extract at 512-1024 μg/mL significantly reduced biofilm forming ability of the organisms up to 42.31-64.58% (p extract at 256 μg/mL was observed (p extract at 50 μg/mL significantly enhanced phagocytosis and killing activity of neutrophils against the organism, compared with the control (p extract displayed a dual mode of action, inhibiting virulence factors of C. albicans and enhancing neutrophil functions. Further pharmaceutical development of the extract might be useful as an alternative therapeutic agent against oral candidiasis. Copyright © 2017. Published by Elsevier Ltd.

  1. Effects of red grape juice polyphenols in NADPH oxidase subunit expression in human neutrophils and mononuclear blood cells.

    Science.gov (United States)

    Dávalos, Alberto; de la Peña, Gema; Sánchez-Martín, Carolina C; Teresa Guerra, M; Bartolomé, Begoña; Lasunción, Miguel A

    2009-10-01

    The NADPH oxidase enzyme system is the main source of superoxide anions in phagocytic and vascular cells. NADPH oxidase-dependent superoxide generation has been found to be abnormally enhanced in several chronic diseases. Evidence is accumulating that polyphenols may have the potential to improve cardiovascular health, although the mechanism is not fully established. Consumption of concentrated red grape juice, rich in polyphenols, has been recently shown to reduce NADPH oxidase activity in circulating neutrophils from human subjects. In the present work we studied whether red grape juice polyphenols affected NADPH oxidase subunit expression at the transcription level. For this, we used human neutrophils and mononuclear cells from peripheral blood, HL-60-derived neutrophils and the endothelial cell line EA.hy926.Superoxide production was measured with 2'7'-dichlorofluorescein diacetate or lucigenin, mRNA expression by real-time RT-PCR and protein expression by Western blot. Each experiment was performed at least three times. In all cell types tested, red grape juice, dealcoholised red wine and pure polyphenols decreased superoxide anion production. Red grape juice and dealcoholised red wine selectively reduced p47phox, p22phox and gp91phox expression at both mRNA and protein levels, without affecting the expression of p67phox. Pure polyphenols, particularly quercetin, also reduced NADPH oxidase subunit expression, especially p47phox, in all cell types tested. The present results showing that red grape juice polyphenols reduce superoxide anion production provide an alternative mechanism by which consumption of grape derivatives may account for a reduction of oxidative stress associated with cardiovascular and/or inflammatory diseases related to NADPH oxidase superoxide overproduction.

  2. The influence of procyanidins isolated from small-leaved lime flowers (Tilia cordata Mill.) on human neutrophils.

    Science.gov (United States)

    Czerwińska, Monika E; Dudek, Marta K; Pawłowska, Karolina A; Pruś, Anna; Ziaja, Maria; Granica, Sebastian

    2018-02-12

    Linden flower is a wildly used plant material among patients in the treatment of common cold symptoms and mucosa inflammations. However, the structure and bioactivity of flavan-3-ol derivatives present in infusions from flowers of Tilia cordata have not been studied so far. The aim of current study was to isolate and identify main procyanidins present in the flowers of small-leaved lime and to evaluate their influence on the inflammatory response of human neutrophils ex vivo. The chemical structure of isolated compounds was established by 1D and 2D NMR experiments. The bioactivity of obtained compounds was tested in human neutrophils model. Cytotoxicity and influence of compounds on apoptosis was established by flow cytometry. The levels of produced cytokines were established by ELISA after stimulation with lipopolysaccharide (LPS). The inhibition of the production of reactive oxygen species was checked by luminol-dependent chemiluminescence method after N-formylmethionyl-leucyl-phenylalanine (f-MLP) induction. The phytochemical work resulted in the isolation of 10 compounds. Compounds were identified as oligomeric procyanidins and their precursor epicatechin. The potential anti-inflammatory activity of compounds was evaluated in the concentration range 5-20 μM. All compounds were able to decrease the production of ROS from f-MLP-stimulated neutrophils. Most of compounds were able to inhibit the LPS-induced release of IL-8. Some trimeric and tetrameric derivatives were also able to decrease the production of MIP-1β. Obtained results partially support the traditional usage of infusion from lime flowers in the treatment of symptoms of inflammation and irritation of mucosa in common cold, pharyngitis and tonsillitis. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Rats and mice immunised with chimeric human/mouse proteinase 3 produce autoantibodies to mouse Pr3 and rat granulocytes

    NARCIS (Netherlands)

    van der Geld, Ymke M.; Hellmark, Thomas; Selga, Daina; Heeringa, Peter; Huitema, Minke G.; Limburg, Pieter C.; Kallenberg, Cees G. M.

    2007-01-01

    Aim: In this study, we employed chimeric human/ mouse Proteinase 3 ( PR3) proteins as tools to induce an autoantibody response to PR3 in rats and mice. Method: Rats and mice were immunised with recombinant human PR3 ( HPR3), recombinant murine PR3 ( mPR3), single chimeric human/ mouse PR3 ( HHm,

  4. Administration of recombinant human granulocyte-colony-stimulating factor does not induce long-lasting detectable epigenetic alterations in healthy donors.

    Science.gov (United States)

    Leitner, Gerda C; Faschingbauer, Martin; Wenda, Sabine; Weigel, Günter; Fischer, Gottfried

    2014-12-01

    The short-term safety profile of recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) in the allogeneic stem cell setting seems acceptable; only few data on long-term safety are available. To further study possible epigenetic alterations, we investigated prospectively the influence of rHuG-CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes in peripheral blood cells of healthy unrelated stem cell donors within an observation period of 1 year. In this study, 20 stem cell donors (14 male/six female; median age, 40 years; range, 22-54 years) and 20 sex- and age-matched blood component donors (controls) were included. Sampling was performed before rHuG-CSF administration; at the time of donation; and on Days (+1), 7, 30, 100, 180, and 360 in both groups. Analysis of DNMT activity in nuclear extracts was performed using a modified radionuclide assay. We performed methylation-specific polymerase chain reaction to detect the methylation status of promoter CpG islands of the genes of the retinoic acid receptor beta (RAR-B) and the Ras association domain family 1A (RASSF1A). DNMT activity increased significantly on the day of donation and 1 day after (p healthy stem cell donors. © 2014 AABB.

  5. Assessment of antioxidant activity of spray dried extracts of Psidium guajava leaves by DPPH and chemiluminescence inhibition in human neutrophils.

    Science.gov (United States)

    Fernandes, M R V; Azzolini, A E C S; Martinez, M L L; Souza, C R F; Lucisano-Valim, Y M; Oliveira, W P

    2014-01-01

    This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE) from Psidium guajava L. leaves. Different drying carriers, namely, maltodextrin, colloidal silicon dioxide, Arabic gum, and β -cyclodextrin at concentrations of 40 and 80% relative to solids content, were added to drying composition. SDE were characterized through determination of the total phenolic, tannins, and flavonoid content. Antioxidant potential of the SDE was assessed by two assays: cellular test that measures the luminol-enhanced chemiluminescence (LumCL) produced by neutrophils stimulated with phorbol myristate acetate (PMA) and the DPPH radical scavenging (DPPH∗ method). In both assays the antioxidant activity of the SDE occurred in a concentration-dependent manner and showed no toxicity to the cells. Using the CLlum method, the IC50 ranged from 5.42 to 6.50 µg/mL. The IC50 of the SDE ranged from 7.96 to 8.11 µg/mL using the DPPH(•) method. Psidium guajava SDE presented significant antioxidant activity; thus they show high potential as an active phytopharmaceutical ingredient. Our findings in human neutrophils are pharmacologically relevant since they indicate that P. guajava SDE is a potential antioxidant and anti-inflammatory agent in human cells.

  6. Direct anti-inflammatory effects of granulocyte colony-stimulating factor (G-CSF) on activation and functional properties of human T cell subpopulations in vitro.

    Science.gov (United States)

    Malashchenko, Vladimir Vladimirovich; Meniailo, Maxsim Evgenievich; Shmarov, Viacheslav Anatolievich; Gazatova, Natalia Dinislamovna; Melashchenko, Olga Borisovna; Goncharov, Andrei Gennadievich; Seledtsova, Galina Victorovna; Seledtsov, Victor Ivanovich

    2018-03-01

    We investigated the direct effects of human granulocyte colony-stimulating factor (G-CSF) on functionality of human T-cell subsets. CD3 + T-lymphocytes were isolated from blood of healthy donors by positive magnetic separation. T cell activation with particles conjugated with antibodies (Abs) to human CD3, CD28 and CD2 molecules increased the proportion of cells expressing G-CSF receptor (G-CSFR, CD114) in all T cell subpopulations studied (CD45RA + /CD197 + naive T cells, CD45RA - /CD197 + central memory T cells, CD45RA - /CD197 - effector memory T cells and CD45RA + /CD197 - terminally differentiated effector T cells). Upon T-cell activation in vitro, G-CSF (10.0 ng/ml) significantly and specifically enhanced the proportion of CD114 + T cells in central memory CD4 + T cell compartment. A dilution series of G-CSF (range, 0.1-10.0 ng/ml) was tested, with no effect on the expression of CD25 (interleukin-2 receptor α-chain) on activated T cells. Meanwhile, G-CSF treatment enhanced the proportion of CD38 + T cells in CD4 + naïve T cell, effector memory T cell and terminally differentiated effector T cell subsets, as well as in CD4 - central memory T cells and terminally differentiated effector T cells. G-CSF did not affect IL-2 production by T cells; relatively low concentrations of G-CSF down-regulated INF-γ production, while high concentrations of this cytokine up-regulated IL-4 production in activated T cells. The data obtained suggests that G-CSF could play a significant role both in preventing the development of excessive and potentially damaging inflammatory reactivity, and in constraining the expansion of potentially cytodestructive T cells. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Priming of the oxidative burst in human neutrophils by physiological agonists or cytochalasin B results from the recruitment of previously non-responsive cells.

    Science.gov (United States)

    Daniels, R H; Elmore, M A; Hill, M E; Shimizu, Y; Lackie, J M; Finnen, M J

    1994-01-01

    Using a sensitive flow cytometric assay, which measures the intracellular oxidation of 2'7' dichlorofluorescein (DCFH) by H2O2, we have assessed, at a single-cell level, the effects of a variety of physiological priming agonists and cytochalasin B (CB) on purified populations of neutrophils stimulated at different points along the signal response transduction pathway. Pretreatment of purified neutrophils with the physiological priming agonists monocyte interleukin-8 (IL-8), granulocyte-monocyte colony-stimulating factor (GM-CSF), platelet-activating factor (PAF), IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6, and non-stimulatory doses of formyl-methionyl-leucyl-phenylalanine (FMLP), resulted in an increased percentage of cells generating an oxidative burst in response to subsequent receptor stimulation with FMLP. CB had a similar but much more pronounced effect on cellular recruitment to a receptor-mediated responsive state. Activation of protein kinase C (PKC) using the phorbol ester phorbol myristate acetate (PMA) resulted in a heterogeneous response, with all cells generating H2O2, but with two populations differing in their magnitude of response. Physiological priming agonists had no effect on the heterogeneity of the PMA response. However, pretreatment with CB dramatically altered the PMA response, producing a homogeneous population highly responsive to stimulation with PKC. In contrast, direct stimulation of G proteins with fluoride (A1F-4) was primed both by physiological priming agonists and by CB. These results demonstrate that priming of neutrophils by physiological agonists involves changes at the level of signal transduction which enable a previously non-responsive cell to respond to a secondary stimulus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7959884

  8. Impaired killing of Candida albicans by granulocytes mobilized for transfusion purposes: a role for granule components.

    Science.gov (United States)

    Gazendam, Roel P; van de Geer, Annemarie; van Hamme, John L; Tool, Anton T J; van Rees, Dieke J; Aarts, Cathelijn E M; van den Biggelaar, Maartje; van Alphen, Floris; Verkuijlen, Paul; Meijer, Alexander B; Janssen, Hans; Roos, Dirk; van den Berg, Timo K; Kuijpers, Taco W

    2016-05-01

    Granulocyte transfusions are used to treat neutropenic patients with life-threatening bacterial or fungal infections that do not respond to anti-microbial drugs. Donor neutrophils that have been mobilized with granulocyte-colony stimulating factor (G-CSF) and dexamethasone are functional in terms of antibacterial activity, but less is known about their fungal killing capacity. We investigated the neutrophil-mediated cytotoxic response against C. albicans and A. fumigatus in detail. Whereas G-CSF/dexamethasone-mobilized neutrophils appeared less mature as compared to neutrophils from untreated controls, these cells exhibited normal ROS production by the NADPH oxidase system and an unaltered granule mobilization capacity upon stimulation. G-CSF/dexamethasone-mobilized neutrophils efficiently inhibited A. fumigatus germination and killed Aspergillus and Candida hyphae, but the killing of C. albicans yeasts was distinctly impaired. Following normal Candida phagocytosis, analysis by mass spectrometry of purified phagosomes after fusion with granules demonstrated that major constituents of the antimicrobial granule components, including major basic protein (MBP), were reduced. Purified MBP showed candidacidal activity, and neutrophil-like Crisp-Cas9 NB4-KO-MBP differentiated into phagocytes were impaired in Candida killing. Together, these findings indicate that G-CSF/dexamethasone-mobilized neutrophils for transfusion purposes have a selectively impaired capacity to kill Candida yeasts, as a consequence of an altered neutrophil granular content. Copyright© Ferrata Storti Foundation.

  9. Clinical symptoms and neutropenia: the balance of neutrophil development, functional activity, and cell death

    NARCIS (Netherlands)

    Kuijpers, Taco W.

    2002-01-01

    Neutrophilic granulocytes form the major type of leukocytes with counts ranging from about 1500-5000 cells/ micro l of blood under normal conditions. Neutrophils protect our body against bacterial and fungal infections. For this purpose, these cells are equipped with a machinery to sense the site of

  10. Suppressors Of Cytokine Signaling in G-CSF-induced neutrophil development

    NARCIS (Netherlands)

    G.J.M. van de Geijn (Gert-Jan)

    2004-01-01

    textabstractG-CSF is the most important growth factor involved in the production of neutrophilic granulocytes. Signaling routes activated upon ligand binding to the G-CSF receptor (G-CSF-R) control survival, proliferation and differentiation of myeloid progenitor cells towards mature neutrophils.

  11. A bovine whey protein extract stimulates human neutrophils to generate bioactive IL-1Ra through a NF-kappaB- and MAPK-dependent mechanism.

    Science.gov (United States)

    Rusu, Daniel; Drouin, Réjean; Pouliot, Yves; Gauthier, Sylvie; Poubelle, Patrice E

    2010-02-01

    Innate immunity depends on the efficiency of neutrophils to be activated rapidly to restore homeostasis. It can benefit from priming agents that enhance neutrophil capacity to respond more efficiently to a subsequent stimulation. Among natural products, a bovine whey protein extract (WPE) has been shown to prime normal human blood neutrophils by enhancing their chemotaxis, phagocytosis, oxidative burst, and degranulation. These leukocytes are also an important source of cytokines, some of which have antiinflammatory functions. We investigated the role of WPE, as well as its mechanisms of action, on the production of interleukin (IL)-1 receptor antagonist (IL-1Ra) by neutrophils in vitro. WPE dose-dependently stimulated de novo synthesis and release of IL-1Ra by normal human blood neutrophils. Among the major proteins present in WPE, beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) were the only active components. They had additive effects that exactly reproduced those of WPE. Similarly to WPE, they also stimulated the accumulation of IL-1beta, IL-8, IL-6, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and tumor necrosis factor-alpha. However, neutrophils incubated with WPE, beta-LG, and alpha-LA produced IL-1Ra in excess of IL-1beta and the ratio IL-1Ra:IL-1beta increased linearly. The amounts of IL-1Ra stimulated by WPE or beta-LG + alpha-LA significantly reduced the IL-1 activity in EL4 cells. Inhibitors of p38 and extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase, and nuclear factor-kappaB cascades reduced neutrophil production of IL-1Ra. Our data suggest that WPE, through beta-LG + alpha-LA, has immunomodulatory properties and the potential to increase host defenses.

  12. Cathepsin G, a Neutrophil Protease, Induces Compact Cell-Cell Adhesion in MCF-7 Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Tomoya Kudo

    2009-01-01

    Full Text Available Cathepsin G is a serine protease secreted by activated neutrophils that play a role in the inflammatory response. Because neutrophils are known to be invading leukocytes in various tumors, their products may influence the characteristics of tumor cells such as the growth state, motility, and the adhesiveness between cells or the extracellular matrix. Here, we demonstrate that cathepsin G induces cell-cell adhesion of MCF-7 human breast cancer cells resulting from the contact inhibition of cell movement on fibronectin but not on type IV collagen. Cathepsin G subsequently induced cell condensation, a very compact cell colony, resulting due to the increased strength of E-cadherin-mediated cell-cell adhesion. Cathepsin G action is protease activity-dependent and was inhibited by the presence of serine protease inhibitors. Cathepsin G promotes E-cadherin/catenin complex formation and Rap1 activation in MCF-7 cells, which reportedly regulates E-cadherin-based cell-cell junctions. Cathepsin G also promotes E-cadherin/protein kinase D1 (PKD1 complex formation, and Go6976, the selective PKD1 inhibitor, suppressed the cathepsin G-induced cell condensation. Our findings provide the first evidence that cathepsin G regulates E-cadherin function, suggesting that cathepsin G has a novel modulatory role against tumor cell-cell adhesion.

  13. Inhibition of neutrophil elastase and metalloprotease-9 of human adenocarcinoma gastric cells by chamomile (Matricaria recutita L.) infusion.

    Science.gov (United States)

    Bulgari, Michela; Sangiovanni, Enrico; Colombo, Elisa; Maschi, Omar; Caruso, Donatella; Bosisio, Enrica; Dell'Agli, Mario

    2012-12-01

    This study investigated whether the antiinflammatory effect of chamomile infusion at gastric level could be ascribed to the inhibition of metalloproteinase-9 and elastase. The infusions from capitula and sifted flowers (250-1500 µg/mL) and individual flavonoids (10 µM) were tested on phorbol 12-myristate 13-acetate-stimulated AGS cells and human neutrophil elastase. The results indicate that the antiinflammatory activity associated with chamomile infusions from both the capitula and sifted flowers is most likely due to the inhibition of neutrophil elastase and gastric metalloproteinase-9 activity and secretion; the inhibition occurring in a concentration dependent manner. The promoter activity was inhibited as well and the decrease of metalloproteinase-9 expression was found to be associated with the inhibition of NF-kB driven transcription. The results further indicate that the flavonoid-7-glycosides, major constituents of chamomile flowers, may be responsible for the antiinflammatory action of the chamomile infusion observed here. Copyright © 2012 John Wiley & Sons, Ltd.

  14. Regulation of Neutrophil Survival/Apoptosis by Mcl-1

    Directory of Open Access Journals (Sweden)

    Eric Milot

    2011-01-01

    Full Text Available Neutrophil granulocytes have the shortest lifespan among leukocytes in the circulation and die via apoptosis. At sites of infection or tissue injury, prolongation of neutrophil lifespan is critical for effective host defense. Apoptosis of inflammatory neutrophils and their clearance are critical control points for termination of the inflammatory response. Evasion of neutrophil apoptosis aggravates local injury and leads to persistent tissue damage. The short-lived prosurvival Bcl-2 family protein, Mcl-1 (myeloid cell leukemia-1, is instrumental in controlling apoptosis and consequently neutrophil lifespan in response to rapidly changing environmental cues during inflammation. This paper will focus on multiple levels of control of Mcl-1 expression and function and will discuss targeting Mcl-1 as a potential therapeutic strategy to enhance the resolution of inflammation through accelerating neutrophil apoptosis.

  15. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    LENUS (Irish Health Repository)

    Little, Mark A

    2012-01-01

    Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener\\'s granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻\\/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  16. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    Directory of Open Access Journals (Sweden)

    Mark A Little

    Full Text Available Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3 antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener's granulomatosis. Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17% more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  17. Scavenging of superoxide anion by phosphorylethanolamine: studies in human neutrophils and in a cell free system.

    Science.gov (United States)

    Gordon, L I; Weiss, D; Prachand, S; Weitzman, S A

    1991-01-01

    On the basis of previous observations, we attempted to characterize the effects of various products of phospholipid hydrolysis on neutrophil (PMN) respiratory burst activity. We studied the effects of phosphorylcholine (PC) and phosphorylethanoline (PE) on superoxide anion production in PMN and in cell free system. We found that PE but not PC inhibited measured superoxide anion, but that this was not due to inhibition of cellular superoxide generation but to scavenging of generated superoxide anion. Further, utilizing a system based upon the photo-oxidation of O-dianisidine sensitized by riboflavin, we were able to determine that the scavenging effect of PE was not superoxide dismutase (SOD)-like but rather a general scavenging or glutathione (GSH)-like effect. These data underscore the importance of identifying the mechanism of inhibition of superoxide generation by putative inhibitors as being due to a direct cellular effect or to a scavenging property.

  18. TLR agonists extend the functional lifespan of professional phagocytic granulocytes in the bony fish gilthead seabream and direct precursor differentiation towards the production of granulocytes.

    Science.gov (United States)

    Sepulcre, María P; López-Muñoz, Azucena; Angosto, Diego; García-Alcazar, Alicia; Meseguer, José; Mulero, Victoriano

    2011-03-01

    Neutrophils are major cells participants in innate host responses. They are short-lived leukocytes, although microbial products activate intracellular signaling cascades that prolong their survival by inhibiting constitutive apoptosis. To gain insight into the phylogeny of this important cell type, we examined the ability of toll-like receptor agonists to extend the lifespan of gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils. The results obtained demonstrated that apoptosis was also the default state of seabream acidophilic granulocytes and that toll-like receptor agonists were able to dramatically extend their functional lifespan (up to 10 days) by inhibiting apoptosis and inducing a long lasting activation of phagocytic and respiratory burst activities, together with the expression of genes coding for several proinflammatory molecules. This process was independent on contaminating cells and interleukin-1β production. In addition, the results showed that p38 mitogen-activated protein kinase, but not nuclear factor κB, c-Jun terminal kinase or phosphatidylinositol 3-kinase, was involved in the inhibition of acidophilic granulocyte apoptosis following toll-like receptor engagement. Finally, stimulation of head kidney hematopoietic precursor cells with toll-like receptor agonists promoted their terminal differentiation to acidophilic granulocytes. These results demonstrated that the extension of neutrophil lifespan by microbial products is conserved in lower vertebrates although the magnitude of the response is much higher in fish. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Platelet lysate and granulocyte-colony stimulating factor serve safe and accelerated expansion of human bone marrow stromal cells for stroke therapy.

    Science.gov (United States)

    Yamauchi, Tomohiro; Saito, Hisayasu; Ito, Masaki; Shichinohe, Hideo; Houkin, Kiyohiro; Kuroda, Satoshi

    2014-12-01

    Autologous human bone marrow stromal cells (hBMSCs) should be expanded in the animal serum-free condition within clinically relevant periods in order to secure safe and effective cell therapy for ischemic stroke. This study was aimed to assess whether the hBMSCs enhance their proliferation capacity and provide beneficial effect in the infarct brain when cultured with platelet lysate (PL) and granulocyte-colony stimulating factor (G-CSF). The hBMSCs were cultured in the fetal calf serum (FCS)-, PL-, or PL/G-CSF-containing medium. Cell growth kinetics was analyzed. The hBMSCs-PL, hBMSC-PL/G-CSF, or vehicle was stereotactically transplanted into the ipsilateral striatum of the rats subjected to permanent middle cerebral artery occlusion 7 days after the insult. Motor function was assessed for 8 weeks, and the fate of transplanted hBMSCs was examined using immunohistochemistry. As the results, the hBMSCs-PL/G-CSF showed more enhanced proliferation than the hBMSCs-FCS and hBMSCs-PL. Transplantation of hBMSCs expanded with the PL- or PL/G-CSF-containing medium equally promoted functional recovery compared with the vehicle group. Histological analysis revealed that there were no significant differences in their migration, survival, and neural differentiation in the infarct brain between the hBMSCs-PL and hBMSCs-PL/G-CSF. These findings strongly suggest that the combination of PL and G-CSF may accelerate hBMSC expansion and serve safe cell therapy for patients with ischemic stroke at clinically relevant timing.

  20. Relationship between native-state solubility and non-native aggregation of recombinant human granulocyte colony stimulating factor: practical implications for protein therapeutic development.

    Science.gov (United States)

    Banks, Douglas D; Zhang, Jun; Siska, Christine C

    2014-10-06

    Prescreening methods are needed in the biotechnology industry for rapid selection of protein therapeutic candidates and formulations of low aggregation propensity. In recent reports solubility measurements have shown promise as one such method, although the connection between protein solubility and non-native aggregation is not well understood. In the present investigation, recombinant human granulocyte colony stimulating factor (rhGCSF) was used to explore this relationship since it was previously shown to rapidly undergo non-native aggregation/precipitation under physiological conditions in a reaction attenuated by the addition of sucrose [Krishnan, S.; et al. Biochemistry 2002, 41, 6422-6431]. Strong correlations were found between rhGCSF non-native aggregation and both solubility and thermal stability as a function of sucrose concentration. We believe these results make sense in the context of an rhGCSF aggregation mechanism where loss of monomer to insoluble aggregate is limited by association to an observable dimer from a less soluble (and aggregation competent) intermediate species that exists in a temperature sensitive pre-equilibrium with the native monomer. Both solubility and measures of conformational stability report on the position of this equilibrium and therefore the concentration of reactive intermediate. Interestingly, aggregation also correlated with rhGCSF solubility as a function of salting-in concentrations of phosphate since both are dependent on the colloidal stability of the reactive intermediate but not with conformational stability. In lieu of a complete understanding of the aggregation processes that limit protein therapeutic shelf life, these results highlight the potential of using simple solubility measurements as an additional tool in the biotechnology prescreening repertoire.

  1. Four-Week Repeated Intravenous Dose Toxicity and Toxicokinetic Study of TS-DP2, a Novel Human Granulocyte Colony Stimulating Factor in Rats.

    Science.gov (United States)

    Lee, JooBuom; Lee, Kyungsun; Choe, Keunbum; Jung, Hyunseob; Cho, Hyunseok; Choi, Kiseok; Kim, Taegon; Kim, Seojin; Lee, Hyeong-Seok; Cha, Mi-Jin; Song, Si-Whan; Lee, Chul Kyu; Chun, Gie-Taek

    2015-12-01

    TS-DP2 is a recombinant human granulocyte colony stimulating factor (rhG-CSF) manufactured by TS Corporation. We conducted a four-week study of TS-DP2 (test article) in repeated intravenous doses in male and female Sprague-Dawley (SD) rats. Lenograstim was used as a reference article and was administered intravenously at a dose of 1000 μg/kg/day. Rats received TS-DP2 intravenously at doses of 250, 500, and 1000 μg/kg/day once daily for 4 weeks, and evaluated following a 2-week recovery period. Edema in the hind limbs and loss of mean body weight and body weight gain were observed in both the highest dose group of TS-DP2 and the lenograstim group in male rats. Fibro-osseous lesions were observed in the lenograstim group in both sexes, and at all groups of TS-DP2 in males, and at doses of TS-DP2 500 μg/kg/day and higher in females. The lesion was considered a toxicological change. Therefore, bone is the primary toxicological target of TS-DP2. The lowest observed adverse effect level (LOAEL) in males was 250 μg/kg/day, and no observed adverse effect level (NOAEL) in females was 250 μg/kg/day in this study. In the toxicokinetic study, the serum concentrations of G-CSF were maintained until 8 hr after administration. The systemic exposures (AUC0-24h and C0) were not markedly different between male and female rats, between the administration periods, or between TS-DP2 and lenograstim. In conclusion, TS-DP2 shows toxicological similarity to lenograstim over 4-weeks of repeated doses in rats.

  2. Interleukin 17A controls Interleukin 17F production and maintains blood neutrophil counts in mice

    OpenAIRE

    von Vietinghoff, Sibylle; Ley, Klaus

    2009-01-01

    Granulocyte colony-stimulating factor (G-CSF), its receptor and interleukin-17 receptor A (IL-17RA) are all required to maintain baseline neutrophil counts in mice. Here, we tested whether IL-17F could compensate and maintain baseline neutrophil counts in the absence of IL-17A. Unlike the reduced neutrophil counts found in IL-17RA deficient mice, neutrophil counts were mildly increased in IL-17A deficient (Il17a-/-) animals. There was no evidence for infection or altered neutrophil function. ...

  3. Silver nanoparticles of 70 nm and 20 nm affect differently the biology of human neutrophils.

    Science.gov (United States)

    Poirier, Michelle; Simard, Jean-Christophe; Girard, Denis

    2016-05-01

    The influence of size of nanoparticles (NP), especially in regard to pulmonary toxicity, has been widely investigated. In general, NP with smaller diameters are more pro-inflammatory in vivo, at least in terms of neutrophil influx. Nevertheless, the influence of size of NP on polymorphonuclear neutrophil (PMN) cell biology is poorly documented. In the study here, it was decided to determine if AgNP with a diameter of 70 nm (AgNP70) will alter the biology of human PMN similarly to AgNP20 previously reported to induce apoptosis and inhibit de novo protein synthesis. The results here indicated that, in contrast to AgNP20, AgNP70 delayed PMN apoptosis. However, both AgNP20 and AgNP70 inhibited de novo protein synthesis. Both forms of AgNP did not significantly increase reactive oxygen species (ROS) production, but AgNP20 significantly increased the cell production of the CXCL8 chemokine (IL-8). In addition, AgNP20, but not AgNP70, induced the release of albumin and matrix metalloproteinase-9 (MMP-9/gelatinase B) into culture supernatants. Consistent with this latter observation, gelatinase activity was increased by AgNP20, as assessed by zymography. From these outcomes, it is concluded that two NP with different initial diameters can possess similar - as well as distinct - biological properties in modulating human PMN functions. These outcomes are testimony to the complexity of the modes of action of NP at the cellular level.

  4. Differential role of CD97 in interleukin-8-induced and granulocyte-colony stimulating factor-induced hematopoietic stem and progenitor cell mobilization

    NARCIS (Netherlands)

    van Pel, Melissa; Hagoort, Henny; Kwakkenbos, Mark J.; Hamann, Jörg; Fibbe, Willem E.

    2008-01-01

    CD97 is broadly expressed on hematopoietic cells and is involved in neutrophil migration. Since neutrophils are key regulators in HSC/HPC mobilization, we studied a possible role for CD97 in interleukin-8 and granulocyte-colony stimulating factor-induced HSC/HPC mobilization. Mobilization was absent

  5. Treatment with granulocyte colony-stimulating factor decreases the capacity of hematopoietic progenitor cells for generation of lymphocytes in human immunodeficiency virus-infected persons

    DEFF Research Database (Denmark)

    Nielsen, Susanne Dam; Clark, D R; Hutchings, M

    1999-01-01

    An obstacle to stem cell gene therapy for AIDS is the limited numbers of hematopoietic progenitors available. Granulocyte colony-stimulating factor (G-CSF) is used for mobilization of progenitors, but little is known about the functional characteristics of mobilized progenitors, and immature and ...

  6. Prostaglandin E2 resistance in granulocytes from patients with aspirin-exacerbated respiratory disease.

    Science.gov (United States)

    Laidlaw, Tanya M; Cutler, Anya J; Kidder, Molly S; Liu, Tao; Cardet, Juan Carlos; Chhay, Heng; Feng, Chunli; Boyce, Joshua A

    2014-06-01

    Aspirin-exacerbated respiratory disease (AERD) is an inflammatory condition of the respiratory tract and is characterized by overproduction of leukotrienes (LT) and large numbers of circulating granulocyte-platelet complexes. LT production can be suppressed by prostaglandin E(2) (PGE(2)) and the cyclic AMP-dependent protein kinase A (PKA). To determine if PGE(2)-dependent control of LT production by granulocytes is dysregulated in AERD. Granulocytes from well-characterized patients with and without AERD were activated ex vivo and subjected to a range of functional and biochemical analyses. Granulocytes from subjects with AERD generated more LTB4 and cysteinyl LTs than did granulocytes from controls with aspirin-tolerant asthma and controls without asthma. When compared with controls, granulocytes from subjects with AERD had comparable levels of EP(2) protein expression and PGE(2)-mediated cAMP accumulation, yet were resistant to PGE(2)-mediated suppression of LT generation. Percentages of platelet-adherent neutrophils correlated positively with LTB4 generation and inversely with responsiveness to PGE(2)-mediated suppression of LTB(4). The PKA inhibitor H89 potentiated LTB4 generation by control granulocytes but was inactive in granulocytes from individuals with AERD and had no effect on platelet P-selectin induction. Both tonic PKA activity and levels of PKA catalytic gamma subunit protein were significantly lower in granulocytes from individuals with AERD relative to those from controls. Impaired granulocyte PKA function in AERD may lead to dysregulated control of 5-lipoxygenase activity by PGE(2), whereas adherent platelets lead to increased production of LTs, which contributes to the features of persistent respiratory tract inflammation and LT overproduction. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  7. YKL-40, a mammalian member of the chitinase family, is a matrix protein of specific granules in human neutrophils

    DEFF Research Database (Denmark)

    Volck, B; Price, P A; Johansen, J S

    1998-01-01

    of patients with RA, and the cells are assumed to play a role in joint destruction in that disorder. Therefore, we examined whether neutrophils are a source of YKL-40. YKL-40 was found to colocalize and comobilize with lactoferrin (the most abundant protein of specific granules) but not with gelatinase...... in subcellular fractionation studies on stimulated and unstimulated neutrophils. Double-labeling immunoelectron microscopy confirmed the colocalization of YKL-40 and lactoferrin in specific granules of neutrophils. Immunohistochemistry on bone marrow cells showed that neutrophil precursors begin to synthesize...

  8. Granulocyte maturation determines ability to release chromatin NETs and loss of DNA damage response; these properties are absent in immature AML granulocytes.

    Science.gov (United States)

    Lukášová, Emilie; Kořistek, Zdeněk; Klabusay, Martin; Ondřej, Vladan; Grigoryev, Sergei; Bačíková, Alena; Řezáčová, Martina; Falk, Martin; Vávrová, Jiřina; Kohútová, Viera; Kozubek, Stanislav

    2013-03-01

    Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1β isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis.

    Science.gov (United States)

    Chouinard, François; Lefebvre, Julie S; Navarro, Pauline; Bouchard, Line; Ferland, Claudine; Lalancette-Hébert, Mélanie; Marsolais, David; Laviolette, Michel; Flamand, Nicolas

    2011-03-01

    Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.

  10. Prognostic Significance of Immunoreactive Neutrophil Elastase in Human Breast Cancer: Long-Term Follow-Up Results in 313 Patients

    Directory of Open Access Journals (Sweden)

    Miwa Akizuki

    2007-03-01

    Full Text Available OBJECTIVE: We have measured the concentration of immunoreactive neutrophil elastase (ir-NE in the tumor extracts of 313 primary human breast cancers. Sufficient time has elapsed, and we are now ready to analyze its prognostic value in human breast cancer. METHODS: ir-NE concentration in tumor extracts was determined with an enzyme-linked immunosorbent assay that enables a rapid measurement of both free-form ir-NE and the α1-protease inhibitor-complexed form of ir-NE. We analyzed the prognostic value of this enzyme in human breast cancer in univariate and multivariate analyses. RESULTS: Patients with breast cancer tissue containing a high concentration of ir-NE had poor survival compared to those with a low concentration of ir-NE at the cutoff point of 9.0 µg/100 mg protein (P = .0012, which had been previously determined in another group of 49 patients. Multivariate stepwise analysis selected lymph node status (P= .0004; relative risk = 1.46 and ir-NE concentration (P= .0013; relative risk = 1.43 as independent prognostic factors for recurrence. CONCLUSIONS: Tumor ir-NE concentration is an independent prognostic factor in patients with breast cancer who undergo curative surgery. This enzyme may play an active role in tumor progression that leads to metastasis in human breast cancer.

  11. Increased proliferation and decreased membrane permeability as defense mechanisms of Fusobacterium nucleatum against human neutrophilic peptide-1.

    Science.gov (United States)

    Keskin, Mutlu; Könönen, Eija; Söderling, Eva; Isik, Gülden; Firatli, Erhan; Uitto, Veli-Jukka; Gürsoy, Ulvi Kahraman

    2014-12-01

    Human neutrophilic peptides (HNPs) constitute a class of host defense molecules, which contribute to the non-oxidative killing of bacteria and other microorganisms. Since the adaptability is crucial to bacterial survival in changing environments, it is of interest to know how Fusobacterium nucleatum, the major bridge organism connecting early and late colonizers in dental biofilms, defends itself against HNPs. This study aimed to examine the planktonic growth, membrane permeability, and biofilm formation characteristics as defense mechanisms of F. nucleatum against HNP-1. In all experiments, the type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. nucleatum AHN 9508 and ssp. polymorphum AHN 9910) were used. Planktonic growth (measured in colony forming units), capsular polysaccharide production (visualized by Ziehl-Neelsen stain), membrane permeability (demonstrated as N-phenyl-1-naphthylamine uptake), biofilm formation, and established biofilm development (measured as total mass and polysaccharide levels) were analyzed in the presence of 0 μg/ml (control), 1 μg/ml, 5 μg/ml, and 10 μg/ml of HNP-1. Planktonic growth of the strains AHN 9508 and ATCC 25586 were significantly (p<0.05) increased in the presence of HNP-1, while their membrane permeability decreased (p<0.005) in the planktonic form. HNP-1 decreased the biofilm formation of the strains ATCC 25586 and AHN 9910, whereas it increased the growth of the strain AHN 9508 in established biofilms. Capsule formation and polysaccharide production were not observed in any strain. We conclude that the inhibition of the membrane permeability and the increase in planktonic and established biofilm growth could act as bacterial defense mechanisms against neutrophilic defensins. In addition, this strain-dependent survival ability against HNP-1 may explain the variation in the virulence of different F. nucleatum strains. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Toll-like receptor-induced reactivity and strongly potentiated IL-8 production in granulocytes mobilized for transfusion purposes.

    Science.gov (United States)

    Drewniak, Agata; Tool, Anton T J; Geissler, Judy; van Bruggen, Robin; van den Berg, Timo K; Kuijpers, Taco W

    2010-06-03

    Transfusion of granulocytes from granulocyte-colony stimulating factor (G-CSF)/dexamethasone (dexa)-treated donors can be beneficial for neutropenic recipients that are refractory to antimicrobial therapy. G-CSF/dexa treatment not only increases the number of circulating neutrophils but also affects their gene expression. Because of the intended transfusion of these granulocytes into patients who are severely ill, it is of importance to establish to what extent mobilization affects the cellular behavior of neutrophils. Here, we studied the effects of mobilization on Toll-like receptor (TLR)-mediated responses. Mobilized granulocytes displayed increased gene and protein expression of TLR2, TLR4, TLR5, and TLR8. Although mobilized granulocytes displayed normal priming of nicotinamide adenine dinucleotide phosphate oxidase activity and a slight increase in adhesion in response to TLR stimulation, these cells produced massive amounts of interleukin-8 (IL-8), in particular to TLR2 and TLR8 stimulation. The increase in IL-8 release occurred despite reduced IL-8 mRNA levels in the donor granulocytes after in vivo G-CSF/dexa treatment, indicating that the enhanced TLR-induced IL-8 production was largely determined by posttranscriptional regulation. In summary, granulocytes mobilized for transfusion purposes show enhanced TLR responsiveness in cytokine production, which is anticipated to be beneficial for the function of these cells on transfusion into patients.

  13. The Natural Stilbenoid Piceatannol Decreases Activity and Accelerates Apoptosis of Human Neutrophils: Involvement of Protein Kinase C

    Directory of Open Access Journals (Sweden)

    Viera Jancinova

    2013-01-01

    Full Text Available Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40phox—a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular membranes. The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC—the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α, βII, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues.

  14. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation

    NARCIS (Netherlands)

    B.J. van Raam (Bram); W.J. Sluiter (Wim); F.R.C. de Wit (Frank); D. Roos (Dirk); A.J. Verhoeven (Arthur); T.W. Kuijpers (Taco W.)

    2008-01-01

    textabstractBackground: Neutrophils depend mainly on glycolysis for their enegry provision. Their mitochondria maintain a membrace potential (ΔΨm), which is usually generated by the repiratory chain complexes. We investigated the source of ΔΨm in neutrophils, as compared to peripheral blood

  15. Sexy again: the renaissance of neutrophils in psoriasis.

    Science.gov (United States)

    Schön, Michael P; Broekaert, Sigrid M C; Erpenbeck, Luise

    2017-04-01

    Notwithstanding their prominent presence in psoriatic skin, the functional role of neutrophilic granulocytes still remains somewhat enigmatic. Sparked by exciting scientific discoveries regarding neutrophil functions within the last years, the interest in these short-lived cells of the innate immune system has been boosted recently. While it had been known for some time that neutrophils produce and respond to a number of inflammatory mediators, recent research has linked neutrophils with the pathogenic functions of IL-17, possibly in conjunction with the formation of NETs (neutrophil extracellular traps). Antipsoriatic therapies exert their effects, at least in part, through interference with neutrophils. Neutrophils also appear to connect psoriasis with comorbid diseases. However, directly tampering with neutrophil functions is not trivial as evinced by the failure of therapeutic approaches targeting redundantly regulated cellular communication networks. It has also become apparent that neutrophils link important pathogenic functions of the innate and the adaptive immune system and that they are intricately involved in regulatory networks underlying the pathophysiology of psoriasis. In order to advocate intensified research into the role of this interesting cell population, we here highlight some features of neutrophils and put them into perspective with our current view of the pathophysiology of psoriasis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Aspirin-triggered lipoxins override the apoptosis-delaying action of serum amyloid A in human neutrophils: a novel mechanism for resolution of inflammation.

    Science.gov (United States)

    El Kebir, Driss; József, Levente; Khreiss, Tarek; Pan, Wanling; Petasis, Nicos A; Serhan, Charles N; Filep, János G

    2007-07-01

    Elevated plasma levels of the acute-phase reactant serum amyloid A (SAA) have been used as a marker and predictor of inflammatory diseases. SAA regulates leukocyte activation; however, it is not known whether it also modulates neutrophil apoptosis, which is critical to the optimal expression and resolution of inflammation. Culture of human neutrophils with SAA (0.1-20 microg/ml) markedly prolonged neutrophil longevity by delaying constitutive apoptosis. SAA evoked concurrent activation of the ERK and PI3K/Akt signaling pathways, leading to phosphorylation of BAD at Ser(112) and Ser(136), respectively, and to prevention of collapse of mitochondrial transmembrane potential, cytochrome c release, and caspase-3 activation. These actions were abrogated by pharmacological inhibition of the formyl peptide receptor, ERK or PI3K. Furthermore, aspirin-triggered 15-epi-lipoxin A(4) (15-epi-LXA(4)) and its stable analog 15-epi-16-p-fluorophenoxy-LXA(4), which binds to the same receptor as SAA, effectively overrode the antiapoptosis signal from SAA even when neutrophils were treated with 15-epi-LXA(4) at either 1 or 4 h postculture with SAA. 15-Epi-LXA(4) itself did not affect neutrophil survival and apoptosis. Our results indicate that SAA at clinically relevant concentrations promotes neutrophil survival by suppressing the apoptotic machinery, an effect that can be opposed by 15-epi-LXA(4). The opposing actions of SAA and aspirin-triggered 15-epi-LXA(4) may contribute to the local regulation of exacerbation and resolution of inflammation, respectively.

  17. Relationship between chemical composition and biological function of Pseudomonas aeruginosa lipopolysaccharide: effect on human neutrophil chemotaxis and oxidative burst

    DEFF Research Database (Denmark)

    Kharazmi, A; Fomsgaard, A; Conrad, R S

    1991-01-01

    of LPS. After preincubation, the chemotaxis and chemiluminescence of neutrophils to various stimuli were determined. It was shown that LPS from different strains did not exert the same degree of regulatory effect on neutrophil functions. LPS from strain 174-O:9 exerted the most pronounced effect...... on neutrophil function seen as inhibition of neutrophil chemotaxis toward the chemotactic peptide f-Met-Leu-Phe and zymosan-activated serum (ZAS) and priming of the cells for less than or equal to 8-fold enhancement of chemiluminescence response to f-Met-Leu-Phe. Conversely, LPS from strain 1118-O:3 had...... no effect on neutrophil chemotaxis and a slight effect on chemiluminescence. The major differences in chemical composition of the LPS from these two strains are in the rhamnose and heptose content of the O side chain and in the alanine content of the core region. These data indicate that chemical...

  18. Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

    Directory of Open Access Journals (Sweden)

    Campbell Kevin P

    2007-08-01

    Full Text Available Abstract Background Polymorphonuclear neutrophils (PMN constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.

  19. Effects of Rhodomyrtus tomentosa Extract on Killing Activity of Human Neutrophils and Membrane Integrity of Enterohaemorrhagic Escherichia coli O157:H7.

    Science.gov (United States)

    Hmoteh, Jutharat; Syed Musthafa, Khadar; Pomwised, Rattanaruji; Voravuthikunchai, Supayang Piyawan

    2016-05-27

    Enterohaemorrhagic Escherichia coli (E. coli) O157:H7 is one of the most virulent causative agents of foodborne disease. Use of antibiotics for the treatment against E. coli O157:H7 infection leads to hemolytic uremic syndrome. The present study evaluated the potential of ethanolic leaf extract of a medicinal plant, Rhodomyrtus tomentosa in enhancing the killing activity of human neutrophils against E. coli O157:H7. In addition, the effects of the extract on membrane permeability of the organisms were studied. In the killing assay, percentage survival of the bacterial cells after being exposed to human neutrophils in the presence of various concentrations of the extract were determined. At 45 min, percentage survival of E. coli O157:H7 and E. coli ATCC 25922 after treated with neutrophils in the presence of the extract at 125-250 µg/mL was 58.48%-50.28% and 69.13%-35.35%, respectively. Furthermore, upon treatment with R. tomentosa at 250 µg/mL uptake of crystal violet by E. coli O157:H7 and E. coli ATCC 25922 was increased to 40.07% and 36.16%, respectively. Therefore, it is suggested that the extract exhibited dual effects as immunostimulant and membrane permeabilizing agent perhaps resulted in enhancing the killing activity of neutrophils against the organisms.

  20. Combination therapy of human umbilical cord blood cells and granulocyte colony stimulating factor reduces histopathological and motor impairments in an experimental model of chronic traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Sandra A Acosta

    Full Text Available Traumatic brain injury (TBI is associated with neuro-inflammation, debilitating sensory-motor deficits, and learning and memory impairments. Cell-based therapies are currently being investigated in treating neurotrauma due to their ability to secrete neurotrophic factors and anti-inflammatory cytokines that can regulate the hostile milieu associated with chronic neuroinflammation found in TBI. In tandem, the stimulation and mobilization of endogenous stem/progenitor cells from the bone marrow through granulocyte colony stimulating factor (G-CSF poses as an attractive therapeutic intervention for chronic TBI. Here, we tested the potential of a combined therapy of human umbilical cord blood cells (hUCB and G-CSF at the acute stage of TBI to counteract the progressive secondary effects of chronic TBI using the controlled cortical impact model. Four different groups of adult Sprague Dawley rats were treated with saline alone, G-CSF+saline, hUCB+saline or hUCB+G-CSF, 7-days post CCI moderate TBI. Eight weeks after TBI, brains were harvested to analyze hippocampal cell loss, neuroinflammatory response, and neurogenesis by using immunohistochemical techniques. Results revealed that the rats exposed to TBI treated with saline exhibited widespread neuroinflammation, impaired endogenous neurogenesis in DG and SVZ, and severe hippocampal cell loss. hUCB monotherapy suppressed neuroinflammation, nearly normalized the neurogenesis, and reduced hippocampal cell loss compared to saline alone. G-CSF monotherapy produced partial and short-lived benefits characterized by low levels of neuroinflammation in striatum, DG, SVZ, and corpus callosum and fornix, a modest neurogenesis, and a moderate reduction of hippocampal cells loss. On the other hand, combined therapy of hUCB+G-CSF displayed synergistic effects that robustly dampened neuroinflammation, while enhancing endogenous neurogenesis and reducing hippocampal cell loss. Vigorous and long-lasting recovery of

  1. Binding of human IgG to single-walled carbon nanotubes accelerated myeloperoxidase-mediated degradation in activated neutrophils.

    Science.gov (United States)

    Ding, Yun; Tian, Rong; Yang, Zhen; Chen, Jianfa; Lu, Naihao

    2016-11-01

    The binding of protein to carboxylated single-walled carbon nanotubes (SWCNTs) was believed to play an important role in the biological effects of nanotubes. However, the effects of protein-SWCNTs interactions on the oxidative degradation of nanotubes were not stressed enough. Here, we investigated the binding of human immunoglobulin G (IgG) to SWCNTs, and found that the preferred binding site was located in the Fc region of IgG. The hydrophobic and electrostatic interactions might be the crucial factors in stabilizing the binding of SWCNTs with IgG. Through the competitive binding of IgG and myeloperoxidase (MPO) to nanotube surfaces, the binding of IgG could impair MPO-induced SWCNTs biodegradation in vitro. However, both SWCNTs and IgG-SWCNTs were significantly degraded in zymosan-stimulated neutrophils, and the degradation degree was more for IgG-SWCNTs. These results suggest that the binding of IgG may be an important determinant for MPO-mediated SWCNTs biodegradation in activated human inflammatory cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Binding of human serum albumin to single-walled carbon nanotubes activated neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes.

    Science.gov (United States)

    Lu, Naihao; Li, Jiayu; Tian, Rong; Peng, Yi-Yuan

    2014-06-16

    Previous studies have shown that carboxylated single-walled carbon nanotubes (SWCNTs) can be catalytically biodegraded by hypochlorite (OCl-) and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase (MPO). However, the importance of protein-SWCNT interactions in the biodegradation of SWCNTs was not stressed. Here, we used both experimental and theoretical approaches to investigate the interactions of SWCNTs with human serum albumin (HSA, one of the most abundant proteins in blood circulation) and found that the binding was involved in the electrostatic interactions of positively charged Arg residues of HSA with the carboxyls on the nanotubes, along with the π-π stacking interactions between SWCNTs and aromatic Tyr residues in HSA. Compared with SWCNTs, the binding of HSA could result in a reduced effect for OCl- (or the human MPO system)-induced SWCNTs degradation in vitro. However, the HSA-SWCNT interactions would enhance cellular uptake of nanotubes and stimulate MPO release and OCl- generation in neutrophils, thereby creating the conditions favorable for the degradation of the nanotubes. Upon zymosan stimulation, both SWCNTs and HSA-SWCNTs were significantly biodegraded in neutrophils, and the degree of biodegradation was more for HSA-SWCNTs under these relevant in vivo conditions. Our findings suggest that the binding of HSA may be an important determinant for MPO-mediated SWCNT biodegradation in human inflammatory cells and therefore shed light on the biomedical and biotechnological applications of safe carbon nanotubes by comprehensive preconsideration of their interactions with human serum proteins.

  3. Production of inflammatory mediators and extracellular traps by carp macrophages and neutrophils in response to lipopolysaccharide and/or interferon-¿2

    NARCIS (Netherlands)

    Pijanowski, L.; Scheer, M.H.; Verburg-van Kemenade, B.M.L.; Chadzinska, M.K.

    2015-01-01

    Neutrophilic granulocytes and macrophages are crucial for the innate immune response against infections. They migrate into the focus of inflammation, where they efficiently bind, engulf and kill bacteria by proteolytic enzymes, antimicrobial peptides, reactive oxygen (ROS) and nitrogen (RNS)

  4. The Granulocyte-colony stimulating factor has a dual role in neuronal and vascular plasticity

    Directory of Open Access Journals (Sweden)

    Stephanie eWallner

    2015-08-01

    Full Text Available Granulocyte-colony stimulating factor (G-CSF is a growth factor that has originally been identified several decades ago as a hematopoietic factor required mainly for the generation of neutrophilic granulocytes, and is in clinical use for that. More recently, it has been discovered that G-CSF also plays a role in the brain as a growth factor for neurons and neural stem cells, and as a factor involved in the plasticity of the vasculature. We review and discuss these dual properties in view of the neuroregenerative potential of this growth factor.

  5. PARTICIPATION OF TLR4 IN ENGULFMENT OF ESCHERICHIA COLI BY HUMAN BLOOD NEUTROPHILS IN PRESENCE OF LIPOPOLYSACCHARIDES

    Directory of Open Access Journals (Sweden)

    S. V. Zubova

    2012-01-01

    Full Text Available Abstract. TLR4 is a key player in signaling system of host cells. Possible role of TLR4 is actively discussed, e.g. its significance for phagocytosis. A capacity of neutrophils to engulf FITC-labeled E. coli bacteria upon activation with LPS of different origin was studied in presence of anti-TLR4 Mab’s (HTA125 clone. It was shown that, in whole blood, TLR4 does not play any essential role in engulfment of bacteria by the neutrophils. Phagocytic activity of neutrophils in blood increases increased after their priming with E. coli endotoxins. LPS from Rb. сapsulatus did not affect phagocytosis. In presence of endotoxins, the degree of TLR4 involvement in neutrophil phagocytosis depends on LPS structure.

  6. Effect of Isolation Techniques on Viability of Bovine Blood Neutrophils

    Directory of Open Access Journals (Sweden)

    P. Sláma

    2006-01-01

    Full Text Available The effect of selected isolation methods on the viability of neutrophil granulocytes (neutrophils from the blood of healthy Holstein x Bohemian Red Pied crossbred heifers was evaluated. Two methods of neutrophil isolation were used: a neutrophil isolation on the basis of hypotonic erythrocyte lysis (in two variants: after the erythrocyte lysis proper, the cells were centrifuged at either 200 g or 1000 g, and b neutrophil isolation with FACS Lysing Solution as the lysing agent. The viability of the isolated neutrophils was evaluated on the basis of apoptosis and necrosis. The results obtained with flow cytometry (FCM suggest that, from the isolation techniques used, the method based on FACS Lysing Solution impaired the neutrophil viability least. After the application of this method, 5.36 ± 2.15% of neutrophils were apoptotic and 0.51 ± 0.12% were necrotic. In contrast, when the hypotonic erythrocyte lysis was used, the proportion of apoptotic neutrophils amounted to 42.14 ± 7.12% and 49.00 ± 14.70%, respectively, and 41.12 ± 5.55% and 36.91 ± 24.38% respectively of necrotic neutrophils (P < 0.01. This was also confirmed by the light microscopy. After the isolation with FASC Lysing Solution, 1.92 ± 1.74% of neutrophils were apoptotic and 1.05 ± 0.76% were necrotic, as distinct from after the hypotonic erythrocyte lysis where 9.43 ± 3.69% of neutrophils were apoptotic and 12.67 ± 4.74% of necrotic after centrifugation at 200 g, while 12.60 ± 4.35 were apoptotic and 14.96 ± 12.64% were necrotic after centrifugation at 1000 g. It follows from the above-mentioned data that hypotonic lysis is not a suitable method for the isolation of neutrophils, as the method itself markedly affects cell viability.

  7. Acetylcholine stimulation of human neutrophil chemotactic activity is directly inhibited by tiotropium involving Gq and ERK-1/2 regulation

    Directory of Open Access Journals (Sweden)

    Kurai M

    2012-01-01

    Full Text Available Tiotropium, a long-acting anticholinergic, may improve chronic obstructive pulmonary disease (COPD by mechanisms beyond bronchodilatation. We tested the hypothesis that tiotropium may act as an anti-inflammatory mediator by directly acting on and inhibiting human neutrophil chemotactic activity (NCA that is promoted by acetylcholine (ACh exposure. ACh treatment increased NCA in a dose dependent manner (p < 0.001 and tiotropium pretreatment reduced ACh stimulation (dose effect; 0 to 1000 nM; p < 0.001. Selective muscarinic receptor inhibitors demonstrated that subtype-3 (M3 receptor plays a role in NCA regulation. In addition, NCA that was stimulated by cevimeline (M3 agonist and pasteurella multocida toxin (PMT, M3 coupled Gq agonist. However, the increased NCA to cevimeline and PMT was reduced by tiotropium pretreatment (p < 0.001. ACh treatment stimulated ERK-1/2 activation by promoting protein phosphorylation and tiotropium reduced this effect (p < 0.01. In addition, pretreatment of the cells with a specific MEK-1/2 kinase inhibitor reduced ACh stimulated NCA (p < 0.01. Together these results demonstrated that cholinergic stimulation of NCA is effectively inhibited by tiotropium and is governed by a mechanism involving M3 coupled Gq signaling and downstream ERK signaling. This study further demonstrates that tiotropium may act as an anti-inflammatory agent in lung disease.

  8. Natural glucocorticoids induce expansion of all developmental stages of murine bone marrow granulocytes without inhibiting function

    Science.gov (United States)

    Trottier, Mark D.; Newsted, Matthew M.; King, Louis E.; Fraker, Pamela J.

    2008-01-01

    Natural glucocorticoids (Gc) produced during stress have profound effects on the immune system. It is well known that Gc induce apoptosis in precursor T and B cells, markedly altering lymphopoiesis. However, it has been noted that marrow myeloid cells expanded both in proportion and absolute numbers in the mouse after Gc exposure. Mice were implanted with a corticosterone (CS) tablet that increased serum Gc and caused atrophied thymuses, both classic signs of activation of the stress axis. Blood neutrophil counts were elevated (4.8×), whereas lymphocyte counts declined. Flow cytometric analysis of the marrow revealed that the phenotypic distribution of the various major classes of cells was shifted by Gc exposure. As expected, marrow lymphocyte numbers declined >40% after 3 days of exposure to Gc. Conversely, in the myeloid compartment, both monocytes and granulocytes increased in number by >40%. Further, all granulocyte developmental stages showed large increases in both total number and percentage of cells. To investigate the functional capacity of mature granulocytes from Gc-treated mice, an improved granulocyte isolation method was developed. Gc exposure had little effect on the ability of granulocytes to produce superoxide or undergo chemotaxis or phagocytose bacteria. These results indicate that Gc treatment shifts bone marrow composition and provides evidence that granulocytes and their progenitors are selectively preserved under stressful conditions without losing function. PMID:18250324

  9. Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with L-lysine

    NARCIS (Netherlands)

    Peschel, A.; Jack, R.W.; Otto, M.; Collins, L.V.; Staubitz, P.; Nicholson, G.; Kalbacher, H.; Nieuwenhuizen, W.F.; Jung, G.; Tarkowski, A.; Kessel, K.P.M. van; Strijp, J.A.G. van

    2001-01-01

    Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance

  10. Peptide conjugated cellulose nanocrystals with sensitive human neutrophil elastase sensor activity

    Science.gov (United States)

    In chronic wounds, elevated human neutorphil elastase (HNE) is a destructive protease that has been proposed as a biomarker. Numerous wound dressing designs have been introduced in an effort to lower HNE levels. The clinical detection of HNE as a point of care biomarker or an in situ colorimetric ...

  11. Effects of pyrrophenone, an inhibitor of group IVA phospholipase A2, on eicosanoid and PAF biosynthesis in human neutrophils

    Science.gov (United States)

    Flamand, N; Picard, S; Lemieux, L; Pouliot, M; Bourgoin, S G; Borgeat, P

    2006-01-01

    Background and Purpose: The biosynthesis of leukotrienes (LT) and platelet-activating factor (PAF) involves the release of their respective precursors, arachidonic acid (AA) and lyso-PAF by the group IVA PLA2 (cPLA2α). This paper aims at characterizing the inhibitory properties of the cPLA2α inhibitor pyrrophenone on eicosanoids and PAF in human neutrophils (PMN). Experimental Approach: Freshly isolated human PMN were activated with physiological and pharmacological agents (fMLP, PAF, exogenous AA, A23187 and thapsigargin) in presence and absence of the cPLA2α inhibitor pyrrophenone and biosynthesis of LT, PAF, and PGE2 was measured. Key Results: Pyrrophenone potently inhibited LT, PGE2 and PAF biosynthesis in PMN with IC50s in the range of 1–20 nM. These inhibitory effects of pyrrophenone were specific (the consequence of substrate deprivation), as shown by the reversal of inhibition by exogenous AA and lyso-PAF. Comparative assessment of pyrrophenone, methyl-arachidonoyl-fluoro-phosphonate (MAFP) and arachidonoyl-trifluoromethylketone (AACOCF3) demonstrated that pyrrophenone was more specific and 100-fold more potent than MAFP and AACOCF3 for the inhibition of LT biosynthesis in A23187-activated PMN. The inhibitory effect of pyrrophenone on LT biosynthesis was reversible as LT biosynthesis was recovered when pyrrophenone-treated PMN were washed with autologous plasma. No alteration of phospholipase D (PLD) activity in fMLP-activated PMN was observed with up to 10 μM pyrrophenone, suggesting that the cPLA2α inhibitor does not directly inhibit PLD. Conclusions and Implications: Pyrrophenone is a more potent and specific cPLA2α inhibitor than MAFP and AACOCF3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of eicosanoids and PAF. PMID:16967052

  12. In vivo kinetics of sup 111 Indium-labelled autologous granulocytes following i. v. administration of granulocyte-macrophage colony-stimulating factor (GM-CSF)

    Energy Technology Data Exchange (ETDEWEB)

    Hovgaard, D.; Mortensen, B.T.; Nissen, N.I. (Department of Hematology, Rigshospitalet, Copenhagen (Denmark)); Schifter, S.; Raboel, A. (Department of Clinical Physiology and Nuclear Medicine, Rigshospitalet, Copenhagen (Denmark))

    1992-01-01

    Administration of both glycosylated and non-glycosylated recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) induces an immediate transient granulocytopenia of 1-3 hours' duration. In order to explore this phenomenon, granulocytes were labelled with {sup 111}Indium and the effect on the kinetics of granulocytes after administration of rhGM-CSF was studied in 10 previously untreated patients with malignant lymphoma. For both types and doses of rhGM-CSF, a significant and dramatic accumulation of the {sup 111}Indium-labelled granulocytes was observed in the lung within a few minutes after i.v. injection of rhGM-CSF. The accumulation of radioactivity coincided with the pronounced and transient granulocytopenia in peripheral blood. The {sup 111}Indium-labelled granulocytes later reappeared in the peripheral blood, indicating reversible pulmonary vascular margination of the granulocytes. Half-life of labelled granulocytes after reappearance was comparable to half-life values under normal conditions. The transient accumulation of granulocytes in the pulmonary vessels seems not to be of clinical importance in the management of patients, but it may to some degree explain previously described side-effects, such as transient hypoxemia (''first-dose'' reaction) following administration of rhGM-CSF. (au).

  13. Changes in gene expression of granulocytes during in vivo granulocyte colony-stimulating factor/dexamethasone mobilization for transfusion purposes

    NARCIS (Netherlands)

    Drewniak, Agata; van Raam, Bram J.; Geissler, Judy; Tool, Anton T. J.; Mook, Olaf R. F.; van den Berg, Timo K.; Baas, Frank; Kuijpers, Taco W.

    2009-01-01

    The treatment of healthy donors with granulocyte colony-stimulating factor (G-CSF) and dexamethasone results in sufficient numbers of circulating granulocytes to prepare granulocyte concentrates for clinical purposes. Granulocytes obtained in this way demonstrate relatively normal functional

  14. Solonamide B Inhibits Quorum Sensing and Reduces Staphylococcus aureus Mediated Killing of Human Neutrophils

    DEFF Research Database (Denmark)

    Nielsen, Anita; Månsson, Maria; Bojer, Martin S.

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a serious human pathogen, and particularly the spread of community associated (CA)-MRSA strains such as USA300 is a concern, as these strains can cause severe infections in otherwise healthy adults. Recently, we reported...... that a cyclodepsipeptide termed Solonamide B isolated from the marine bacterium, Photobacterium halotolerans strongly reduces expression of RNAIII, the effector molecule of the agr quorum sensing system. Here we show that Solonamide B interferes with the binding of S. aureus autoinducing peptides (AIPs) to sensor......A controlled virulence gene expression in S. aureus....

  15. Granulocyte transfusion combined with granulocyte colony stimulating factor in severe infection patients with severe aplastic anemia: a single center experience from China.

    Directory of Open Access Journals (Sweden)

    Huaquan Wang

    Full Text Available OBJECTIVE: To investigate the efficacy and safety of granulocyte transfusion combined with granulocyte colony stimulating factor (G-CSF in severe infection patients with severe aplastic anemia (SAA. METHODS: Fifty-six patients in severe infections with SAA who had received granulocyte transfusions combined with G-CSF from 2006 to 2012 in our department were analyzed. A retrospective analysis was undertaken to investigate the survival rates (at 30 days, 90 days and 180 days, the responses to treatment (at 7 days and 30 days, including microbiological, radiographic and clinical responses, the neutrophil count and adverse events after transfusion. RESULTS: All SAA patients with severe infections were treated with granulocyte transfusions combined with G-CSF. Forty-seven patients had received antithymocyte globulin/antilymphocyte globulin and cyclosporine A as immunosuppressive therapy. The median number of granulocyte components transfused was 18 (range, 3-75. The survival at 30 days, 90 days and 180 days were 50(89%, 39(70% and 37(66% respectively. Among 31 patients who had invasive fungal infections, the survival at 30 days, 90 days and 180 days were 27(87%, 18(58% and 16(52% respectively. Among the 25 patients who had refractory severe bacterial infections, the survival at 30 days, 90 days and 180 days were 23(92%, 21(84% and 21(84% respectively. Survival rate was correlated with hematopoietic recovery. Responses of patients at 7 and 30 days were correlated with survival rate. Common adverse effects of granulocyte transfusion included mild to moderate fever, chills, allergy and dyspnea. CONCLUSION: Granulocyte transfusions combined with G-CSF could be an adjunctive therapy for treating severe infections of patients with SAA.

  16. Crystalline inclusions in granulocytic sarcoma.

    Science.gov (United States)

    Strauchen, James A; Gordon, Ronald E

    2002-01-01

    Two cases of granulocytic sarcoma were found to contain numerous crystalline inclusions identified on hematoxylin-eosin-stained sections as clusters of pointed needlelike crystals present in foci of necrosis or within macrophages. The crystals were negative for chloroacetate esterase and myeloperoxidase. Electron microscopy demonstrated homogeneously dense, bipyramidal structures, indistinguishable from Charcot-Leyden crystals. Granulocytic sarcomas may contain crystalline inclusions similar to Charcot-Leyden crystals; these structures should be distinguished from crystalline immunoglobulin inclusions occurring in cases of plasma cell myeloma and lymphoplasmacytic lymphoma, which may have a similar appearance.

  17. The effect of ethanol and N-nitrosodimethylamine on the iNOS-dependent NO production in human neutrophils. Role of NF-κB.

    Science.gov (United States)

    Nowak, Karolina; Ratajczak-Wrona, Wioletta; Garley, Marzena; Jabłońska, Ewa

    2017-06-30

    1. The objective of study was to determine the influence of ethanol and/or N-nitrosodimethyloamine (NDMA) on the inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by human neutrophils and determination of the role of NF-κB in this process. 2. Isolated polymorphonuclear leukocytes (PMNs) derived from 15 human volunteers were incubated in the presence of ethanol and/or NDMA. Expression of the tested proteins were evaluated using the Western blot method. Total NO metabolites was assayed in the cell cultures by Griess reaction. 3. In neutrophils exposed to ethanol or NDMA was observed an increased NF-κB-dependent NO production mediated by iNOS with the contribution of MAP kinases: p38 and JNK. An inhibiting effect of NF-κB signaling pathway on the MAP kinases was observed, which are involved in the iNOS-dependent NO production. By the simultaneous effect, ethanol and NDMA caused stronger generation of NO by neutrophils without the contribution of iNOS. Inhibition of NF-κB in cells simultaneously exposed to the xenobiotics caused a decreased expression of MAP kinases. 4. Individual and simultaneous effect of ethanol and NDMA may cause disorders in the response of immune system. However, the joint effect of the tested substances results in uncontrolled interactions, leading to cascading disorders of signal transduction.

  18. Unsaturated long-chain fatty acids induce the respiratory burst of human neutrophils and monocytes in whole blood

    Directory of Open Access Journals (Sweden)

    Osthaus Wilhelm A

    2008-07-01

    Full Text Available Abstract Background It is increasingly recognized that infectious complications in patients treated with total parenteral nutrition (TPN may be caused by altered immune responses. Neutrophils and monocytes are the first line of defence against bacterial and fungal infection through superoxide anion production during the respiratory burst. To characterize the impact of three different types of lipid solutions that are applied as part of TPN formulations, we investigated the unstimulated respiratory burst activation of neutrophils and monocytes in whole blood. Methods Whole blood samples were incubated with LCT (Intralipid®, LCT/MCT (Lipofundin® and LCT-MUFA (ClinOleic® in three concentrations (0.06, 0.3 and 0.6 mg ml-1 for time periods up to one hour. Hydrogen peroxide production during the respiratory burst of neutrophils and monocytes was measured by flow cytometry. Results LCT and LCT-MUFA induced a hydrogen peroxide production in neutrophils and monocytes without presence of a physiological stimulus in contrast to LCT/MCT. Conclusion We concluded that parenteral nutrition containing unsaturated oleic (C18:1 and linoleic (C18:2 acid can induce respiratory burst of neutrophils and monocytes, resulting in an elevated risk of tissue damage by the uncontrolled production of reactive oxygen species. Contradictory observations reported in previous studies may in part be the result of different methods used to determine hydrogen peroxide production.

  19. Presence of cytidine 5'-monophospho-N-acetylneuraminic acid:Gal beta 1-3GalNAc-R alpha(2-3)-sialyltransferase in normal human leukocytes and increased activity of this enzyme in granulocytes from chronic myelogenous leukemia patients.

    Science.gov (United States)

    Baker, M A; Kanani, A; Brockhausen, I; Schachter, H; Hindenburg, A; Taub, R N

    1987-06-01

    We have examined granulocytes from patients with chronic myelogenous leukemia (CML) and from normal subjects to determine whether activity of a specific sialyltransferase might account for the aberrant sialylation of O-linked membrane oligosaccharides in CML cells. Total membrane preparations of morphologically mature CML and normal granulocytes were tested for sialyltransferase activity using the substrates galactosyl-beta 1-3-N-acetyl-D-galactosamine-alpha-O-nitrophenyl and N-acetyl-D-galactosamine-alpha-phenyl. N-Acetyl-D-galactosamine-alpha-phenyl was not an acceptor with either CML or normal cells. With galactosyl-beta 1-3-N-acetyl-D-galactosamine-alpha-O-nitrophenyl, sialyltransferase activity was 2.8 times higher in CML cells compared to normal cells. Product identification by high performance liquid chromatography showed that enzyme from both normal and CML granulocytes linked sialic acid to galactosyl-beta 1-3-N-acetyl-D-galactosamine-R by the alpha(2-3) and not the alpha(2-6) linkage. The enzyme CMP-N-acetylneuraminic acid: galactosyl-beta 1-3-N-acetyl-D-galactosamine-R alpha(2-3)-sialyltransferase has not previously been described in human granulocytes. The marked increase in activity of this enzyme in CML and the resulting increase in sialylation may contribute to the pathophysiological behavior of CML granulocytes.

  20. Ezrin/Exocyst complex regulates mucin 5AC secretion induced by neutrophil elastase in human airway epithelial cells.

    Science.gov (United States)

    Li, Qi; Li, Na; Liu, Chun-Yi; Xu, Rui; Kolosov, Victor P; Perelman, Juliy M; Zhou, Xiang-Dong

    2015-01-01

    Increased mucin secretion is a characteristic feature of many chronic airway diseases, particularly during periods of exacerbation; however, the exact mechanism of mucin secretion remains unclear. Ezrin, which is a specific marker of apical membranes, is predominantly concentrated in exocyst-rich cell surface structures, crosslinking the actin cytoskeleton with the plasma membrane. In the present study, we examined whether Ezrin is involved in mucin 5AC (MUC5AC) secretion after neutrophil elastase (NE) attack, and we investigated the role of the exocyst complex docking protein Sec3 in this process. NE was used as a stimulator in a 16HBE14o- cell culture model. The expression and location of Ezrin and Sec3 were investigated, and the interaction between Ezrin and Sec3 in 16HBE14o-cells was assayed after treatment with NE, Ezrin siRNA, Sec3 siRNA, neomycin or PIP2-Ab. We found that Ezrin was highly expressed in the bronchi of humans with chronic airway diseases. NE induced robust MUC5AC protein secretion. The Ezrin siRNA, Sec3 siRNA, and neomycin treatments led to impaired MUC5AC secretion in cells. Both Ezrin and Sec3 were recruited primarily to the cytoplasmic membrane after NE stimulation, and the neomycin and PIP2-Ab treatments abrogated this effect. Immunoprecipitation analysis revealed that Ezrin and Sec3 combined to form complexes; however, these complexes could not be detected in Ezrin∆1-333 mutant-transfected cells, even when PIP2 was added. These results demonstrate that Ezrin/Sec3 complexes are essential for MUC5AC secretion in NE-stimulated airway epithelial cells and that PIP2 is of critical importance in the formation of these complexes. © 2015 S. Karger AG, Basel.

  1. Ezrin/Exocyst Complex Regulates Mucin 5AC Secretion Induced by Neutrophil Elastase in Human Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Qi Li

    2015-01-01

    Full Text Available Background/Aim: Increased mucin secretion is a characteristic feature of many chronic airway diseases, particularly during periods of exacerbation; however, the exact mechanism of mucin secretion remains unclear. Ezrin, which is a specific marker of apical membranes, is predominantly concentrated in exocyst-rich cell surface structures, crosslinking the actin cytoskeleton with the plasma membrane. In the present study, we examined whether Ezrin is involved in mucin 5AC (MUC5AC secretion after neutrophil elastase (NE attack, and we investigated the role of the exocyst complex docking protein Sec3 in this process. Methods: NE was used as a stimulator in a 16HBE14o- cell culture model. The expression and location of Ezrin and Sec3 were investigated, and the interaction between Ezrin and Sec3 in 16HBE14o-cells was assayed after treatment with NE, Ezrin siRNA, Sec3 siRNA, neomycin or PIP2-Ab. Results: We found that Ezrin was highly expressed in the bronchi of humans with chronic airway diseases. NE induced robust MUC5AC protein secretion. The Ezrin siRNA, Sec3 siRNA, and neomycin treatments led to impaired MUC5AC secretion in cells. Both Ezrin and Sec3 were recruited primarily to the cytoplasmic membrane after NE stimulation, and the neomycin and PIP2-Ab treatments abrogated this effect. Immunoprecipitation analysis revealed that Ezrin and Sec3 combined to form complexes; however, these complexes could not be detected in Ezrin∆1-333 mutant-transfected cells, even when PIP2 was added. Conclusions: These results demonstrate that Ezrin/Sec3 complexes are essential for MUC5AC secretion in NE-stimulated airway epithelial cells and that PIP2 is of critical importance in the formation of these complexes.

  2. Human mucosal leishmaniasis: neutrophils infiltrate areas of tissue damage that express high levels of Th17-related cytokines.

    Science.gov (United States)

    Boaventura, Viviane S; Santos, Claire S; Cardoso, Cristina R; de Andrade, José; Dos Santos, Washington L C; Clarêncio, Jorge; Silva, João S; Borges, Valeria M; Barral-Netto, Manoel; Brodskyn, Claudia I; Barral, Aldina

    2010-10-01

    Mucosal leishmaniasis (ML) is characterised by severe tissue destruction. Herein, we evaluated the involvement of the IL-17-type response in the inflammatory infiltrate of biopsy specimens from 17 ML patients. IL-17 and IL-17-inducing cytokines (IL-1β, IL-23, IL-6 and TGF-β) were detected by immunohistochemistry in ML patients. IL-17(+) cells exhibited CD4(+), CD8(+) or CD14(+) phenotypes, and numerous IL-17(+) cells co-expressed the CC chemokine receptor 6 (CCR6). Neutrophils, a hallmark of Th17-mediated inflammation, were regularly detected in necrotic and perinecrotic areas and stained positive for neutrophil elastase, myeloperoxidase and MMP-9. Taken together, these observations demonstrate the existence of Th17 cells in ML lesions associated with neutrophils in areas of tissue injury and suggest that IL-17 is involved in ML pathogenesis.

  3. Nutritionally relevant concentrations of resveratrol and hydroxytyrosol mitigate oxidative burst of human granulocytes and monocytes and the production of pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.

    Science.gov (United States)

    Bigagli, Elisabetta; Cinci, Lorenzo; Paccosi, Sara; Parenti, Astrid; D'Ambrosio, Mario; Luceri, Cristina

    2017-02-01

    The health benefits of bio-active phenolic compounds have been largely investigated in vitro at concentrations which exceed those reachable in vivo. We investigated and compared the anti-inflammatory effects of resveratrol, hydroxytyrosol and oleuropein at physiologically relevant concentrations by using in vitro models of inflammation. Human granulocytes and monocytes were stimulated with phorbol myristate acetate (PMA) and the ability of resveratrol, hydroxytyrosol and oleuropein to inhibit the oxidative burst and CD11b expression was measured. Nitric oxide (NO), prostaglandin E2 (PGE2) levels, COX-2, iNOS, TNFα, IL-1β and miR-146a expression and activation of the transcription factor Nrf2 were evaluated in macrophages RAW 264.7 stimulated with LPS (1μg/ml) for 18h, exposed to resveratrol, hydroxytyrosol and oleuropein (5 and 10μM). Synergistic effects were explored as well, together with the levels of PGE2, COX-2 and IL-1β expression in macrophages after 6h of LPS stimulation. PGE2 and COX-2 expression were also assessed on human monocytes. All the tested compounds inhibited granulocytes oxidative burst in a concentration dependent manner and CD11b expression was also significantly counteracted by resveratrol and hydroxytyrosol. The measurement of oxidative burst in human monocytes produced similar effects being resveratrol more active. Hydroxytyrosol and resveratrol inhibited the production of NO and PGE2 but did not reduce iNOS, TNFα or IL-1β gene expression in LPS-stimulated RAW 264.7 for 18h. Resveratrol slightly decreased COX-2 expression after 18h but not after 6h, but reduced PGE2 levels after 6h. Resveratrol and hydroxytyrosol 10μM induced NRf2 nuclear translocation and reduced miR-146a expression in LPS treated RAW 264.7. Overall, we reported an anti-inflammatory effect of resveratrol and hydroxytyrosol at low, nutritionally relevant concentrations, involving the inhibition of granulocytes and monocytes activation, the modulation of miR-146a

  4. Efficacy of transfusion with granulocytes from G-CSF/dexamethasone-treated donors in neutropenic patients with infection.

    Science.gov (United States)

    Price, Thomas H; Boeckh, Michael; Harrison, Ryan W; McCullough, Jeffrey; Ness, Paul M; Strauss, Ronald G; Nichols, W Garrett; Hamza, Taye H; Cushing, Melissa M; King, Karen E; Young, Jo-Anne H; Williams, Eliot; McFarland, Janice; Holter Chakrabarty, Jennifer; Sloan, Steven R; Friedman, David; Parekh, Samir; Sachais, Bruce S; Kiss, Joseph E; Assmann, Susan F

    2015-10-29

    High-dose granulocyte transfusion therapy has been available for 20 years, yet its clinical efficacy has never been conclusively demonstrated. We report here the results of RING (Resolving Infection in Neutropenia with Granulocytes), a multicenter randomized controlled trial designed to address this question. Eligible subjects were those with neutropenia (absolute neutrophil count granulocyte transfusions from donors stimulated with granulocyte colony-stimulating factor (G-CSF) and dexamethasone. The primary end point was a composite of survival plus microbial response, at 42 days after randomization. Microbial response was determined by a blinded adjudication panel. Fifty-six subjects were randomized to the granulocyte arm and 58 to the control arm. Transfused subjects received a median of 5 transfusions. Mean transfusion dose was 54.9 × 10(9) granulocytes. Overall success rates were 42% and 43% for the granulocyte and control groups, respectively (P > .99), and 49% and 41%, respectively, for subjects who received their assigned treatments (P = .64). Success rates for granulocyte and control arms did not differ within any infection type. In a post hoc analysis, subjects who received an average dose per transfusion of ≥0.6 × 10(9) granulocytes per kilogram tended to have better outcomes than those receiving a lower dose. In conclusion, there was no overall effect of granulocyte transfusion on the primary outcome, but because enrollment was half that planned, power to detect a true beneficial effect was low. RING was registered at www.clinicaltrials.gov as #NCT00627393. © 2015 by The American Society of Hematology.

  5. Elevated glucose concentrations promote receptor-independent activation of adherent human neutrophils: an experimental and computational approach

    DEFF Research Database (Denmark)

    Kummer, Ursula; Zobeley, Jürgen; Brasen, Jens Christian

    2007-01-01

    of NO and superoxide formation were observed. However, these changes were not observed for sorbitol, a nonmetabolizable carbohydrate. Glucose transport appears to be important in this process as phloretin interferes with the glucose-specific receptor-independent activation of neutrophils. However, LY83583...

  6. Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils.

    Science.gov (United States)

    Ledderose, Carola; Heyn, Jens; Limbeck, Elisabeth; Kreth, Simone

    2011-10-20

    The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR). This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes. The mRNA expression of 17 (T cells), 7 (neutrophils) or 8 (unselected leukocytes) potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells. The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

  7. Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

    Directory of Open Access Journals (Sweden)

    Ledderose Carola

    2011-10-01

    Full Text Available Abstract Background The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR. This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes. Results The mRNA expression of 17 (T cells, 7 (neutrophils or 8 (unselected leukocytes potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells. Conclusions The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

  8. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

    Directory of Open Access Journals (Sweden)

    Prajna Jena

    Full Text Available Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

  9. Neutrophil-to-lymphocyte ratio, calprotectin and YKL-40 in patients with chronic obstructive pulmonary disease

    DEFF Research Database (Denmark)

    Sørensen, Allan Klitgaard; Holmgaard, Dennis Back; Mygind, Lone Hagens

    2015-01-01

    BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and progressive decline in pulmonary function. Neutrophil-to-lymphocyte ratio (NLR), YKL-40 and calprotectin are biomarkers of inflammation and predict mortality in patients with different inflammatory...... diseases. We aimed to investigate the correlation between levels of these three biomarkers and neutrophil granulocyte and lymphocyte count in patients with moderate to very severe COPD stratified by use of systemic glucocorticoids. Furthermore, we studied the ability of these biomarkers to predict all...

  10. [Effects of recombinant human granulocyte-macrophage colony-stimulating factor on wound healing and microRNA expression in diabetic rats].

    Science.gov (United States)

    Liu, Yifeng; Liu, Dewu; Guo, Guanghua; Mao, Yuangui; Wang, Xianlin

    2014-06-01

    To investigate the effects of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) on wound healing and microRNA expression in diabetic rats. Eighteen male SD rats of clean grade were used to reproduce diabetes model. Four weeks later, a total of 64 full-thickness skin wounds were created on the back of 16 rats with established diabetes, with 4 wounds on each rat. Two symmetrical wounds on either side of the spine were created as a pair according to paired design. Then the wounds were divided into groups A and B according to the random number table and blind method (red and blue tags on the rhGM-CSF or the gel vehicle), with 32 wounds in each group. The ointment with red tag was applied on the wounds of group A and the blue one on group B. The application was conducted once a day, with a thickness of 3 mm, up to post injury day (PID) 14. Gross observation of wound healing was conducted on PID 3, 7, 14. The wound healing rate was determined on PID 3 and 7. On PID 3, 7, 14, tissues from 2, 4, and 8 wounds were harvested from each group respectively for the observation of the histopathological changes with HE staining, and also for analyzing the expression of proliferating cell nuclear antigen (PCNA) and CD31 with immunohistochemical staining (denoted as absorbance value). On PID 7, tissues from 6 wounds in each group were harvested for microarray gene chip to screen the differentially expressed microRNAs. Enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway on the differentially expressed microRNAs were performed after the microRNA screening results were validated by real-time fluorescent quantitative RT-PCR. Data were processed with paired t test or two-sample t test. (1) On PID 3, the wound area was significantly decreased, and the wound granulation was significantly proliferated in both groups. On PID 7, the wound area was further decreased, and the wound area was almost filled by granulation in both

  11. Original paper The effect of leptin on the respiratory burst of human neutrophils cultured in synovial fluid

    Directory of Open Access Journals (Sweden)

    Michał Gajewski

    2015-04-01

    Full Text Available Objectives: Leptin is a hormone responsible for nutritional status and immune competence coordination. In rheumatoid arthritis (RA increased leptin levels were observed in both serum and synovial fluid. Its influence on development of the disease still remains unclear. So far, research on leptin’s influence on the emission of reactive oxygen intermediates (ROI measured with chemiluminescence (CL has provided unclear and contradictory results. In this study, we evaluated the influence of leptin on oxidative activity of neutrophils isolated from blood of healthy volunteers and cultured in different amounts of synovial fluid (SF from patients with RA. Material and methods: Neutrophils’ oxidative metabolism was measured by two types of CL. The first one, luminol-dependent CL (CL-lum, allows one to determine phagocytic activity and the level of ROI generated in a myeloperoxidase-dependent manner. The second method used was lucigenin-dependent CL (CL-luc, which monitors ROI production dependent on the NADPH oxidase enzyme complex located in the cell membranes of neutrophils and enables one to determine the scope of extracellular ROI emission. Results: Neutrophils stimulated by opsonized zymosan show a decrease in the level of CL-lum, proportional to the increasing concentration of both SF and serum collected from healthy donors. The observed effect of decreased CL-lum may, therefore, be dependent on the physical conditions (viscosity of fluids used. None of these experiments showed any effect of leptin on the level of CL-lum. Conclusions : The present study showed that leptin does not affect the level of any of the CL types in inactive neutrophils incubated in normal serum, and it does not affect the level of oxidative activity in resting neutrophils incubated with SF. However, leptin influences extracellular ROI emission (measured by CL-luc. Leptin reduces extracellular emission of ROI, and this effect is dependent on concentration and duration

  12. Neutrophil Segmentation Index Anomaly in Acquired ...

    African Journals Online (AJOL)

    Neutrophil lobe count was conducted on the blood films of 262 patients with the Acquired Immunodeficiency Syndrome Virus (AIDS) and 204 Human Immunodeficiency Virus (HIV) antibody-negative apparently healthy controls. The count for each group was evaluated for neutrophil segmentation index by standard method.

  13. Distinct neutrophil subpopulations phenotype by flow cytometry in myelodysplastic syndromes.

    Science.gov (United States)

    Vikentiou, Myrofora; Psarra, Katerina; Kapsimali, Violetta; Liapis, Konstantinos; Michael, Michalis; Tsionos, Konstantinos; Lianidou, Evi; Papasteriades, Chryssa

    2009-03-01

    The cardinal feature of myelodysplastic syndromes (MDS) is dysplasia involving one or more myeloid cell lineages. In the present study, we used 4-color flow cytometric analysis to investigate dysgranulopoiesis in bone marrow specimens from 65 patients with MDS. The antigen expression patterns of total neutrophil granulocytes (TNG) and of the two distinct neutrophil granulocytic subpopulations (NGSs), NGS-1 (dimmer CD45 expression) and NGS-2 (stronger CD45 expression) identified on the side scatter (SS) vs. CD45-intensity plot, were studied. The neutrophil granulocytes from patients with MDS showed characteristic antigen expression aberrancies which were more pronounced in NGS-2 subpopulation. Studying separately the NGS-2 subpopulation with the CD16/MPO/LF combination, the low CD16(+)/MPO(+) and low CD16(+)/LF(+) percentages seemed to discriminate between lower-risk and higher-risk patients with MDS in most occasions. Furthermore, a detailed assessment of the NGS-1 and NGS-2 immunophenotypic patterns revealed early dysplastic changes, not otherwise observed by standard TNG analysis, especially in cases of lower-risk MDS.

  14. Granulocytes of reptilian sauropsids contain beta-defensin-like peptides: a comparative ultrastructural survey.

    Science.gov (United States)

    Alibardi, Lorenzo

    2013-08-01

    The ability of lizards to withstand infections after wounding or amputation of the tail or limbs has suggested the presence of antimicrobial peptides in their tissues. Previous studies on the lizard Anolis carolinensis have identified several beta-defensin-like peptides that may potentially be involved in protection from infections. The present ultrastructural immunocytochemical study has analyzed tissues in different reptilian species in order to localize the cellular source of one of the more expressed beta-defensins previously sequenced in lizard indicated as AcBD15. Beta-defensin-like immunoreactivity is present in some of the larger, nonspecific granules of granulocytes in two lizard species, a snake, the tuatara, and a turtle. The ultrastructural study indicates that only heterophilic and basophilic granulocytes contain this defensin while other cell types from the epidermis, mesenchyme, and dermis, muscles, nerves, cartilage or bone are immunonegative. The study further indicates that not all granules in reptilian granulocytes contain the beta-defensin peptide, suggesting the presence of granules with different content as previously indicated for mammalian neutrophilic leucocytes. No immunolabeling was instead observed in granulocytes of the alligator and chick using this antibody. The present immunocytochemical observations suggest a broad cross-reactivity and conservation of beta-defensin-like sequence or steric motif across lepidosaurians and likely in turtles while archosaurian granulocytes may contain different beta-defensin-like or other peptides. Copyright © 2013 Wiley Periodicals, Inc.

  15. The granulocytes in neutropenia 1 (GIN 1) study: a safety study of granulocytes collected from whole blood and stored in additive solution and plasma.

    Science.gov (United States)

    Massey, Edwin; Harding, Kay; Kahan, Brennan C; Llewelyn, Charlotte; Wynn, Robert; Moppett, John; Robinson, Stephen P; Green, Ann; Lucas, Geoff; Sadani, Deepak; Liakopoulou, Effie; Bolton-Maggs, Paula; Marks, David I; Stanworth, Simon

    2012-08-01

    To evaluate the safety of transfusing pooled, whole blood-derived granulocytes in additive solution and plasma (GASP) in 30 recipients. Demand for granulocytes in England has increased five-fold. With the advantages of reduced red cell, plasma and overall volume, GASP maintains function in vitro. Observations were recorded prior to and post transfusion. Increments were recorded at 1 h and the following morning. Leucocyte antibody screening was undertaken prior to and at 1-6 months following transfusion. Thirty patients aged between 8 months and 68 years received 221 GASP in 148 transfusion episodes. GASP contained an average of 1.0 × 10(10) granulocytes in 207 mL. Adults usually received two packs and children 10-20 mL kg(-1). Children and adults received a median [interquartile range (IQR)] dose of 12.5 (9.1-25.3) and 19.7 (12.0-25.8) × 10(9) granulocytes per transfusion, respectively. There was one episode of transfusion-associated circulatory overload (TACO) in a patient with chronic cardiac failure following 600 mL of unpooled granulocytes, other fluids and one GASP. New leucocyte alloimmunisation occurred in 3/30 recipients 10%. No other significant reactions were reported. Median peripheral blood neutrophil increments at 1 h post transfusion were 0.06 (IQR, 0.01-0.17) in children and (0.03) (IQR, 0-0.16) in adults. GASP has a similar safety profile to other sources of granulocytes for patients with refractory infection or in need of secondary prophylactic transfusion. Further studies are required to clarify the role of GASP in the treatment of neutropenic patients. © 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society.

  16. Characterization of neutrophil adhesion to different titanium surfaces

    Indian Academy of Sciences (India)

    Home; Journals; Bulletin of Materials Science; Volume 37; Issue 1. Characterization of neutrophil adhesion to different titanium surfaces ... The aim of the present study is to compare the behaviours of neutrophils in direct contact with different Ti surfaces. Isolated human neutrophils were placed into contact with Ti discs, ...

  17. CFP-10 from Mycobacterium tuberculosis Selectively Activates Human Neutrophils through a Pertussis Toxin-Sensitive Chemotactic Receptor

    OpenAIRE

    Welin, Amanda; Björnsdottir, Halla; Winther, Malene; Christenson, Karin; Oprea, Tudor; Karlsson, Anna; Forsman, Huamei; Dahlgren, Claes; Bylund, Johan

    2014-01-01

    Upon infection with Mycobacterium tuberculosis, neutrophils are massively recruited to the lungs, but the role of these cells in combating the infection is poorly understood. Through a type VII secretion system, M. tuberculosis releases a heterodimeric protein complex, containing a 6-kDa early secreted antigenic target (ESAT-6) and a 10-kDa culture filtrate protein (CFP-10), that is essential for virulence. Whereas the ESAT-6 component possesses multiple virulence-related activities, no direc...

  18. Identification and characterization of CKLiK, a novel granulocyteCa^(++)/calmodulin-dependent kinase

    NARCIS (Netherlands)

    Verploegen, Sandra; Lammers, J.W.J.; Koenderman, L.; Coffer, P.J.

    2000-01-01

    Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction- based strategy was used to identify

  19. Neutrophil mediated IFN activation in the bone marrow alters B cell development in human and murine SLE1

    Science.gov (United States)

    Palanichamy, Arumugam; Bauer, Jason W; Yalavarthi, Srilakshmi; Meednu, Nida; Barnard, Jennifer; Owen, Teresa; Cistrone, Christopher; Bird, Anna; Rabinovich, Alfred; Nevarez, Sarah; Knight, Jason S.; Dedrick, Russell; Rosenberg, Alexander; Wei, Chungwen; Rangel-Moreno, Javier; Liesveld, Jane; Sanz, Inaki; Baechler, Emily; Kaplan, Mariana J.; Anolik, Jennifer H

    2014-01-01

    Inappropriate activation of type I interferon (IFN) plays a key role in the pathogenesis of autoimmune disease, including systemic lupus erythematosus (SLE). Here we report the presence of IFN activation in SLE bone marrow (BM), as measured by an IFN gene signature, increased IFN regulated chemokines, and direct production of IFN by BM resident cells, associated with profound changes in B cell development. The majority of SLE patients had an IFN signature in the BM that was more pronounced than the paired peripheral blood (PB) and correlated with both higher autoantibodies and disease activity. Pronounced alterations in B cell development were noted in SLE in the presence of an IFN signature with a reduction in the fraction of pro/pre B cells suggesting an inhibition in early B cell development and an expansion of B cells at the transitional (T2) stage. These B cell changes strongly correlated with an increase in BAFF and APRIL expression in the IFN high BM. Furthermore, we found that BM neutrophils in SLE were prime producers of IFN-α and B cell factors. In NZM lupus-prone mice similar changes in B cell development were observed and mediated by IFN, given abrogation in NZM mice lacking type I IFN receptor. BM neutrophils were abundant, responsive to and producers of IFN, in close proximity to B cells. These results indicate that the BM is an important but previously unrecognized target organ in SLE with neutrophil mediated IFN activation and alterations in B cell ontogeny and selection. PMID:24379124

  20. Eosinophil Granulocytes Account for Indoleamine 2,3-Dioxygenase-Mediated Immune Escape in Human Non-Small Cell Lung Cancer1

    Science.gov (United States)

    Astigiano, Simonetta; Morandi, Barbara; Costa, Roberta; Mastracci, Luca; D'Agostino, Antonella; Battista Ratto, Giovanni; Melioli, Giovanni; Frumento, Guido

    2005-01-01

    Abstract Indoleamine 2,3-dioxygenase (IDO), a catabolizing enzyme of tryptophan, is supposed to play a role in tumor immune escape. Its expression in solid tumors has not yet been well elucidated: IDO can be expressed by the tumor cells themselves, or by ill-defined infiltrating cells, possibly depending on tumor type. We have investigated IDO expression in 25 cases of non-small cell lung cancer (NSCLC). Using histochemistry and immunohistochemistry, we found that IDO was expressed not by tumor cells, but by normal cells infiltrating the peritumoral stroma. These cells were neither macrophages nor dendritic cells, and were identified as eosinophil granulocytes. The amount of IDO-positive eosinophils varied in different cases, ranging from a few cells to more than 50 per field at x200 magnification. IDO protein in NSCLC was enzymatically active. Therefore, at least in NSCLC cases displaying a large amount of these cells in the inflammatory infiltrate, IDO-positive eosinophils could exert an effective immunosuppressive action. On analyzing the 17 patients with adequate follow-up, a significant relationship was found between the amount of IDO-positive infiltrate and overall survival. This finding suggests that the degree of IDO-positive infiltrate could be a prognostic marker in NSCLC. PMID:15967116

  1. Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes.

    Science.gov (United States)

    Vowells, S J; Sekhsaria, S; Malech, H L; Shalit, M; Fleisher, T A

    1995-01-13

    Chronic granulomatous disease (CGD) is a rare recessive disorder caused by defects in the NADPH oxidase enzyme complex of phagocytes (neutrophils, eosinophils and monocytes). CGD phagocytes fail to produce superoxide and other reactive oxygen species following cell activation (Malech, 1993). The products of oxidase activation can be measured in individual cells by flow cytometry using specific fluorescent probes that increase fluorescence upon oxidation (Trinkle et al., 1987). This approach can be used to confirm a diagnosis of CGD, and to detect the normal/abnormal phagocyte mixture that characterizes the X-linked CGD carrier state. Three fluorescent probes have been described as useful for this purpose: 2'7'-dichlorofluorescin diacetate (DCF) (Bass et al., 1983), 5,6-carboxy-2'7'-dichlorofluorescein diacetate, bis(acetoxymethyl) ester (C-DCF) (Hockenbery et al., 1993) and dihydrorhodamine 123 (DHR) (Rothe et al., 1988; Kinsey et al., 1987). A direct comparison between these three probes has not been reported. In this study we performed a direct comparison between these three probes, evaluating their ability in flow cytometric analysis to maximize fluorescent separation between activated CGD patient and normal granulocytes. Using a whole blood technique with phorbol myristate acetate (PMA) as an activator, it was found that DHR loaded normal granulocytes had a fluorescence intensity which, upon activation, was 48-fold higher than that of C-DCF loaded granulocytes and seven-fold higher than DCF loaded granulocytes (P < 0.001). Use of sodium azide to decrease the catabolism of H2O2 enhanced the fluorescence of DCF by 140%, C-DCF by 45% and DHR by 25%, suggesting that DCF is primarily sensitive to H2O2. DCF and DHR were then evaluated for sensitivity in the detection of small percentages of normal cells in a CGD/normal granulocyte mixture. Normal sub-populations as small as 0.1% could clearly be distinguished using DHR, while DCF was insensitive at this level. Based

  2. Sweet’s Syndrome Successfully Treated with Granulocyte and Monocyte Adsorption Apheresis

    Directory of Open Access Journals (Sweden)

    Asami Fujii

    2017-05-01

    Full Text Available Sweet’s syndrome is a neutrophilic dermatosis characterized by an abrupt onset of painful erythematous lesions showing neutrophilic infiltrates in the dermis. Fever and an elevated neutrophil level are generally observed. Sweet’s syndrome may be idiopathic, malignancy-associated, or drug-induced (mainly involving granulocyte colony-stimulating factor (G-CSF administration. Although systemic corticosteroids are usually effective, the symptoms of Sweet’s syndrome recur in some refractory cases. Herein, we report a case of a 55-year-old Japanese woman with recurrent symptoms of fever (>39°C and painful erythematous lesions on her four extremities, trunk, and neck. Laboratory findings revealed leukocytosis and high levels of C-reactive protein (CRP and G-CSF. She was diagnosed with a recurrence of Sweet’s syndrome, and was exclusively treated with granulocyte and monocyte adsorption apheresis (GMA therapy once a week for 3 consecutive weeks. After the first session of GMA therapy, all symptoms including the erythematous lesions and fever were completely resolved, and serum G-CSF level was reduced. Leukocyte count, neutrophil count, serum amyloid A protein, and CRP levels were restored within normal ranges by 2 weeks. Thus, GMA therapy can successfully treat a patient with recurrent Sweet’s syndrome, potentially related to the restoration of elevated serum G-CSF levels.

  3. Increased FasL expression correlates with apoptotic changes in granulocytes cultured with oxidized clozapine.

    Science.gov (United States)

    Husain, Zaheed; Almeciga, Ingrid; Delgado, Julio C; Clavijo, Olga P; Castro, Januario E; Belalcazar, Viviana; Pinto, Clara; Zuñiga, Joaquin; Romero, Viviana; Yunis, Edmond J

    2006-08-01

    Clozapine has been associated with a 1% incidence of agranulocytosis. The formation of an oxidized intermediate clozapine metabolite has been implicated in direct polymorphonuclear (PMN) toxicity. We utilized two separate systems to analyze the role of oxidized clozapine in inducing apoptosis in treated cells. Human PMN cells incubated with clozapine (0-10 microM) in the presence of 0.1 mM H2O2 demonstrated a progressive decrease of surface CD16 expression along with increased apoptosis. RT-PCR analysis showed decreased CD16 but increased FasL gene expression in clozapine-treated PMN cells. No change in constitutive Fas expression was observed in treated cells. In HL-60 cells induced to differentiate with retinoic acid (RA), a similar increase in FasL expression, but no associated changes in CD16 gene expression, was observed following clozapine treatments. Our results demonstrate increased FasL gene expression in oxidized clozapine-induced apoptotic neutrophils suggesting that apoptosis in granulocytes treated with clozapine involves Fas/FasL interaction that initiates a cascade of events leading to clozapine-induced agranulocytosis.

  4. Dimethyl fumarate modulates neutrophil extracellular trap formation in a glutathione- and superoxide-dependent manner.

    Science.gov (United States)

    Hoffmann, J H O; Schaekel, K; Hartl, D; Enk, A H; Hadaschik, E N

    2018-01-01

    Neutrophil (polymorphonuclear) granulocytes (PMN) have been shown to contribute to the pathogenesis of psoriasis by releasing interleukin-17 and LL37-DNA complexes via neutrophil extracellular traps (NETs), webs of chromatin strands decorated with antimicrobial peptides, in psoriatic skin. Fumaderm ® , a fumaric acid ester (FAE) formulation consisting of different FAE salts, has been successfully used to treat psoriasis for decades. Most recently, FAE treatment was reported to inhibit NET formation in murine epidermolysis bullosa acquisita. To elucidate the effect of FAE treatment on human psoriasis and healthy donor NET formation. Among the compounds present in the FAE formulation, dimethyl fumarate (DMF) pretreatment of human psoriasis and healthy donor PMN resulted in a consistent inhibitory effect on NET formation in response to phorbol 12-myristate 13-acetate but not to platelet activating factor and ionomycin. This effect was l-glutathione (GSH) dependent and involved a decrease in reactive oxygen species (ROS) production, a key event in NET formation. In contrast, G-protein-coupled signalling and protein synthesis were not involved. Monomethyl fumarate (MMF) was found to slightly reduce ROS production without affecting NET formation. We report DMF as a potent, stimulus-specific, GSH- and ROS-dependent modulator of NET formation. Our results support the notion that modulation of NET formation contributes to the beneficial effects of FAEs in a variety of inflammatory conditions. © 2017 British Association of Dermatologists.

  5. ATRA and As₂O₃ regulate differentiation of human hematopoietic stem cells into granulocyte progenitor via alteration of HoxB8 expression.

    Science.gov (United States)

    Liu, W-J; Jiang, N-J; Guo, Q-L; Xu, Q

    2015-01-01

    This study aimed to investigate the effect of all-trans retinoic acid (ATRA) and/or arsenic trioxide (As2O3) on homeobox B8 (HOXB8) mRNA and protein expressions during the differentiation and proliferation of hematopoietic stem cells (HSCs) to colony forming unit-granulocyte (CFU-G) in order to explore the pathogenesis of leukemia mediated by HOXB8 at mRNA and protein level. Twelve cord blood samples were collected from the fetal placenta umbilical vein and cultured in vitro. The proliferation and differentiation of cord blood HSCs into CFU-G was continuously disrupted with 10 nmol/l of ATRA and/or 10 nmol/l of As2O3. The expression of HOXB8 mRNA and protein were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western-blot, respectively. HOXB8 mRNA/protein expression was detected in control, ATRA, As2O3 and ATRA +As2O3 groups on days 3, 7, and 12 of culture. HOXB8 mRNA/protein expression was detectable on day 3, reached its highest level on day 7 and decreased on day 12. HOXB8 mRNA/protein expression in ATRA, As2O3 and ATRA+As2O3 was upregulated compared with control group (p ATRA/As2O3 up-regulate the expression of HOXB8 mRNA/protein, and treatment of leukemia with ATRA/As2O3 may regulate HOX gene expression.

  6. Angiopoietin-1 upregulates de novo expression of IL-1β and Il1-Ra, and the exclusive release of Il1-Ra from human neutrophils.

    Science.gov (United States)

    Haddad, Lydia E; Sirois, Martin G

    2014-01-01

    The expression of the angiopoietin (Ang) receptor, Tie2, on both endothelial and inflammatory cells supports the idea that Ang signaling may play a fundamental role in initiating and maintaining the inflammatory response. We have previously shown that Ang1 and/or Ang2 alter the innate immune response by enhancing human neutrophil survival, chemotaxis and production of inflammatory cytokine interleukin-8 (IL-8) in vitro. Thus, we hypothesized that Ang1 and Ang2 could modulate other inflammatory signals in neutrophils, a possibility we explored through a gene-based assay looking at changes in the mRNA expression of 84 inflammatory cytokines and their receptors. We observed that Ang1 (10(-8) M), but not Ang2, increased mRNA expression of prominent pro-inflammatory cytokine IL-1β and its natural antagonist IL-1RA, by up to 32.6- and 10.0-fold respectively, compared to PBS-control. The effects of Ang1 extended to the proteins, as Ang1 increased intracellular levels of precursor and mature IL-1β, and extracellular levels of IL-1RA proteins, by up to 4.2-, 5.0- and 4.4-fold respectively, compared to PBS-control. Interestingly, Ang1 failed at inducing IL-1β protein release or at increasing intracellular IL-1RA, but the ratio of IL-1RA to mature IL-1β remained above 100-fold molar excess inside and outside the cells. The above-noted effects of Ang1 were mediated by MAP kinases, whereby inhibiting MEK1/2 lead to up to 70% effect reduction, whereas the blockade of p38MAPK activity doubled Ang1's effect. These findings suggest that Ang1 selectively alters the balance of neutrophil-derived inflammatory cytokines, favoring the blockade of IL-1 activity, a consideration for future therapies of inflammatory diseases.

  7. Angiopoietin-1 upregulates de novo expression of IL-1β and Il1-Ra, and the exclusive release of Il1-Ra from human neutrophils.

    Directory of Open Access Journals (Sweden)

    Lydia E Haddad

    Full Text Available The expression of the angiopoietin (Ang receptor, Tie2, on both endothelial and inflammatory cells supports the idea that Ang signaling may play a fundamental role in initiating and maintaining the inflammatory response. We have previously shown that Ang1 and/or Ang2 alter the innate immune response by enhancing human neutrophil survival, chemotaxis and production of inflammatory cytokine interleukin-8 (IL-8 in vitro. Thus, we hypothesized that Ang1 and Ang2 could modulate other inflammatory signals in neutrophils, a possibility we explored through a gene-based assay looking at changes in the mRNA expression of 84 inflammatory cytokines and their receptors. We observed that Ang1 (10(-8 M, but not Ang2, increased mRNA expression of prominent pro-inflammatory cytokine IL-1β and its natural antagonist IL-1RA, by up to 32.6- and 10.0-fold respectively, compared to PBS-control. The effects of Ang1 extended to the proteins, as Ang1 increased intracellular levels of precursor and mature IL-1β, and extracellular levels of IL-1RA proteins, by up to 4.2-, 5.0- and 4.4-fold respectively, compared to PBS-control. Interestingly, Ang1 failed at inducing IL-1β protein release or at increasing intracellular IL-1RA, but the ratio of IL-1RA to mature IL-1β remained above 100-fold molar excess inside and outside the cells. The above-noted effects of Ang1 were mediated by MAP kinases, whereby inhibiting MEK1/2 lead to up to 70% effect reduction, whereas the blockade of p38MAPK activity doubled Ang1's effect. These findings suggest that Ang1 selectively alters the balance of neutrophil-derived inflammatory cytokines, favoring the blockade of IL-1 activity, a consideration for future therapies of inflammatory diseases.

  8. Immunosenescence of Polymorphonuclear Neutrophils

    Directory of Open Access Journals (Sweden)

    Inga Wessels

    2010-01-01

    Full Text Available All immune cells are affected by aging, contributing to the high susceptibility to infections and increased mortality observed in the elderly. The effect of aging on cells of the adaptive immune system is well documented. In contrast, knowledge concerning age-related defects of polymorphonuclear neutrophils (PMN is limited. During the past decade, it has become evident that in addition to their traditional role as phagocytes, neutrophils are able to secrete a wide array of immunomodulating molecules. Their importance is underlined by the finding that genetic defects that lead to neutropenia increase susceptibility to infections. Whereas there is consistence about the constant circulating number of PMN throughout aging, the abilities of tissue infiltration, phagocytosis, and oxidative burst of PMN from aged donors are discussed controversially. Furthermore, there are numerous discrepancies between in vivo and in vitro results, as well as between results for murine and human PMN. Most of the reported functional changes can be explained by defective signaling pathways, but further research is required to get a detailed insight into the underlying molecular mechanisms. This could form the basis for drug development in order to prevent or treat age-related diseases, and thus to unburden the public health systems.

  9. α-Linoleic acid enhances the capacity of α-1 antitrypsin to inhibit lipopolysaccharide-induced IL-1β in human blood neutrophils.

    Science.gov (United States)

    Aggarwal, Nupur; Korenbaum, Elena; Mahadeva, Ravi; Immenschuh, Stephan; Grau, Veronika; Dinarello, Charles A; Welte, Tobias; Janciauskiene, Sabina

    2016-07-13

    Alpha1-antitrypsin (A1AT, SERPINA1), a major circulating inhibitor of neutrophil elastase (NE) and proteinase-3 (PRN3), has been proposed to reduce the processing and release of IL-1β. Since the anti-inflammatory properties of A1AT are influenced by the presence of polyunsaturated fatty acids, we compared effects of fatty acid-free (A1AT-0) and α-linoleic acid-bound (A1AT-LA) forms of A1AT on lipopolysaccharide (LPS)-induced synthesis of IL-1β precursor and the release of IL-1β from human blood neutrophils. The presence of A1AT-LA or A1AT-0 significantly reduced LPS-induced release of mature IL-1β. However, only A1A-LA reduced both steady state mRNA levels of IL-1β and the secretion of mature IL-1β. In LPS stimulated neutrophils, mRNA levels of TLR2/4, NFKBIA, P2RX7, NLRP3, and CASP1 decreased significantly in the presence of A1AT-LA but not A1AT-0. A1AT-0 and A1AT-LA did not inhibit the direct enzymatic activity of caspase-1, but we observed complexes of either form of A1AT with NE and PR3. Consistent with the effect on TLR and IL-1β gene expression, only A1AT-LA inhibited LPS-induced gene expression of NE and PR3. Increased gene expression of PPAR-γ was observed in A1AT-LA-treated neutrophils without of LPS stimulation, and the selective PPAR-γ antagonist (GW9662) prevented the reduction in IL-1β by A1AT-LA. We conclude from our data, that the ability of A1AT to reduce TLR and IL-1β gene expression depends on its association with LA. Moreover, the anti-inflammatory properties of A1AT-LA are likely to be mediated by the activation of PPAR-γ.

  10. Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

    Directory of Open Access Journals (Sweden)

    Vinicius Canato Santana

    2015-01-01

    Full Text Available T-cell based vaccines against human immunodeficiency virus (HIV generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+ T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

  11. Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses.

    Science.gov (United States)

    Santana, Vinicius Canato; Almeida, Rafael Ribeiro; Ribeiro, Susan Pereira; Ferreira, Luís Carlos de Souza; Kalil, Jorge; Rosa, Daniela Santoro; Cunha-Neto, Edecio

    2015-12-01

    T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

  12. Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrezza C Chagas

    2014-02-01

    Full Text Available Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

  13. Two-photon excited endogenous fluorescence for label-free in vivo imaging ingestion of disease-causing bacteria by human leukocytes

    Science.gov (United States)

    Zeng, Yan; Yan, Bo; Sun, Qiqi; Teh, Seng Khoon; Zhang, Wei; Wen, Zilong; Qu, Jianan Y.

    2013-02-01

    Real time and in vivo monitoring leukocyte behavior provides unique information to understand the physiological and pathological process of infection. In this study, we demonstrate that two-photon excited reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides imaging contrast to distinguish granulocyte and agranulocyte. By using spectral and time-resolved NADH fluorescence, we study the immune response of human neutrophils against bacterial infection (Escherichia coli). The two-photon excited NADH fluorescence images clearly review the morphological changes from resting neutrophils (round shape) to activated neutrophils (ruffle shape) during phagocytosis. The free-tobound NADH ratio of neutrophils decreases after ingesting disease-causing pathogen: Escherichia coli. This finding may provide a new optical tool to investigate inflammatory processes by using NADH fluorescence in vivo.

  14. Anti-polymorphonuclear neutrophil antibodies in patients with leukopenia or neutropenia.

    Science.gov (United States)

    Riera, N E; Rosso Saltó, M; Galán, V; Canalejo, K; Khoury, M; Aixalá, M; Kantor, G L; Vermeulen, M; Bengió, R; De Bracco, M M E

    2010-02-01

    Immune humoral neutropenia (Np) could be the consequence of anti-polymorphonuclear neutrophil (PMN) antibodies, circulating immune complexes (CIC) and/or antibodies against myeloid precursors. Granulocyte immunofluorescence test (GIFT) and a leukoagglutination technique (LAGT) assays are recommended for its diagnosis. Fifty adult patients with secondary Np were screened for anti-PMN. GIFT by flow cytometry from viable PMN and LAGT were employed. In addition, CIC levels, low expression of CD16(b) (CD16 (b)(low)), PMN phenotype and sera tumor necrosis factor-alpha (TNF-alpha) were also evaluated. Direct IgG-PMN binding (dir-GIFT) was positive in 16% of the patients. Antibodies against autologous PMN were detected in 32% of the samples by indirect (ind)-GIFT and demonstrated in 70% of the sera by both ind-GIFT and/or LAGT. Predominance of human neutrophil alloantigen (HNA)-1b and HNA-2 expression was confirmed. CD16(b)(low) was detected in 16% of the patient's PMN and TNF-alpha in 68% of sera patients. Our results suggest that diagnosis of immune Np in the laboratory may be improved by focusing on patient's PMN together with the assessment of cellular markers.

  15. Reversible granulocyte killing defect in anorexia nervosa.

    Science.gov (United States)

    Gotch, F M; Spry, C J; Mowat, A G; Beeson, P B; Maclennan, I C

    1975-08-01

    Three patients are described with anorexia nervosa in whom malnutrition was present with neutropenia and a granulocyte bactericidal degect. Their peripheral blood granulocytes were found to have a reduced rate of killing of Staphylococcus aureus and E. coli in vitro. The opsonic activity of the patients' sera towards Staphylococcus aureus was normal. One of these patients had recurrent episodes of infection which stopped after she had gained 13 kg in weight. Clinical recovery was associated with a return of granulocyte function to normal. It is concluded that granulocyte bactericidal capacity towards a variety of bacteria may be reduced in patients with anorexia nervosa who have malnutrition. This type of acquired granulocyte bactericidal deficiency appears to be reversible.

  16. Bacterial lipoprotein delays apoptosis in human neutrophils through inhibition of caspase-3 activity: regulatory roles for CD14 and TLR-2.

    LENUS (Irish Health Repository)

    Power, Colm P

    2012-02-03

    The human sepsis syndrome resulting from bacterial infection continues to account for a significant proportion of hospital mortality. Neutralizing strategies aimed at individual bacterial wall products (such as LPS) have enjoyed limited success in this arena. Bacterial lipoprotein (BLP) is a major constituent of the wall of diverse bacterial forms and profoundly influences cellular function in vivo and in vitro, and has been implicated in the etiology of human sepsis. Delayed polymorphonuclear cell (PMN) apoptosis is a characteristic feature of human sepsis arising from Gram-negative or Gram-positive bacterial infection. Bacterial wall product ligation and subsequent receptor-mediated events upstream of caspase inhibition in neutrophils remain incompletely understood. BLP has been shown to exert its cellular effects primarily through TLR-2, and it is now widely accepted that lateral associations with the TLRs represent the means by which CD14 communicates intracellular messages. In this study, we demonstrate that BLP inhibits neutrophil mitochondrial membrane depolarization with a subsequent reduction in caspase-3 processing, ultimately leading to a significant delay in PMN apoptosis. Pretreatment of PMNs with an anti-TLR-2 mAb or anti-CD14 mAb prevented BLP from delaying PMN apoptosis to such a marked degree. Combination blockade using both mAbs completely prevented the effects of BLP (in 1 and 10 ng\\/ml concentrations) on PMN apoptosis. At higher concentrations of BLP, the antiapoptotic effects were observed, but were not as pronounced. Our findings therefore provide the first evidence of a crucial role for both CD14 and TLR-2 in delayed PMN apoptosis arising from bacterial infection.

  17. Effect of granulocyte colony-stimulating factor (G-CSF) in human immunodeficiency virus-infected patients: increase in numbers of naive CD4 cells and CD34 cells makes G-CSF a candidate for use in gene therapy or to support antiretroviral therapy

    DEFF Research Database (Denmark)

    Nielsen, S D; Afzelius, P; Dam-Larsen, S

    1998-01-01

    The potential of granulocyte colony-stimulating factor (G-CSF) to mobilize CD4 cells and/or CD34 cells for use in gene therapy or to support antiretroviral therapy was examined. Ten human immunodeficiency virus-infected patients were treated with G-CSF (300 microg/day) for 5 days. Numbers of CD4....... Furthermore, the fraction of naive CD4 cells increased. These findings have implications for the design of immunotherapy or gene therapy protocols....

  18. Dynamic interactions of neutrophils and biofilms

    Directory of Open Access Journals (Sweden)

    Josefine Hirschfeld

    2014-12-01

    Full Text Available Background: The majority of microbial infections in humans are biofilm-associated and difficult to treat, as biofilms are highly resistant to antimicrobial agents and protect themselves from external threats in various ways. Biofilms are tenaciously attached to surfaces and impede the ability of host defense molecules and cells to penetrate them. On the other hand, some biofilms are beneficial for the host and contain protective microorganisms. Microbes in biofilms express pathogen-associated molecular patterns and epitopes that can be recognized by innate immune cells and opsonins, leading to activation of neutrophils and other leukocytes. Neutrophils are part of the first line of defense and have multiple antimicrobial strategies allowing them to attack pathogenic biofilms. Objective/design: In this paper, interaction modes of neutrophils with biofilms are reviewed. Antimicrobial strategies of neutrophils and the counteractions of the biofilm communities, with special attention to oral biofilms, are presented. Moreover, possible adverse effects of neutrophil activity and their biofilm-promoting side effects are discussed. Results/conclusion: Biofilms are partially, but not entirely, protected against neutrophil assault, which include the processes of phagocytosis, degranulation, and formation of neutrophil extracellular traps. However, virulence factors of microorganisms, microbial composition, and properties of the extracellular matrix determine whether a biofilm and subsequent microbial spread can be controlled by neutrophils and other host defense factors. Besides, neutrophils may inadvertently contribute to the physical and ecological stability of biofilms by promoting selection of more resistant strains. Moreover, neutrophil enzymes can degrade collagen and other proteins and, as a result, cause harm to the host tissues. These parameters could be crucial factors in the onset of periodontal inflammation and the subsequent tissue breakdown.

  19. Recombinant rat stem cell factor synergizes with recombinant human granulocyte colony-stimulating factor in vivo in mice to mobilize peripheral blood progenitor cells that have enhanced repopulating potential.

    Science.gov (United States)

    Briddell, R A; Hartley, C A; Smith, K A; McNiece, I K

    1993-09-15

    Splenectomized mice treated for 7 days with pegylated recombinant rat stem cell factor (rrSCF-PEG) showed a dose-dependent increase in peripheral blood progenitor cells (PBPC) that have enhanced in vivo repopulating potential. A dose of rrSCF-PEG at 25 micrograms/kg/d for 7 days produced no significant increase in PBPC. However, when this dose of rrSCF-PEG was combined with an optimal dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 200 micrograms/kg/d), a synergistic increase in PBPC was observed. Compared with treatment with rhG-CSF alone, the combination of rrSCF-PEG plus rhG-CSF resulted in a synergistic increase in peripheral white blood cells, in the incidence and absolute numbers of PBPC, and in the incidence and absolute numbers of circulating cells with in vivo repopulating potential. These data suggest that low doses of SCF, which would have minimal, if any, effects in vivo, can synergize with optimal doses of rhG-CSF to enhance the mobilization of PBPC stimulated by rhG-CSF alone.

  20. Characterization of Total Phenolic Constituents from the Stems of Spatholobus suberectus Using LC-DAD-MSn and Their Inhibitory Effect on Human Neutrophil Elastase Activity

    Directory of Open Access Journals (Sweden)

    Yiming Li

    2013-06-01

    Full Text Available Spatholobus suberectus Dunn, belonging to the legume family (Fabaceae, has been used as a Traditional Chinese Medicine for the treatment of anemia, menoxenia and rheumatism. A limited number of studies report that various types of flavonoids are the main characteristic constituents of this herb. We have now found that S. suberectus contains about 2% phenolic components and characterized the major phenolic components as homogeneous B-type procyanidin conjugates using a liquid chromatography with diode-array detection-ESI mass spectrometry (LC-DAD/ESI-MS method. This is the first report on occurrence of most B-type procyanidins in this herb. Moreover, the total phenolics extract was assayed for inhibitory activity on human neutrophil elastase and its IC50 was found to be 1.33 μg/mL.

  1. Synthesis and evaluation of the potential deleterious effects of ZnO nanomaterials (nanoneedles and nanoflowers) on blood components, including albumin, erythrocytes and human isolated primary neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Pastrello, Bruna [São Paulo State University (UNESP), Department of Chemistry, Faculty of Sciences (Brazil); Paracatu, Luana Chiquetto [São Paulo State University (UNESP), Department of Clinical Analysis, School of Pharmaceutical Sciences (Brazil); Carvalho Bertozo, Luiza de [São Paulo State University (UNESP), Department of Chemistry, Faculty of Sciences (Brazil); Paino, Iêda Maria Martinez [University of São Paulo (USP), Nanomedicine and Nanotoxicology Group, Physics Institute of São Carlos (IFSC) (Brazil); Lisboa-Filho, Paulo Noronha [São Paulo State University (UNESP), Department of Physics, Faculty of Sciences (Brazil); Ximenes, Valdecir Farias, E-mail: vfximenes@fc.unesp.br [São Paulo State University (UNESP), Department of Chemistry, Faculty of Sciences (Brazil)

    2016-07-15

    The application of zinc oxide (ZnO) nanoparticles in biomaterials has increased significantly in the recent years. Here, we aimed to study the potential deleterious effects of ZnO on blood components, including human serum albumin (HSA), erythrocytes and human isolated primary neutrophils. To test the influence of the morphology of the nanomaterials, ZnO nanoneedles (ZnO-nn) and nanoflowers (ZnO-nf) were synthesized. The zeta potential and mean size of ZnO-nf and ZnO-nn suspensions in phosphate-buffered saline were −10.73 mV and 3.81 nm and −5.27 mV and 18.26 nm, respectively. The incubation of ZnO with HSA did not cause its denaturation as verified by the absence of significant alterations in the intrinsic and extrinsic fluorescence and in the circular dichroism spectrum of the protein. The capacity of HSA as a drug carrier was not affected as verified by employing site I and II fluorescent markers. Neither type of ZnO was able to provoke the activation of neutrophils, as verified by lucigenin- and luminol-dependent chemiluminescence and by the extracellular release of hydrogen peroxide. ZnO-nf, but not ZnO-nn, induced the haemolysis of erythrocytes. In conclusion, our results reinforce the concept that ZnO nanomaterials are relatively safe for usage in biomaterials. A potential exception is the capacity of ZnO-nf to promote the lysis of erythrocytes, a discovery that shows the importance of the morphology in the toxicity of nanoparticles.

  2. Quantitative structure-activity relationship analysis of human neutrophil elastase inhibitors using shuffling classification and regression trees and adaptive neuro-fuzzy inference systems.

    Science.gov (United States)

    Asadollahi-Baboli, M

    2012-07-01

    The purpose of this study was to develop quantitative structure-activity relationship models for N-benzoylindazole derivatives as inhibitors of human neutrophil elastase. These models were developed with the aid of classification and regression trees (CART) and an adaptive neuro-fuzzy inference system (ANFIS) combined with a shuffling cross-validation technique using interpretable descriptors. More than one hundred meaningful descriptors, representing various structural characteristics for all 51 N-benzoylindazole derivatives in the data set, were calculated and used as the original variables for shuffling CART modelling. Five descriptors of average Wiener index, Kier benzene-likeliness index, subpolarity parameter, average shape profile index of order 2 and folding degree index selected by the shuffling CART technique have been used as inputs of the ANFIS for prediction of inhibition behaviour of N-benzoylindazole derivatives. The results of the developed shuffling CART-ANFIS model compared to other techniques, such as genetic algorithm (GA)-partial least square (PLS)-ANFIS and stepwise multiple linear regression (MLR)-ANFIS, are promising and descriptive. The satisfactory results r2p = 0.845, Q2(LOO) = 0.861, r2(L25%O) = 0.829, RMSE(LOO)  = 0.305 and RMSE(L25%O)  = 0.336) demonstrate that shuffling CART-ANFIS models present the relationship between human neutrophil elastase inhibitor activity and molecular descriptors, and they yield predictions in excellent agreement with the experimental values.

  3. Synthesis and evaluation of the potential deleterious effects of ZnO nanomaterials (nanoneedles and nanoflowers) on blood components, including albumin, erythrocytes and human isolated primary neutrophils

    Science.gov (United States)

    Pastrello, Bruna; Paracatu, Luana Chiquetto; de Carvalho Bertozo, Luiza; Paino, Iêda Maria Martinez; Lisboa-Filho, Paulo Noronha; Ximenes, Valdecir Farias

    2016-07-01

    The application of zinc oxide (ZnO) nanoparticles in biomaterials has increased significantly in the recent years. Here, we aimed to study the potential deleterious effects of ZnO on blood components, including human serum albumin (HSA), erythrocytes and human isolated primary neutrophils. To test the influence of the morphology of the nanomaterials, ZnO nanoneedles (ZnO-nn) and nanoflowers (ZnO-nf) were synthesized. The zeta potential and mean size of ZnO-nf and ZnO-nn suspensions in phosphate-buffered saline were -10.73 mV and 3.81 nm and -5.27 mV and 18.26 nm, respectively. The incubation of ZnO with HSA did not cause its denaturation as verified by the absence of significant alterations in the intrinsic and extrinsic fluorescence and in the circular dichroism spectrum of the protein. The capacity of HSA as a drug carrier was not affected as verified by employing site I and II fluorescent markers. Neither type of ZnO was able to provoke the activation of neutrophils, as verified by lucigenin- and luminol-dependent chemiluminescence and by the extracellular release of hydrogen peroxide. ZnO-nf, but not ZnO-nn, induced the haemolysis of erythrocytes. In conclusion, our results reinforce the concept that ZnO nanomaterials are relatively safe for usage in biomaterials. A potential exception is the capacity of ZnO-nf to promote the lysis of erythrocytes, a discovery that shows the importance of the morphology in the toxicity of nanoparticles.

  4. The lipidated peptidomimetic Lau-[(S)-Aoc]-(Lys-βNphe)6-NH2 is a novel formyl peptide receptor 2 agonist that activates both human and mouse neutrophil NADPH-oxidase

    DEFF Research Database (Denmark)

    Holdfeldt, Andre; Skovbakke, Sarah Line; Winther, Malene

    2016-01-01

    2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca2+ concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated...

  5. Effect of interleukin 10 on the release of the CXC chemokines growth related oncogene GRO-alpha and epithelial cell-derived neutrophil activating peptide (ENA)-78 during human endotoxemia

    NARCIS (Netherlands)

    Olszyna, D. P.; Pajkrt, D.; van Deventer, S. J.; van der Poll, T.

    2001-01-01

    Pretreatment with interleukin (IL)-10 inhibited the release of growth-related oncogene GRO-alpha but not of epithelial-cell derived neutrophil activating protein (ENA)-78, after injection of lipopolysaccharide (LPS) into healthy humans. In vitro, IL-10 dose-dependently attenuated LPS-induced release

  6. Decreased urokinase receptor expression on granulocytes in HIV-infected patients

    DEFF Research Database (Denmark)

    Storgaard, M; Obel, N; Black, F T

    2002-01-01

    PAR) expression in granulocytes (3.9 x 104 +/- 0.9 x 104 sites/cell). Modulation of uPAR expression was assessed in the presence of chemoattractant gradients. Our findings demonstrate that interleukin (IL)-8, leukotriene B4(LTB4) and formyl-methionyl-leucyl-phenylalanine (f MLP) caused a dose......-dependent upregulation of uPAR on granulocytes in healthy controls. Modulation of uPAR expression is known to regulate chemotactic response. As determined by flow cytometry, uPAR expression by granulocytes from human immunodeficiency virus (HIV)-infected patients was distinctly lower than that of healthy control cells...... (P HIV-infected patients, the uPAR expression on granulocytes correlated (P

  7. The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils

    DEFF Research Database (Denmark)

    Plesner, T; Ploug, M; Ellis, V

    1994-01-01

    including cell migration and resolution of thrombi. We have previously shown that uPAR is expressed on the plasma membrane of circulating neutrophils, and we now report that stimulation with phorbol myristate acetate (PMA), FMLP, or tumor necrosis factor-alpha results in a rapid increase in the expression...... for plasminogen activation. These processes may be important for neutrophil extravasation and migration through extracellular matrix and for the contribution of neutrophils to resolution of thrombi....

  8. Activation of bovine neutrophils by Brucella spp.

    Science.gov (United States)

    Keleher, Lauren L; Skyberg, Jerod A

    2016-09-01

    Brucellosis is a globally important zoonotic infectious disease caused by gram negative bacteria of the genus Brucella. While many species of Brucella exist, Brucella melitensis, Brucella abortus, and Brucella suis are the most common pathogens of humans and livestock. The virulence of Brucella is largely influenced by its ability to evade host factors, including phagocytic killing mechanisms, which are critical for the host response to infection. The aim of this study was to characterize the bovine neutrophil response to virulent Brucella spp. Here, we found that virulent strains of smooth B. abortus, B. melitensis, B. suis, and virulent, rough, strains of Brucella canis possess similar abilities to resist killing by resting, or IFN-γ-activated, bovine neutrophils. Bovine neutrophils responded to infection with a time-dependent oxidative burst that varied little between Brucella spp. Inhibition of TAK1, or SYK kinase blunted the oxidative burst of neutrophils in response to Brucella infection. Interestingly, Brucella spp. did not induce robust death of bovine neutrophils. These results indicate that bovine neutrophils respond similarly to virulent Brucella spp. In addition, virulent Brucella spp., including naturally rough strains of B. canis, have a conserved ability to resist killing by bovine neutrophils. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice

    Energy Technology Data Exchange (ETDEWEB)

    Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.; Pinheiro-Torres, A.S.; Roncalho-Buck, I.A. [Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), Jundiai, SP (Brazil); Squebola-Cola, D.M.; Mello, G.C.; Anhê, G.F.; Antunes, E. [Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP (Brazil); DeSouza, I.A., E-mail: ivanidesouza@uol.com.br [Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), Jundiai, SP (Brazil)

    2015-09-15

    Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. - Highlights: • Airway exposure to SEA causes acute lung inflammation. • SEA induces accumulation of bone marrow (BM) in immature and mature neutrophils. • SEA increases BM granulocyte or BM PMN adhesion to ICAM-1 and VCAM-1, and MAC-1 expression. • SEA induces BM elevations of CXCL-1, INF-γ, TNF-α, GM-CSF, G-CSF and

  10. Abnormal telomere shortening of peripheral blood mononuclear cells and granulocytes in patients with chronic idiopathic neutropenia

    Science.gov (United States)

    Pavlaki, Konstantia I.; Kastrinaki, Maria-Christina; Klontzas, Michail; Velegraki, Maria; Mavroudi, Irene; Papadaki, Helen A.

    2012-01-01

    Background Chronic idiopathic neutropenia is characterized by immune-mediated suppression of neutrophil production. Because patients with immune-mediated bone marrow failure syndromes display age-inappropriate telomere shortening in leukocytes, we investigated telomere lengths in peripheral blood mononuclear cells and granulocytes of patients with chronic idiopathic neutropenia. Design and Methods We studied 37 patients with chronic idiopathic neutropenia and 68 age- and sex-matched healthy controls. Relative telomere length and telomerase reverse transcriptase expression were assessed by a quantitative real time polymerase chain reaction. Telomerase activity was determined by a polymerase chain reaction-based immunoassay. Results The mean relative telomere values of peripheral blood mononuclear cells and granulocytes were significantly lower in patients compared to controls, and significantly lower than expected on the basis of the age-adjusted healthy control distribution. The difference in the relative telomere lengths between patients and controls in both peripheral blood mononuclear cells and granulocytes was prominent in those under the age of 50 years. Contrary to the peripheral blood mononuclear cells, in which an inverse correlation was observed between relative telomere values and age, no significant correlation was noted between granulocyte telomere values and patient age. A significant correlation was observed between individual relative telomere values and absolute neutrophil counts. There was no difference in expression of telomerase reverse transcriptase in peripheral blood mononuclear cells between patients and controls but telomerase activity was identified at a significantly higher frequency in controls than in patients. No correlation was found between telomerase activity or telomerase reverse transcriptase expression and relative telomere lengths of peripheral blood mononuclear cells. Conclusions Patients with chronic idiopathic neutropenia

  11. Separation of granulocytes from whole blood by leukoadhesion, phase 1

    Science.gov (United States)

    1976-01-01

    Capillary glass tubes are investigated for the separation and retrieval of large quantities of viable granulocytes and monocytes from whole blood on a continuous basis from a single donor. This effort represented the feasibility demonstration of a three phase program for development of a capillary tube cell separation device. The activity included the analysis and parametric laboratory testing with subscale models required to design a prototype device. Capillary tubes 40 cm long with a nominal 0.030 cm internal diameter yielded the highest total process efficiency. Recovery efficiencies as high as 89% of the adhering cell population were obtained. Granulocyte phagocytosis of latex particles indicated approximately 90% viability. Monocytes recovered from the separation column retained their capability to stimulate human bone marrow colony growth, as demonstrated in an in vitro cell culture assay.

  12. Identification of Neutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors of Neutrophil Exocytosis and Inflammation

    Science.gov (United States)

    Johnson, Jennifer L.; Ramadass, Mahalakshmi; He, Jing; Brown, Steven J.; Zhang, Jinzhong; Abgaryan, Lusine; Biris, Nikolaos; Gavathiotis, Evripidis; Rosen, Hugh; Catz, Sergio D.

    2016-01-01

    Neutrophils constitute the first line of cellular defense in response to bacterial and fungal infections and rely on granular proteins to kill microorganisms, but uncontrolled secretion of neutrophil cargos is injurious to the host and should be closely regulated. Thus, increased plasma levels of neutrophil secretory proteins, including myeloperoxidase and elastase, are associated with tissue damage and are hallmarks of systemic inflammation. Here, we describe a novel high-throughput screening approach to identify small molecule inhibitors of the interaction between the small GTPase Rab27a and its effector JFC1, two central regulators of neutrophil exocytosis. Using this assay, we have identified small molecule inhibitors of Rab27a-JFC1 binding that were also active in cell-based neutrophil-specific exocytosis assays, demonstrating the druggability of Rab GTPases and their effectors. These compounds, named Nexinhibs (neutrophil exocytosis inhibitors), inhibit exocytosis of azurophilic granules in human neutrophils without affecting other important innate immune responses, including phagocytosis and neutrophil extracellular trap production. Furthermore, the compounds are reversible and potent inhibitors of the extracellular production of superoxide anion by preventing the up-regulation of the granule membrane-associated subunit of the NADPH oxidase at the plasma membrane. Nexinhibs also inhibit the up-regulation of activation signature molecules, including the adhesion molecules CD11b and CD66b. Importantly, by using a mouse model of endotoxin-induced systemic inflammation, we show that these inhibitors have significant activity in vivo manifested by decreased plasma levels of neutrophil secretory proteins and significantly decreased tissue infiltration by inflammatory neutrophils. Altogether, our data present the first neutrophil exocytosis-specific inhibitor with in vivo anti-inflammatory activity, supporting its potential use as an inhibitor of systemic

  13. Immunomodulatory effects of testosterone evaluated in all-trans retinoic acid differentiated HL-60 cells, granulocytes, and monocytes

    DEFF Research Database (Denmark)

    Boje, Alex; Moesby, Lise; Timm, Michael

    2012-01-01

    The sex hormones are known to affect innate immunity in humans. In this study we evaluated the immunomodulatory effects of testosterone in a model system comprising of all-trans retinoic acid differentiated HL-60 cells, and confirmed the results in human granulocytes and monocytes. Results showed...... species and interleukin-8 in human granulocytes and monocytes, respectively, to a similar extent as observed in differentiated HL-60 cells....

  14. Neutrophils at work

    DEFF Research Database (Denmark)

    Nauseef, William M; Borregaard, Niels

    2014-01-01

    blood to tissues in models of blood-borne infections versus bacterial invasion through epithelial linings. We examine data on novel aspects of the activation of NADPH oxidase and the heterogeneity of phagosomes and, finally, consider the importance of two neutrophil-derived biological agents: neutrophil...

  15. Apoptosis of neutrophils

    NARCIS (Netherlands)

    Maianski, N. A.; Maianski, A. N.; Kuijpers, T. W.; Roos, D.

    2004-01-01

    Regulation of the neutrophil life span by apoptosis provides a fine balance between their function as effector cells of host defense and a safe turnover of these potentially harmful cells. Alterations of neutrophil apoptosis are associated with a number of diseases. As do other cell types,

  16. Human neutrophils mediate trogocytosis rather than phagocytosis of CLL B cells opsonized with anti-CD20 antibodies.

    Science.gov (United States)

    Valgardsdottir, Rut; Cattaneo, Irene; Klein, Christian; Introna, Martino; Figliuzzi, Marina; Golay, Josée

    2017-05-11

    Polymorphonuclear neutrophils (PMNs) have previously been reported to mediate phagocytosis of anti-CD20-opsonized B cells from patients with chronic lymphocytic leukemia (CLL). However, recent data have suggested that PMNs, like macrophages, can also mediate trogocytosis. We have performed experiments to more precisely investigate this point and to discriminate between trogocytosis and phagocytosis. In live-cell time-lapse microscopy experiments, we could not detect any significant phagocytosis by purified PMNs of anti-CD20-opsonized CLL B cells, but could detect only the repeated close contact between effectors and targets, which suggested trogocytosis. Similarly, in flow cytometry assays using CLL B-cell targets labeled with the membrane dye PKH67 and opsonized with rituximab or obinutuzumab, we observed that a mean of 50% and 75% of PMNs had taken a fraction of the dye from CLL B cells at 3 and 20 hours, respectively, with no significant decrease in absolute live or total CLL B-cell numbers, confirming that trogocytosis occurs, rather than phagocytosis. Trogocytosis was accompanied by loss of membrane CD20 from CLL B cells, which was evident with rituximab but not obinutuzumab. We conclude that PMNs mediate mostly trogocytosis rather than phagocytosis of anti-CD20-opsonized CLL B cells, and we discuss the implications of this finding in patients with CLL treated with rituximab or obinutuzumab in vivo. © 2017 by The American Society of Hematology.

  17. Kinetics of LFA-1 mediated adhesion of human neutrophils to ICAM-1-role of E-selectin signaling post-activation.

    Science.gov (United States)

    LFA-1 and Mac-1 are the two integrins involved in the arrest and firm adhesion of neutrophils. LFA-1 plays a role in the early stage of cell arrest while Mac-1 stabilizes firm adhesion. Here, we further elucidated the kinetics of LFA-1 activation and its role in mediating neutrophil adhesion to ICAM...

  18. The Anti-Apoptotic Effect of Respiratory Syncytial Virus on Human Peripheral Blood Neutrophils is Mediated by a Monocyte Derived Soluble Factor.

    Science.gov (United States)

    Coleman, Christopher M; Plant, Karen; Newton, Susan; Hobson, Lynsey; Whyte, Moira K B; Everard, Mark L

    2011-01-01

    Respiratory Syncytial Virus (RSV) causes annual epidemics of respiratory disease particularly affecting infants. The associated airway inflammation is characterized by an intense neutrophilia. This neutrophilic inflammation appears to be responsible for much of the pathology and symptoms. Previous work from our group had shown that there are factors within the airways of infants with RSV bronchiolitis that inhibit neutrophil apoptosis. This study was undertaken to determine if RSV can directly affect neutrophil survival.NEUTROPHILS WERE ISOLATED FROM CITRATED VENOUS BLOOD (COLLECTED FROM HEALTHY ADULT VOLUNTEERS) BY DISCONTINUOUS PLASMA: Percoll gradient centrifugation and, in some experiments, further purified by negative immunomagnetic bead selection. The effect of RSV on neutrophil survival was measured by Annexin V-PE /To-Pro-3 staining and by morphological changes, using Dif-Quick staining of cytospins.Inhibition of neutrophil apoptosis was observed in neutrophils isolated by standard plasma:Percoll gradient when exposed to RSV but not in ultra pure neutrophil preparations. Adding monocytes back to ultra purified preparations restored the effect. The inhibition of apoptosis was observed with both active and UV inactivated virus. The effect is dependent on a soluble factor and appears to be dependent on CD14 receptors on the monocytes.

  19. Granulocytic invasion of the central nervous system after hematopoietic stem cell transplantation for systemic lupus erythematosus.

    Science.gov (United States)

    Muraro, Paolo A; Nikolov, Nikolay P; Butman, John A; Abati, Andrea; Gea-Banacloche, Juan; Gress, Ronald; Lipsky, Peter; Illei, Gabor; Pavletic, Steven

    2006-06-01

    We report on the likely mechanism of an exacerbation of neurological symptoms developed during immune reconstitution after autologous non-myeloablative hematopoietic stem cell transplantation in a 33-year-old man with systemic lupus erythematosus- associated recurrent transverse myelitis. Cerebrospinal fluid examination revealed prominent neutrophilic pleocytosis and no evidence of infection or of reactivation of lupus. Following a course of corticosteroid treatment the exacerbation resolved completely and the patient's neurological function continued to improve, resulting in net gain above pre-treatment for over 1 year follow-up without maintenance immunosuppression. Granulocytic invasion of the central nervous system represents a novel and possibly preventable cause of neurological complications during haematologic reconstitution.

  20. Dissociations in the effects of β2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity.

    Directory of Open Access Journals (Sweden)

    Irena Brunskole Hummel

    Full Text Available In neutrophils, activation of the β2-adrenergic receptor (β2AR, a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3',5'-cyclic monophosphate (cAMP accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP-induced superoxide anion (O2(•- production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2(•- formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2(•- production than in stimulating cAMP accumulation. (--Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2(•- production. Moreover, (--adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2(•- assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2(•- production after β2AR-stimulation although cAMP-increasing substances inhibited O2(•- production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2(•- production in a cAMP-independent manner.

  1. Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing.

    Science.gov (United States)

    Palić, Dušan; Beck, Linda S; Palić, Jelena; Andreasen, Claire B

    2011-02-01

    Studies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diff-quick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of lymphocytes, monocytes, platelets/thrombocytes and granulocytes was determined in all fish species. Arctic grayling, June sucker, and cutthroat trout had MPO positive granulocytes, while shovelnose sturgeon heterophils had positive reaction for leukocyte AP, but not MPO. Presence of MPO indicated potential to measure oxidative burst and degranulation of neutrophil primary granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO in shovelnose sturgeon suggested use of different enzyme marker (AP) in degranulation assay for this species. Standardization of cytochemical techniques allowed for rapid screening of leukocyte types, reducing the number of fish, time and effort to select adequate neutrophil function assays to be used in studies of health status in species of special concern. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yoon Shin; Lim, Goh-Woon [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung [Department of Microbiology, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Yoo, Eun-Sun [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Chan Ra, Jeong [Stem Cell Research Center, RNL BIO, Seoul 153-768 (Korea, Republic of); Ryu, Kyung-Ha, E-mail: ykh@ewha.ac.kr [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  3. Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation.

    Directory of Open Access Journals (Sweden)

    Bjoern F Kraemer

    2011-11-01

    Full Text Available Human β-defensins (hBD are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus, forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET formation by target polymorphonuclear leukocytes (PMNs, which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.

  4. Detection of human neutrophil elastase by aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence using specified site fluorescently labeled aptamer.

    Science.gov (United States)

    Bai, Yunlong; Wang, Hailin; Zhao, Qiang

    2017-11-01

    As a multifunctional serine protease, human neutrophil elastase (HNE) plays critical roles in a variety of physiopathological processes, such as acute lung injury, emphysema, atherosclerosis, and arthritis. The quantification of HNE is important in many applications. In this paper, we report an aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) assay for detection of HNE using a tetramethylrhodamine (TMR)-labeled DNA aptamer probe. The affinity complex of HNE and DNA aptamer probe was well separated from the unbound aptamer probe in CE separation based on the difference of electrophoretic mobility. Broad complex peaks appeared due to possible multiple binding. The 45-mer aptamer having TMR labeling on the 40th T base was used as affinity probe, as larger complex peaks were obtained. We investigated the effects of various metal cations (Na + , K + , and Mg 2+ ) in sample buffer on the binding of HNE and the aptamer in CE-LIF analysis. The presence of Na + , K + , or Mg 2+ in sample buffer caused a decrease of complex peaks, and Mg 2+ showed a larger effect. Under optimized conditions, this aptamer CE-LIF assay enabled the detection of HNE at 0.5 nM. This assay showed good specificity and allowed for detection of HNE spiked in diluted human serum sample. Graphical abstract The complex of HNE and DNA aptamer probe was isolated from the unbound aptamer probe in CE separation due to difference of electrophoretic mobility, allowing a CE-LIF assay for HNE.

  5. Down-regulation of surface receptors for TNF and IL-1 on circulating monocytes and granulocytes during human endotoxemia: effect of neutralization of endotoxin-induced TNF activity by infusion of a recombinant dimeric TNF receptor

    NARCIS (Netherlands)

    van der Poll, T.; Coyle, S. M.; Kumar, A.; Barbosa, K.; Agosti, J. M.; Lowry, S. F.

    1997-01-01

    Leukocytes rapidly lose their surface receptors for TNF and IL-1 upon exposure to various stimuli in vitro. We sought to determine by FACS analysis changes in the expression of TNF receptors (TNFR) and type II IL-1R on circulating monocytes and granulocytes during endotoxemia in vivo, and the role

  6. Leukotriene B4 production by blood neutrophils in allergic rhinitis--effects of cetirizine.

    Science.gov (United States)

    Cheria-Sammari, S; Aloui, R; Gormand, F; Chabannes, B; Gallet, H; Grosclaude, M; Melac, M; Rihoux, J P; Perrin-Fayolle, M; Lagarde, M

    1995-08-01

    Mucosal inflammatory processes in late phase of allergic diseases involve cytokine production, cell adhesion molecule overexpression and release of inflammatory mediators with chemotactic activity, such as leukotriene B4 (LTB4). We had previously observed increased production of LTB4 by neutrophils in patients with allergic rhinitis and discussed the role of granulocyte macrophage-colony stimulating factor (GM-CSF) priming. Some antihistaminic compounds were shown to diminish the production of leukotrienes by neutrophils. In a first step, we evaluated in ex vivo and in vitro studies, the effects of cetirizine on LTB4 production by blood neutrophils from allergic and healthy subjects. In a second step, we studied the in vitro effect of cetirizine on LTB4 production by neutrophils from healthy subjects during GM-CSF priming of these cells. Neutrophils from both populations were purified from venous blood and LTB4 production was measured using high performance liquid cromatography (HPLC) method. In ex vivo studies, cetirizine treatment induced a decreased LTB4 production by neutrophils in allergic rhinitis. This effect of decreased LTB4 production was reproduced in vitro with 10(-8)-10(-6)M cetirizine. Nevertheless, this anti-H1 compound had no effect on neutrophil priming with GM-CSF. As LTB4 is an important chemotactic factor, Cetirizine could act on inflammatory cell recruitment by inhibiting LTB4 production by neutrophils.

  7. Granulocyte colony-stimulating factor increases CD4+ T cell counts of human immunodeficiency virus-infected patients receiving stable, highly active antiretroviral therapy

    DEFF Research Database (Denmark)

    Aladdin, H; Ullum, H; Dam Nielsen, S.

    2000-01-01

    Thirty human immunodeficiency virus (HIV)-infected patients with CD4+ T cell counts factor (G-CSF; 0.3 mg/mL 3 times...... a week) for 12 weeks. Blood samples were collected at specified time points. G-CSF treatment enhanced the total lymphocyte count (P=.002) and increased CD3+ (P=.005), CD4+ (P=.03), and CD8+ (P=.004) T cell counts as well as numbers of CD3-CD16+CD56+ NK cells (P=.001). The increases in CD4+ and CD8+ cell...... counts resulted from increases in CD45RO+ memory T cells and cells expressing the CD38 activation marker. Lymphocyte proliferative responses to phytohemagglutinin and Candida antigen decreased, whereas NK cell activity and plasma HIV RNA did not change during G-CSF treatment. After 24 weeks, all immune...

  8. Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

    NARCIS (Netherlands)

    Maianski, N. A.; Geissler, J.; Srinivasula, S. M.; Alnemri, E. S.; Roos, D.; Kuijpers, T. W.

    2004-01-01

    Mitochondria are known to combine life-supporting functions with participation in apoptosis by controlling caspase activity. Here, we report that in human blood neutrophils the mitochondria are different, because they preserve mainly death-mediating abilities. Neutrophil mitochondria hardly

  9. Neutrophil perversion in demyelinating autoimmune diseases: Mechanisms to medicine.

    Science.gov (United States)

    Casserly, Courtney S; Nantes, Julia C; Whittaker Hawkins, Ryder F; Vallières, Luc

    2017-03-01

    Neutrophils are essential to a healthy life, yet pose a threat if improperly controlled. Neutrophil perversion is well documented in a variety of inflammatory disorders (e.g. arthritis, lupus, psoriasis), but is only beginning to be demystified in autoimmune demyelination, the most common cause of neurological disability in young adults. Using the animal model experimental autoimmune encephalomyelitis (EAE), several molecules that help neutrophils invade the central nervous system (CNS) have been identified. Mechanisms by which neutrophils may contribute to demyelination have also been proposed (e.g. secretion of endothelial/leukocytic modulators, antigen presentation to T cells, myelin degradation and phagocytosis). In human, neutrophils are seen in the CNS of people with neuromyelitis optica spectrum disorder and other severe variants of autoimmune demyelinating diseases. At the time of autopsy for multiple sclerosis (MS) - often many years after its onset - neutrophils appear to have escaped the scene of the crime. However, new clues implicate neutrophils in MS relapses and progression. This warrants further investigating 1) the differential importance of neutrophils among demyelinating diseases, 2) the largely unknown effects of current MS therapies on neutrophils, and 3) the potential of neutrophil proteins as clinical biomarkers or therapeutic targets. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Transmission of Anaplasma phagocytophilum from endothelial cells to peripheral granulocytes in vitro under shear flow conditions.

    Science.gov (United States)

    Wang, Jinyong; Dyachenko, Viktor; Munderloh, Ulrike G; Straubinger, Reinhard K

    2015-10-01

    Anaplasma phagocytophilum (Ap) is a tick-borne pathogen, which can cause granulocytic anaplasmosis in humans and animals. In vivo this obligate intracellular pathogen is primarily located in circulating mature granulocytes, but it also infects endothelial cells. In order to study the interaction between Ap-infected endothelial cells and human granulocytes under conditions similar to those found naturally in the infected host, an in vitro model that mimics physiological flow conditions in the microvasculature was established. Cell-to-cell interactions were then visualized by microscopy, which showed that granulocytes adhered strongly to Ap-infected endothelial cells at a shear stress of 0.5 dyne/cm(2). In addition, Ap-transmission assays under flow conditions showed that the bacteria transferred from infected endothelial cells to circulating granulocytes and were able to establish infection in constantly moving granulocytes. Cell surface analysis showed that Ap induced up-regulation of the cell adhesion molecules ICAM-1 and VCAM-1 on infected endothelial cells in a dose-dependent manner. Furthermore, IL-8 secretion by endothelial cells indicated that the presence of Ap induced a pro-inflammatory response. In summary, the results of this study suggest that endothelial cells of the microvasculature (1) provide an excellent site for Ap dissemination to peripheral blood granulocytes under flow conditions and therefore may play a crucial role in the development of persistent infection, and (2) are stimulated by Ap to express surface molecules and cytokines that may lead to inflammatory responses at the site of the infection.

  11. Immunomodulatory impact of the A2A adenosine receptor on the profile of chemokines produced by neutrophils

    Science.gov (United States)

    McColl, Shaun R.; St-Onge, Mireille; Dussault, Andrée-Anne; Laflamme, Cynthia; Bouchard, Line; Boulanger, Jean; Pouliot, Marc

    2010-01-01

    In LPS-stimulated human neutrophils, engagement of the adenosine A2A receptor selectively prevented the expression and release of TNF-α, MIP-1α/CCL3, MIP-1β/CCL4, MIP-2α/CXCL2, and MIP-3α/CCL20. In mice lacking the A2A receptor, granulocytes that migrated into the air pouch 4 h after LPS injection expressed higher mRNA levels of TNF-α, MIP-1α, and MIP-1β than PMNs from wild-type mice. In mononuclear cells present in the air pouch 72 h after LPS injection, expression of IL-1β, TNF-α, IL-6, and MCP-2/CCL6 was higher in A2AR knockout mice. In addition to highlighting neutrophils as an early and pivotal target for mediating adenosine anti-inflammatory activities, these results identify TNF-α and the MIP chemokine family as gene products whose expression is pivotally affected by activation of A2AR in LPS-activated PMNs. Modulation by A2AR in the production of inflammatory signals by PMNs may thus influence the evolution of an inflammatory response by reducing the activation status of inflammatory cells. PMID:16280366

  12. Autophagy Primes Neutrophils for Neutrophil Extracellular Trap Formation during Sepsis.

    Science.gov (United States)

    Park, So Young; Shrestha, Sanjeeb; Youn, Young-Jin; Kim, Jun-Kyu; Kim, Shin-Yeong; Kim, Hyun Jung; Park, So-Hee; Ahn, Won-Gyun; Kim, Shin; Lee, Myung Goo; Jung, Ki-Suck; Park, Yong Bum; Mo, Eun-Kyung; Ko, Yousang; Lee, Suh-Young; Koh, Younsuck; Park, Myung Jae; Song, Dong-Keun; Hong, Chang-Won

    2017-09-01

    Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined. We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia. Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis. Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism. These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.

  13. Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

    KAUST Repository

    Zhu, Jiang

    2014-06-11

    Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Human Neutrophil Lipocalin in Activated Whole Blood Is a Specific and Rapid Diagnostic Biomarker of Bacterial Infections in the Respiratory Tract.

    Science.gov (United States)

    Venge, Per; Eriksson, Ann-Katrin; Douhan-Håkansson, Lena; Pauksen, Karlis

    2017-07-01

    The distinction between bacterial and viral causes of infections of the respiratory tract is a major but important clinical challenge. We investigated the diagnostic performance of human neutrophil lipocalin (HNL) in respiratory tract infections compared to those of C-reactive protein (CRP) and procalcitonin (PCT). Patients were recruited from the emergency department and from a primary care unit (n = 162). The clinical diagnosis with regard to bacterial or viral cause of infection was complemented with objective microbiological/serological testing. HNL was measured in whole blood after preactivation with the neutrophil activator formyl-methionine-leucine-phenylalanine (fMLP) (B-HNL), and CRP and PCT were measured in plasma. Head-to-head comparisons of the three biomarkers showed that B-HNL was a superior diagnostic means to distinguish between causes of infections, with areas under the concentration-time curve (AUCs) of receiver operating characteristic (ROC) analysis for HNL of 0.91 (95% confidence interval [CI], 0.83 to 0.96) and 0.92 (95% CI, 0.82 to 0.97) for all respiratory infections and for upper respiratory infections, respectively, compared to 0.72 (95% CI, 0.63 to 0.80) and 0.68 (95% CI, 0.56 to 0.79) for CRP, respectively (P = 0.001). In relation to major clinical symptoms of respiratory tract infections (cough, sore throat, stuffy nose, and signs of sinusitis), AUCs varied between 0.88 and 0.93 in those patients with likely etiology (i.e., etiology is likely determined) of infection, compared to 0.63 and 0.71 for CRP, respectively, and nonsignificant AUCs for PCT. The diagnostic performance of B-HNL is superior to that of plasma CRP (P-CRP) and plasma PCT (P-PCT) in respiratory tract infections, and the activity specifically reflects bacterial challenge in the body. The rapid and accurate analysis of HNL by point-of-care technologies should be a major advancement in the diagnosis and management of respiratory infections with respect to antibiotic

  15. Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3 in colon cancer serum and tumours: a biomarker study

    Directory of Open Access Journals (Sweden)

    Olsen Jesper

    2005-01-01

    Full Text Available Abstract Background Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer. Methods By Surface Enhanced Laser Desorption/Ionisation – Time Of Flight / Mass spectrometry (SELDI-TOF/MS we compare the protein profiles of colon cancer serum with serum from healthy individuals and the protein profiles of colon tumours with normal colon tissue. By size exclusion chromatography, we investigate the binding of HNP 1-3 to high mass plasma proteins. By microflow we investigate the effect of HNP 1-3 on mammalian cells. Results Human Neutrophil Peptides -1, -2 and -3 (HNP 1-3, also known as alfa-defensin-1, -2 and -3, are present in elevated concentrations in serum from colon cancer patients and in protein extracts from colon tumours. A fraction of HNP 1-3 in serum is bound to unidentified high mass plasma proteins. HNP 1-3 purified from colon tumours are lethal to mammalian cells. Conclusions HNP 1-3 may serve as blood markers for colon cancer in combination with other diagnostic tools. We propose that HNP 1-3 are carried into the bloodstream by attaching to high mass plasma proteins in the tumour microenvironment. We discuss the effect of HNP 1-3 on tumour progression.

  16. Inhibitory Effects of Standardized Extracts of Phyllanthus amarus and Phyllanthus urinaria and Their Marker Compounds on Phagocytic Activity of Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Yuandani

    2013-01-01

    Full Text Available The standardized methanol extracts of Phyllanthus amarus and P. urinaria, collected from Malaysia and Indonesia, and their isolated chemical markers, phyllanthin and hypophyllanthin, were evaluated for their effects on the chemotaxis, phagocytosis and chemiluminescence of human phagocytes. All the plant extracts strongly inhibited the migration of polymorphonuclear leukocytes (PMNs with the Malaysian P. amarus showing the strongest inhibitory activity (IC50 value, 1.1 µg/mL. There was moderate inhibition by the extracts of the bacteria engulfment by the phagocytes with the Malaysian P. amarus exhibiting the highest inhibition (50.8% of phagocytizing cells. The Malaysian P. amarus and P. urinaria showed strong reactive oxygen species (ROS inhibitory activity, with both extracts exhibiting IC50 value of 0.7 µg/mL. Phyllanthin and hypophyllanthin exhibited relatively strong activity against PMNs chemotaxis, with IC50 values slightly lower than that of ibuprofen (1.4 µg/mL. Phyllanthin exhibited strong inhibitory activity on the oxidative burst with an IC50 value comparable to that of aspirin (1.9 µg/mL. Phyllanthin exhibited strong engulfment inhibitory activity with percentage of phagocytizing cells of 14.2 and 27.1% for neutrophils and monocytes, respectively. The strong inhibitory activity of the extracts was due to the presence of high amounts of phyllanthin and hypophyllanthin although other constituents may also contribute.

  17. In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection

    Directory of Open Access Journals (Sweden)

    Le Fichoux Yves

    2001-08-01

    Full Text Available Abstract Background The role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum. Results Promastigote phagocytosis and killing occurs very early after infection, as demonstrated by electron microscopy analyses which show in BALB/c mouse spleen, but not in liver, numerous PN harbouring ultrastructurally degraded parasites. It is shown, using mAb RB6-8C5 directed against mature mouse granulocytes, that in chronically infected mice, long-term PN depletion did not enhance parasite counts neither in liver nor in spleen, indicating that these cells are not involved in the late phase of L. infantum infection. In acute stage of infection, in mouse liver, where L. infantum load is initially larger than that in spleen but resolves spontaneously, there was no significant effect of neutrophils depletion. By contrast, early in infection the neutrophil cells crucially contributed to parasite killing in spleen, since PN depletion, performed before and up to 7 days after the parasite inoculation, resulted in a ten-fold increase of parasite burden. Conclusions Taken together these data show that neutrophil cells contribute to the early control of the parasite growth in spleen but not in liver and that these cells have no significant effect late in infection in either of these target organs.

  18. Interactions between Neutrophils and Pseudomonas aeruginosa in Cystic Fibrosis

    Science.gov (United States)

    Rada, Balázs

    2017-01-01

    Cystic fibrosis (CF) affects 70,000 patients worldwide. Morbidity and mortality in CF is largely caused by lung complications due to the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. Cystic fibrosis airway inflammation is mediated by robust infiltration of polymorphonuclear neutrophil granulocytes (PMNs, neutrophils). Neutrophils are not capable of clearing lung infections and contribute to tissue damage by releasing their dangerous cargo. Pseudomonas aeruginosa is an opportunistic pathogen causing infections in immunocompromised individuals. P. aeruginosa is a main respiratory pathogen in CF infecting most patients. Although PMNs are key to attack and clear P. aeruginosa in immunocompetent individuals, PMNs fail to do so in CF. Understanding why neutrophils cannot clear P. aeruginosa in CF is essential to design novel therapies. This review provides an overview of the antimicrobial mechanisms by which PMNs attack and eliminate P. aeruginosa. It also summarizes current advances in our understanding of why PMNs are incapable of clearing P. aeruginosa and how this bacterium adapts to and resists PMN-mediated killing in the airways of CF patients chronically infected with P. aeruginosa. PMID:28282951

  19. Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

    Directory of Open Access Journals (Sweden)

    Go Kamoshida

    2018-02-01

    Full Text Available Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs, has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA, and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

  20. Early diagnostic method for sepsis based on neutrophil MR imaging

    Directory of Open Access Journals (Sweden)

    Shanhua Han

    2015-06-01

    Conclusion: Mouse and human neutrophils could be more effectively labelled by Mannan-coated SPION in vitro than Feridex. Sepsis analog neutrophils labelled by Mannan-coated SPIONs could be efficiently detected on MR images, which may serve as an early diagnostic method for sepsis.

  1. Generalized intramuscular granulocytic sarcoma mimicking polymyositis

    Energy Technology Data Exchange (ETDEWEB)

    Fritz, Jan; Claussen, Claus D.; Pereira, Philippe L.; Horger, Marius S. [Eberhard-Karls-University, Department of Diagnostic Radiology, Tuebingen (Germany); Vogel, Wichard [Eberhard-Karls-University, Department of Internal Medicine-Oncology, Tuebingen (Germany); Wehrmann, Martin [Eberhard-Karls-University, Department of Pathology, Tuebingen (Germany)

    2007-10-15

    We report a case of granulocytic sarcoma exclusively manifesting as diffuse intramuscular infiltration of the proximal upper and lower limb girdle and the torso muscles in a patient with previous history of acute myelogenous leukemia 5a. Whole-body CT showed widespread distribution of ill-defined intramuscular, homogeneously enhancing lesions. On whole-body MRI, lesions were homogeneously hyperintense on fat saturated T2-weighted images, isointense on T1-weighted images and strongly enhancing after intravenous gadolinium contrast administration. Histopathology revealed muscular infiltration of blast cells with identical immunochemistry to the initial manifestation of leukemia, diagnostic for an extramedullary relapse manifesting as granulocytic sarcoma. CT and MRI characteristics of this previously undocumented manifestation of granulocytic sarcoma should assist in the identification of such cases. (orig.)

  2. Red blood cell sedimentation of Apheresis Granulocytes.

    Science.gov (United States)

    Lodermeier, Michelle A; Byrne, Karen M; Flegel, Willy A

    2017-10-01

    Sedimentation of Apheresis Granulocyte components removes red blood cells. It is used to increase the blood donor pool when blood group-compatible donors cannot be recruited for a patient because of a major ABO incompatibility or incompatible red blood cell antibodies in the recipient. Because granulocytes have little ABO and few other red blood cell antigens on their membrane, such incompatibility lies mostly with the contaminating red blood cells. Video Clip S1 shows the process of red blood cell sedimentation of an Apheresis Granulocyte component. This video was filmed with a single smart phone attached to a commercial tripod and was edited on a tablet computer with free software by an amateur videographer without prior video experience. © 2017 AABB.

  3. Experimental and Human Evidence for Lipocalin-2 (Neutrophil Gelatinase-Associated Lipocalin [NGAL]) in the Development of Cardiac Hypertrophy and heart failure.

    Science.gov (United States)

    Marques, Francine Z; Prestes, Priscilla R; Byars, Sean G; Ritchie, Scott C; Würtz, Peter; Patel, Sheila K; Booth, Scott A; Rana, Indrajeetsinh; Minoda, Yosuke; Berzins, Stuart P; Curl, Claire L; Bell, James R; Wai, Bryan; Srivastava, Piyush M; Kangas, Antti J; Soininen, Pasi; Ruohonen, Saku; Kähönen, Mika; Lehtimäki, Terho; Raitoharju, Emma; Havulinna, Aki; Perola, Markus; Raitakari, Olli; Salomaa, Veikko; Ala-Korpela, Mika; Kettunen, Johannes; McGlynn, Maree; Kelly, Jason; Wlodek, Mary E; Lewandowski, Paul A; Delbridge, Lea M; Burrell, Louise M; Inouye, Michael; Harrap, Stephen B; Charchar, Fadi J

    2017-06-14

    Cardiac hypertrophy increases the risk of developing heart failure and cardiovascular death. The neutrophil inflammatory protein, lipocalin-2 (LCN2/NGAL), is elevated in certain forms of cardiac hypertrophy and acute heart failure. However, a specific role for LCN2 in predisposition and etiology of hypertrophy and the relevant genetic determinants are unclear. Here, we defined the role of LCN2 in concentric cardiac hypertrophy in terms of pathophysiology, inflammatory expression networks, and genomic determinants. We used 3 experimental models: a polygenic model of cardiac hypertrophy and heart failure, a model of intrauterine growth restriction and Lcn2 -knockout mouse; cultured cardiomyocytes; and 2 human cohorts: 114 type 2 diabetes mellitus patients and 2064 healthy subjects of the YFS (Young Finns Study). In hypertrophic heart rats, cardiac and circulating Lcn2 was significantly overexpressed before, during, and after development of cardiac hypertrophy and heart failure. Lcn2 expression was increased in hypertrophic hearts in a model of intrauterine growth restriction, whereas Lcn2 -knockout mice had smaller hearts. In cultured cardiomyocytes, Lcn2 activated molecular hypertrophic pathways and increased cell size, but reduced proliferation and cell numbers. Increased LCN2 was associated with cardiac hypertrophy and diastolic dysfunction in diabetes mellitus. In the YFS, LCN2 expression was associated with body mass index and cardiac mass and with levels of inflammatory markers. The single-nucleotide polymorphism, rs13297295, located near LCN2 defined a significant cis -eQTL for LCN2 expression. Direct effects of LCN2 on cardiomyocyte size and number and the consistent associations in experimental and human analyses reveal a central role for LCN2 in the ontogeny of cardiac hypertrophy and heart failure. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  4. Utility of Immature Granulocyte Percentage in Pediatric Appendicitis

    Science.gov (United States)

    Mathews, Eleanor K.; Griffin, Russell L.; Mortellaro, Vincent; Beierle, Elizabeth A.; Harmon, Carroll M.; Chen, Mike K.; Russell, Robert T.

    2014-01-01

    Background Acute appendicitis is the most common cause of abdominal surgery in children. Adjuncts are utilized to help clinicians predict acute or perforated appendicitis, which may affect treatment decisions. Automated hematologic analyzers can perform more accurate automated differentials including immature granulocyte percentages (IG%). Elevated IG% has demonstrated improved accuracy for predicting sepsis in the neonatal population than traditional immature to total neutrophil count (I/T) ratios. We intended to assess the additional discriminatory ability of IG% to traditionally assessed parameters in the differentiation between acute and perforated appendicitis. Materials and Methods We identified all patients with appendicitis from July 2012 to June 2013 by ICD-9 code. Charts were reviewed for relevant demographic, clinical, and outcome data, which were compared between acute and perforated appendicitis groups using Fischer’s exact and t-test for categorical and continuous variables, respectively. We utilized an adjusted logistic regression model utilizing clinical lab values to predict the odds of perforated appendicitis. Results 251 patients were included in the analysis. Those with perforated appendicitis had a higher white blood cell (WBC) count (p=0.0063), C-reactive protein (CRP) (pappendicitis. The c-statistic of the final model was 0.70, suggesting fair discriminatory ability in predicting perforated appendicitis. Conclusions IG% did not provide any additional benefit to elevated CRP and presence of left shift in the differentiation between acute and perforated appendicitis. PMID:24793450

  5. Purification of human recombinant granulocyte colony stimulating ...

    African Journals Online (AJOL)

    In Escherichia coli, recombinant proteins were produced either as three dimensionally folded forms or as unfolded forms, inclusion body (IB). The formation of IB was a frequent consequence of high-level protein production and inadequacy of folding agents namely chaperones in the cytoplasm. The structure of the protein in ...

  6. The redistribution of granulocytes following E. coli endotoxin induced sepsis