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Sample records for arrests human monocytic

  1. The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line

    DEFF Research Database (Denmark)

    Suzuki, Harukazu; Forrest, Alistair R R; van Nimwegen, Erik

    2009-01-01

    Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites...

  2. Human monocyte differentiation stage affects response to arachidonic acid.

    Science.gov (United States)

    Escobar-Alvarez, Elizabeth; Pelaez, Carlos A; García, Luis F; Rojas, Mauricio

    2010-01-01

    AA-induced cell death mechanisms acting on human monocytes and monocyte-derived macrophages (MDM), U937 promonocytes and PMA-differentiated U937 cells were studied. Arachidonic acid induced apoptosis and necrosis in monocytes and U937 cells but only apoptosis in MDM and U937D cells. AA increased both types of death in Mycobacterium tuberculosis-infected cells and increased the percentage of TNFalpha+ cells and reduced IL-10+ cells. Experiments blocking these cytokines indicated that AA-mediated death was TNFalpha- and IL-10-independent. The differences in AA-mediated cell death could be explained by high ROS, calpain and sPLA-2 production and activity in monocytes. Blocking sPLA-2 in monocytes and treatment with antioxidants favored M. tuberculosis control whereas AA enhanced M. tuberculosis growth in MDM. Such evidence suggested that AA-modulated effector mechanisms depend on mononuclear phagocytes' differentiation stage.

  3. Design of phosphorylated dendritic architectures to promote human monocyte activation.

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    Poupot, Mary; Griffe, Laurent; Marchand, Patrice; Maraval, Alexandrine; Rolland, Olivier; Martinet, Ludovic; L'Faqihi-Olive, Fatima-Ezzahra; Turrin, Cédric-Olivier; Caminade, Anne-Marie; Fournié, Jean-Jacques; Majoral, Jean-Pierre; Poupot, Rémy

    2006-11-01

    As first defensive line, monocytes are a pivotal cell population of innate immunity. Monocyte activation can be relevant to a range of immune conditions and responses. Here we present new insights into the activation of monocytes by a series of phosphonic acid-terminated, phosphorus-containing dendrimers. Various dendritic or subdendritic structures were synthesized and tested, revealing the basic structural requirements for monocyte activation. We showed that multivalent character and phosphonic acid capping of dendrimers are crucial for monocyte targeting and activation. Confocal videomicroscopy showed that a fluorescein-tagged dendrimer binds to isolated monocytes and gets internalized within a few seconds. We also found that dendrimers follow the phagolysosomial route during internalization by monocytes. Finally, we performed fluorescence resonance energy transfer (FRET) experiments between a specifically designed fluorescent dendrimer and phycoerythrin-coupled antibodies. We showed that the typical innate Toll-like receptor (TLR)-2 is clearly involved, but not alone, in the sensing of dendrimers by monocytes. In conclusion, phosphorus-containing dendrimers appear as precisely tunable nanobiotools able to target and activate human innate immunity and thus prove to be good candidates to develop new drugs for immunotherapies.

  4. Lactic acid delays the inflammatory response of human monocytes

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    Peter, Katrin, E-mail: katrin.peter@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); Rehli, Michael, E-mail: michael.rehli@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); RCI Regensburg Center for Interventional Immunology, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); Singer, Katrin, E-mail: katrin.singer@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); Renner-Sattler, Kathrin, E-mail: kathrin.renner-sattler@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); Kreutz, Marina, E-mail: marina.kreutz@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); RCI Regensburg Center for Interventional Immunology, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany)

    2015-02-13

    Lactic acid (LA) accumulates under inflammatory conditions, e.g. in wounds or tumors, and influences local immune cell functions. We previously noted inhibitory effects of LA on glycolysis and TNF secretion of human LPS-stimulated monocytes. Here, we globally analyze the influence of LA on gene expression during monocyte activation. To separate LA-specific from lactate- or pH-effects, monocytes were treated for one or four hours with LPS in the presence of physiological concentrations of LA, sodium lactate (NaL) or acidic pH. Analyses of global gene expression profiles revealed striking effects of LA during the early stimulation phase. Up-regulation of most LPS-induced genes was significantly delayed in the presence of LA, while this inhibitory effect was attenuated in acidified samples and not detected after incubation with NaL. LA targets included genes encoding for important monocyte effector proteins like cytokines (e.g. TNF and IL-23) or chemokines (e.g. CCL2 and CCL7). LA effects were validated for several targets by quantitative RT-PCR and/or ELISA. Further analysis of LPS-signaling pathways revealed that LA delayed the phosphorylation of protein kinase B (AKT) as well as the degradation of IκBα. Consistently, the LPS-induced nuclear accumulation of NFκB was also diminished in response to LA. These results indicate that the broad effect of LA on gene expression and function of human monocytes is at least partially caused by its interference with immediate signal transduction events after activation. This mechanism might contribute to monocyte suppression in the tumor environment. - Highlights: • Lactic acid broadly delays LPS-induced gene expression in human monocytes. • Expression of important monocyte effector molecules is affected by lactic acid. • Interference of lactic acid with TLR signaling causes the delayed gene expression. • The profound effect of lactic acid might contribute to immune suppression in tumors.

  5. Derivation of multipotent progenitors from human circulating CD14+ monocytes.

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    Seta, Noriyuki; Kuwana, Masataka

    2010-07-01

    Circulating CD14(+) monocytes are originated from hematopoietic stem cells in the bone marrow and believed to be committed precursors for phagocytes, such as macrophages. Recently, we have reported a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which has a fibroblast-like morphology in culture and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from circulating CD14(+) monocytes, but circulating precursors for MOMCs still remain undetermined. Comparative analysis of gene expression profiles of MOMCs and other monocyte-derived cells has revealed that embryonic stem cell markers, Nanog and Oct-4, are specifically expressed by MOMCs. In vitro generation of MOMCs requires binding to fibronectin and exposure to soluble factors derived from activated platelets. MOMCs contain progenitors with capacity to differentiate into a variety of nonphagocytes, including bone, cartilage, fat, skeletal and cardiac muscle, neuron, and endothelium, indicating that circulating monocytes are more multipotent than previously thought. In addition, MOMCs are capable of promoting ex vivo expansion of human hematopoietic progenitor cells through direct cell-to-cell contact and secretion of a variety of hematopoietic growth factors. These findings obtained from the research on MOMCs indicate that CD14(+) monocytes in circulation are involved in a variety of physiologic functions other than innate and acquired immune responses, such as repair and regeneration of the damaged tissue.

  6. Maturation and demise of human primary monocytes by carbon nanotubes

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    De Nicola, Milena, E-mail: milena.de.nicola@uniroma2.it [University of Rome ' Tor Vergata' , Department of Biology (Italy); Mirabile Gattia, Daniele, E-mail: daniele.mirabile@enea.it [UTTMAT, ENEA-C.R. Casaccia (Italy); Traversa, Enrico, E-mail: Enrico.Traversa@kaust.edu.sa [King Abdullah University of Science and Technology (KAUST), Division of Physical Science and Engineering (Saudi Arabia); Ghibelli, Lina, E-mail: ghibelli@uniroma2.it [University of Rome ' Tor Vergata' , Department of Biology (Italy)

    2013-06-15

    The possibility of exploiting carbon nanotubes (CNT) in biomedical practices requires thorough analysis of the chemical or bulk effects they may exert on the immune system, the complex network that recognizes and eliminates foreign particles. In particular, the phagocytosing ability of cells belonging to the monocyte/macrophage lineage may render these immune cells an ideal toxicological target of pristine CNT, which may form aggregates of size exceeding monocyte/macrophage phagocytosing plasticity. To shed light on this issue, we analyzed the effects that pristine multi-walled CNT (MWCNT) without metal or biological impurities exert on survival and activation of freshly explanted human peripheral blood monocytes, analyzing in parallel the non-phagocytosing lymphocytes, and using graphite as control carbon material. MWCNT (diameter 10-50 nm, length up to 10 {mu}m) exert two different toxic effects on mononuclear leukocytes: a minor apoptogenic effect (on lymphocytes > monocytes), and a major, apoptosis-independent effect that exclusively and deeply affect monocyte homeostasis. Analysis of monocyte number, adhesion, redox equilibrium, and the differentiation markers CD14 and CD11b reveals that MWCNT cause the selective disappearance of phagocytosis-competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes, and their differentiation toward a peculiar maturation asset. These observations point out novel mechanisms of CNT toxicity, renewing concerns that they may impair the innate immune system deranging the inflammatory responses.

  7. Maturation and demise of human primary monocytes by carbon nanotubes

    KAUST Repository

    De Nicola, Milena D.

    2013-05-17

    The possibility of exploiting carbon nanotubes (CNT) in biomedical practices requires thorough analysis of the chemical or bulk effects they may exert on the immune system, the complex network that recognizes and eliminates foreign particles. In particular, the phagocytosing ability of cells belonging to the monocyte/macrophage lineage may render these immune cells an ideal toxicological target of pristine CNT, which may form aggregates of size exceeding monocyte/macrophage phagocytosing plasticity. To shed light on this issue, we analyzed the effects that pristine multi-walled CNT (MWCNT) without metal or biological impurities exert on survival and activation of freshly explanted human peripheral blood monocytes, analyzing in parallel the non-phagocytosing lymphocytes, and using graphite as control carbon material. MWCNT (diameter 10-50 nm, length up to 10 μm) exert two different toxic effects on mononuclear leukocytes: a minor apoptogenic effect (on lymphocytes > monocytes), and a major, apoptosis-independent effect that exclusively and deeply affect monocyte homeostasis. Analysis of monocyte number, adhesion, redox equilibrium, and the differentiation markers CD14 and CD11b reveals that MWCNT cause the selective disappearance of phagocytosis-competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes, and their differentiation toward a peculiar maturation asset. These observations point out novel mechanisms of CNT toxicity, renewing concerns that they may impair the innate immune system deranging the inflammatory responses. © 2013 Springer Science+Business Media Dordrecht.

  8. Immunomodulation of human monocytes following exposure to Lutzomyia intermedia saliva

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    Barral Aldina

    2008-04-01

    Full Text Available Abstract Background Sand fly saliva contains potent and complex pharmacologic molecules that are able to modulate the host's hemostatic, inflammatory, and immune systems. In this study, we evaluated the effects of salivary gland sonicate (SGS of Lutzomyia intermedia, the natural vector of Leishmania braziliensis, on monocytes obtained from the peripheral blood mononuclear cells (PBMC of healthy volunteers. We investigated the effects of sand fly saliva on cytokine production and surface molecule expression of LPS-stimulated human monocytes uninfected or infected with L. braziliensis. Results Pre-treatment of non-infected human monocytes with L. intermedia SGS followed by LPS-stimulation led to a significant decrease in IL-10 production accompanied by a significant increase in CD86, CD80, and HLA-DR expression. Pre-treatment with SGS followed by LPS stimulation and L. braziliensis infection led to a significant increase in TNF-α, IL-6, and IL-8 production without significant alterations in co-stimulatory molecule expression. However, pre-treatment with L. intermedia SGS did not result in significant changes in the infection rate of human monocytes. Conclusion Our data indicate that L. intermedia saliva is able to modulate monocyte response, and, although this modulation is dissociated from enhanced infection with L. braziliensis, it may be associated with successful parasitism.

  9. Induction of reactive oxygen intermediates in human monocytes by tumour cells and their role in spontaneous monocyte cytotoxicity

    Science.gov (United States)

    Mytar, B; Siedlar, M; Woloszyn, M; Ruggiero, I; Pryjma, J; Zembala, M

    1999-01-01

    The present study examined the ability of human monocytes to produce reactive oxygen intermediates after a contact with tumour cells. Monocytes generated oxygen radicals, as measured by luminol-enhanced chemiluminescence and superoxide anion production, after stimulation with the tumour, but not with untransformed, cells. The use of specific oxygen radical scavengers and inhibitors, superoxide dismutase, catalase, dimethyl sulphoxide and deferoxamine as well as the myeloperoxidase inhibitor 4-aminobenzoic acid hydrazide, indicated that chemiluminescence was dependent on the production of superoxide anion and hydroxyl radical and the presence of myeloperoxidase. The tumour cell-induced chemiluminescent response of monocytes showed different kinetics from that seen after activation of monocytes with phorbol ester. These results indicate that human monocytes can be directly stimulated by tumour cells for reactive oxygen intermediate production. Spontaneous monocyte-mediated cytotoxicity towards cancer cells was inhibited by superoxide dismutase, catalase, deferoxamine and hydrazide, implicating the role of superoxide anion, hydrogen peroxide, hydroxyl radical and hypohalite. We wish to suggest that so-called ‘spontaneous’ tumoricidal capacity of freshly isolated human monocytes may in fact be an inducible event associated with generation of reactive oxygen intermediates and perhaps other toxic mediators, resulting from a contact of monocytes with tumour cells. © 1999 Cancer Research Campaign PMID:10070862

  10. DMPD: LPS induction of gene expression in human monocytes. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 11257452 LPS induction of gene expression in human monocytes. Guha M, Mackman N. Ce...ll Signal. 2001 Feb;13(2):85-94. (.png) (.svg) (.html) (.csml) Show LPS induction of gene expression in huma...n monocytes. PubmedID 11257452 Title LPS induction of gene expression in human monocytes. Authors Guha M, Ma

  11. The outcome of Cryptococcus neoformans intracellular pathogenesis in human monocytes

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    Pirofski Liise-anne

    2009-03-01

    Full Text Available Abstract Background Cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen. The interaction between macrophages and C. neoformans is critical for extrapulmonary dissemination of this pathogenic yeast. C. neoformans can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. However, most studies of intracellular pathogenesis have been made with mouse cells and their relevance to human infection is uncertain. In this study we extended studies of C. neoformans-macrophage cellular interaction/s to human peripheral blood monocytes. Results This study demonstrated that C. neoformans can shed polysaccharide within human monocytes, spread from cell to cell, and be extruded from them. Furthermore, human monocytes responded to ingestion of C. neoformans with cell cycle progression from G1 to S. Conclusion Similarities between mouse and human cells support the suitability of mouse cells for the study of intracellular pathogenesis mechanisms. Given that these hosts diverged over 70 million years ago, the similar pathogenic strategies for C. neoformans in murine and human cells supports the hypothesis that the mechanism that underlies the mammalian intracellular pathogenesis of C. neoformans originated from interactions with a third host, possibly soil amoeboid predators, before the mammalian radiation.

  12. Glucocorticoids enhance the in vivo migratory response of human monocytes.

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    Yeager, Mark P; Pioli, Patricia A; Collins, Jane; Barr, Fiona; Metzler, Sara; Sites, Brian D; Guyre, Paul M

    2016-05-01

    Glucocorticoids (GCs) are best known for their potent anti-inflammatory effects. However, an emerging model for glucocorticoid (GC) regulation of in vivo inflammation also includes a delayed, preparatory effect that manifests as enhanced inflammation following exposure to an inflammatory stimulus. When GCs are transiently elevated in vivo following exposure to a stressful event, this model proposes that a subsequent period of increased inflammatory responsiveness is adaptive because it enhances resistance to a subsequent stressor. In the present study, we examined the migratory response of human monocytes/macrophages following transient in vivo exposure to stress-associated concentrations of cortisol. Participants were administered cortisol for 6h to elevate in vivo cortisol levels to approximate those observed during major systemic stress. Monocytes in peripheral blood and macrophages in sterile inflammatory tissue (skin blisters) were studied before and after exposure to cortisol or placebo. We found that exposure to cortisol induced transient upregulation of monocyte mRNA for CCR2, the receptor for monocyte chemotactic protein-1 (MCP-1/CCL2) as well as for the chemokine receptor CX3CR1. At the same time, mRNA for the transcription factor IκBα was decreased. Monocyte surface expression of CCR2 but not CX3CR1 increased in the first 24h after cortisol exposure. Transient exposure to cortisol also led to an increased number of macrophages and neutrophils in fluid derived from a sterile inflammatory site in vivo. These findings suggest that the delayed, pro-inflammatory effects of cortisol on the human inflammatory responses may include enhanced localization of effector cells at sites of in vivo inflammation.

  13. A curated compendium of monocyte transcriptome datasets of relevance to human monocyte immunobiology research.

    Science.gov (United States)

    Rinchai, Darawan; Boughorbel, Sabri; Presnell, Scott; Quinn, Charlie; Chaussabel, Damien

    2016-01-01

    Systems-scale profiling approaches have become widely used in translational research settings. The resulting accumulation of large-scale datasets in public repositories represents a critical opportunity to promote insight and foster knowledge discovery. However, resources that can serve as an interface between biomedical researchers and such vast and heterogeneous dataset collections are needed in order to fulfill this potential. Recently, we have developed an interactive data browsing and visualization web application, the Gene Expression Browser (GXB). This tool can be used to overlay deep molecular phenotyping data with rich contextual information about analytes, samples and studies along with ancillary clinical or immunological profiling data. In this note, we describe a curated compendium of 93 public datasets generated in the context of human monocyte immunological studies, representing a total of 4,516 transcriptome profiles. Datasets were uploaded to an instance of GXB along with study description and sample annotations. Study samples were arranged in different groups. Ranked gene lists were generated based on relevant group comparisons. This resource is publicly available online at http://monocyte.gxbsidra.org/dm3/landing.gsp.

  14. Delayed hippocampal damage in humans following cardiorespiratory arrest.

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    Petito, C K; Feldmann, E; Pulsinelli, W A; Plum, F

    1987-08-01

    Transient ischemia in animals produces delayed cell death in vulnerable hippocampal neurons. To see if this occurs in humans, we reexamined brain slides from all patients with anoxic-ischemic encephalopathy and a well-documented cardiorespiratory arrest. Eight patients dying 18 hours or less after cardiac arrest had minimal damage in hippocampus and moderate damage in cerebral cortex and putamen. Six patients living 24 hours or more had severe damage in all four regions. The increase in damage with time postarrest was significant only in the hippocampus. Delayed hippocampal injury now documented in humans provides a target for possible therapy that can be initiated after cardiopulmonary resuscitation.

  15. Human recombinant macrophage inflammatory protein-1 alpha and -beta and monocyte chemotactic and activating factor utilize common and unique receptors on human monocytes.

    Science.gov (United States)

    Wang, J M; Sherry, B; Fivash, M J; Kelvin, D J; Oppenheim, J J

    1993-04-01

    The human macrophage inflammatory proteins-1 alpha and -beta (MIP-1 alpha and -beta), which are also known as LD78 and ACT2, respectively, are distinct but highly related members of the chemoattractant cytokine (chemokine) family. rMIP-1 alpha and -beta labeled with 125I specifically bind to human peripheral blood monocytes, the monocytic cell line THP-1, peripheral blood T cells, and the YT cell line. Steady state binding experiments revealed approximately 3000 high affinity binding sites/cell for MIP-1 alpha on human monocytes and on THP-1 cells, with Kd values of 383 pM and 450 pM, respectively. Human MIP-1 alpha and -beta had nearly identical affinities for the binding sites and each competed equally well for binding. Human monocyte chemotactic and activating factor (MCAF), a member of the same chemokine family, consistently displaced about 25% of human MIP-1 alpha and -beta binding on monocytes but not on YT cells, which did not bind MCAF. On the other hand, human rMIP-1 alpha and -beta partially inhibited binding of radiolabeled MCAF to monocytes. Both MIP-1 alpha and -beta were chemotactic for human monocytes. Preincubation of monocytes with human rMIP-1 alpha or -beta markedly reduced cell migration towards the other cytokine, whereas preincubation with human rMCAF only partially desensitized the monocyte chemotaxis response to human rMIP-1 alpha or -beta. These data suggest the existence of three subtypes of receptors, i.e., one unique receptor shared by MIP-1 alpha and -beta, a second unique receptor for MCAF, and a third species that recognizes both MCAF and MIP-1 peptides.

  16. A case of human monocytic ehrlichiosis in Serbia

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    Arsić Bogdan

    2014-01-01

    Full Text Available Introduction. Ehrlichiosis is a bacterial zoonosis transmitted by hematophagous arthropods - ticks. In humans, it occurs as monocytic, granulocytic, and ewingii ehrlichiosis. Pathological process is based on parasitic presence of Ehrlichia organisms within peripheral blood cells - monocytes and granulocytes. Case Outline. Fifty-two year old patient was admitted to hospital due to high fever of over 40°C that lasted two days, accompanied with chills, muscle aches, malaise, loss of appetite, headache, confusion, breathing difficulties, and mild dry cough. The history suggested tick bite that occurred seven days before the onset of disease. Doxycycline was introduced and administered for 14 days, causing the disease to subside. Indirect immunofluorescence assay was used to analyze three serum samples obtained from this patient for Ehrlichia chaffeensis antibodies, and peripheral blood smear was evaluated for the presence of Ehrlichia and Ehrlichia aggregation into morulae. Conclusion. Ehrlichiosis should be considered in each case where there is a history of tick bite together with the clinical picture (high fever, chills, muscle aches, headache, generalized weakness and malaise, and possible maculopapular rash. The presence of Ehrlichia chaffeensis antibodies was confirmed in a patient with the history of tick bite, appropriate clinical picture and indirect immunofluorescence assay. This confirmed the presence of human monocytotropic ehrlichiosis, a disease that is uncommonly identified in our country.

  17. Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation– A Short Protocol

    OpenAIRE

    Clarke, Elizabeth V.; Benoit, Marie E.; Tenner, Andrea J.

    2013-01-01

    Investigations of the activation processes involved in human monocytes and monocyte-derived macrophages and dendritic cells often required large numbers of cells that have not been possibly altered or activated by adherence to surfaces, by binding of antibodies to surface antigens during positive selection, or by release of activators by platelets or other non myeloid cells during isolation or co-culture. Human peripheral blood monocytes as well as lymphocytes from the same blood donor can be...

  18. Momilactone B induces apoptosis and G1 arrest of the cell cycle in human monocytic leukemia U937 cells through downregulation of pRB phosphorylation and induction of the cyclin-dependent kinase inhibitor p21Waf1/Cip1.

    Science.gov (United States)

    Park, Cheol; Jeong, Na Young; Kim, Gi-Young; Han, Min Ho; Chung, Ill-Min; Kim, Wun-Jae; Yoo, Young Hyun; Choi, Yung Hyun

    2014-04-01

    Momilactone B, a terpenoid phytoalexin present in rice bran, has been shown to exhibit several biological activities. The present study was conducted using cultured human leukemia U937 cells to elucidate the possible mechanisms by which momilactone B exerts its anticancer activity, which to date has remained poorly understood. Momilactone B treatment of U937 cells resulted in a dose-dependent inhibition of cell growth and induced apoptotic cell death as detected by chromatin condensation, DNA fragmentation, the cleavage of poly(ADP-ribose) polymerase and Annexin V-FITC staining. Flow cytometric analysis revealed that momilactone B resulted in G1 arrest in cell cycle progression, which was associated with the dephosphorylation of retinoblastoma protein (pRB) and enhanced binding of pRB with the E2F transcription factor family proteins. Treatment with momilactone B also increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1 in a p53-independent manner, without any noticeable changes in G1 cyclins and cyclin-dependent kinases (Cdks), except a slight decrease in cyclin E. Moreover, in vitro kinase assay indicated that momilactone B significantly decreased Cdk4- and Cdk6-associated kinase activities through a notably increased binding of p21 to Cdk4 and Cdk6. Our results demonstrated that momilactone B caused G1 cell cycle arrest and apoptosis in U937 cells through the induction of p21 expression, inhibition of Cdk/cyclin-associated kinase activities, and reduced phosphorylation of pRB, which may be related to anticancer activity.

  19. High glucose-induced oxidative stress increases transient receptor potential channel expression in human monocytes

    DEFF Research Database (Denmark)

    Wuensch, Tilo; Thilo, Florian; Krueger, Katharina;

    2010-01-01

    Transient receptor potential (TRP) channel-induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose-induced oxidative stress on TRP channel expression in human monocytes....

  20. Sphingosylphosphorylcholine stimulates human monocyte-derived dendritic cell chemotaxis

    Institute of Scientific and Technical Information of China (English)

    Ha-young LEE; Eun-ha SHIN; Yoe-sik BAE

    2006-01-01

    Aim: To investigate the effects of Sphingosylphosphorylcholine (SPC) on human monocyte-derived dendritic cell (DC) chemotaxis. Methods: Human DC were generated from peripheral blood monocytes by culturing them with granulocyte macrophage-colony stimulating factor and interleukin-4. The effect of SPC on the DC chemotactic migration was measured by chemotaxis assay. Intracellular signaling event involved in the SPC-induced DC chemotaxis was investigated with several inhibitors for specific kinase. The expression of the SPC receptors was examined by reverse transcription polymerase chain reaction. Results: We found that SPC induced chemotactic migration in immature DC (iDC) and mature DC (mDC). In terms of SPC-induced signaling events, mitogen activated protein kinase activation and Akt activation in iDC and mDC were stimulated. SPC-induced chemotaxis was mediated by extracellular signal-regulated protein kinase and phosphoino-sitide-3-kinase, but not by calcium in both iDC and mDC. Although mDC express ovarian cancer G protein-coupled receptor 1, but not G protein-coupled receptor 4, iDC do not express any of these receptors. To examine the involvement of sphin-gosine-1-phosphate (SIP) receptors, we checked the effect of an SIP receptor antagonist (VPC23019) on SPC-induced DC chemotaxis. VPC23019 did not affect SPC-induced DC chemotaxis. Conclusion: The results suggest that SPC may play a role in regulating DC trafficking during phagocytosis and the T cell-stimulating phase, and the unique SPC receptor, which is different from SIP receptors, is involved in SPC-induced chemotaxis.

  1. Expression and regulation of Schlafen (SLFN family members in primary human monocytes, monocyte-derived dendritic cells and T cells

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    Alexander Puck

    2015-01-01

    Full Text Available Schlafen (SLFN/Slfn family members have been investigated for their involvement in fundamental cellular processes including growth regulation, differentiation and control of viral replication. However, most research has been focused on the characterization of Slfns within the murine system or in human cell lines. Since little is known about SLFNs in primary human immune cells, we set out to analyze the expression and regulation of the six human SLFN genes in monocytes, monocyte-derived dendritic cells (moDCs and T cells. Comparison of SLFN gene expression across these three cell types showed high mRNA expression of SLFN11 in monocytes and moDCs and high SLFN5 expression in T cells, indicating functional importance within these cell types. Differentiation of monocytes to moDCs leads to the gradual upregulation of SLFN12L and SLFN13 while SLFN12 levels were decreased by differentiation stimuli. Stimulation of moDCs via human rhinovirus, lipopolysaccharide, or IFN-α lead to strong upregulation of SLFN gene expression, while peptidoglycan poorly stimulated regulation of both SLFNs and the classical interferon-stimulated gene MxA. T cell activation was found to downregulate the expression of SLFN5, SLFN12 and SLFN12L, which was reversible upon addition of exogenous IFN-α. In conclusion, we demonstrate, that SLFN gene upregulation is mainly dependent on autocrine type I interferon signaling in primary human immune cells. Rapid decrease of SLFN expression levels following T cell receptor stimulation indicates a role of SLFNs in the regulation of human T cell quiescence.

  2. A novel in vitro human microglia model: characterization of human monocyte-derived microglia.

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    Etemad, Samar; Zamin, Rasheeda Mohd; Ruitenberg, Marc J; Filgueira, Luis

    2012-07-30

    Microglia are the innate immune cells of the central nervous system. They help maintaining physiological homeostasis and contribute significantly to inflammatory responses in the course of infection, injury and degenerative processes. To date, there is no standardized simple model available to investigate the biology of human microglia. The aim of this study was to establish a new human microglia model. For that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of M-CSF, GM-CSF, NGF and CCL2 to generate monocyte-derived microglia (M-MG). M-MG were clearly different in morphology, phenotype and function from freshly isolated monocytes, cultured monocytes in the absence of the cytokines and monocyte-derived dendritic cells (M-DC) cultured in the presence of GM-CSF and IL-4. M-MG acquired a ramified morphology with primary and secondary processes. M-MG displayed a comparable phenotype to the human microglia cell line HMC3, expressing very low levels of CD45, CD14 and HLA-DR, CD11b and CD11c; and undetectable levels of CD40, CD80 and CD83, and a distinct pattern of chemokine receptors (positive for CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR3, CX3CR1; negative for CCR6 and CCR7). In comparison with M-DC, M-MG displayed lower T-lymphocyte stimulatory capacity, as well as lower phagocytosis activity. The described protocol for the generation of human monocyte-derived microglia is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. This protocol will certainly be very helpful for future studies investigating the biology and pathology of human microglia.

  3. A functional study on the migration of human monocytes to human leukemic cell lines and the role of monocyte chemoattractant protein-1

    NARCIS (Netherlands)

    Legdeur, MCJC; Beelen, RHJ; Schuurhuis, GJ; Broekhoven, MG; vandeLoosdrecht, AA; Tekstra, J; Langenhuijsen, MMAC; Ossenkoppele, GJ

    1997-01-01

    In the present study the migration of human monocytes towards the supernatants of five different human myeloid leukemic cell lines, four different human lymphatic leukemic cell lines and blasts derived from three different patients with acute myeloid leukemia (AML) was studied and the role of monocy

  4. Modulation of the expression of chondroitin sulfate proteoglycan in stimulated human monocytes

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    Uhlin-Hansen, L.; Eskeland, T.; Kolset, S.O. (Univ. of Tromso (Norway))

    1989-09-05

    Proteoglycan biosynthesis was studied in human monocytes and monocyte-derived macrophages (MDM) after exposure to typical activators of the monocyte/macrophage system: interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). By morphological examination, both monocytes and MDM were stimulated by these activators. Treatment with IFN-gamma resulted in a slight decrease in the expression of (35S)chondroitin sulfate proteoglycan (CSPG) in both monocytes and MDM, whereas LPS treatment increased the (35S)CSPG expression 1.8 and 2.2 times, respectively. PMA, in contrast, decreased the CSPG expression 0.4 times in monocytes, whereas MDM were stimulated to increase the biosynthesis 1.9 times. An increase in the sulfate density of the chondroitin sulfate chains was evident following differentiation of monocytes into MDM due to the expression of disulfated disaccharide units of the chondroitin sulfate E type (CS-E). However, monocytes exposed to PMA did also express disaccharides of the chondroitin sulfate E type. Furthermore, the expression of CS-E in MDM was increased 2 times following PMA treatment. An inactive phorbol ester, phorbol 12,13-diacetate, did not affect the expression of CS-E in either monocytes or MDM when compared with control cultures, suggesting that protein kinase C-dependent signal pathways may be involved in the regulation of sulfation of CSPG. Exposure to LPS or IFN-gamma did not lead to any changes in the sulfation of the chondroitin sulfate chains.

  5. Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

    OpenAIRE

    Kontny, U.; Kurane, I; Ennis, F A

    1988-01-01

    It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexe...

  6. Species difference of CD137 ligand signaling in human and murine monocytes.

    Directory of Open Access Journals (Sweden)

    Qianqiao Tang

    Full Text Available BACKGROUND: Stimulation of CD137 ligand on human monocytes has been shown to induce DC differentiation, and these CD137L-DCs are more potent than classical DCs, in stimulating T cell responses in vitro. To allow an in vivo evaluation of the potency of CD137L-DCs in murine models we aimed at generating murine CD137L-DCs. METHODOLOGY/PRINCIPAL FINDINGS: When stimulated through CD137 ligand murine monocytes responded just as human monocytes with an increased adherence, morphological changes, proliferation and an increase in viable cell numbers. But CD137 ligand signaling did not induce expression of inflammatory cytokines and costimulatory molecules in murine monocytes and these cells had no T cell stimulatory activity. Murine monocytes did not differentiate to inflammatory DCs upon CD137 ligand signaling. Furthermore, while CD137 ligand signaling induces maturation of human immature classical DCs it failed to do so with murine immature classical DCs. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that both human and murine monocytes become activated by CD137 ligand signaling but only human and not murine monocytes differentiate to inflammatory DCs.

  7. Alpha 1-antitrypsin does not inhibit human monocyte caspase-1.

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    Mohd Akhlakur Rahman

    Full Text Available Alpha 1-antitrypsin (A1AT is a 52 kDa serine protease inhibitor produced largely by hepatocytes but also by mononuclear phagocytes. A1AT chiefly inhibits neutrophil elastase and proteinase-3 but has also been reported to have immune modulatory functions including the ability to inhibit caspases. Its clinical availability for infusion suggests that A1AT therapy might modulate caspase related inflammation. Here we tested the ability of A1AT to modulate caspase-1 function in human mononuclear phagocytes.Purified plasma derived A1AT was added to active caspase-1 in a cell-free system (THP-1 lysates as well as added exogenously to cell-culture models and human whole blood models of caspase-1 activation. Functional caspase-1 activity was quantified by the cleavage of the caspase-1 specific fluorogenic tetrapeptide substrate (WEHD-afc and the release of processed IL-18 and IL-1β.THP-1 cell lysates generated spontaneous activation of caspase-1 both by WEHD-afc cleavage and the generation of p20 caspase-1. A1AT added to this cell free system was unable to inhibit caspase-1 activity. Release of processed IL-18 by THP-1 cells was also unaffected by the addition of exogenous A1AT prior to stimulation with LPS/ATP, a standard caspase-1 activating signal. Importantly, the A1AT exhibited potent neutrophil elastase inhibitory capacity. Furthermore, A1AT complexed to NE (and hence conformationally modified also did not affect THP-1 cell caspase-1 activation. Finally, exogenous A1AT did not inhibit the ability of human whole blood samples to process and release IL-1β.A1AT does not inhibit human monocyte caspase-1.

  8. IL-4 induces cAMP and cGMP in human monocytic cells

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    B. Dugas

    1995-01-01

    Full Text Available Human monocytes, preincubated with IFN-γ respond to IL-4 by a cGMP increase through activation of an inducible NO synthase. Here, IL-4 was found to induce an accumulation of cGMP (1 – 3 min and cAMP (20 – 25 min in unstimulated monocytes. This was impaired with NOS inhibitors, but also with EGTA and calcium/calmodulin inhibitors. These results suggest that: (1 IL-4 may stimulate different NOS isoforms in resting and IFN-γ activated monocytes, and (2 cAMP accumulation may be partially dependent on the NO pathway. By RT-PCR, a type III constitutive NOS mRNA was detected in U937 monocytic cells. IL-4 also increased the [Ca2+]i in these cells. Different NOS may thus be expressed in monocytic cells depending on their differentiation and the signals they receive.

  9. Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components.

    Science.gov (United States)

    Delfino, D; Cianci, L; Lupis, E; Celeste, A; Petrelli, M L; Curró, F; Cusumano, V; Teti, G

    1997-06-01

    In order to ascertain if Cryptococcus neoformans components can induce interleukin-6 (IL-6) production, we stimulated human whole blood with purified capsular products. Their potencies in stimulating IL-6 release were mannoproteins > galactoxylomannan = glucuronoxylomannan > alpha(1-3)glucan. IL-6 production was tumor necrosis factor alpha independent and required the presence of monocytes and plasma. Since IL-6 can stimulate replication of the human immunodeficiency virus in monocytic cells, these findings may be clinically relevant.

  10. The subcellular particulate NADPH-dependent O2.(-)-generating oxidase from human blood monocytes: comparison to the neutrophil system.

    Science.gov (United States)

    Chaudhry, A N; Santinga, J T; Gabig, T G

    1982-10-01

    Highly purified preparations of normal human monocytes obtained from peripheral blood were shown to contain a subcellular particulate O2.(-)-generating oxidase system. This O2.(-)-generating activity was present in particulate preparations from monocytes that had been previously stimulated with phorbol myristate acetate but was low or absent in control preparations from unstimulated monocytes or stimulated monocytes from a patient with chronic granulomatous disease. In the stimulated preparations from normal monocytes, O2.(-)-generation was linearly proportional to cell protein concentration, insensitive to inhibition by azide, and dependent on NADPH as substrate. These characteristics are similar to the O2.(-)-generating oxidase system from human neutrophils. A significant difference in the apparent Km for NADPH was shown between preparations from stimulated monocytes and neutrophils (monocyte 83 +/- 16 microM, neutrophil 31 +/- 5 microM, mean +/- SE). Additionally, affinity of the stimulated monocyte particulate preparation for NADH was unmeasurably low.

  11. Release of salusin-beta from human monocytes/macrophages.

    Science.gov (United States)

    Sato, Kengo; Fujimoto, Kazumi; Koyama, Takatoshi; Shichiri, Masayoshi

    2010-06-01

    the release of salusin-beta. These data demonstrate that salusin-beta, which induces macrophage foam cell formation, is secreted in its authentic form from human monocytes/macrophages.

  12. Isolation and cryopreservation of human peripheral blood monocytes.

    Science.gov (United States)

    Tsvetkov, T; Nickolov, C; Buckureshtliev, A; Mincheff, M; Tsoney, L; Terziev, R; Popov, D

    1986-12-01

    A modification of the Freundlich and Avdalovic method (J. Immunol. Methods 62, 31 (1983] is reported. Buffy coats, separated and pooled together, are used for isolation of monocytes (70% yield, 100% purity). Cell density of working suspension is increased up to 0.65 X 10(9) cells/75 cm2 surface by multiplication of the active fibronectin sites. For the purpose, cryoprecipitate is used instead of plasma for coating the glass-gelatin surface. Monocytes, isolated by that procedure, could be successfully cryopreserved with dimethyl sulfoxide cryoprotective solution.

  13. Anti-tumour cytotoxin produced by human monocytes: studies on its mode of action.

    OpenAIRE

    Matthews, N.

    1983-01-01

    Human monocytes can be induced to synthesize a cytotoxin which affects certain tumour cell lines. The interaction of monocyte cytotoxin with a susceptible cell line (L929) has been studied to obtain clues to the mode of action of the cytotoxin. The cytotoxin acts directly on the cells rather than on the culture medium and is cytotoxic at higher concentrations and cytostatic at lower concentrations. First signs of cell damage appear about 20 h after contact with the cytotoxin which must be pre...

  14. Interactions of human monocytes with TMVs (tumour-derived microvesicles).

    Science.gov (United States)

    Baj-Krzyworzeka, Monika; Baran, Jarosław; Szatanek, Rafał; Mytar, Bożenna; Siedlar, Maciej; Zembala, Marek

    2013-02-01

    The tumour microenvironment represents a dynamic complex milieu, which includes tumour cells, cells of the immune system and other (cellular and non-cellular) components. The role of these particular 'puzzle pieces' may change substantially due to their mutual interactions. The present review concerns different opinions on interactions that occur between monocytes, tumour cells and TMVs (tumour-derived microvesicles).

  15. Decreased glucose uptake by hyperglycemia is regulated by different mechanisms in human cancer cells and monocytes

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    Kim, Chae Kyun; Chung, June Key; Lee, Yong Jin; Hong, Mee Kyoung; Jeong, Jae Min; Lee, Dong Soo; Lee, Myung Chul [College of Medicine, Seoul National Univ., Seoul (Korea, Republic of)

    2002-04-01

    To clarify the difference in glucose uptake between human cancer cells and monocytes, we studied ({sup 18}F) fluorodeoxyglucose (FDG) uptake in three human colon cancer cell lines (SNU-C2A, SNU-C4, SNU-C5), one human lung cancer cell line (NCI-H522), and human peripheral blood monocytes. The FDG uptake of both cancer cells and monocytes was increased in glucose-free medium, but decreased in the medium containing 16.7 mM glucose (hyperglycemic). The level of Glut1 mRNA decreased in human colon cancer cells and NCI-H522 under hyperglycemic condition. Glut1 protein expression was also decreased in the four human cancer cell lines under hyperglycemic condition, whereas it was consistently undetectable in monocytes. SNU-C2A, SNU-C4 and NCI-H522 showed a similar level of hexokinase activity (7.5-10.8 mU/mg), while SNU-C5 and moncytes showed lower range of hexokinase activity (4.3-6.5 mU/mg). These data suggest that glucose uptake is regulated by different mechanisms in human cancer cells and monocytes.

  16. Expression of the cytochrome P450 epoxygenase CYP2J2 in human monocytic leukocytes.

    Science.gov (United States)

    Nakayama, Kaeko; Nitto, Takeaki; Inoue, Teruo; Node, Koichi

    2008-08-29

    CYP2J2 is one of the cytochrome P450 epoxygenases involved in the metabolism of arachidonic acid. CYP2J2 has been identified in several tissues, especially cardiovascular tissues. CYP2J2 has cardiovascular effects, as epoxyeicosatrienoic acid, one of its metabolites, has anti-inflammatory and vasodilative activities. We investigated the expression of CYP2J2 in human leukocytes using reverse transcription-polymerase chain reaction, immunoblotting and immunostaining. Human monocytic cells, but not human neutrophils, exhibited constitutive expression of CYP2J2. Furthermore, the expression of CYP2J2 mRNA increased when the human monocytic cell line THP-1 cells and human monocytes were stimulated with phorbol 12-myristate 13-acetate and macrophage-colony stimulating factor in combination with granulocyte/macrophage-colony stimulating factor, respectively. These results suggest that expression of CYP2J2 was up-regulated when human monocytes differentiated into macrophages and that human monocytic cells and macrophages have a pathway to metabolize arachidonic acid using CYP epoxygenases.

  17. Viral infection triggers rapid differentiation of human blood monocytes into dendritic cells.

    Science.gov (United States)

    Hou, Wanqiu; Gibbs, James S; Lu, Xiuju; Brooke, Christopher B; Roy, Devika; Modlin, Robert L; Bennink, Jack R; Yewdell, Jonathan W

    2012-03-29

    Surprisingly little is known about the interaction of human blood mononuclear cells with viruses. Here, we show that monocytes are the predominant cell type infected when peripheral blood mononuclear cells are exposed to viruses ex vivo. Remarkably, infection with vesicular stomatitis virus, vaccinia virus, and a variety of influenza A viruses (including circulating swine-origin virus) induces monocytes to differentiate within 18 hours into CD16(-)CD83(+) mature dendritic cells with enhanced capacity to activate T cells. Differentiation into dendritic cells does not require cell division and occurs despite the synthesis of viral proteins, which demonstrates that monocytes counteract the capacity of these highly lytic viruses to hijack host cell biosynthetic capacity. Indeed, differentiation requires infectious virus and viral protein synthesis. These findings demonstrate that monocytes are uniquely susceptible to viral infection among blood mononuclear cells, with the likely purpose of generating cells with enhanced capacity to activate innate and acquired antiviral immunity.

  18. A curated compendium of monocyte transcriptome datasets of relevance to human monocyte immunobiology research [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Darawan Rinchai

    2016-04-01

    Full Text Available Systems-scale profiling approaches have become widely used in translational research settings. The resulting accumulation of large-scale datasets in public repositories represents a critical opportunity to promote insight and foster knowledge discovery. However, resources that can serve as an interface between biomedical researchers and such vast and heterogeneous dataset collections are needed in order to fulfill this potential. Recently, we have developed an interactive data browsing and visualization web application, the Gene Expression Browser (GXB. This tool can be used to overlay deep molecular phenotyping data with rich contextual information about analytes, samples and studies along with ancillary clinical or immunological profiling data. In this note, we describe a curated compendium of 93 public datasets generated in the context of human monocyte immunological studies, representing a total of 4,516 transcriptome profiles. Datasets were uploaded to an instance of GXB along with study description and sample annotations. Study samples were arranged in different groups. Ranked gene lists were generated based on relevant group comparisons. This resource is publicly available online at http://monocyte.gxbsidra.org/dm3/landing.gsp.

  19. HMG-CoA reductase inhibition aborts functional differentiation and triggers apoptosis in cultured primary human monocytes: a potential mechanism of statin-mediated vasculoprotection

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    Vamvakopoulos Joannis E

    2003-07-01

    Full Text Available Abstract Background Statins effectively lower blood cholesterol and the risk of cardiovascular death. Immunomodulatory actions, independent of their lipid-lowering effect, have also been ascribed to these compounds. Since macrophages participate in several vascular pathologies, we examined the effect of statin treatment on the survival and differentiation of primary human monocytes. Methods Peripheral blood mononuclear cells (PBMCs from healthy individuals were cultured in the presence or absence of mevastatin. Apoptosis was monitored by annexin V / PI staining and flow cytometry. In parallel experiments, cultures were stimulated with LPS in the presence or absence of mevastatin and the release of IL-1β and IL-1Ra was measured by ELISA. Results Among PBMCs, mevastatin-treated monocytes were particularly susceptible to apoptosis, which occurred at doses >1 microM and was already maximal at 5 microM. However, even at the highest mevastatin dose used (10 microM, apoptosis occurred only after 24 h of culture, possibly reflecting a requirement for cell commitment to differentiation. After 72 h of treatment the vast majority (>50% of monocytes were undergoing apoptosis. Stimulation with LPS revealed that mevastatin-treated monocytes retained the high IL-1β output characteristic of undifferentiated cells; conversely, IL-1Ra release was inhibited. Concurrent treatment with mevalonolactone prevented the induction of apoptosis and suppressed both IL-1β and IL-1Ra release in response to LPS, suggesting a rate-limiting role for HMG-CoA reductase in monocyte differentiation. Conclusions Our findings indicate that statins arrest the functional differentiation of monocytes into macrophages and steer these cells into apoptosis, suggesting a novel mechanism for the vasculoprotective properties of HMG-CoA reductase inhibitors.

  20. Heparin-like polymers modulate proinflammatory cytokine production by lipopolysaccharide-stimulated human monocytes.

    Science.gov (United States)

    Anastase-Ravion, Sylvie; Carreno, Marie-Paule; Blondin, Catherine; Ravion, Olivier; Champion, Jacqueline; Chaubet, Frédéric; Haeffner-Cavaillon, Nicole; Letourneur, Didier

    2002-06-05

    The search for heparin-like materials remains an intensive field of research. In this context, we studied the immunomodulatory properties of semisynthetic dextran derivatives and naturally occurring sulfated polysaccharides present in brown seaweed (fucans). In this study, we investigated the functional potencies of fucan and dextran derivatives by analyzing their effects on the release of proinflammatory cytokines by resting or lipopolysaccharide (LPS)-stimulated human monocytes and their interactions on monocyte surfaces. The results showed that fucan, dextran derivatives, and heparin differentially (1) triggered interleukin-1alpha, tumor necrosis factor alpha, interleukin-6, and interleukin-8 production by monocytes in a dose-dependent manner, (2) modulated cytokine production by LPS-stimulated monocytes, and (3) specifically inhibited the binding of biotinylated LPS to monocyte membranes. Taken together, these data indicated that fucan and dextran derivatives displayed interesting immunomodulatory effects on human blood cells that could be relevant as new drugs or biomaterial coatings. Indeed, such polysaccharides, by regulating monocyte activation, could contribute to the improved biocompatibility of implants.

  1. In Vitro Brucella suis Infection Prevents the Programmed Cell Death of Human Monocytic Cells

    Science.gov (United States)

    Gross, Antoine; Terraza, Annie; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre; Dornand, Jacques

    2000-01-01

    During the complex interaction between an infectious agent and a host organism, the pathogen can interfere with the host cell's programmed death to its own benefit. Induction or prevention of host cell apoptosis appears to be a critical step for determining the infection outcome. Members of the gram-negative bacterial genus Brucella are intracellular pathogens which preferentially invade monocytic cells and develop within these cells. We investigated the effect of Brucella suis infection on apoptosis of human monocytic phagocytes. The present study provides evidence that Brucella infection inhibited spontaneously occurring apoptosis in human monocytes. Prevention of monocyte apoptosis was not mediated by Brucella lipopolysaccharide and required bacterial survival within infected cells. Both invaded and noninvaded cells were protected, indicating that soluble mediators released during infection were involved in the phenomenon. Analysis of Brucella-infected monocytes revealed specific overexpression of the A1 gene, a member of the bcl-2 family implicated in the survival of hematopoietic cells. Brucella infection also rendered macrophage-like cells resistant to Fas ligand- or gamma interferon-induced apoptosis, suggesting that Brucella infection protected host cells from several cytotoxic processes occurring at different steps of the immune response. The present data clearly show that Brucella suis modulated the monocyte/macrophage's apoptotic response to the advantage of the pathogen, thus preventing host cell elimination. This might represent a strategy for Brucella development in infected hosts. PMID:10603407

  2. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes

    Science.gov (United States)

    Yeap, Wei Hseun; Wong, Kok Loon; Shimasaki, Noriko; Teo, Esmeralda Chi Yuan; Quek, Jeffrey Kim Siang; Yong, Hao Xiang; Diong, Colin Phipps; Bertoletti, Antonio; Linn, Yeh Ching; Wong, Siew Cheng

    2016-01-01

    Antibody-dependent cellular cytotoxicity (ADCC) is exerted by immune cells expressing surface Fcγ receptors (FcγRs) against cells coated with antibody, such as virus-infected or transformed cells. CD16, the FcγRIIIA, is essential for ADCC by NK cells, and is also expressed by a subset of human blood monocytes. We found that human CD16− expressing monocytes have a broad spectrum of ADCC capacities and can kill cancer cell lines, primary leukemic cells and hepatitis B virus-infected cells in the presence of specific antibodies. Engagement of CD16 on monocytes by antibody bound to target cells activated β2-integrins and induced TNFα secretion. In turn, this induced TNFR expression on the target cells, making them susceptible to TNFα-mediated cell death. Treatment with TLR agonists, DAMPs or cytokines, such as IFNγ, further enhanced ADCC. Monocytes lacking CD16 did not exert ADCC but acquired this property after CD16 expression was induced by either cytokine stimulation or transient transfection. Notably, CD16+ monocytes from patients with leukemia also exerted potent ADCC. Hence, CD16+ monocytes are important effectors of ADCC, suggesting further developments of this property in the context of cellular therapies for cancer and infectious diseases. PMID:27670158

  3. The effect of short-chain fatty acids on human monocyte-derived dendritic cells

    DEFF Research Database (Denmark)

    Nastasi, Claudia; Candela, Marco; Bonefeld, Charlotte Menné

    2015-01-01

    negligible effects, while both butyrate and propionate strongly modulated gene expression in both immature and mature human monocyte-derived DC. An Ingenuity pathway analysis based on the differentially expressed genes suggested that propionate and butyrate modulate leukocyte trafficking, as SCFA strongly......The gut microbiota is essential for human health and plays an important role in the pathogenesis of several diseases. Short-chain fatty acids (SCFA), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients that distribute systemically via the blood....... The aim of this study was to investigate the transcriptional response of immature and LPS-matured human monocyte-derived DC to SCFA. Our data revealed distinct effects exerted by each individual SCFA on gene expression in human monocyte-derived DC, especially in the mature ones. Acetate only exerted...

  4. Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes.

    Science.gov (United States)

    Lee, Sahmin; Lee, Hyun-Chae; Kwon, Yoo-Wook; Lee, Sang Eun; Cho, Youngjin; Kim, Joonoh; Lee, Soobeom; Kim, Ju-Young; Lee, Jaewon; Yang, Han-Mo; Mook-Jung, Inhee; Nam, Ky-Youb; Chung, Junho; Lazar, Mitchell A; Kim, Hyo-Soo

    2014-03-04

    Human resistin is a cytokine that induces low-grade inflammation by stimulating monocytes. Resistin-mediated chronic inflammation can lead to obesity, atherosclerosis, and other cardiometabolic diseases. Nevertheless, the receptor for human resistin has not been clarified. Here, we identified adenylyl cyclase-associated protein 1 (CAP1) as a functional receptor for human resistin and clarified its intracellular signaling pathway to modulate inflammatory action of monocytes. We found that human resistin directly binds to CAP1 in monocytes and upregulates cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, and NF-κB-related transcription of inflammatory cytokines. Overexpression of CAP1 in monocytes enhanced the resistin-induced increased activity of the cAMP-dependent signaling. Moreover, CAP1-overexpressed monocytes aggravated adipose tissue inflammation in transgenic mice that express human resistin from their monocytes. In contrast, suppression of CAP1 expression abrogated the resistin-mediated inflammatory activity both in vitro and in vivo. Therefore, CAP1 is the bona fide receptor for resistin leading to inflammation in humans.

  5. β2-Agonist clenbuterol hinders human monocyte differentiation into dendritic cells.

    Science.gov (United States)

    Giordani, Luciana; Cuzziol, Noemi; Del Pinto, Tamara; Sanchez, Massimo; Maccari, Sonia; Massimi, Alessia; Pietraforte, Donatella; Viora, Marina

    2015-12-10

    Clenbuterol (CLB) is a beta2-adrenergic agonist commonly used in asthma therapy, but is also a non-steroidal anabolic drug often abused in sport doping practices. Here we evaluated the in vitro impact of CLB on the physiology and function of human monocytes and dendritic cells (DCs), instrumental in the development of immune responses. We demonstrate that CLB inhibits the differentiation of monocytes into DCs and this effect is specific and dependent on β2-adrenergic receptor (AR) activation. We found that CLB treatment reduced the percentage of CD1a(+) immature DCs, while increasing the frequency of monocytes retaining CD14 surface expression. Moreover, CLB inhibited tumor necrosis factor-alpha (TNF-alpha) enhanced IL-(interleukin)-10 and IL-6 production. In contrast, CLB did not modulate the phenotypic and functional properties of monocytes and DCs, such as the surface expression of HLA-DR, CD83, CD80 and CD86 molecules, cytokine production, immunostimulatory activity and phagocytic activity. Moreover, we found that CLB did not modulate the activation of NF-kB in DCs. Moreover, we found that the differentiation of monocytes into DCs was associated with a significant decrease of β2-ARs mRNA expression. These results provide new insights on the effect of CLB on monocyte differentiation into DCs. Considering the frequent illegal use of CLB in doping, our work suggests that this drug is potentially harmful to immune responses decreasing the supply of DCs, thus subverting immune surveillance.

  6. Differential Oxidative Stress Induced by Dengue Virus in Monocytes from Human Neonates, Adult and Elderly Individuals

    Science.gov (United States)

    Valero, Nereida; Mosquera, Jesús; Añez, Germán; Levy, Alegria; Marcucci, Rafael; de Mon, Melchor Alvarez

    2013-01-01

    Changes in immune response during lifespan of man are well known. These changes involve decreased neonatal and elderly immune response. In addition, it has been shown a relationship between immune and oxidative mechanisms, suggesting that altered immune response could be associated to altered oxidative response. Increased expression of nitric oxide (NO) has been documented in dengue and in monocyte cultures infected with different types of dengue virus. However, there is no information about the age-dependent NO oxidative response in humans infected by dengue virus. In this study, monocyte cultures from neonatal, elderly and adult individuals (n = 10 each group) were infected with different dengue virus types (DENV- 1 to 4) and oxidative/antioxidative responses and apoptosis were measured at days 1 and 3 of culture. Increased production of NO, lipid peroxidation and enzymatic and nonenzymatic anti-oxidative responses in dengue infected monocyte cultures were observed. However, neonatal and elderly monocytes had lower values of studied parameters when compared to those in adult-derived cultures. Apoptosis was present in infected monocytes with higher values at day 3 of culture. This reduced oxidant/antioxidant response of neonatal and elderly monocytes could be relevant in the pathogenesis of dengue disease. PMID:24069178

  7. Differential oxidative stress induced by dengue virus in monocytes from human neonates, adult and elderly individuals.

    Directory of Open Access Journals (Sweden)

    Nereida Valero

    Full Text Available Changes in immune response during lifespan of man are well known. These changes involve decreased neonatal and elderly immune response. In addition, it has been shown a relationship between immune and oxidative mechanisms, suggesting that altered immune response could be associated to altered oxidative response. Increased expression of nitric oxide (NO has been documented in dengue and in monocyte cultures infected with different types of dengue virus. However, there is no information about the age-dependent NO oxidative response in humans infected by dengue virus. In this study, monocyte cultures from neonatal, elderly and adult individuals (n = 10 each group were infected with different dengue virus types (DENV- 1 to 4 and oxidative/antioxidative responses and apoptosis were measured at days 1 and 3 of culture. Increased production of NO, lipid peroxidation and enzymatic and nonenzymatic anti-oxidative responses in dengue infected monocyte cultures were observed. However, neonatal and elderly monocytes had lower values of studied parameters when compared to those in adult-derived cultures. Apoptosis was present in infected monocytes with higher values at day 3 of culture. This reduced oxidant/antioxidant response of neonatal and elderly monocytes could be relevant in the pathogenesis of dengue disease.

  8. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells.

    OpenAIRE

    Cushing, S D; Berliner, J A; Valente, A. J.; Territo, M C; Navab, M; Parhami, F; Gerrity, R; Schwartz, C J; Fogelman, A M

    1990-01-01

    After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2- to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human ...

  9. Differential Activation of Human Monocytes and Lymphocytes by Distinct Strains of Trypanosoma cruzi

    Science.gov (United States)

    Magalhães, Luísa M. D.; Viana, Agostinho; Chiari, Egler; Galvão, Lúcia M. C.; Gollob, Kenneth J.; Dutra, Walderez O.

    2015-01-01

    Background Trypanosoma cruzi strains are currently classified into six discrete typing units (DTUs) named TcI to VI. It is known that these DTUs have different geographical distribution, as well as biological features. TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively. Our hypothesis is that upon infection of human peripheral blood cells, Y strain (Tc II) and Col cl1.7 (Tc I), cause distinct immunological changes, which might influence the clinical course of Chagas disease. Methodology/Principal Findings We evaluated the infectivity of CFSE-stained trypomastigotes of Col cl1.7 and Y strain in human monocytes for 15 and 72 hours, and determined the immunological profile of lymphocytes and monocytes exposed to the different isolates using multiparameter flow cytometry. Our results showed a similar percentage and intensity of monocyte infection by Y and Col cl1.7. We also observed an increased expression of CD80 and CD86 by monocytes infected with Col cl1.7, but not Y strain. IL-10 was significantly higher in monocytes infected with Col cl1.7, as compared to Y strain. Moreover, infection with Col cl1.7, but not Y strain, led to an increased expression of IL-17 by CD8+ T cells. On the other hand, we observed a positive correlation between the expression of TNF-alpha and granzyme A only after infection with Y strain. Conclusion/Significance Our study shows that while Col cl1.7 induces higher monocyte activation and, at the same time, production of IL-10, infection with Y strain leads to a lower monocyte activation but higher inflammatory profile. These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression. PMID:26147698

  10. Increased synthesis of eicosanoids by human monocytes following leucine and methionine enkephalin administration

    Energy Technology Data Exchange (ETDEWEB)

    Wiederhold, M.D.; Ou, D.W.

    1986-03-05

    Regulation of eicosanoid biosynthesis by neuropeptides was investigated in human peripheral blood monocytes from normal donors. Metabolites of /sup 3/H-arachidonic acid (/sup 3/H-AA) were analyzed by thin layer and high pressure liquid chromatography following exposure to 0.2 ..mu..gm/ml and 2.0 ..mu..gm/ml of leucine (L-ENK) and methionine (M-ENK) enkephalin. Supernatants of cultured cells were analyzed. The data indicate that both leucine and methionine enkephalin can stimulate eicosanoid biosynthesis in human monocytes, and may indicate a possible regulatory mechanism between the central nervous system and the reticuloendothelial system.

  11. Regulation of nutrition-associated receptors in blood monocytes of normal weight and obese humans.

    Science.gov (United States)

    Pivovarova, Olga; Hornemann, Silke; Weimer, Sandra; Lu, Ye; Murahovschi, Veronica; Zhuk, Sergei; Seltmann, Anne-Cathrin; Malashicheva, Anna; Kostareva, Anna; Kruse, Michael; Busjahn, Andreas; Rudovich, Natalia; Pfeiffer, Andreas F H

    2015-03-01

    Obesity, type 2 diabetes and associated metabolic diseases are characterized by low-grade systemic inflammation which involves interplay of nutrition and monocyte/macrophage functions. We suggested that some factors such as nutrient components, neuropeptides involved in the control of gastrointestinal functions, and gastrointestinal hormones might influence immune cell functions and in this way contribute to the disease pathogenesis. The aim of this study was to investigate the mRNA expression of twelve nutrition-associated receptors in peripheral blood mononuclear cells (PBMC), isolated monocytes and monocyte-derived macrophages and their regulation under the switching from the high-carbohydrate low-fat diet to the low-carbohydrate high-fat (LC/HFD) isocaloric diet in healthy humans. The mRNA expression of receptors for short chain fatty acids (GPR41, GPR43), bile acids (TGR5), incretins (GIPR, GLP1R), cholecystokinin (CCKAR), neuropeptides VIP and PACAP (VIPR1, VIPR2), and neurotensin (NTSR1) was detected in PBMC and monocytes, while GPR41, GPR43, GIPR, TGR5, and VIPR1 were found in macrophages. Correlations of the receptor expression in monocytes with a range of metabolic and inflammatory markers were found. In non-obese subjects, the dietary switch to LC/HFD induced the increase of GPR43 and VIPR1 expression in monocytes. No significant differences of receptor expression between normal weight and moderately obese subjects were found. Our study characterized for the first time the expression pattern of nutrition-associated receptors in human blood monocytes and its dietary-induced changes linking metabolic responses to nutrition with immune functions in health and metabolic diseases.

  12. Monocyte-expressed urokinase regulates human vascular smooth muscle cell migration in a coculture model.

    Science.gov (United States)

    Kusch, Angelika; Tkachuk, Sergey; Lutter, Steffen; Haller, Hermann; Dietz, Rainer; Lipp, Martin; Dumler, Inna

    2002-01-01

    Interactions of vascular smooth muscle cells (VSMC) with monocytes recruited to the arterial wall at a site of injury, with resultant modulation of VSMC growth and migration, are central to the development of vascular intimal thickening. Urokinase-type plasminogen activator (uPA) expressed by monocytes is a potent chemotactic factor for VSMC and might serve for the acceleration of vascular remodeling. In this report, we demonstrate that coculture of human VSMC with freshly isolated peripheral blood-derived human monocytes results in significant VSMC migration that increases during the coculture period. Accordingly, VSMC adhesion was inhibited with similar kinetics. VSMC proliferation, however, was not affected and remained at the same basal level during the whole period of coculture. The increase of VSMC migration in coculture was equivalent to the uPA-induced migration of monocultured VSMC and was blocked by addition into coculture of soluble uPAR (suPAR). Analysis of uPA and uPAR expression in cocultured cells demonstrated that monocytes are a major source of uPA, whose expression increases in coculture five-fold, whereas VSMC display an increased expression of cell surface-associated uPAR. These findings indicate that upregulated uPA production by monocytes following vascular injury acts most likely as an endogenous activator of VSMC migration contributing to the remodeling of vessel walls.

  13. Monocyte human leukocyte antigen-DR transcriptional downregulation by cortisol during septic shock.

    Science.gov (United States)

    Le Tulzo, Yves; Pangault, Celine; Amiot, Laurence; Guilloux, Valérie; Tribut, Olivier; Arvieux, Cédric; Camus, Christophe; Fauchet, Renée; Thomas, Rémi; Drénou, Bernard

    2004-05-15

    Monocyte deactivation has been identified as a major factor of immunosuppression in sepsis and is associated with a loss of surface human leukocyte antigen-DR (HLA-DR) expression on circulating monocytes. Using flow cytometry, quantitative reverse transcription-polymerase chain reaction, we investigated this phenomenon in septic patients. We confirmed the early loss of monocyte HLA-DR expression in all infected patients and demonstrated that this persistent lowered expression at Day 6 correlated with severity scores, secondary infection, and death. This phenomenon occurred at a transcriptional level via a decrease in the class II transactivator A (CIITA) transcription. Furthermore, these abnormalities correlated with the high cortisol levels observed in sepsis and not with those of other putative factors such as catecholamines or interleukin-10. Finally, in vitro studies evidenced that glucocorticoids decrease HLA-DR expression at a transcriptional level via a decrease in CIITA mRNA levels, mainly by down modulating its isoforms I and III. We conclude that in human sepsis, the loss of HLA-DR expression on circulating monocytes is associated with a poor outcome. We suggest that the high endogenous cortisol level observed in septic shock may be a possible new factor involved in the loss of HLA-DR expression on monocytes via its effect on HLA-DR and CIITA transcription.

  14. Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells

    Science.gov (United States)

    Mazumder, B.; Mukhopadhyay, C. K.; Prok, A.; Cathcart, M. K.; Fox, P. L.

    1997-01-01

    Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.

  15. A new method of preparing monocyte suspensions from human whole blood.

    Science.gov (United States)

    Stoll, H P; Krämer, S; Oberhausen, E

    1986-01-01

    A method of isolating monocytes from human whole blood is described. The technique is primarily based on simple centrifugation steps that follow Tylose-sedimentation as well as on the use of the new density gradient medium Nycodens. Counterflow centrifugation is not involved. The final monocyte suspension is free of platelets. The contaminating cells are predominantly lymphocytes. As a whole, the method is a modification of the Nycodens technique published by Boyum in 1983, which leads to a total elimination of platelet contamination in the final cell suspension.

  16. In-Vitro differentiation of mature dendritic cells from human blood monocytes

    OpenAIRE

    Robert Gieseler; Dirk Heise; Afsaneh Soruri; Peter Schwartz; J. Hinrich Peters

    1998-01-01

    Representing the most potent antigen-presenting cells, dendritic cells (DC) can now be generated from human blood monocytes. We recently presented a novel protocol employing GM-CSF, IL-4, and IFN-γ to differentiate monocyte-derived DC in vitro. Here, such cells are characterized in detail. Cells in culture exhibited both dendritic and veiled morphologies, the former being adherent and the latter suspended. Phenotypically, they were CD1a-/dim, CD11a+, CD11b++, CD11c+, CD14dim/-, CD16a-/dim, CD...

  17. Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1.

    Science.gov (United States)

    Mewhort, Holly E M; Lipon, Brodie D; Svystonyuk, Daniyil A; Teng, Guoqi; Guzzardi, David G; Silva, Claudia; Yong, V Wee; Fedak, Paul W M

    2016-03-15

    Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1. These data implicate inflammation as a potential driver of cardiac fibrosis.

  18. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    Science.gov (United States)

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-09-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

  19. The role of monocyte-lineage cells in human immuno-deficiency virus persistence: mechanisms and progress

    Institute of Scientific and Technical Information of China (English)

    WU Li

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1) persistence is a major barrier to the successful treatment and eradication of acquired immunodeficiency syndrome (AIDS). In addition to resting CD4+ T cells, a significant long-lived compartment of HIV-1 infection in vivo includes blood monocytes and tissue macrophages. Studying HIV-1 persistence in monocyte-lineage cells is critical because these cells are important HIV-1 target cells in vivo. Monocyte-lineage cells, including monocytes, dendritic cells (DCs) and macrophages, play a significant role in HIV-1 infection and transmission. These cells have been implicated as viral reservoirs that facilitate HIV-1 latency and persistence. A better understanding of HIV-1 interactions with monocyte-lineage cells can potentially aid in the development of new approaches for intervention. This minireview highlights the latest advances in understanding the role of monocyte-lineage cells in HIV-1 persistence and emphasizes new insights into the mechanisms underlying viral persistence.

  20. In vitro production of tumor necrosis factor-alpha by human monocytes stimulated with lipopolysaccharide is positively correlated with increased blood monocytes after exposure to a swine barn.

    Science.gov (United States)

    Willson, P J; Khozani, T Talaei; Juurlink, B H J; Senthilselvan, A; Rennie, D C; Gerdts, V; Gawaziuk, J; Schneberger, D; Burch, Lauranell H; Dosman, J A

    2008-01-01

    Recently there has been interest in the air quality in and around intensive livestock production facilities, such as modern swine production barns, where agricultural workers and surrounding residents may be exposed to elevated levels of organic dusts. The health effects of these exposures are not completely understood. The study that is reported here is a component of a larger investigation of the relationships among the acute effects of high-concentration endotoxin exposure (swine barn dust), polymorphisms in the TLR4 gene, and respiratory outcomes following exposure to swine confinement buildings. The relationships among a mediator of acute lung inflammation, tumor necrosis factor alpha (TNF-alpha), and clinical responses to acute swine barn exposure were characterized. Analysis of the results showed that in vitro stimulation of human monocytes with as little as 1 ng/ml of lipopolysaccharide (LPS) produced a significant increase in the monocytes that produced TNF-alpha. Although the proportion of TNF-alpha-positive monocytes after in vitro stimulation with 1 ng/ml of LPS was not associated with gender or TLR4 genotype, it was positively associated with the concentration of monocytes in blood after barn exposure. Thus, these two responses to different forms of LPS exposure are significantly correlated, and more responsive monocytes in vitro indicate a forthcoming relative monocytosis, post barn exposure, which may initiate a cascade of chronic inflammation.

  1. 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.

  2. Immunomodulating and antiviral activities of Uncaria tomentosa on human monocytes infected with Dengue Virus-2.

    Science.gov (United States)

    Reis, Sonia Regina I N; Valente, Ligia M M; Sampaio, André L; Siani, Antonio C; Gandini, Mariana; Azeredo, Elzinandes L; D'Avila, Luiz A; Mazzei, José L; Henriques, Maria das Graças M; Kubelka, Claire F

    2008-03-01

    Uncaria tomentosa (Willd.) DC., a large woody vine native to the Amazon and Central American rainforests has been used medicinally by indigenous peoples since ancient times and has scientifically proven immunomodulating, anti-inflammatory, cytotoxic and antioxidant activities. Several inflammatory mediators that are implicated in vascular permeability and shock are produced after Dengue Virus (DENV) infection by monocytes, the primary targets for virus replication. Here we assessed the immunoregulatory and antiviral activities from U. tomentosa-derived samples, which were tested in an in vitro DENV infection model. DENV-2 infected human monocytes were incubated with U. tomentosa hydro-alcoholic extract or either its pentacyclic oxindole alkaloid-enriched or non-alkaloid fractions. The antiviral activity was determined by viral antigen (DENV-Ag) detection in monocytes by flow cytometry. Our results demonstrated an in vitro inhibitory activity by both extract and alkaloidal fraction, reducing DENV-Ag+ cell rates in treated monocytes. A multiple microbead immunoassay was applied for cytokine determination (TNF-alpha, IFN-alpha, IL-6 and IL-10) in infected monocyte culture supernatants. The alkaloidal fraction induced a strong immunomodulation: TNF-alpha and IFN-alpha levels were significantly decreased and there was a tendency towards IL-10 modulation. We conclude that the alkaloidal fraction was the most effective in reducing monocyte infection rates and cytokine levels. The antiviral and immunomodulating in vitro effects from U. tomentosa pentacyclic oxindole alkaloids displayed novel properties regarding therapeutic procedures in Dengue Fever and might be further investigated as a promising candidate for clinical application.

  3. Binding of recombinant HIV coat protein gp120 to human monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Finbloom, D.S.; Hoover, D.L.; Meltzer, M.S. (Food and Drug Administration, Bethesda, MD (USA))

    1991-02-15

    Inasmuch as the exact level of CD4 Ag expression on macrophages is controversial and because HIV may interact with macrophages in a manner different from that on T cells, we analyzed the binding of gp120 to freshly isolated and cultured monocytes. rgp120 was iodinated using the lactoperoxidase method to a sp. act. of 600 Ci/mmol. Highly purified monocytes (greater than 90%) were isolated from the leukapheresed blood of normal volunteers by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation and cultured 7 days in DMEM supplemented with 1000 U/ml macrophage CSF in 10% human serum. Whereas MOLT/4 cells consistently bound freshly prepared 125I-rgp120 at 80% specificity with 5100 +/- 700 mol/cell, MCSF cultured monocytes bound rgp120 at only 0 to 20% specificity and 420 +/- 200 mol/cell. Most of the radioactivity bound by these cells could not be blocked by the addition of unlabeled rgp120. In contrast, the U937 myeloid cell line bound rgp120 with 50% specificity and about 2500 mol/cell. Whereas the antibody OKT4a (anti-CD4) blocked 80% of the binding on MOLT/4 cells and 50% on U937 cells, binding was only inhibited on the average of 6% on cultured monocytes. When soluble rCD4 was used as an inhibitor, binding to MOLT/4 cells was blocked by 80%. In contrast, binding to cultured monocytes was inhibited by 28%. HIV infectivity was blocked by similar concentrations of OKT4a. These observations suggest that although most binding of gp120 to cultured monocytes is not to the CD4 determinant, several hundred molecules do bind to a CD4-like molecule which promotes virus entry and replication.

  4. Neurogenic potential of progenitors derived from human circulating CD14+ monocytes.

    Science.gov (United States)

    Kodama, Hiroaki; Inoue, Takafumi; Watanabe, Ryuichi; Yasutomi, Daisuke; Kawakami, Yutaka; Ogawa, Satoshi; Mikoshiba, Katsuhiko; Ikeda, Yasuo; Kuwana, Masataka

    2006-04-01

    We previously reported a primitive cell fraction derived from human circulating CD14+ monocytes, named monocyte-derived multipotential cells (MOMC), that can differentiate along mesenchymal lineages, including bone, cartilage, fat, skeletal muscle and cardiac muscle. In this study, we investigated whether MOMC can differentiate into the neuronal lineage. MOMC were fluorescently labelled and cocultivated with a primary culture of rat neurons for up to 4 weeks. The protein and gene expressions of neuron-specific markers in the human MOMC were evaluated over time using immunohistochemistry, in situ hybridization and reverse transcription followed by PCR. Shortly after cocultivation with rat neurons, nearly all the MOMC expressed early neuroectodermal markers, Mash1, Neurogenin2 and NeuroD, together with nestin, an intermediate filament expressed in neurogenesis. After 14 days of coculture, a subpopulation of MOMC displayed a multipolar morphology with elongated neurites and expressed mature neuron-specific markers, including neurofilament, microtubule-associated protein type 2, beta3-tubulin, NeuN and Hu. Transdifferentiation of monocytes into the neuroectodermal lineage was shown by the simultaneous expression of proneural markers and CD45/CD14 early in the differentiation process. The cocultivated MOMC retained their proliferative capacity for at least 16 days. Finally, the neuronal differentiation of MOMC was observed when they were cultured with neurons without cell-to-cell contact. The capacity of MOMC to differentiate into both mesodermal and neuroectodermal lineages suggests that circulating CD14+ monocytes are more multipotential than previously thought.

  5. Variation in dietary salt intake induces coordinated dynamics of monocyte subsets and monocyte-platelet aggregates in humans: implications in end organ inflammation.

    Directory of Open Access Journals (Sweden)

    Xin Zhou

    Full Text Available BACKGROUND: Monocyte activation and tissue infiltration are quantitatively associated with high-salt intake induced target organ inflammation. We hypothesized that high-salt challenge would induce the expansion of CD14++CD16+ monocytes, one of the three monocyte subsets with a pro-inflammatory phenotype, that is associated with target organ inflammation in humans. METHODOLOGY/PRINCIPAL FINDINGS: A dietary intervention study was performed in 20 healthy volunteers, starting with a 3-day usual diet and followed with a 7-day high-salt diet (≥15 g NaCl/day, and a 7-day low-salt diet (≤5 g NaCl/day. The amounts of three monocyte subsets ("classical" CD14++CD16-, "intermediate" CD14++CD16+ and "non-classical" CD14+CD16++ and their associations with monocyte-platelet aggregates (MPAs were measured by flow cytometry. Blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI was used to evaluate renal hypoxia. Switching to a high-salt diet resulted in CD14++ monocyte activation and a rapid expansion of CD14++CD16+ subset and MPAs, with a reciprocal decrease in the percentages of CD14++CD16- and CD14+CD16++ subsets. In vitro study using purified CD14++ monocytes revealed that elevation in extracellular [Na(+] could lead to CD14++CD16+ expansion via a ROS dependent manner. In addition, high-salt intake was associated with progressive hypoxia in the renal medulla (increased R2* signal and enhanced urinary monocyte chemoattractant protein-1 (MCP-1 excretion, indicating a temporal and spatial correlation between CD14++CD16+ subset and renal inflammation. The above changes could be completely reversed by a low-salt diet, whereas blood pressure levels remained unchanged during dietary intervention. CONCLUSIONS/SIGNIFICANCE: The present work demonstrates that short-term increases in dietary salt intake could induce the expansion of CD14++CD16+ monocytes, as well as an elevation of MPAs, which might be the underlying cellular basis of high-salt induced

  6. Proteomic profile of human monocytic cells infected with dengue virus

    Institute of Scientific and Technical Information of China (English)

    Viviana Martnez-Betancur; Marlen Martnez-Gutierrez

    2016-01-01

    Objective: To identify the changes in the proteome of U937 cells infected with dengue virus (DENV). Methods: In this study, differentiated U937 cultures were infected with two DENV-2 strains, one of which was associated with dengue (DENV-2/NG) and the other one with severe dengue (DENV-2/16681), with the aim of determining the cellular proteomic profiles under different infection conditions. Cellular proteins were extracted and sepa-rated by two-dimensional electrophoresis, and those proteins with differential expression profiles were identified by mass spectrometry. The obtained results were correlated with cellular viability, the number of infectious viral particles, and the viral DNA/protein quantity. Results: In comparison with non-infected cultures, in the cells infected with the DENV-2/NG strain, nine proteins were expressed differentially (five were upregulated and four were downregulated); in those cultures infected with the DENV-2/16681 strain, six proteins were differentially expressed (two were downregulated and four were upregu-lated). The downregulated proteins included fatty acid-binding protein, heterogeneous nuclear ribonucleoprotein 1, protein disulfide isomerase, enolase 1, heat shock 70 kDa protein 9, phosphotyrosyl phosphatase, and annexin IV. The upregulated proteins included heat shock 90 kDa protein AA1, tubulin beta, enolase 1, pyruvate kinase, transaldolase and phospholipase C-alpha. Conclusions: Because the monocyte/macrophage lineage is critical for disease patho-genicity, additional studies on these proteins could provide a better understanding of the cellular response to DENV infection and could help identify new therapeutic targets against infection.

  7. Comparative Analysis of the Interaction of Helicobacter pylori with Human Dendritic Cells, Macrophages, and Monocytes

    Science.gov (United States)

    Fehlings, Michael; Drobbe, Lea; Moos, Verena; Renner Viveros, Pablo; Hagen, Jana; Beigier-Bompadre, Macarena; Pang, Ervinna; Belogolova, Elena; Churin, Yuri; Schneider, Thomas; Meyer, Thomas F.; Aebischer, Toni

    2012-01-01

    Helicobacter pylori may cause chronic gastritis, gastric cancer, or lymphoma. Myeloid antigen-presenting cells (APCs) are most likely involved in the induction and expression of the underlying inflammatory responses. To study the interaction of human APC subsets with H. pylori, we infected monocytes, monocyte-derived dendritic cells (DCs), and monocyte-derived (classically activated; M1) macrophages with H. pylori and analyzed phenotypic alterations, cytokine secretion, phagocytosis, and immunostimulation. Since we detected CD163+ (alternatively activated; M2) macrophages in gastric biopsy specimens from H. pylori-positive patients, we also included monocyte-derived M2 macrophages in the study. Upon H. pylori infection, monocytes secreted interleukin-1β (IL-1β), IL-6, IL-10, and IL-12p40 (partially secreted as IL-23) but not IL-12p70. Infected DCs became activated, as shown by the enhanced expression of CD25, CD80, CD83, PDL-1, and CCR7, and secreted IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, and IL-23. However, infection led to significantly downregulated CD209 and suppressed the constitutive secretion of macrophage migration inhibitory factor (MIF). H. pylori-infected M1 macrophages upregulated CD14 and CD32, downregulated CD11b and HLA-DR, and secreted mainly IL-1β, IL-6, IL-10, IL-12p40, and IL-23. Activation of DCs and M1 macrophages correlated with increased capacity to induce T-cell proliferation and decreased phagocytosis of dextran. M2 macrophages upregulated CD14 and CD206 and secreted IL-10 but produced less of the proinflammatory cytokines than M1 macrophages. Thus, H. pylori affects the functions of human APC subsets differently, which may influence the course and the outcome of H. pylori infection. The suppression of MIF in DCs constitutes a novel immune evasion mechanism exploited by H. pylori. PMID:22615251

  8. Virulent Type A Francisella tularensis actively suppresses cytokine responses in human monocytes

    Directory of Open Access Journals (Sweden)

    Devyn D Gilette

    2014-04-01

    Full Text Available Francisella tularensis is a Gram-negative facultative bacterium that can cause the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. Previous work has shown that monocytes infected with highly virulent F. tularensis subsp. tularensis strain Schu S4 responded with a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes and cytokine production in comparison to those infected with the less virulent related F. novicida. However, it has been unclear whether the virulent Schu S4 was merely evading or actively suppressing monocyte responses. By using mixed infection assays with F. tularensis and F. novicida, we show that F. tularensis actively suppresses monocyte pro-inflammatory responses. Additional experiments show that this suppression occurs in a dose-dependent manner and is dependent upon the viability of F. tularensis. Importantly, F. tularensis was able to suppress pro-inflammatory responses to earlier infections with F. novicida. These results lend support that F. tularensis actively dampens human monocyte responses and this likely contributes to its enhanced pathogenicity.

  9. Human monocytes/macrophages are a target of Neisseria meningitidis Adhesin A (NadA).

    Science.gov (United States)

    Franzoso, Susanna; Mazzon, Cristina; Sztukowska, Maryta; Cecchini, Paola; Kasic, Tihana; Capecchi, Barbara; Tavano, Regina; Papini, Emanuele

    2008-05-01

    Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA(Delta351-405), a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.

  10. Induction of Chemokine Secretion and Monocyte Migration by Human Choroidal Melanocytes in Response to Proinflammatory Cytokines

    DEFF Research Database (Denmark)

    Jehs, Tina; Faber, Carsten; Udsen, Maja S.

    2016-01-01

    Purpose: To determine to which extent inflammatory cytokines affect chemokine secretion by primary human choroidal melanocytes (HCMs), their capacity to attract monocytes, and whether HCMs are able to influence the proliferation of activated T cells. Methods: Primary cultures of HCMs were...... established from eyes of 13 donors. Human choroidal melanocytes were stimulated with IFN-γ and TNF-α or with supernatant from activated T cells (T-cell–conditioned media [TCM]). Gene expression analysis was performed by using microarrays. Protein levels were quantified with ELISA or cytometric bead array....... Supernatants of HCMs were assessed for the capability to attract monocytes in a transwell plate. Proliferation of activated T cells was assessed in a direct coculture with HCMs by a [3H]-thymidine incorporation assay. Results: Stimulation of HCMs with TCM or IFN-γ and TNF-α resulted in increased expression...

  11. Differential Constitutive and Cytokine-Modulated Expression of Human Toll-like Receptors in Primary Neutrophils, Monocytes, and Macrophages

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    D. Shane O'Mahony, Uyenvy Pham, Ramesh Iyer, Thomas R. Hawn, W. Conrad Liles

    2008-01-01

    Full Text Available Human Toll-like receptors (TLRs comprise a family of proteins that recognizes pathogen-associated molecular patterns (PAMPs and initiates host innate immune responses. Neutrophils, monocytes, and macrophages are critical cellular components of the human innate immune system. Proinflammatory cytokines, such as granulocyte colony-stimulating factor (G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF, macrophage colony-stimulating factor (M-CSF, and interferon-γ (IFN-γ, have been shown to up-regulate microbicidal activity in these effector cells of innate immunity. Currently, the cellular and molecular mechanisms responsible for these effects are not completely understood. We hypothesized that these cytokines may up-regulate TLR expression as a mechanism to facilitate microbial recognition and augment the innate immune response. Using quantitative realtime rt-PCR technology, we examined constitutive expression of TLR2, TLR4, TLR5, and TLR9 mRNA and the effects of G-CSF, GM-CSF, M-CSF, and IFN-γ on TLR mRNA expression in purified populations of normal human neutrophils, monocytes, and monocyte-derived macrophages. Relative constitutive expression of TLR2, TLR4, and TLR9 was similar in neutrophils and monocytes. Constitutive expression of TLR5 was less in neutrophils compared to monocytes. Constitutive expression of TLR4 was greater and that of TLR9 lower in monocyte-derived macrophages compared to monocytes. Of the cytokines examined, IFN-γ and GM-CSF caused the greatest effects on TLR expression. IFN- γ up-regulated TLR2 and TLR4 in neutrophils and monocytes. GM-CSF up-regulated expression of TLR2 and TLR4 in neutrophils and TLR2 in monocytes. TLR5 was down-regulated by inflammatory cytokines in monocytes. These results suggest a potential role for IFN- γ and/or GM-CSF as therapeutic immunomodulators of the host defense to infection.

  12. Cell-to-cell contact of human monocytes with infected arterial smooth-muscle cells enhances growth of Chlamydia pneumoniae.

    Science.gov (United States)

    Puolakkainen, Mirja; Campbell, Lee Ann; Lin, Tsun-Mei; Richards, Theresa; Patton, Dorothy L; Kuo, Cho-Chou

    2003-02-01

    Chlamydia pneumoniae can infect arterial cells. It has been shown that coculture of human monocytes (U937) and endothelial cells promotes infection of C. pneumoniae in endothelial cells and that the enhancement was mediated by a soluble factor (insulin-like growth factor 2) secreted by monocytes. In this study, it is shown that coculture of monocytes with C. pneumoniae enhances infection of C. pneumoniae in arterial smooth-muscle cells 5.3-fold at a monocyte-to-smooth-muscle cell ratio of 5. However, unlike endothelial cells, no enhancement was observed if monocytes were placed in cell culture inserts or if conditioned medium from monocyte cultures was used, which suggests that cell-to-cell contact is critical. The addition of mannose 6-phosphate or octyl glucoside, a nonionic detergent containing a sugar group, to cocultures inhibited the enhancement. These findings suggest that the monocyte-smooth-muscle cell interaction may be mediated by mannose 6-phosphate receptors present on monocytes.

  13. Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts

    Science.gov (United States)

    Zhao, Lei; Shao, Qianqian; Zhang, Yun; Zhang, Lin; He, Ying; Wang, Lijie; Kong, Beihua; Qu, Xun

    2016-01-01

    Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal–placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN+CD14+CD1a− phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4+CD25+Foxp3+ Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal–fetal interface. PMID:26857012

  14. Factor H Binds to Extracellular DNA Traps Released from Human Blood Monocytes in Response to Candida albicans

    Science.gov (United States)

    Halder, Luke D.; Abdelfatah, Mahmoud A.; Jo, Emeraldo A. H.; Jacobsen, Ilse D.; Westermann, Martin; Beyersdorf, Niklas; Lorkowski, Stefan; Zipfel, Peter F.; Skerka, Christine

    2017-01-01

    Upon systemic infection with human pathogenic yeast Candida albicans (C. albicans), human monocytes and polymorph nuclear neutrophilic granulocytes are the first immune cells to respond and come into contact with C. albicans. Monocytes exert immediate candidacidal activity and inhibit germination, mediate phagocytosis, and kill fungal cells. Here, we show that human monocytes spontaneously respond to C. albicans cells via phagocytosis, decondensation of nuclear DNA, and release of this decondensed DNA in the form of extracellular traps (called monocytic extracellular traps: MoETs). Both subtypes of monocytes (CD14++CD16−/CD14+CD16+) formed MoETs within the first hours upon contact with C. albicans. MoETs were characterized by the presence of citrullinated histone, myeloperoxidase, lactoferrin, and elastase. MoETs were also formed in response to Staphylococcus aureus and Escherichia coli, indicating a general reaction of monocytes to infectious microbes. MoET induction differs from extracellular trap formation in macrophages as MoETs are not triggered by simvastatin, an inhibitor of cholesterol synthesis and inducer of extracellular traps in macrophages. Extracellular traps from both monocytes and neutrophils activate complement and C3b is deposited. However, factor H (FH) binds via C3b to the extracellular DNA, mediates cofactor activity, and inhibits the induction of the inflammatory cytokine interleukin-1 beta in monocytes. Altogether, the results show that human monocytes release extracellular DNA traps in response to C. albicans and that these traps finally bind FH via C3b to presumably support clearance without further inflammation. PMID:28133459

  15. Human pregnancy-specific glycoprotein 1a (PSG1a) induces alternative activation in human and mouse monocytes and suppresses the accessory cell-dependent T cell proliferation.

    Science.gov (United States)

    Motrán, Claudia Cristina; Díaz, Fernando López; Gruppi, Adriana; Slavin, Daniela; Chatton, Bruno; Bocco, José Luis

    2002-09-01

    It has been proposed that pregnancy-specific factors induce the suppression of a specific arm of the maternal response accompanied by activation of the nonspecific, innate immune system. The aim of this study was to determine whether pregnancy-specific glycoprotein 1a (PSG1a), the major variant of PSG polypeptides, is able to modulate the monocyte/macrophage (Mo) metabolism to regulate T cell activation and proliferation. Using the recombinant form of this glycoprotein (rec-PSG1a), expressed in mammalian cells with a vaccinia-based expression vector, we have demonstrated that human PSG1a induces arginase activity in peripheral blood human Mo and human and murine Mo cell lines. In addition, rec-PSG1a is able to induce alternative activation because it up-regulates the arginase activity and inhibits the nitric oxide production in Mo activated by lipopolysaccharides. We also observed that rec-PSG1a is an important accessory cells-dependent T cell suppressor factor that causes partial growth arrest at the S/G2/M phase of the cell cycle. Additionally, an impaired T cell proliferative response induced by mitogens and specific antigen was observed in BALB/c mice upon in vivo expression of PSG1a. Our results suggest that PSG1a function contributes to the immunomodulation during pregnancy, having opposite effects on maternal innate and adaptative systems.

  16. Interaction between human monocytes and vascular smooth muscle cells induces vascular endothelial growth factor expression.

    Science.gov (United States)

    Hojo, Y; Ikeda, U; Maeda, Y; Takahashi, M; Takizawa, T; Okada, M; Funayama, H; Shimada, K

    2000-05-01

    The objective of this study was to investigate whether synthesis of vascular endothelial growth factor (VEGF), a major mitogen for vascular endothelial cells, was induced by a cell-to-cell interaction between monocytes and vascular smooth muscle cells (VSMCs). Human VSMCs and THP-1 cells (human monocytoid cell) were cocultured. VEGF levels in the coculture medium were determined by enzyme-linked immunosorbent assay. Northern blot analysis of VEGF mRNA was performed using a specific cDNA probe. Immunohistochemistry was performed to determine which types of cell produce VEGF. Adding THP-1 cells to VSMCs for 24 h increased VEGF levels of the culture media, 8- and 10-fold relative to those of THP-1 cells and VSMCs alone, respectively. Northern blot analysis showed that VEGF mRNA expression was induced in the cocultured cells and peaked after 12 h. Immunohistochemistry disclosed that both types of cell in the coculture produced VEGF. Separate coculture experiments revealed that both direct contact and a soluble factor(s) contributed to VEGF production. Neutralizing anti-interleukin (IL)-6 antibody inhibited VEGF production by the coculture of THP-1 cells and VSMCs. A cell-to-cell interaction between monocytes and VSMCs induced VEGF synthesis in both types of cell. An IL-6 mediated mechanism is at least partially involved in VEGF production by the cocultures. Local VEGF production induced by a monocyte-VSMC interaction may play an important role in atherosclerosis and vascular remodeling.

  17. Dengue-2 infection and the induction of apoptosis in human primary monocytes

    Directory of Open Access Journals (Sweden)

    Amanda Torrentes-Carvalho

    2009-12-01

    Full Text Available Monocytes/macrophages are important targets for dengue virus (DENV replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i., decayed afterwards and produced the apoptosis-related cytokines TNF-α and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

  18. Dengue-2 infection and the induction of apoptosis in human primary monocytes.

    Science.gov (United States)

    Torrentes-Carvalho, Amanda; Azeredo, Elzinandes L; Reis, Sonia R I; Miranda, Alessandro S; Gandini, Mariana; Barbosa, Luciana S; Kubelka, Claire F

    2009-12-01

    Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-alpha and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

  19. In vitro immunotoxicity assessment of culture-derived extracellular vesicles in human monocytes

    Science.gov (United States)

    Rosas, Lucia E.; Elgamal, Ola A.; Mo, Xiaokui; Phelps, Mitch A.; Schmittgen, Thomas D.; Papenfuss, Tracey L.

    2016-01-01

    The potential to engineer extracellular vesicles (EV) that target specific cells and deliver a therapeutic payload has propelled a growing interest in their development as promising therapeutics. These EV are often produced from cultured cells. Very little is known about the interaction of cell culture-derived EV with cells of the immune system and their potential immunomodulatory effects. The present study evaluated potential immunotoxic effects of HEK293T-derived EV on the human monocytic cell lines THP-1 and U937. Incubation of cells with different doses of EV for 16–24 h was followed by assessment of cytotoxicity and cell function by flow cytometry. Changes in cell functionality were evaluated by the capacity of cells to phagocytize fluorescent microspheres. In addition, the internalization of labeled EV in THP-1 and U937 cells was evaluated. Exposure to EV did not affect the viability of THP-1 or U937 cells. Although lower doses of the EV increased phagocytic capacity in both cell lines, phagocytic efficiency of individual cells was not affected by EV exposure at any of the doses evaluated. This study also demonstrated that THP-1 and U937 monocytic cells are highly permissive to EV entry in a dose-response manner. These results suggest that, although HEK293T-derived EV are efficiently internalized by human monocytic cells, they do not exert a cytotoxic effect or alter phagocytic efficiency on the cell lines evaluated. PMID:27075513

  20. Saturated fatty acids activate caspase-4/5 in human monocytes, triggering IL-1β and IL-18 release.

    Science.gov (United States)

    Pillon, Nicolas J; Chan, Kenny L; Zhang, Shitian; Mejdani, Marios; Jacobson, Maya R; Ducos, Alexandre; Bilan, Philip J; Niu, Wenyan; Klip, Amira

    2016-11-01

    Obesity is associated with metabolic tissue infiltration by monocyte-derived macrophages. Saturated fatty acids contribute to proinflammatory gene induction in tissue-embedded immune cells. However, it is unknown how circulating monocytes, the macrophage precursors, react to high-fat environments. In macrophages, saturated fatty acids activate inflammatory pathways and, notably, prime caspase-associated inflammasomes. Inflammasome-activated IL-1β contributes to type 2 diabetes. We hypothesized that 1) human monocytes from obese patients show caspase activation, and 2) fatty acids trigger this response and consequent release of IL-1β/IL-18. Human peripheral blood monocytes were sorted by flow cytometry, and caspase activity was measured with a FLICA dye-based assay. Blood monocytes from obese individuals exhibited elevated caspase activity. To explore the nature and consequence of this activity, human THP1 monocytes were exposed to saturated or unsaturated fatty acids. Caspase activity was revealed by isoform-specific cleavage and enzymatic activity; cytokine expression/release was measured by qPCR and ELISA. Palmitate, but not palmitoleate, increased caspase activity in parallel to the release of IL-1β and IL-18. Palmitate induced eventual monocyte cell death with features of pyroptosis (an inflammation-linked cell death program involving caspase-4/5), scored through LDH release, vital dye influx, cell volume changes, and nuclear morphology. Notably, selective gene silencing or inhibition of caspase-4/5 reduced palmitate-induced release of IL-1β and IL-18. In summary, monocytes from obese individuals present elevated caspase activity. Mechanistically, palmitate activates a pyroptotic program in monocytes through caspase-4/5, causing inflammatory cytokine release, additional to inflammasomes. These caspases represent potential, novel, therapeutic targets to taper obesity-associated inflammation.

  1. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Cushing, S.D.; Berliner, J.A.; Valente, A.J.; Territo, M.C.; Navab, M.; Parhami, F.; Gerrity, R.; Schwartz, C.J.; Fogelman, A.M.

    1990-07-01

    After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2- to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human glioma U-105MG cell line. Antibody that had been prepared against cultured baboon smooth muscle cell chemotactic factor (anti-SMCF) did not inhibit monocyte migration induced by the potent bacterial chemotactic factor f-Met-Leu-Phe. However, anti-SMCF completely inhibited the monocyte chemotactic activity found in the media of U-105MG cells, EC, and SMC before and after exposure to MM-LDL. Moreover, monocyte migration into the subendothelial space of a coculture of EC and SMC that had been exposed to MM-LDL was completely inhibited by anti-SMCF. Anti-SMCF specifically immunoprecipitated 10-kDa and 12.5-kDa proteins from EC. Incorporation of (35S)methionine into the immunoprecipitated proteins paralleled the monocyte chemotactic activity found in the medium of MM-LDL stimulated EC and the levels of MCP-1 mRNA found in the EC. We conclude that SMCF is in fact MCP-1 and MCP-1 is induced by MM-LDL.

  2. cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells

    Science.gov (United States)

    Paijo, Jennifer; Döring, Marius; Spanier, Julia; Grabski, Elena; Nooruzzaman, Mohammed; Schmidt, Tobias; Witte, Gregor; Messerle, Martin; Hornung, Veit; Kaever, Volkhard; Kalinke, Ulrich

    2016-01-01

    Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages. PMID:27058035

  3. Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

    KAUST Repository

    Schmeier, Sebastian

    2009-12-10

    Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

  4. Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Orcel, P.; Bielakoff, J.; De Vernejoul, M.C. (INSERM U18, Hopital Lariboisiere, Paris (France))

    1990-02-01

    Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes.

  5. Interleukin-18 Increases TLR4 and Mannose Receptor Expression and Modulates Cytokine Production in Human Monocytes

    Directory of Open Access Journals (Sweden)

    Luciane Alarcão Dias-Melicio

    2015-01-01

    Full Text Available Interleukin-18 is a proinflammatory cytokine belonging to the interleukin-1 family of cytokines. This cytokine exerts many unique biological and immunological effects. To explore the role of IL-18 in inflammatory innate immune responses, we investigated its impact on expression of two toll-like receptors (TLR2 and TLR4 and mannose receptor (MR by human peripheral blood monocytes and its effect on TNF-α, IL-12, IL-15, and IL-10 production. Monocytes from healthy donors were stimulated or not with IL-18 for 18 h, and then the TLR2, TLR4, and MR expression and intracellular TNF-α, IL-12, and IL-10 production were assessed by flow cytometry and the levels of TNF-α, IL-12, IL-15, and IL-10 in culture supernatants were measured by ELISA. IL-18 treatment was able to increase TLR4 and MR expression by monocytes. The production of TNF-α and IL-10 was also increased by cytokine treatment. However, IL-18 was unable to induce neither IL-12 nor IL-15 production by these cells. Taken together, these results show an important role of IL-18 on the early phase of inflammatory response by promoting the expression of some pattern recognition receptors (PRRs that are important during the microbe recognition phase and by inducing some important cytokines such as TNF-α and IL-10.

  6. Cytokine and Eicosanoid Production by Cultured Human Monocytes Exposed to Titanium Particulate Debris

    Science.gov (United States)

    Robinson, Timothy M.; Manley, Paul A.; Sims, Paul A.; Albrecht, Ralph; Darien, Benjamin J.

    1999-10-01

    Phagocytosis of particulate wear debris from arthroplasties by macrophages induces an inflammatory response that has been linked to implant loosening and premature failure of artificial joints. Inflammatory mediators released by phagocytic macrophages such as tumor necrosis factor-a (TNF-[alpha]), interleukin-1[beta] (IL-1[beta]), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) are believed to play a central role in the pathogenesis of aseptic loosening. The objective of this study was to characterize titanium alloy particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by cultured monocytes. Peripheral blood monocytes were isolated from healthy human volunteers. Monocytes were cultured in 96-well plates for 24 h, washed, and exposed to three concentrations of titanium particulates and controls from 18Ð24 h. Supernatants were assayed for TNF-[alpha], IL-1[beta], IL-6, and PGE2 activity. Energy dispersive X-ray spectroscopy (EDX) verified the titanium alloy to be Ti6A14V. Scanning electron microscopy (SEM) analysis showed significant titanium particulate heterogeneity with approximately 95% of the particles TNF-[alpha], IL-1[beta], and PGE2.

  7. Ultrafine carbon particles down-regulate CYP1B1 expression in human monocytes

    Directory of Open Access Journals (Sweden)

    Ziegler-Heitbrock Loems

    2009-10-01

    Full Text Available Abstract Background Cytochrome P450 monoxygenases play an important role in the defence against inhaled toxic compounds and in metabolizing a wide range of xenobiotics and environmental contaminants. In ambient aerosol the ultrafine particle fraction which penetrates deeply into the lungs is considered to be a major factor for adverse health effects. The cells mainly affected by inhaled particles are lung epithelial cells and cells of the monocyte/macrophage lineage. Results In this study we have analyzed the effect of a mixture of fine TiO2 and ultrafine carbon black Printex 90 particles (P90 on the expression of cytochrome P450 1B1 (CYP1B1 in human monocytes, macrophages, bronchial epithelial cells and epithelial cell lines. CYP1B1 expression is strongly down-regulated by P90 in monocytes with a maximum after P90 treatment for 3 h while fine and ultrafine TiO2 had no effect. CYP1B1 was down-regulated up to 130-fold and in addition CYP1A1 mRNA was decreased 13-fold. In vitro generated monocyte-derived macrophages (MDM, epithelial cell lines, and primary bronchial epithelial cells also showed reduced CYP1B1 mRNA levels. Benzo[a]pyrene (BaP is inducing CYB1B1 but ultrafine P90 can still down-regulate gene expression at 0.1 μM of BaP. The P90-induced reduction of CYP1B1 was also demonstrated at the protein level using Western blot analysis. Conclusion These data suggest that the P90-induced reduction of CYP gene expression may interfere with the activation and/or detoxification capabilities of inhaled toxic compounds.

  8. Manumycin A downregulates release of proinflammatory cytokines from TNF alpha stimulated human monocytes.

    Science.gov (United States)

    Cecrdlova, Eva; Petrickova, Katerina; Kolesar, Libor; Petricek, Miroslav; Sekerkova, Alena; Svachova, Veronika; Striz, Ilja

    2016-01-01

    Macrolide antibiotics such as azithromycin or clarithromycin are known to have potent anti-inflammatory and immunomodulatory effects but these properties cannot be widely used due to a risk of bacterial resistance. We studied another polyketide antibiotic, structurally related manumycin A known as a streptomycete derived farnesyltransferase inhibitor with limited antibacterial effects, with respect to its potential regulation of mRNA expression of several genes associated with proinflammatory responses. Downregulation of mRNA for IL-6, TLR-8, IL-1 beta and IL-10 was found in THP-1 cells after 4h stimulation with TNF alpha in the presence of manumycin A and downregulated TLR-8 and EGR-1 genes were observed after 8h. Among the genes upregulated in response to manumycin were HMOX-1, TNFRSF10A, IL-1R1, TICAM2, NLRP12 after 4h and only IL-1R1 after 8h. Furthermore, manumycin A was found to inhibit IL-1beta, IL-6, and IL-8 production in TNF alpha stimulated THP-1 cells and peripheral blood monocytes in a dose dependent manner (0.25-1 μM of manumycin A) without affecting cell viability. Cell viability of blood monocytes decreased by about 30% at manumycin A doses of 2-5 μM. Manumycin A also inhibited IL-18 release from THP-1 cells, while in cultures of blood monocytes, this cytokine was not detectable. That manumycin A mediated downregulation of proinflammatory genes in human monocytes confirmed by a measurement of cytokine levels in culture supernatants, together with a very limited effect on cell viability, might suggest potential anti-inflammatory properties of this polyketide antibiotic.

  9. Microvascular hemodynamics in human hypothermic circulatory arrest and selective antegrade cerebral perfusion

    NARCIS (Netherlands)

    P.W.G. Elbers; A. Ozdemir; R.H. Heijmen; J. Heeren; M. van Iterson; E.P.A. van Dongen; C. Ince

    2010-01-01

    Objective: The behavior of the human microcirculation in the setting of cardiac arrest is largely unknown. Animal experiments have consistently revealed that global hemodynamics do not necessarily reflect microvascular perfusion. In addition, the time it takes for capillary blood flow to stop after

  10. Monocyte migration into the subendothelial space of a coculture of adult human aortic endothelial and smooth muscle cells.

    OpenAIRE

    Navab, M; Hough, G P; Stevenson, L W; Drinkwater, D C; Laks, H; Fogelman, A M

    1988-01-01

    Human aortic endothelial cells (EC) and smooth muscle cells (SMC) were isolated and used to form a multilayer of EC-SMC separated by a layer of collagen. SMC and/or collagen layers exerted minimal effects on Na+ transport but impeded the transport of LDL. The presence of an endothelial monolayer markedly reduced the transport of Na+ and LDL. When monocytes were presented to the complete coculture, in the absence of added chemoattractant, one monocyte entered the subendothelial space for every...

  11. Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

    Science.gov (United States)

    Kuffer, Christian; Kuznetsova, Anastasia Yurievna; Storchová, Zuzana

    2013-08-01

    Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

  12. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    OpenAIRE

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-01-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its o...

  13. Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes

    OpenAIRE

    Obermaier Bianca; Dauer Marc; Herten Jan; Schad Katharina; Endres Stefan; Eigler Andreas

    2003-01-01

    We developed a new 2-day protocol for the generation of dendritic cells (DCs) from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of...

  14. Epstein-Barr virus induces MCP-1 secretion by human monocytes via TLR2.

    Science.gov (United States)

    Gaudreault, Eric; Fiola, Stéphanie; Olivier, Martin; Gosselin, Jean

    2007-08-01

    Epstein-Barr virus (EBV) is a gammaherpesvirus infecting the majority of the human adult population in the world. TLR2, a member of the Toll-like receptor (TLR) family, has been implicated in the immune responses to different viruses including members of the herpesvirus family, such as human cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. In this report, we demonstrate that infectious and UV-inactivated EBV virions lead to the activation of NF-kappaB through TLR2 using HEK293 cells cotransfected with TLR2-expressing vector along with NF-kappaB-Luc reporter plasmid. NF-kappaB activation in HEK293-TLR2 cells (HEK293 cells transfected with TLR2) by EBV was not enhanced by the presence of CD14. The effect of EBV was abrogated by pretreating HEK293-TLR2 cells with blocking anti-TLR2 antibodies or by preincubating viral particles with neutralizing anti-EBV antibodies 72A1. In addition, EBV infection of primary human monocytes induced the release of MCP-1 (monocyte chemotactic protein 1), and the use of small interfering RNA targeting TLR2 significantly reduced such a chemokine response to EBV. Taken together, these results indicate that TLR2 may be an important pattern recognition receptor in the immune response directed against EBV infection.

  15. Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes

    Institute of Scientific and Technical Information of China (English)

    TANG Ling-ling; ZHANG Zhe; ZHENG Jie-sheng; SHENG Ji-fang; LIU Ke-zhou

    2005-01-01

    Objective: This study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro. Methods: PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFα) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFα or lipopolysaccharide (LPS) stimulations for 24 h. Results: After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD 1 a, CD80 and CD86, features of DCs. TNFα treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity. Conclusion: This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.

  16. Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment.

    Science.gov (United States)

    Clark, R A; Wikner, N E; Doherty, D E; Norris, D A

    1988-08-25

    Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell

  17. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein.

    OpenAIRE

    Navab, M; Imes, S S; Hama, S Y; Hough, G P; Ross, L.A.; Bork, R W; Valente, A. J.; Berliner, J A; Drinkwater, D C; Laks, H

    1991-01-01

    Incubation of cocultures of human aortic endothelial (HAEC) and smooth muscle cells (HASMC) with LDL in the presence of 5-10% human serum resulted in a 7.2-fold induction of mRNA for monocyte chemotactic protein 1 (MCP-1), a 2.5-fold increase in the levels of MCP-1 protein in the coculture supernatants, and a 7.1-fold increase in the transmigration of monocytes into the subendothelial space of the cocultures. Monocyte migration was inhibited by 91% by antibody to MCP-1. Media collected from t...

  18. Nordihydroguaiaretic Acid Attenuates the Oxidative Stress-Induced Decrease of CD33 Expression in Human Monocytes

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    Silvia Guzmán-Beltrán

    2013-01-01

    Full Text Available Nordihydroguaiaretic acid (NDGA is a natural lignan with recognized antioxidant and beneficial properties that is isolated from Larrea tridentata. In this study, we evaluated the effect of NDGA on the downregulation of oxidant stress-induced CD33 in human monocytes (MNs. Oxidative stress was induced by iodoacetate (IAA or hydrogen peroxide (H2O2 and was evaluated using reactive oxygen species (ROS production, and cell viability. NDGA attenuates toxicity, ROS production and the oxidative stress-induced decrease of CD33 expression secondary to IAA or H2O2 in human MNs. It was also shown that NDGA (20 μM attenuates cell death in the THP-1 cell line that is caused by treatment with either IAA or H2O2. These results suggest that NDGA has a protective effect on CD33 expression, which is associated with its antioxidant activity in human MNs.

  19. Nordihydroguaiaretic acid attenuates the oxidative stress-induced decrease of CD33 expression in human monocytes.

    Science.gov (United States)

    Guzmán-Beltrán, Silvia; Pedraza-Chaverri, José; Gonzalez-Reyes, Susana; Hernández-Sánchez, Fernando; Juarez-Figueroa, Ulises E; Gonzalez, Yolanda; Bobadilla, Karen; Torres, Martha

    2013-01-01

    Nordihydroguaiaretic acid (NDGA) is a natural lignan with recognized antioxidant and beneficial properties that is isolated from Larrea tridentata. In this study, we evaluated the effect of NDGA on the downregulation of oxidant stress-induced CD33 in human monocytes (MNs). Oxidative stress was induced by iodoacetate (IAA) or hydrogen peroxide (H(2)O(2)) and was evaluated using reactive oxygen species (ROS) production, and cell viability. NDGA attenuates toxicity, ROS production and the oxidative stress-induced decrease of CD33 expression secondary to IAA or H(2)O(2) in human MNs. It was also shown that NDGA (20  μ M) attenuates cell death in the THP-1 cell line that is caused by treatment with either IAA or H(2)O(2). These results suggest that NDGA has a protective effect on CD33 expression, which is associated with its antioxidant activity in human MNs.

  20. Differentiation and function of human monocyte-derived dendritic cells under the influence of leflunomide

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    Stojić-Vukanić Z.

    2011-01-01

    Full Text Available Leflunomide is an immunosuppressive drug effective in experimental models of transplantation and autoimmune diseases and in the treatment of active rheumatoid arthritis (RA. Having in mind that it has been shown that some other immunosuppressive drugs (glucocorticoids, mycophenolate mofetil, sirolimus etc. impair dendritic cell (DC phenotype and function, we investigated the effect of A77 1726, an active metabolite of leflunomide, on the differentiation and function of human monocyte-derived dendritic cells (MDDC in vitro. Immature MDDC were generated by cultivating monocytes in medium supplemented with GM-CSF and IL-4. To induce maturation, immature MDDC were cultured for 2 additional days with LPS. A77 1726 (100 μM was added at the beginning of cultivation. Flow cytometric analysis showed that MDDC differentiated in the presence of A77 1726 exhibited an altered phenotype, with a down-regulated surface expression of CD80, CD86, CD54 and CD40 molecules. Furthermore, the continuous presence of A77 1726 during differentiation and maturation prevented successful maturation, judging by the decreased expression of maturation marker CD83, costimulatory and adhesive molecules on A77 1726-treated mature MDDC. In addition, A77 1726-pretreated MDDC exhibited a poor stimulatory capacity of the allogeneic T cells and a low production of IL-10 and IL-18. These data suggest that leflunomide impairs the differentiation, maturation and function of human MDDC in vitro, which is an additional mechanism of its immunosuppressive effect.

  1. Monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues

    Energy Technology Data Exchange (ETDEWEB)

    Zwadlo, G.; Schlegel, R.; Sorg, C.

    1986-07-15

    A monoclonal antibody is described that was generated by immunizing mice with cultured human blood monocytes. The antibody (27E10) belongs to the IgG1 subclass and detects a surface antigen at M/sub r/ 17,000 that is found on 20% of peripheral blood monocytes. The antigen is increasingly expressed upon culture of monocytes, reaching a maximum between days 2 and 3. Stimulation of monocytes with interferon-..gamma.. (IFN-..gamma..), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and lipopolysaccharide (LPS) Ylalanine (fMLP) increased the 27E10 antigen density. The amount of 27E10-positive cells is not or is only weakly affected. The antigen is absent from platelets, lymphotyces, and all tested human cell lines, yet it cross-reacts with 15% of freshly isolated granulocytes. By using the indirect immunoperoxidase technique, the antibody is found to be negative on cryostat sections of normal human tissue (skin, lung, and colon) and positive on only a few monocyte-like cells in liver and on part of the cells of the splenic red pulp. In inflammatory tissue, however, the antibody is positive on monocytes/macrophages and sometimes on endothelial cells and epidermal cells, depending on the stage and type of inflammation, e.g., BCG ranulomas are negative, whereas psoriasis vulgaris, atopic dermatitis, erythrodermia, pressure urticaria, and periodontitis contain positively staining cells. In contact eczemas at different times after elicitation (6 hr, 24 hr, and 72 hr), the 27E10 antigen is seen first after 24 hr on a few infiltrating monocytes/macrophages, which increase in numbers after 72 hr.

  2. Costunolide causes mitotic arrest and enhances radiosensitivity in human hepatocellular carcinoma cells

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    Chen Chih-Jen

    2011-05-01

    Full Text Available Abstract Purpose This work aimed to investigate the effect of costunolide, a sesquiterpene lactone isolated from Michelia compressa, on cell cycle distribution and radiosensitivity of human hepatocellular carcinoma (HCC cells. Methods The assessment used in this study included: cell viability assay, cell cycle analysis by DNA histogram, expression of phosphorylated histone H3 (Ser 10 by flow cytometer, mitotic index by Liu's stain and morphological observation, mitotic spindle alignment by immunofluorescence of alpha-tubulin, expression of cell cycle-related proteins by Western blotting, and radiation survival by clonogenic assay. Results Our results show that costunolide reduced the viability of HA22T/VGH cells. It caused a rapid G2/M arrest at 4 hours shown by DNA histogram. The increase in phosphorylated histone H3 (Ser 10-positive cells and mitotic index indicates costunolide-treated cells are arrested at mitosis, not G2, phase. Immunofluorescence of alpha-tubulin for spindle formation further demonstrated these cells are halted at metaphase. Costunolide up-regulated the expression of phosphorylated Chk2 (Thr 68, phosphorylated Cdc25c (Ser 216, phosphorylated Cdk1 (Tyr 15 and cyclin B1 in HA22T/VGH cells. At optimal condition causing mitotic arrest, costunolide sensitized HA22T/VGH HCC cells to ionizing radiation with sensitizer enhancement ratio up to 1.9. Conclusions Costunolide could reduce the viability and arrest cell cycling at mitosis in hepatoma cells. Logical exploration of this mitosis-arresting activity for cancer therapeutics shows costunolide enhanced the killing effect of radiotherapy against human HCC cells.

  3. Human monocytes undergo excessive apoptosis following temozolomide activating the ATM/ATR pathway while dendritic cells and macrophages are resistant.

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    Martina Bauer

    Full Text Available Immunodeficiency is a severe therapy-limiting side effect of anticancer chemotherapy resulting from sensitivity of immunocompetent cells to DNA damaging agents. A central role in the immune system is played by monocytes that differentiate into macrophages and dendritic cells (DCs. In this study we compared human monocytes isolated from peripheral blood and cytokine matured macrophages and DCs derived from them and assessed the mechanism of toxicity of the DNA methylating anticancer drug temozolomide (TMZ in these cell populations. We observed that monocytes, but not DCs and macrophages, were highly sensitive to the killing effect of TMZ. Studies on DNA damage and repair revealed that the initial DNA incision was efficient in monocytes while the re-ligation step of base excision repair (BER can not be accomplished, resulting in an accumulation of DNA single-strand breaks (SSBs. Furthermore, monocytes accumulated DNA double-strand breaks (DSBs following TMZ treatment, while DCs and macrophages were able to repair DSBs. Monocytes lack the DNA repair proteins XRCC1, ligase IIIα and PARP-1 whose expression is restored during differentiation into macrophages and DCs following treatment with GM-CSF and GM-CSF plus IL-4, respectively. These proteins play a key role both in BER and DSB repair by B-NHEJ, which explains the accumulation of DNA breaks in monocytes following TMZ treatment. Although TMZ provoked an upregulation of XRCC1 and ligase IIIα, BER was not enhanced likely because PARP-1 was not upregulated. Accordingly, inhibition of PARP-1 did not sensitize monocytes, but monocyte-derived DCs in which strong PARP activation was observed. TMZ induced in monocytes the DNA damage response pathways ATM-Chk2 and ATR-Chk1 resulting in p53 activation. Finally, upon activation of the Fas-receptor and the mitochondrial pathway apoptosis was executed in a caspase-dependent manner. The downregulation of DNA repair in monocytes, resulting in their selective

  4. Δ9-tetrahydrocannabinol treatment during human monocyte differentiation reduces macrophage susceptibility to HIV-1 infection

    Science.gov (United States)

    Williams, Julie C.; Appelberg, Sofia; Goldberger, Bruce A.; Klein, Thomas W.; Sleasman, John W.; Goodenow, Maureen M.

    2014-01-01

    The major psychoactive component of marijuana, Δ9-tetrahydrocannabinol (THC), also acts to suppress inflammatory responses. Receptors for THC, CB1, CB2, and GPR55, are differentially expressed on multiple cell types including monocytes and macrophages, which are important modulators of inflammation in vivo and target cells for HIV-1 infection. Use of recreational and medicinal marijuana is increasing, but the consequences of marijuana exposure on HIV-1 infection are unclear. Ex vivo studies were designed to investigate effects on HIV-1 infection in macrophages exposed to THC during or following differentiation. THC treatment of primary human monocytes during differentiation reduced HIV-1 infection of subsequent macrophages by replication competent or single cycle CCR5 using viruses. In contrast, treatment of macrophages with THC immediately prior to or continuously following HIV-1 exposure failed to alter infection. Specific receptor agonists indicated that the THC effect during monocyte differentiation was mediated primarily through CB2. THC reduced the number of p24 positive cells with little to no effect on virus production per infected cell, while quantitation of intracellular viral gag pinpointed the THC effect to an early event in the viral life cycle. Cells treated during differentiation with THC displayed reduced expression of CD14, CD16, and CD163 and donor dependent increases in mRNA expression of selected viral restriction factors, suggesting a fundamental alteration in phenotype. Ultimately, the mechanism of THC suppression of HIV-1 infection was traced to a reduction in cell surface HIV receptor (CD4, CCR5 and CXCR4) expression that diminished entry efficiency. PMID:24562630

  5. Δ(9)-Tetrahydrocannabinol treatment during human monocyte differentiation reduces macrophage susceptibility to HIV-1 infection.

    Science.gov (United States)

    Williams, Julie C; Appelberg, Sofia; Goldberger, Bruce A; Klein, Thomas W; Sleasman, John W; Goodenow, Maureen M

    2014-06-01

    The major psychoactive component of marijuana, Δ(9)-tetrahydrocannabinol (THC), also acts to suppress inflammatory responses. Receptors for THC, CB1, CB2, and GPR55, are differentially expressed on multiple cell types including monocytes and macrophages, which are important modulators of inflammation in vivo and target cells for HIV-1 infection. Use of recreational and medicinal marijuana is increasing, but the consequences of marijuana exposure on HIV-1 infection are unclear. Ex vivo studies were designed to investigate effects on HIV-1 infection in macrophages exposed to THC during or following differentiation. THC treatment of primary human monocytes during differentiation reduced HIV-1 infection of subsequent macrophages by replication competent or single cycle CCR5 using viruses. In contrast, treatment of macrophages with THC immediately prior to or continuously following HIV-1 exposure failed to alter infection. Specific receptor agonists indicated that the THC effect during monocyte differentiation was mediated primarily through CB2. THC reduced the number of p24 positive cells with little to no effect on virus production per infected cell, while quantitation of intracellular viral gag pinpointed the THC effect to an early event in the viral life cycle. Cells treated during differentiation with THC displayed reduced expression of CD14, CD16, and CD163 and donor dependent increases in mRNA expression of selected viral restriction factors, suggesting a fundamental alteration in phenotype. Ultimately, the mechanism of THC suppression of HIV-1 infection was traced to a reduction in cell surface HIV receptor (CD4, CCR5 and CXCR4) expression that diminished entry efficiency.

  6. Tacaribe virus but not junin virus infection induces cytokine release from primary human monocytes and macrophages.

    Directory of Open Access Journals (Sweden)

    Allison Groseth

    Full Text Available The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV and the hemorrhagic fever-causing Junin virus (JUNV, in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation.

  7. Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions.

    Science.gov (United States)

    Klimchenko, Olena; Di Stefano, Antonio; Geoerger, Birgit; Hamidi, Sofiane; Opolon, Paule; Robert, Thomas; Routhier, Mélanie; El-Benna, Jamel; Delezoide, Anne-Lise; Boukour, Siham; Lescure, Bernadette; Solary, Eric; Vainchenker, William; Norol, Françoise

    2011-03-17

    The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14(low)CD16(-) precursor to form CD14(high)CD16(+) cells without producing the CD14(high)CD16(-) cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

  8. FUCOIDIN INHIBITS OXIDIZED LOW DENSITY LIPOPROTEIN FROM INDUCING HUMAN PERIPHERAL BLOOD MONOCYTE EXPRESSION OF PROINFLAMMATORY CYTOKINES mRNA

    Institute of Scientific and Technical Information of China (English)

    雷新军; 马爱群; 任冰稳; 耿涛; 张葳; 白玲

    2003-01-01

    Objective To study the significance of scavenger receptor class A(SR-A)in mediating human peripheral blood monocyte to uptake oxidized low density lipoprotein(OxLDL) and promoting the atherosclerotic immuno-pathological lesion in the local blood vessel. Methods With the Digoxenin-labeled Oligonucleotide-probes In situ Hybridization, this research investigated the effects of OxLDL on the mRNA expression of proinflammatory cytokines including MCP-1, bFGF, PDGF and IL-10 in the human peripheral blood monocyte and whether fucoidin, a peculiarly inhibitory ligand for SR-A, would influence this process. Results Monocyte was significantly increased the mRNA expression of MCP-1, bFGF, PDGF and IL-10 in a dose-dependent manner after incubating with OxLDL (10,15,20,25,30·mg·L-1, respectively)for 24 hours(P<0.001). Fucoidin(50,100,150,200,250·mg·mL-1, respectively)completely inhibited OxLDL(20·mg·L-1)from inducing monocyte the mRNA expression of above proinflammatory cytokines(P<0.001). Conclusion OxLDL can stimulate human peripheral blood monocyte to give expression to proinflammatory cytokines mRNA in a dose-dependent manner, while a peculiarly inhibitory ligand for SR-A-fucoidin has an obviously opposed role.

  9. Inhibitory effect of esculentoside A on tumour necrosis factor α production by human monocytes

    Directory of Open Access Journals (Sweden)

    H-B. Wang

    1996-01-01

    Full Text Available Esculentoside A (EsA is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments have shown that it has strong anti-inflammatory effects. Tumour necrosis factor (TNF is a very important inflammatory mediator. It is known that there are two types of TNF—TNFα is from macrophages/monocytes and TNFβ is from activated lymphocytes. In order to study the mechanism of the anti-inflammatory effect of EsA, it was determined whether TNFα production from human peripheral monocytes was altered by EsA under lipopolysaccharide (LPS-stimulated conditions. EsA was found to decrease TNFα production in a dose-dependent manner at concentrations higher than 1 μmol/l EsA. Recent studies have shown that EsA has a curative effect on chocolate cyst and other inflammatory diseases. Our previous studies have shown that EsA could reduce the release of platelet activating factor (PAF from rat macrophages, and inhibit interleukin-1 and interleukin-6 production from routine macrophages. The reducing effects of EsA on the release of TNFα, IL-1, IL-6 and PAF may explain its anti-inflammatory effect.

  10. Human Monocyte-Derived Osteoclasts Are Targeted by Staphylococcal Pore-Forming Toxins and Superantigens.

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    Sacha Flammier

    Full Text Available Staphylococcus aureus is the leading cause of bone and joint infections (BJIs. Staphylococcal pathogenesis involves numerous virulence factors including secreted toxins such as pore-forming toxins (PFTs and superantigens. The role of these toxins on BJI outcome is largely unknown. In particular, few studies have examined how osteoclasts, the bone-resorbing cells, respond to exposure to staphylococcal PFTs and superantigens. We investigated the direct impact of recombinant staphylococcal toxins on human primary mature monocyte-derived osteoclasts, in terms of cytotoxicity and cell activation with cell death and bone resorption assays, using macrophages of the corresponding donors as a reference. Monocyte-derived osteoclasts displayed similar toxin susceptibility profiles compared to macrophages. Specifically, we demonstrated that the Panton-Valentine leukocidin, known as one of the most powerful PFT which lyses myeloid cells after binding to the C5a receptor, was able to induce the death of osteoclasts. The archetypal superantigen TSST-1 was not cytotoxic but enhanced the bone resorption activity of osteoclasts, suggesting a novel mechanism by which superantigen-producing S. aureus can accelerate the destruction of bone tissue during BJI. Altogether, our data indicate that the diverse clinical presentations of BJIs could be related, at least partly, to the toxin profiles of S. aureus isolates involved in these severe infections.

  11. Suppression of human monocyte interleukin-1beta production by ajulemic acid, a nonpsychoactive cannabinoid.

    Science.gov (United States)

    Zurier, Robert B; Rossetti, Ronald G; Burstein, Sumner H; Bidinger, Bonnie

    2003-02-15

    Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, reduces joint tissue damage in rats with adjuvant arthritis. Because interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) are central to the progression of inflammation and joint tissue injury in patients with rheumatoid arthritis, we investigated human monocyte IL-1beta and TNFalpha responses after the addition of AjA to cells in vitro. Peripheral blood and synovial fluid monocytes (PBM and SFM) were isolated from healthy subjects and patients with inflammatory arthritis, respectively, treated with AjA (0-30 microM) in vitro, and then stimulated with lipopolysaccharide. Cells were harvested for mRNA, and supernatants were collected for cytokine assay. Addition of AjA to PBM and SFM in vitro reduced both steady-state levels of IL-1beta mRNA and secretion of IL-1beta in a concentration-dependent manner. Suppression was maximal (50.4%) at 10 microM AjA (Parthritis. Development of nonpsychoactive therapeutically useful synthetic analogs of Cannabis constituents, such as AjA, may help resolve the ongoing debate about the use of marijuana as medicine.

  12. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  13. Oxygen-Loaded Nanodroplets Effectively Abrogate Hypoxia Dysregulating Effects on Secretion of MMP-9 and TIMP-1 by Human Monocytes

    Directory of Open Access Journals (Sweden)

    Giulia Rossana Gulino

    2015-01-01

    Full Text Available Monocytes play a key role in the inflammatory stage of the healing process. To allow monocyte migration to injured tissues, the balances between secreted matrix metalloproteinases (MMPs and their inhibitors (TIMPs must be finely modulated. However, a reduction of blood supply and local oxygen tension can modify the phenotype of immune cells. Intriguingly, hypoxia might be targeted by new effective oxygenating devices such as 2H,3H-decafluoropentane- (DFP- based oxygen-loaded nanodroplets (OLNs. Here, hypoxia effects on gelatinase/TIMP release from human peripheral monocytes were investigated, and the therapeutic potential of dextran-shelled OLNs was evaluated. Normoxic monocytes constitutively released ~500 ng/mL MMP-9, ~1.3 ng/mL TIMP-1, and ~0.6 ng/mL TIMP-2 proteins. MMP-2 was not detected. After 24 hours, hypoxia significantly altered MMP-9/TIMP-1 balance by reducing MMP-9 and increasing TIMP-1, without affecting TIMP-2 secretion. Interestingly OLNs, not displaying toxicity to human monocytes after cell internalization, effectively counteracted hypoxia, restoring a normoxia-like MMP-9/TIMP-1 ratio. The action of OLNs was specifically dependent on time-sustained oxygen diffusion up to 24 h from their DFP-based core. Therefore, OLNs appear as innovative, nonconventional, cost-effective, and nontoxic therapeutic tools, to be potentially employed to restore the physiological invasive phenotype of immune cells in hypoxia-associated inflammation.

  14. Whole-blood culture is a valid low-cost method to measure monocytic cytokines - A comparison of cytokine production in cultures of human whole-blood, mononuclear cells and monocytes

    DEFF Research Database (Denmark)

    Damsgaard, Camilla T.; Lauritzen, Lotte; Calder, Philip C.

    2009-01-01

    assessed the intra- and inter-individual variation in cytokine production. In 64 healthy men (age 19-40 years) IL-6, TNF and IL-10 were measured by enzyme-linked immunosorbent assay in supernatants from whole-blood, PBMC and monocytes cultured 24 h with lipopolysaccharide (LPS) or UV-killed L acidophilus......Whole-blood and peripheral blood mononuclear cell (PBMC) cultures are used as non-validated surrogate measures of monocytic cytokine production. The aim of this investigation was to compare ex vivo cytokine production from human whole-blood and PBMC with that from isolated monocytes. We also...

  15. Macrophage Inflammatory Protein-1alpha mediates Matrix Metalloproteinase-9 enhancement in human adherent monocytes fed with malarial pigment

    Institute of Scientific and Technical Information of China (English)

    Giuliana Giribaldi; Elena Valente; Amina Khadjavi; Manuela Polimeni; Mauro Prato

    2011-01-01

    Objective:To investigate the role of macrophage inflammatory protein-1alpha (MIP-1alpha) in the detrimental enhancement of matrix metalloproteinase-9 (MMP-9)expression, release and activity induced by phagocytosis of malarial pigment (haemozoin,HZ) in human monocytes. Methods: Human adherent monocytes were unfed/fed with nativeHZ for 2 h. After 24 hours, MIP-1alpha production was evaluated by ELISA in cell supernatants. Alternatively,HZ-unfed/fed monocytes were treated in presence/absence of anti-humanMIP-1alpha blocking antibodies or recombinant humanMIP-1alpha for15 h (RNA studies) or 24 h (protein studies); therefore,MMP-9mRNA expression was evaluated in cell lysates by Real TimeRT-PCR, whereas proMMP-9and activeMMP-9protein release were measured in cell supernatants by Western blotting and gelatin zymography.Results: Phagocytosis ofHZ by human monocytes increased production ofMIP-1alpha, mRNA expression ofMMP-9and protein release of proMMP-9 and activeMMP-9. All theHZ-enhancing effects onMMP-9 were abrogated by anti-humanMIP-1alpha blocking antibodies and mimicked by recombinant humanMIP-1alpha.Conclusions:The present work suggests a role for MIP-1alpha in theHZ-dependent enhancement ofMMP-9 expression, release and activity observed in human monocytes, highlighting new detrimental effects ofHZ-triggered proinflammatory response by phagocytic cells in falciparum malaria.

  16. Activated human mast cells induce LOX-1-specific scavenger receptor expression in human monocyte-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Mervi Alanne-Kinnunen

    Full Text Available Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs.Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1 mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α, and transforming growth factor beta (TGF-β1, which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell -induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.

  17. Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors.

    Science.gov (United States)

    Dauer, Marc; Obermaier, Bianca; Herten, Jan; Haerle, Carola; Pohl, Katrin; Rothenfusser, Simon; Schnurr, Max; Endres, Stefan; Eigler, Andreas

    2003-04-15

    It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5-7 days of differentiation with GM-CSF and IL-4, followed by 2-3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1alpha. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-gamma. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-gamma production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.

  18. Respecting Human Rights during the Execution of the European Arrest Warrant

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    Ion Rusu

    2012-05-01

    Full Text Available In this paper we examine a topic in the field of European law, which is of particular interest, beingalways a novel subject, that is the protection and human rights within the complex activity of executing theEuropean arrest warrant by the judicial authorities of each Member State. The paper continues previousresearch materialized in some in studies and articles published in national or international specialized journalsor proceedings. The examination of the European arrest warrant institution has led to some conclusions,which ultimately need to certify the completion of some provisions, notably the ones regarding the mutualrecognition and increase of efficiency. The work can be useful both for practitioners and theorists in the field,the essential contribution consisting of the examination and the expressed critical opinions, which may lead tothe amendment and completion of the European legislative act.

  19. Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Kanayo [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Sakaguchi, Minoru, E-mail: sakaguti@gly.oups.ac.jp [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Tanaka, Satoshi [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Yoshimoto, Tadashi [Department of Life Science, Setsunan University, 17-8 Ikeda-Nakamachi, Neyagawa, Osaka 572-8508 (Japan); Takaoka, Masanori [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan)

    2014-01-03

    Highlights: •We examined the effects of prolyl oligopeptidase (POP) inhibition on p53 null gastric cancer cell growth. •POP inhibition-induced cell growth suppression was associated with an increase in a quiescent G{sub 0} state. •POP might regulate the exit from and/or reentry into the cell cycle. -- Abstract: Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G{sub 0}/G{sub 1} cell cycle arrest and increased levels of the CDK inhibitor p27{sup kip1} and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-((4-[2-(E)-styrylphenoxy]butanoyl)-L-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G{sub 0}/G{sub 1} cell cycle phase arrest and increased levels of p27{sup kip1} in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G{sub 0} state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.

  20. In vitro interaction of Stenotrophomonas maltophilia with human monocyte-derived dendritic cells

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    Emanuela eRoscetto

    2015-07-01

    Full Text Available Stenotrophomonas maltophilia is increasingly identified as an opportunistic pathogen in immunocompromised, cancer and cystic fibrosis (CF patients. Knowledge on innate immune responses to S. maltophilia and its potential modulation is poor. The present work investigated the ability of 12 clinical S. maltophilia strains (5 from CF patients, 7 from non-CF patients and one environmental strain to survive inside human monocyte-derived dendritic cells (DCs. The effects of the bacteria on maturation of and cytokine secretion by DCs were also measured. S. maltophilia strains presented a high degree of heterogeneity in internalization and intracellular replication efficiencies as well as in the ability of S. maltophilia to interfere with normal DCs maturation. By contrast, all S. maltophilia strains were able to activate DCs, as measured by increase in the expression of surface maturation markers and proinflammatory cytokines secretion.

  1. Isolation of IL-12p70-competent human monocyte-derived dendritic cells

    DEFF Research Database (Denmark)

    Søndergaard, Jonas Nørskov; Pedersen, Susanne Brix

    2012-01-01

    that moDCs generated under standard conditions develop into two subsets based on CD1a-expression with the CD1a+ moDCs being the main IL-12p70 producers. This has however not been generally accepted, which we show here because the subset described as CD1a-negative does express CD1a, but at a lower level......Diverse methodologies ranging from experimental immunological studies to immunotherapy involve the application of human monocyte-derived dendritic cells (moDCs). Considerable donor-dependent variations in the moDC production of IL-12p70 affect the outcome of these methodologies. It has been shown...

  2. Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes.

    Science.gov (United States)

    Obermaier, Bianca; Dauer, Marc; Herten, Jan; Schad, Katharina; Endres, Stefan; Eigler, Andreas

    2003-01-01

    We developed a new 2-day protocol for the generation of dendritic cells (DCs) from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of total culture period, expression of activation markers was more pronounced in cells generated by the 2-step differentiation and activation method. Our new protocol for 2-day DC differentiation reduces labor, cost and time and also reliably renders high numbers of mature and viable DCs.

  3. Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes

    Directory of Open Access Journals (Sweden)

    Obermaier Bianca

    2003-01-01

    Full Text Available We developed a new 2-day protocol for the generation of dendritic cells (DCs from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of total culture period, expression of activation markers was more pronounced in cells generated by the 2-step differentiation and activation method. Our new protocol for 2-day DC differentiation reduces labor, cost and time and also reliably renders high numbers of mature and viable DCs.

  4. [EVALUATION OF THE HUMAN SENSITIVITY TO SMALLPOX VIRUS BY THE PRIMARY CULTURES OF THE MONOCYTE-MACROPHAGES].

    Science.gov (United States)

    Zamedyanskaya, A S; Titova, K A; Sergeev, Al A; Kabanov, A S; Bulychev, L E; Sergeev, Ar A; Galakhova, D O; Nesterov, A E; Nosareva, O V; Shishkina, L N; Taranov, O S; Omigov, V V; Agafonov, A P; Sergeev, A N

    2016-01-01

    Studies of the primary cultures of granulocytes, mononuclear, and monocyte-macrophage cells derived from human blood were performed using variola virus (VARV) in the doses of 0.001-0.021 PFU/cell (plaques-forming units per cell). Positive dynamics of the virus accumulation was observed only in the monocyte-macrophages with maximum values of virus concentration (5.0-5.5 Ig PFU/ml) mainly within six days after the infection. The fact of VARV replication in the monocyte-macrophages was confirmed by the data of electron microscopy. At the same time, virus vaccines when tested in doses 3.3 and 4.2 Ig PFU/ml did not show the ability to reproduce in these human cells. The people sensitivity to VARV as assessed from the data obtained on human monocyte-macrophages corresponded to -1 PFU (taking into account the smooth interaction of the virus in the body to the cells of this type), which is consistent to previously found theoretical data on the virus sensitivity. The human susceptibility to VARV assessed experimentally can be used to predict the adequacy of developed smallpox models (in vivo) based on susceptible animals. This is necessary for reliable assessment of the efficiency of development of drugs for treatment and prophylaxis of the smallpox.

  5. 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...

  6. Induction of cyclooxygenase-2 expression during HIV-1-infected monocyte-derived macrophage and human brain microvascular endothelial cell interactions

    NARCIS (Netherlands)

    Pereira, CF; Boven, LA; Middel, J; Verhoef, J; Nottet, HSLM

    2000-01-01

    Human immunodeficiency virus type-1 (HIV-1)-associated dementia (HAD) is a neurodegenerative disease characterized by HIV infection and replication in brain tissue. HIV-1-infected monocytes overexpress inflammatory molecules that facilitate their entry into the brain. Prostanoids are lipid mediators

  7. Cell surface expression and function of the macromolecular C1 complex on the surface of human monocytes

    Directory of Open Access Journals (Sweden)

    Kinga K Hosszu

    2012-03-01

    Full Text Available The synthesis of the subunits of the C1 complex (C1q, C1s, C1r, and its regulator C1 inhibitor (C1-Inh by human monocytes has been previously established. However, surface expression of these molecules by monocytes has not been shown. Using flow cytometry and antigen-capture ELISA, we show here for the first time that, in addition to C1q, PB monocytes and the monocyte-derived U937 cells express C1s and C1r, as well as Factor B and C1-Inh on their surface. C1s and C1r immunoprecipitated with C1q, suggesting that at least some of the C1q on these cells is part of the C1 complex. Furthermore, the C1 complex on U937 cells was able to trigger complement activation via the classical pathway. The presence of C1-Inh may ensure that an unwarranted autoactivation of the C1 complex does not take place. Since C1-Inh closely monitors the activation of the C1 complex in a sterile or infectious inflammatory environment, further elucidation of the role of C1 complex is crucial to dissect its function in monocyte, DC and T cell activities, and its implications in host defense and tolerance.

  8. Flagella from five Cronobacter species induce pro-inflammatory cytokines in macrophage derivatives from human monocytes.

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    Ariadnna Cruz-Córdova

    Full Text Available Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10 in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng induced the release of IL-8 (3314-6025 pg/ml, TNF-α (39-359 pg/ml, and IL-10 (2-96 pg/ml, in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200 suppressed the secretion of IL-8, TNF-α, and IL-10 between 95-100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria.

  9. Flagella from Five Cronobacter Species Induce Pro-Inflammatory Cytokines in Macrophage Derivatives from Human Monocytes

    Science.gov (United States)

    Cruz-Córdova, Ariadnna; Rocha-Ramírez, Luz M.; Ochoa, Sara A.; Gónzalez-Pedrajo, Bertha; Espinosa, Norma; Eslava, Carlos; Hernández-Chiñas, Ulises; Mendoza-Hernández, Guillermo; Rodríguez-Leviz, Alejandra; Valencia-Mayoral, Pedro; Sadowinski-Pine, Stanislaw; Hernández-Castro, Rigoberto; Estrada-García, Iris; Muñoz-Hernández, Onofre; Rosas, Irma; Xicohtencatl-Cortes, Juan

    2012-01-01

    Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314–6025 pg/ml), TNF-α (39–359 pg/ml), and IL-10 (2–96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95–100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria. PMID:23284883

  10. Salvianolic acid B suppresses maturation of human monocyte-derived dendritic cells by activating PPARγ

    Science.gov (United States)

    Sun, Aijun; Liu, Hongying; Wang, Shijun; Shi, Dazhuo; Xu, Lei; Cheng, Yong; Wang, Keqiang; Chen, Keji; Zou, Yunzeng; Ge, Junbo

    2011-01-01

    BACKGROUND AND PURPOSE Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is known to be effective in the prevention of atherosclerosis. Here, we tested the hypothesis that the anti-atherosclerotic effect of Sal B might be mediated by suppressing maturation of human monocyte-derived dendritic cells (h-monDC). EXPERIMENTAL APPROACH h-monDC were derived by incubating purified human monocytes with GM-CSF and IL-4. h-monDC were pre-incubated with or without Sal B and stimulated by oxidized low-density lipoprotein (ox-LDL) in the presence or absence of PPARγ siRNA. Expression of h-monDC membrane molecules (CD40, CD86, CD1a, HLA-DR) were analysed by FACS, cytokines were measured by elisa and the TLR4-associated signalling pathway was determined by Western blotting. KEY RESULTS Ox-LDL promoted h-monDC maturation, stimulated CD40, CD86, CD1a, HLA-DR expression and IL-12, IL-10, TNF-α production; and up-regulated TLR4 signalling. These effects were inhibited by Sal B. Sal B also triggered PPARγ activation and promoted PPARγ nuclear translocation, attenuated ox-LDL-induced up-regulation of TLR4 and myeloid differentiation primary-response protein 88 and inhibited the downstream p38-MAPK signalling cascade. Knocking down PPARγ with the corresponding siRNA blocked these effects of Sal B. CONCLUSIONS AND IMPLICATIONS Our data suggested that Sal B effectively suppressed maturation of h-monDC induced by ox-LDL through PPARγ activation. PMID:21649636

  11. 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

    on neutrophils. The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100 degrees C. Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused...... 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...... chromatography revealed spherical particles with diameters ranging from 5 to 20 nm. Biochemical characterization and SDS polyacrylamide gel electrophoresis suggested that these particles consisted of protein and carbohydrate including lipopolysaccharide with the major enzyme activity being lipase. Various...

  12. HIV-1 Vpr induces interferon-stimulated genes in human monocyte-derived macrophages.

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    Muhammad Atif Zahoor

    Full Text Available Macrophages act as reservoirs of human immunodeficiency virus type 1 (HIV-1 and play an important role in its transmission to other cells. HIV-1 Vpr is a multi-functional protein involved in HIV-1 replication and pathogenesis; however, its exact role in HIV-1-infected human macrophages remains poorly understood. In this study, we used a microarray approach to explore the effects of HIV-1 Vpr on the transcriptional profile of human monocyte-derived macrophages (MDMs. More than 500 genes, mainly those involved in the innate immune response, the type I interferon pathway, cytokine production, and signal transduction, were differentially regulated (fold change >2.0 after infection with a recombinant adenovirus expressing HIV-1 Vpr protein. The differential expression profiles of select interferon-stimulated genes (ISGs and genes involved in the innate immune response, including STAT1, IRF7, MX1, MX2, ISG15, ISG20, IFIT1, IFIT2, IFIT3, IFI27, IFI44L, APOBEC3A, DDX58 (RIG-I, TNFSF10 (TRAIL, and RSAD2 (viperin were confirmed by real-time quantitative PCR and were consistent with the microarray data. In addition, at the post-translational level, HIV-1 Vpr induced the phosphorylation of STAT1 at tyrosine 701 in human MDMs. These results demonstrate that HIV-1 Vpr leads to the induction of ISGs and expand the current understanding of the function of Vpr and its role in HIV-1 immune pathogenesis.

  13. Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts.

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    Sascha Schäuble

    Full Text Available Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD, fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P via reversibly cell cycle arrested (C to irreversibly arrested senescent cells (S. In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.

  14. MIF inhibition reverts the gene expression profile of human melanoma cell line-induced MDSCs to normal monocytes

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    Sabine Waigel

    2016-03-01

    Full Text Available Myeloid-derived suppressor cells (MDSCs are potently immunosuppressive innate immune cells that accumulate in advanced cancer patients and actively inhibit anti-tumor T lymphocyte responses [1]. Increased numbers of circulating MDSCs directly correlate with melanoma patient morbidity and reduced anti-tumor immune responses [2,3]. Previous studies have revealed that monocyte-derived macrophage migration inhibitory factor (MIF is necessary for the immune suppressive function of MDSCs in mouse models of melanoma [4,5]. To investigate whether MIF participates in human melanoma-induced MDSC differentiation and/or suppressive function, we have established an in vitro MDSC induction model using primary, normal human monocytes co-cultured with human melanoma cell lines in the presence or absence of the MIF antagonist—4-IPP [4,6–9]. To identify potential mechanistic effectors, we have performed transcriptome analyses on cultured monocytes and on melanoma-induced MDSCs obtained from either untreated or 4-IPP-treated A375:monocyte co-cultures. Here, we present a detailed protocol, which can facilitate easy reproduction of the microarray results (NCBI GEO accession number GSE73333 published by Yaddanapudi et al. (2015 in Cancer Immunology Research [10].

  15. Early activation of MyD88-mediated autophagy sustains HSV-1 replication in human monocytic THP-1 cells

    Science.gov (United States)

    Siracusano, Gabriel; Venuti, Assunta; Lombardo, Daniele; Mastino, Antonio; Esclatine, Audrey; Sciortino, Maria Teresa

    2016-01-01

    Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy, and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes. PMID:27509841

  16. Evodiamine induces caspase-dependent apoptosis and S phase arrest in human colon lovo cells.

    Science.gov (United States)

    Zhang, Chun; Fan, Xia; Xu, Xiang; Yang, Xue; Wang, Xi; Liang, Hua-Ping

    2010-09-01

    Evodiamine, one of the major bioactive components derived from Wu-Chu-Yu, a long-standing Chinese herb, was reported to possess anticancer activity. In this study, we investigated the in-vitro and in-vivo anticancer effects of evodiamine on human colon lovo cells and their potential mechanisms. The 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay showed that the in-vitro proliferation of lovo cells was inhibited by evodiamine of various concentrations. Flow cytometry showed a time-dependent increase in the percentage of apoptotic cells and cells arrested in the S phase after treatment with 60 micromol/l evodiamine. Western blot indicated that evodiamine treatment decreased the expression of procaspase-8, procaspase-9, and procaspase-3 in lovo cells, accompanied by the activation of caspase-8, caspase-9, and caspase-3. However, the translocation of apoptosis-inducing factor and endonuclease G was not affected by evodiamine. Moreover, western blot assay also suggested that evodiamine-induced S phase arrest in lovo cells was associated with a marked decrease in the protein expression of cyclinA, cyclinA-dependent kinase 2, and cdc25c. In-vivo antineoplastic characteristics of evodiamine were examined in a human colon carcinoma lovo xenograft model and results showed that evodiamine increased the number of TUNEL-positive cells accompanied by the downregulated expression of procaspase-8, procaspase-9, and procaspase-3. In conclusion, these findings indicated that evodiamine could inhibit the in-vitro and in-vivo proliferation of human colon lovo cells by inducing caspase-dependent apoptosis and S phase arrest.

  17. Reprogramming of human peripheral blood monocytes to erythroid lineage by blocking of the PU-1 gene expression.

    Science.gov (United States)

    Nouri, Masoumeh; Deezagi, Abdolkhalegh; Ebrahimi, Marzieh

    2016-03-01

    In hematopoietic system development, PU.1 and GATA-1 as lineage-specific transcription factors (TF) are expressed in common myeloid progenitors. The cross antagonism between them ascertains gene expression programs of monocytic and erythroid cells, respectively. This concept in transdifferentiation approaches has not been well considered yet, especially in intralineage conversion systems. To demonstrate whether PU.1 suppression induces monocyte lineage conversion into red blood cells, a combination of three PU.1-specific siRNAs was implemented to knock down PU.1 gene expression and generate the balance in favor of GATA-1 expression to induce erythroid differentiation. For this purpose, monocytes were isolated from human peripheral blood and transfected by PU.1 siRNAs. In transfected monocytes, the rate of PU.1 expression in mRNA level was significantly decreased until 0.38 ± 0.118 when compared to untreated monocytes at 72 h (p value ≤0.05) which resulted in significant overexpression of GATA1 of 16.1 ± 0.343-fold compared to the untreated group (p value ≤0.01). Subsequently, overexpression of hemoglobin (α 13.26 ± 1.34-fold; p value≤0.0001) and β-globin (37.55 ± 16.56-fold; p value≤0.0001) was observed when compared to control groups. The results of western immunoblotting confirm those findings too. While, reduced expression of monocyte, CD14 gene, was observed in qRT-PCR and flow cytometry results. Our results suggest that manipulating the ratio of the two TFs in bifurcation differentiation pathways via applying siRNA technology can possibly change the cells' fate as a safe way for therapeutics application.

  18. Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Lintao Wang; Yanyan Peng; Kaikai Shi; Haixiao Wang; Jianlei Lu; Yanli Li; Changyan Ma

    2015-01-01

    Recent studies have revealed that osthole,an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson,a traditional Chinese medicine,possesses anticancer activity.However,its effect on breast cancer cells so far has not been elucidated clearly.In the present study,we evaluated the effects of osthole on the proliferation,cell cycle and apoptosis of human breast cancer cells MDA-MB 435.We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells,The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole,as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation.The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Were observed taken together,these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.

  19. Rapid detection of dendritic cell and monocyte disorders using CD4 as a lineage marker of the human peripheral blood antigen presenting cell compartment

    Directory of Open Access Journals (Sweden)

    Laura eJardine

    2013-12-01

    Full Text Available Dendritic cells (DCs and monocytes are critical regulators and effectors of innate and adaptive immune responses. Monocyte expansion has been described in many pathological states while monocyte and DC deficiency syndromes are relatively recent additions to the catalogue of human primary immunodeficiency disorders. Clinically applicable screening tests to diagnose and monitor these conditions are lacking. Conventional strategies for identifying human DCs and monocytes have been based on the use of a lineage gate to exclude lymphocytes, thus preventing simultaneous detection of DCs, monocytes and lymphocyte subsets. Here we demonstrate that CD4 is a reliable lineage marker for the human peripheral blood antigen presenting cell compartment that can be used to identify DCs and monocytes in parallel with lymphocytes. Based on this principle, simple modification of a standard lymphocyte phenotyping assay permits simultaneous enumeration of four lymphocyte and five DC/monocyte populations from a single sample. This approach is applicable to clinical samples and facilitates the diagnosis of DC and monocyte disorders in a wide range of clinical settings, including genetic deficiency, neoplasia and inflammation.

  20. The transcriptome of Legionella pneumophila-infected human monocyte-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Christopher T D Price

    Full Text Available Legionella pneumophila is an intracellular bacterial pathogen that invades and replicates within alveolar macrophages through injection of ∼ 300 effector proteins by its Dot/Icm type IV translocation apparatus. The bona fide F-box protein, AnkB, is a nutritional virulence effector that triggers macrophages to generate a surplus of amino acids, which is essential for intravacuolar proliferation. Therefore, the ankB mutant represents a novel genetic tool to determine the transcriptional response of human monocyte-derived macrophages (hMDMs to actively replicating L. pneumophila.Here, we utilized total human gene microarrays to determine the global transcriptional response of hMDMs to infection by wild type or the ankB mutant of L. pneumophila. The transcriptomes of hMDMs infected with either actively proliferating wild type or non-replicative ankB mutant bacteria were remarkably similar. The transcriptome of infected hMDMs was predominated by up-regulation of inflammatory pathways (IL-10 anti-inflammatory, interferon signaling and amphoterin signaling, anti-apoptosis, and down-regulation of protein synthesis pathways. In addition, L. pneumophila modulated diverse metabolic pathways, particularly those associated with bio-active lipid metabolism, and SLC amino acid transporters expression.Taken together, the hMDM transcriptional response to L. pneumophila is independent of intra-vacuolar replication of the bacteria and primarily involves modulation of the immune response and metabolic as well as nutritional pathways.

  1. Isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags.

    Science.gov (United States)

    Menck, Kerstin; Behme, Daniel; Pantke, Mathias; Reiling, Norbert; Binder, Claudia; Pukrop, Tobias; Klemm, Florian

    2014-09-09

    Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

  2. Berberine inhibits growth and induces G1 arrest and apoptosis in human cholangiocarcinoma QBC939 cells.

    Science.gov (United States)

    He, Wei; Wang, Bin; Zhuang, Yun; Shao, Dong; Sun, Kewen; Chen, Jianping

    2012-01-01

    The chemotherapeutic approach using non-toxic natural products may be one of the strategies for the management of the cholangiocarcinoma. Here we report that in vitro treatment of human cholangiocarcinoma QBC939 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability and induced cell death in a dose-dependent manner, which was associated with an increase in G1 arrest. Our western blot analysis showed that berberine-induced G1 cell cycle arrest was mediated through the increased expression of cyclin-dependent kinase inhibitors (Cdki) proteins (Cip1/p21 and Kip1/p27); a simultaneous decrease in Cdk2 and Cdk4 and cyclins D1, and reduced activity of the Cyclins-Cdk complex. In additional studies, treatment of QBC939 cells with different concentrations (10, 40, 80 μM) of berberine for 48 h resulted in a significant dose-dependent increase in apoptosis compared to the non-berberine-treated control, which was associated with an increased expression of pro-apoptotic protein Bax and decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xL. Together, this study for the first time identified berberine as a chemotherapeutic agent against human cholangiocarcinoma cells QBC939 cells in vitro. Further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of cholangiocarcinoma.

  3. Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest

    Institute of Scientific and Technical Information of China (English)

    Long-Zhu Li; Hong-Xia Deng; Wen-Zhu Lou; Xue-Yan Sun; Meng-Wan Song; Jing Tao; Bing-Xiu Xiao; Jun-Ming Guo

    2012-01-01

    AIM: To investigate the growth effects of 4-phenyl butyric acid (PBA) on human gastric carcinoma cells and their mechanisms. METHODS: Moderately-differentiated human gastric carcinoma SGC-7901 and lowly-differentiated MGC-803 cells were treated with 5, 10, 20, 40, and 60 μmol/L PBA for 1-4 d. Cell proliferation was detected using the MTT colorimetric assay. Cell cycle distributions were examined using flow cytometry. RESULTS: The proliferation of gastric carcinoma cells was inhibited by PBA in a dose- and time-dependent fashion. Flow cytometry showed that SGC-7901 cells treated with low concentrations of PBA were arrested at the G0/G1 phase, whereas cells treated with high concentrations of PBA were arrested at the G2/M phase. Although MGC-803 cells treated with low concentrations of PBA were also arrested at the G0/G1 phase, cells treated with high concentrations of PBA were arrested at the S phase. CONCLUSION: The growth inhibitory effect of PBA on gastric cancer cells is associated with alteration of the cell cycle. For moderately-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and G2/M phases. For lowly-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and S phases.

  4. Amelioration of Glucolipotoxicity-Induced Endoplasmic Reticulum Stress by a “Chemical Chaperone” in Human THP-1 Monocytes

    Directory of Open Access Journals (Sweden)

    Raji Lenin

    2012-01-01

    Full Text Available Chronic ER stress is emerging as a trigger that imbalances a number of systemic and arterial-wall factors and promote atherosclerosis. Macrophage apoptosis within advanced atherosclerotic lesions is also known to increase the risk of atherothrombotic disease. We hypothesize that glucolipotoxicity might mediate monocyte activation and apoptosis through ER stress. Therefore, the aims of this study are (a to investigate whether glucolipotoxicity could impose ER stress and apoptosis in THP-1 human monocytes and (b to investigate whether 4-Phenyl butyric acid (PBA, a chemical chaperone could resist the glucolipotoxicity-induced ER stress and apoptosis. Cells subjected to either glucolipotoxicity or tunicamycin exhibited increased ROS generation, gene and protein (PERK, GRP-78, IRE1α, and CHOP expression of ER stress markers. In addition, these cells showed increased TRPC-6 channel expression and apoptosis as revealed by DNA damage and increased caspase-3 activity. While glucolipotoxicity/tunicamycin increased oxidative stress, ER stress, mRNA expression of TRPC-6, and programmed the THP-1 monocytes towards apoptosis, all these molecular perturbations were resisted by PBA. Since ER stress is one of the underlying causes of monocyte dysfunction in diabetes and atherosclerosis, our study emphasize that chemical chaperones such as PBA could alleviate ER stress and have potential to become novel therapeutics.

  5. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

    Science.gov (United States)

    Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

    2015-12-22

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p cell death in Pb(NO₃)₂-treated cells, indicative of membrane rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects.

  6. Recognition and uptake of free and nanoparticle‐bound betalactoglobulin – a food allergen – by human monocytes

    DEFF Research Database (Denmark)

    Marengo, Mauro; Bonomi, Francesco; Iametti, Stefania

    2011-01-01

    was covalently conjugated to dextran‐coated magnetic nanoparticles (MNPs) without affecting its structure and immunoreactivity. BLG‐conjugated MNPs were taken up by human monocytes much more efficiently than non‐conjugated MNPs, allowing easy magnetic separation of cells that had adsorbed the allergen. BLG, OM......, and HSA were conjugated to MNPs also labeled with a fluorescent probe. The uptake of these materials by human monocytes was monitored through flow cytometry, and compared with fluorescent MNPs and the free fluorescently labeled proteins, confirming higher uptake of the BLG‐conjugated MNPs versus non......‐conjugated MNPs. OM but not HSA conjugation to particles enhanced uptake of the MNPs. Confocal microscopy provided direct evidence of the actual internalization of BLG–MNP conjugates into the cytoplasm. Conclusions: These results contribute to the current understanding of the interaction between food allergens...

  7. Human β-Defensin 3 Reduces TNF-α-Induced Inflammation and Monocyte Adhesion in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Tianying Bian

    2017-01-01

    Full Text Available The aim of this study was to investigate the role of human β-defensin 3 (hBD3 in the initiation stage of atherosclerosis with human umbilical vein endothelial cells (HUVECs triggered by tumor necrosis factor- (TNF- α. The effects of hBD3 on TNF-α-induced endothelial injury and inflammatory response were evaluated. Our data revealed that first, hBD3 reduced the production of interleukin-6 (IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1, and macrophage migration inhibitory factor (MIF in HUVECs in a dose-dependent manner. In addition, hBD3 significantly prevented intracellular reactive oxygen species (ROS production by HUVECs. Second, western blot analysis demonstrated that hBD3 dose-dependently suppressed the protein levels of intracellular adhesion molecule-1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1 in TNF-α-induced HUVECs. As a result, hBD3 inhibited monocyte adhesion to TNF-α-treated endothelial cells. Additionally, hBD3 suppressed TNF-α-induced F-actin reorganization in HUVECs. Third, hBD3 markedly inhibited NF-κB activation by decreasing the phosphorylation of IKK-α/β, IκB, and p65 subunit within 30 min. Moreover, the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK in the mitogen-activated protein kinase (MAPK pathway were also inhibited by hBD3 in HUVECs. In conclusion, hBD3 exerts anti-inflammatory and antioxidative effects in endothelial cells in response to TNF-α by inhibiting NF-κB and MAPK signaling.

  8. Human β-Defensin 3 Reduces TNF-α-Induced Inflammation and Monocyte Adhesion in Human Umbilical Vein Endothelial Cells

    Science.gov (United States)

    Bian, Tianying; Li, Houxuan; Zhou, Qian; Ni, Can; Zhang, Yangheng

    2017-01-01

    The aim of this study was to investigate the role of human β-defensin 3 (hBD3) in the initiation stage of atherosclerosis with human umbilical vein endothelial cells (HUVECs) triggered by tumor necrosis factor- (TNF-) α. The effects of hBD3 on TNF-α-induced endothelial injury and inflammatory response were evaluated. Our data revealed that first, hBD3 reduced the production of interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1 (MCP-1), and macrophage migration inhibitory factor (MIF) in HUVECs in a dose-dependent manner. In addition, hBD3 significantly prevented intracellular reactive oxygen species (ROS) production by HUVECs. Second, western blot analysis demonstrated that hBD3 dose-dependently suppressed the protein levels of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in TNF-α-induced HUVECs. As a result, hBD3 inhibited monocyte adhesion to TNF-α-treated endothelial cells. Additionally, hBD3 suppressed TNF-α-induced F-actin reorganization in HUVECs. Third, hBD3 markedly inhibited NF-κB activation by decreasing the phosphorylation of IKK-α/β, IκB, and p65 subunit within 30 min. Moreover, the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK) in the mitogen-activated protein kinase (MAPK) pathway were also inhibited by hBD3 in HUVECs. In conclusion, hBD3 exerts anti-inflammatory and antioxidative effects in endothelial cells in response to TNF-α by inhibiting NF-κB and MAPK signaling.

  9. Unsaturated fatty acids prevent activation of NLRP3 inflammasome in human monocytes/macrophages[S

    Science.gov (United States)

    L'homme, Laurent; Esser, Nathalie; Riva, Laura; Scheen, André; Paquot, Nicolas; Piette, Jacques; Legrand-Poels, Sylvie

    2013-01-01

    The NLRP3 inflammasome is involved in many obesity-associated diseases, such as type 2 diabetes, atherosclerosis, and gouty arthritis, through its ability to induce interleukin (IL)-1β release. The molecular link between obesity and inflammasome activation is still unclear, but free fatty acids have been proposed as one triggering event. Here we reported opposite effects of saturated fatty acids (SFAs) compared with unsaturated fatty acids (UFAs) on NLRP3 inflammasome in human monocytes/macrophages. Palmitate and stearate, both SFAs, triggered IL-1β secretion in a caspase-1/ASC/NLRP3-dependent pathway. Unlike SFAs, the UFAs oleate and linoleate did not lead to IL-1β secretion. In addition, they totally prevented the IL-1β release induced by SFAs and, with less efficiency, by a broad range of NLRP3 inducers, including nigericin, alum, and monosodium urate. UFAs did not affect the transcriptional effect of SFAs, suggesting a specific effect on the NLRP3 activation. These results provide a new anti-inflammatory mechanism of UFAs by preventing the activation of the NLRP3 inflammasome and, therefore, IL-1β processing. By this way, UFAs might play a protective role in NLRP3-associated diseases. PMID:24006511

  10. Lobohedleolide induces interleukin-8 production in LPS-stimulated human monocytic cell line THP-1

    Directory of Open Access Journals (Sweden)

    T Oda

    2011-09-01

    Full Text Available Summary: Lobohedleolide (1 has been isolated from a soft coral, Sarcophyton sp., as one of three responsible substances for observed inhibitory activity against TNF-a production in lipopolysaccharide (LPS-stimulated murine macrophage-like RAW 264.7 cells. During the examination of other inflammatory cytokines, we found that only 1 induced the production of interleukin (IL-8 in LPS-stimulated human monocytic THP-1 cells, while the other two compounds did not affect the production of IL-8. Although 1 showed an inhibitory effect on nuclear factor-kappaB (NF-kB activation, this compound induced IL-8 promoter activity, which led to the induction of IL-8 production in LPS-stimulated THP-1 cells. Industrial Relevance: Marine organisms are a rich resource of biologically active secondary metabolites. Two marine natural products and two derivatives of marine sponge compounds have been utilized for medical treatment, and marked numbers of marine natural products and their derivatives are now in clinical and pre-clinical trials. Therefore, marine natural products are attractive sources of medicines and their lead compounds, and elucidation of new bioactivity and the mechanism of action of marine natural products are also a very important study for drug discovery. This report describes a new bioactivity of a diterpene previously obtained from an Indonesian soft corral.

  11. Up-Regulation of CCR5 and CXCR4 Expression on Human Monocytes by Interferon Gamma

    Institute of Scientific and Technical Information of China (English)

    陆韵; 刘祖强; 陈应华

    2003-01-01

    Chemokine receptors, mainly CCR5 and CXCR4, have been proved to be the important coreceptors in HIV-1 entry.HIV-1 disease progression is, in general, characterized by an initial predominance of CCR5 using macrophage tropic, non-syncytium-inducing (NSI) isolates, switching later to CXCR4 using T-cell tropic, syncytium-inducing (SI) isolates.How this shift occurs and how the shift can be controlled are still unclear.Since patients with rapid decline of T cell counts have constantly high levels of IFN-γ in the sera and lymphoid nodes, we investigated the influence of this cytokine on the expression of the HIV-1 coreceptors CCR5 and CXCR4 on the cell surfaces of human monocytic cell line U937 and promonocyte NB4.IFN-γ could intensively enhance the expression of both, while a low level of CCR5 expression was detected in two cell lines before stimulation.The results of semiquantitative RT-PCR also confirm the up-regulation.As the newly generated X4-strains have been demonstrated to be insensitive to chemokine in some reports, IFN-γ may play an important role in selecting CXCR4-used strains.

  12. TLR2 ligands induce NF-κB activation from endosomal compartments of human monocytes.

    Directory of Open Access Journals (Sweden)

    Karim J Brandt

    Full Text Available Localization of Toll-like receptors (TLR in subcellular organelles is a major strategy to regulate innate immune responses. While TLR4, a cell-surface receptor, signals from both the plasma membrane and endosomal compartments, less is known about the functional role of endosomal trafficking upon TLR2 signaling. Here we show that the bacterial TLR2 ligands Pam3CSK4 and LTA activate NF-κB-dependent signaling from endosomal compartments in human monocytes and in a NF-κB sensitive reporter cell line, despite the expression of TLR2 at the cell surface. Further analyses indicate that TLR2-induced NF-κB activation is controlled by a clathrin/dynamin-dependent endocytosis mechanism, in which CD14 serves as an important upstream regulator. These findings establish that internalization of cell-surface TLR2 into endosomal compartments is required for NF-κB activation. These observations further demonstrate the need of endocytosis in the activation and regulation of TLR2-dependent signaling pathways.

  13. Human endotoxin tolerance is associated with enrichment of the CD14+ CD16+ monocyte subset.

    Science.gov (United States)

    Domínguez-Nieto, Aimée; Zentella, Alejandro; Moreno, José; Ventura, José L; Pedraza, Sigifredo; Velázquez, Juan R

    2015-01-01

    Prior exposure to lipopolysaccharides (LPS) induces a state of cell resistance to subsequent LPS restimulation, known as endotoxin tolerance, mainly by repressing the expression of pro-inflammatory cytokines. We established an endotoxin tolerance model in human monocytes Endotoxin-tolerant cells showed a decrease in IκBα degradation and diminished expression of Tumor necrosis factor (TNF) (both messenger RNA [mRNA] and protein content). The myeloid differentiation factor 88 (MyD88)/MyD88 splice variant (MyD88s) ratio, an indirect way to test the Toll-like receptor 4 (TLR4) MyD88-dependent signaling cascade, did not change in endotoxin-tolerant cells when compared to LPS-stimulated or -unstimulated ones. Remarkably, cell population analysis indicated a significant increase of the CD14+ CD16+ subset only under the endotoxin-tolerant condition. Furthermore, endotoxin-tolerant cells produced higher amounts of C-X-C motif chemokine 10 (CXCL10), a typical MyD88-independent cytokine.

  14. The Immuno-Regulatory Effects of Schisandra chinensis and Its Constituents on Human Monocytic Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Mei-Hsien Lee

    2011-06-01

    Full Text Available Many diseases occur when the immune system is weakened. Intracellular signals activate immuno-responsive cells to produce cytokines that modulate the immune response. Schisandra chinensis has been used traditionally to treat general fatigue, neurasthenia, and spontaneous sweating. In the present study, the effect of constituents of S. chinensis on cytokine release by human monocytic leukemia cells (THP-1 was tested using microparticle-based flow cytometric analysis. Two major lignans, schizandrin (Sch and gomisin A (Gom A, were identified and shown to induce interleukin (IL-8, macrophage inflammatory protein-1β (MIP-1β, and granulocyte-macrophage-colony stimulating factor (GM-CSF release by THP-1 cells. By reverse transcription polymerase chain reaction (RT-PCR or quantitative real-time PCR, there was a dose-dependent increase of IL-8, MIP-1β and GM-CSF mRNA levels. Thus, Sch and Gom A from S. chinensis enhance cytokine release by THP-1 cells and this effect occurs through mRNA upregulation. Upregulation of MIP-1β and GM-CSF in particular may have clinical applications. Therefore, S. chinensis may be therapeutically beneficial by promoting humoral and cell-mediated immune responses.

  15. Influence of sex and genetic variability on expression of X-linked genes in human monocytes.

    Science.gov (United States)

    Castagné, Raphaële; Zeller, Tanja; Rotival, Maxime; Szymczak, Silke; Truong, Vinh; Schillert, Arne; Trégouët, David-Alexandre; Münzel, Thomas; Ziegler, Andreas; Cambien, François; Blankenberg, Stefan; Tiret, Laurence

    2011-11-01

    In humans, the fraction of X-linked genes with higher expression in females has been estimated to be 5% from microarray studies, a proportion lower than the 25% of genes thought to escape X inactivation. We analyzed 715 X-linked transcripts in circulating monocytes from 1,467 subjects and found an excess of female-biased transcripts on the X compared to autosomes (9.4% vs 5.5%, pgenes not previously known to escape inactivation, the most significant one was EFHC2 whose 20% of variability was explained by sex. We also investigated cis expression quantitative trait loci (eQTLs) by analyzing 15,703 X-linked SNPs. The frequency and magnitude of X-linked cis eQTLs were quite similar in males and females. Few genes exhibited a stronger genetic effect in females than in males (ARSD, DCX, POLA1 and ITM2A). These genes would deserve further investigation since they may contribute to sex pathophysiological differences.

  16. Effect of size of man-made and natural mineral fibers on chemiluminescent response in human monocyte-derived macrophages.

    OpenAIRE

    2001-01-01

    Fiber size is an important factor in the tumorigenicity of various mineral fibers and asbestos fibers in animal experiments. We examined the time course of the ability to induce lucigenin-dependent chemiluminescence (CL) from human monocyte-derived macrophages exposed to Japan Fibrous Material standard reference samples (glass wool, rock wool, micro glass fiber, two types of refractory ceramic fiber, refractory mullite fiber, potassium titanium whisker, silicon carbide whisker, titanium oxide...

  17. Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes.

    Science.gov (United States)

    López-López, Cintya; Jaramillo-Polanco, Josue; Portales-Pérez, Diana P; Gómez-Coronado, Karen S; Rodríguez-Meléndez, Jessica G; Cortés-García, Juan D; Espinosa-Luna, Rosa; Montaño, Luis M; Barajas-López, Carlos

    2016-12-15

    To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.

  18. Mechanisms involved in ceramide-induced cell cycle arrest in human hepatocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Xiao-Wen Lv; Jie-Ping Shi; Xiao-Song Hu

    2007-01-01

    AIM:To investigate the effect of ceramide on the cell cycle in human hepatocarcinoma Bel7402 cells.Possible molecular mechanisms were explored.METHODS:[3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT)assay,plasmid transfection,reporter assay,FACS and Western blotting analyses were employed to investigate the effect and the related molecular mechanisms of C2-ceramide on the cell cycle of Bel7402 cells.RESULTS:C2-ceramide was found to inhibit the growth of Bel7402 cells by inducing cell cycle arrest.During the process,the expression of p21 protein increased,while that of cyclinD1,phospho-ERK1/2 and c-myc decreased.Furthermore,the level of CDK7 was downregulated,while the transcriptional activity of PPARγ was upregulated.Addition of GW9662,which is a PPARγ specific antagonist,could reserve the modulation action on CDK7.CONCLUSION:Our results support the hypothesis that cell cycle arrest induced by C2-ceramide may be mediated via accumulation of p21 and reduction of cyclinD1 and CDK7,at least partly,through PPARγ activation.The ERK signaling pathway was involved in this process.

  19. Interactions of TANGO and leukocyte integrin CD11c/CD18 regulate the migration of human monocytes.

    Science.gov (United States)

    Arndt, Stephanie; Melle, Christian; Mondal, Krishna; Klein, Gerd; von Eggeling, Ferdinand; Bosserhoff, Anja-Katrin

    2007-12-01

    The TANGO gene was originally identified as a new member of the MIA gene family. It codes for a protein of yet unknown function. TANGO revealed a very broad expression pattern in contrast to the highly restricted expression pattern determined for the other family members. The only cells lacking TANGO expression are cells of the hematopoietic system. One of the major differences between mature hematopoietic cells and other tissue cells is the lack of adhesion until these cells leave the bloodstream. In this study, we observed that TANGO expression was induced after adhesion of human monocytic cells to substrate. To understand the mechanism of TANGO function during monocyte adhesion we isolated interacting proteins and found an interaction between TANGO and the leukocyte-specific integrin CD11c. In functional assays, we observed reduced attachment of human monocytic cells to fibrinogen, ICAM-1 and to human microvascular endothelial cells (HMECs) after stimulation with recombinant TANGO protein. Additionally, the migrating capacity of premonocytic cells through fibrinogen or HMECs was increased after stimulation of these cells with recombinant TANGO. Therefore, we suggest that TANGO reduced the attachment to fibrinogen or other cell adhesion molecules. As TANGO does not compete for CD11c ligand binding directly, we hypothesize TANGO function by modulation of integrin activity. Taken together, the results from this study present TANGO as a novel ligand for CD11c, regulating migratory processes of hematopoietic cells.

  20. Effects of an interleukin-1 receptor antagonist on human sleep, sleep-associated memory consolidation, and blood monocytes.

    Science.gov (United States)

    Schmidt, Eva-Maria; Linz, Barbara; Diekelmann, Susanne; Besedovsky, Luciana; Lange, Tanja; Born, Jan

    2015-07-01

    Pro-inflammatory cytokines like interleukin-1 beta (IL-1) are major players in the interaction between the immune system and the central nervous system. Various animal studies report a sleep-promoting effect of IL-1 leading to enhanced slow wave sleep (SWS). Moreover, this cytokine was shown to affect hippocampus-dependent memory. However, the role of IL-1 in human sleep and memory is not yet understood. We administered the synthetic IL-1 receptor antagonist anakinra (IL-1ra) in healthy humans (100mg, subcutaneously, before sleep; n=16) to investigate the role of IL-1 signaling in sleep regulation and sleep-dependent declarative memory consolidation. Inasmuch monocytes have been considered a model for central nervous microglia, we monitored cytokine production in classical and non-classical blood monocytes to gain clues about how central nervous effects of IL-1ra are conveyed. Contrary to our expectation, IL-1ra increased EEG slow wave activity during SWS and non-rapid eye movement (NonREM) sleep, indicating a deepening of sleep, while sleep-associated memory consolidation remained unchanged. Moreover, IL-1ra slightly increased prolactin and reduced cortisol levels during sleep. Production of IL-1 by classical monocytes was diminished after IL-1ra. The discrepancy to findings in animal studies might reflect species differences and underlines the importance of studying cytokine effects in humans.

  1. Berberine induces p53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Liu Zhaojian; Liu Qiao; Xu Bing; Wu Jingjing [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Guo Chun; Zhu Faliang [Institute of Immunology, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Yang Qiaozi [Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States); Gao Guimin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Gong Yaoqin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China)], E-mail: yxg8@sdu.edu.cn; Shao Changshun [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States)], E-mail: shao@biology.rutgers.edu

    2009-03-09

    Alkaloid berberine is widely used for the treatment of diarrhea and other diseases. Many laboratory studies showed that it exhibits anti-proliferative activity against a wide spectrum of cancer cells in culture. In this report we studied the mechanisms underlying the inhibitory effects of berberine on human osteosarcoma cells and on normal osteoblasts. The inhibition was largely attributed to cell cycle arrest at G1 and G2/M, and to a less extent, to apoptosis. The G1 arrest was dependent on p53, as G1 arrest was abolished in p53-deficient osteosarcoma cells. The induction of G1 arrest and apoptosis was accompanied by a p53-dependent up-regulation of p21 and pro-apoptotic genes. However, the G2/M arrest could be induced by berberine regardless of the status of p53. Interestingly, DNA double-strand breaks, as measured by the phosphorylation of H2AX, were remarkably accumulated in berberine-treated cells in a dose-dependent manner. Thus, one major mechanism by which berberine exerts its growth-inhibitory effect is to inflict genomic lesions on cells, which in turn trigger the activation of p53 and the p53-dependent cellular responses including cell cycle arrest and apoptosis.

  2. Induction of G2/M arrest and apoptosis by sesquiterpene lactones in human melanoma cell lines.

    Science.gov (United States)

    Rozenblat, Sharon; Grossman, Shlomo; Bergman, Margalit; Gottlieb, Hugo; Cohen, Yigal; Dovrat, Sara

    2008-01-15

    Malignant melanoma is a highly aggressive tumor which frequently resists chemotherapy, therefore, the search for new agents for its treatment is of great importance. In this study, we purified the sesquiterpene lactones (SLs), Tomentosin and Inuviscolide from Inula viscosa (Compositae) leaves and studied their anti-cancer potency against human melanoma cell lines in order to develop new agents for melanoma treatment. SLs inhibited the proliferation of three human melanoma cell lines: SK-28, 624 mel and 1363 mel in a dose-dependent manner. We further investigated SLs mechanism of action using SK-28 as a representative cell line model. SLs caused cell-cycle arrest at G(2)/M, accompanied by the appearance of a sub-G0 fraction, indicative of apoptotic cell death. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential (DeltaPsi) and by detection of Caspase-3 activity. Rapid inhibitory phosphorylation of Cdc2 (Thr14 and Tyr15) was seen early after treatment, followed by a later decrease in the expression level of both Cyclin b1 and Cdc2. Induction of p53 and p21(waf1) proteins and phosphorylation of p53 at Ser15 were also detected early after treatment. The anti-apoptotic proteins, p65 subunit of nuclear factor kappaB (NF-kappaB), and Survivin were reduced in a dose-dependent manner. Taken together, these changes partially explain the ability of the SLs to induce G(2)/M arrest and apoptosis. Induction of apoptosis by Tomentosin and Inuviscolide in human aggressive melanoma cell lines has high pharmacological value and implies that SLs might be developed as new agents for melanoma treatment.

  3. KR-31543 reduces the production of proinflammatory molecules in human endothelial cells and monocytes and attenuates atherosclerosis in mouse model.

    Science.gov (United States)

    Choi, Jae-Hoon; Yoo, Ji-Young; Kim, Sun-Ok; Yoo, Sung-Eun; Oh, Goo Taeg

    2012-12-31

    KR-31543, (2S, 3R, 4S)-6-amino-4-[N-(4-chlorophenyl)- N-(2-methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro- 2-dimethyoxymethyl-3-hydroxy-2-methyl-2H-1-benz opyran is a new neuroprotective agent for ischemiareperfusion damage. It has also been reported that KR-31543 has protective effects on lipid peroxidation and H₂O₂-induced reactive oxygen species production. In this study, we investigated the anti-inflammatory and anti-atherogenic properties of KR-31543. We observed that KR-31543 treatment reduced the production of MCP-1, IL-8, and VCAM-1 in HUVECs, and of MCP-1 and IL-6 in THP-1 human monocytes. We also examined the effect of KR-31543 on monocytes migration in vitro. KR-31543 treatment effectively reduced the migration of THP-1 human monocytes to the HUVEC monolayer in a dose-dependent manner. We next examined the effects of this compound on atherogenesis in LDL receptor deficient (Ldlr ⁻/⁻) mice. After 10 weeks of western diet, the formation of atherosclerotic lesion in aorta was reduced in the KR-31543-treated group compared to the control group. The accumulation of macrophages in lesion was also reduced in KR-31543 treated group. However, the plasma levels of total cholesterol, HDL, LDL, and triglyceride were not affected by KR-31543 treatment. Taken together, these results show that KR-31543 has anti-inflammatory properties on human monocytes and endothelial cells, and inhibits fatty streak lesion formation in mouse model of atherosclerosis, suggesting the potential of KR-31543 for the treatment for atherosclerosis.

  4. DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines.

    Science.gov (United States)

    Sahingur, S E; Xia, X-J; Alamgir, S; Honma, K; Sharma, A; Schenkein, H A

    2010-04-01

    Toll-like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP-1). THP-1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA-initiated cytokine production was determined either by blocking TLR9 signaling in THP-1 cells with chloroquine or by measuring IL-8 production and nuclear factor-kappaB (NF-kappaB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL-1beta, IL-6, and TNF-alpha) was increased significantly in bDNA-stimulated cells compared with controls. Chloroquine treatment of THP-1 cells decreased cytokine production, suggesting that TLR9-mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9-transfected cells demonstrated significantly increased IL-8 production (P < 0.001) and NF-kappaB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF-kappaB genes in response to periodontal bDNA in THP-1 cells, suggesting that cytokine induction is through NF-kappaB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.

  5. OSCAR Is a Receptor for Surfactant Protein D That Activates TNF-α Release from Human CCR2+ Inflammatory Monocytes

    DEFF Research Database (Denmark)

    Barrow, Alexander D; Palarasah, Yaseelan; Bugatti, Mattia;

    2015-01-01

    Surfactant protein D (SP-D) is critical for maintenance of lung homeostasis and provides a first line of defense to pathogens at mucosal surfaces. Polymorphisms in the SP-D-encoding gene SFTPD have been associated with chronic obstructive pulmonary disease and ulcerative colitis. Identification...... of recombinant SP-D and captured native SP-D from human bronchoalveolar lavage. OSCAR localized in an intracellular compartment of alveolar macrophages together with SP-D. Moreover, we found OSCAR on the surface of interstitial lung and blood CCR2(+) inflammatory monocytes, which secreted TNF-α when exposed...... therapeutic target in chronic inflammatory diseases of the lung as well as other diseases involving tissue accumulation of SP-D, infiltration of inflammatory monocytes, and release of TNF-α....

  6. STAT3 activation in monocytes accelerates liver cancer progression

    Directory of Open Access Journals (Sweden)

    Wu Wen-Yong

    2011-12-01

    Full Text Available Abstract Background Signal transducer and activator of transcription 3 (STAT3 is an important transcription factor ubiquitously expressed in different cell types. STAT3 plays an essential role in cell survival, proliferation, and differentiation. Aberrantly hyper-activated STAT3 signaling in cancer cells and in the tumor microenvironment has been detected in a wide variety of human cancers and is considered an important factor for cancer initiation, development, and progression. However, the role of STAT3 activation in monocytes in the development of HCC has not been well understood. Methods Immunohistochemical analysis of phosphorylated STAT3 was performed on tissue microarray from HCC patients. Using a co-culture system in vivo, HCC cell growth was determined by the MTT assay. In vivo experiments were conducted with mice given diethylinitrosamine (DEN, which induces HCC was used to investigate the role of STAT3 expression in monocytes on tumor growth. Real-time PCR was used to determine the expression of cell proliferation and cell arrest associated genes in the tumor and nontumor tissue from liver. Results Phosphorylated STAT3 was found in human hepatocellular carcinoma tissue samples and was expressed in tumor cells and also in monocytes. Phosphorylated STAT3 expression in monocyte was significantly correlated to advanced clinical stage of HCC and a poor prognosis. Using a co-culture system in vivo, monocytes promoted HCC cell growth via the IL-6/STAT3 signaling pathway. The STAT3 inhibitor, NSC 74859, significantly suppressed tumor growth in vivo in mice with diethylinitrosamine (DEN-induced HCC. In this animal model, blockade of STAT3 with NSC 74859 induced tumor cell apoptosis, while inhibiting both tumor cells and monocytes proliferation. Furthermore, NSC 74859 treatment suppressed cancer associated inflammation in DEN-induce HCC. Conclusion Our data suggest constitutively activated STAT3 monocytes promote liver tumorigenesis in clinical

  7. Role of ATM in bystander signaling between human monocytes and lung adenocarcinoma cells.

    Science.gov (United States)

    Ghosh, Somnath; Ghosh, Anu; Krishna, Malini

    2015-12-01

    The response of a cell or tissue to ionizing radiation is mediated by direct damage to cellular components and indirect damage mediated by radiolysis of water. Radiation affects both irradiated cells and the surrounding cells and tissues. The radiation-induced bystander effect is defined by the presence of biological effects in cells that were not themselves in the field of irradiation. To establish the contribution of the bystander effect in the survival of the neighboring cells, lung carcinoma A549 cells were exposed to gamma-irradiation, 2Gy. The medium from the irradiated cells was transferred to non-irradiated A549 cells. Irradiated A549 cells as well as non-irradiated A549 cells cultured in the presence of medium from irradiated cells showed decrease in survival and increase in γ-H2AX and p-ATM foci, indicating a bystander effect. Bystander signaling was also observed between different cell types. Phorbol-12-myristate-13-acetate (PMA)-stimulated and gamma-irradiated U937 (human monocyte) cells induced a bystander response in non-irradiated A549 (lung carcinoma) cells as shown by decreased survival and increased γ-H2AX and p-ATM foci. Non-stimulated and/or irradiated U937 cells did not induce such effects in non-irradiated A549 cells. Since ATM protein was activated in irradiated cells as well as bystander cells, it was of interest to understand its role in bystander effect. Suppression of ATM with siRNA in A549 cells completely inhibited bystander effect in bystander A549 cells. On the other hand suppression of ATM with siRNA in PMA stimulated U937 cells caused only a partial inhibition of bystander effect in bystander A549 cells. These results indicate that apart from ATM, some additional factor may be involved in bystander effect between different cell types.

  8. Gene expression profiling of human monocyte-derived dendritic cells - Searching for molecular regulators of tolerogenicity

    Directory of Open Access Journals (Sweden)

    Katina eSchinnerling

    2015-10-01

    Full Text Available The ability of dendritic cells (DCs to initiate and modulate antigen-specific immune responses has made them attractive targets for immunotherapy. Since DC research in humans is limited by the scarcity of DC populations in the blood circulation, most of our knowledge about DC biology and function has been obtained in vitro from monocyte-derived DCs (moDCs, which can be readily generated in sufficient numbers and are able to differentiate into distinct functional subsets depending on the nature of stimulus. In particular, moDCs with tolerogenic properties (tolDCs possess great therapeutic potential for the treatment of autoimmune diseases. Several protocols have been developed to generate tolDCs in vitro, able to reinstruct auto-reactive T cells and to promote regulatory cells. While ligands and soluble mediators, by which DCs shape immune responses, have been vastly studied, the intracellular pathways and transcriptional regulators that govern tolDC differentiation and function are poorly understood. Whole-genome microarrays and proteomics provide useful strategies to dissect the complex molecular processes that promote tolerogenicity. Only few attempts have been made to understand tolDC biology through a global view on ‘omics’ profiles. So far, the identification of a common regulator of tolerogenicity has been hampered by the fact that each protocol, used for tolDC generation, targets distinct signaling pathways. Here we review the progress in understanding the transcriptional regulation of moDC differentiation, with a special focus on tolDCs, and highlight candidate molecules that might be associated with DC tolerogenicity.

  9. PSP activates monocytes in resting human peripheral blood mononuclear cells: immunomodulatory implications for cancer treatment.

    Science.gov (United States)

    Sekhon, Bhagwant Kaur; Sze, Daniel Man-Yuen; Chan, Wing Keung; Fan, Kei; Li, George Qian; Moore, Douglas Edwin; Roubin, Rebecca Heidi

    2013-06-15

    Polysaccharopeptide (PSP), from Coriolus versicolor, has been used as an adjuvant to chemotherapy, and has demonstrated anti-tumor and immunomodulating effects. However its mechanism remains unknown. To elucidate how PSP affects immune populations, we compared PSP treatments both with and without prior incubation in phytohaemagglutinin (PHA) - a process commonly used in immune population experimentation. We first standardised a capillary electrophoresis fingerprinting technique for PSP identification and characterisation. We then established the proliferative capability of PSP on various immune populations in peripheral blood mononuclear cells, using flow cytometry, without prior PHA treatment. It was found that PSP significantly increased the number of monocytes (CD14(+)/CD16(-)) compared to controls without PHA. This increase in monocytes was confirmed using another antibody panel of CD14 and MHCII. In contrast, proliferations of T-cells, NK, and B-cells were not significantly changed by PSP. Thus, stimulating monocyte/macrophage function with PSP could be an effective therapeutic intervention in targeting tumors.

  10. NF-κB Mediated Transcription in Human Monocytic Cells and Endothelial Cells.

    Science.gov (United States)

    Parry, G C; Mackman, N

    1998-04-01

    Monocytes and endothelial cells become activated at sites of inflammation and contribute to the pathology of many diseases, including septic shock and atherosclerosis. In these cells, induction of genes expressing various inflammatory mediators, such as adhesion molecules, cytokines, and growth factors, is regulated by NF-κB/Rel transcription factors. Recent studies have identified components of the signal transduction pathways leading to the activation of NF-κB/Rel proteins. Inhibition of these signaling pathways provides a novel therapeutic approach to prevent inducible gene expression in both monocytes and endothelial cells. (Trends Cardiovasc Med 1998;8:138-142). © 1998, Elsevier Science Inc.

  11. 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

    The host response to Plasmodia includes the production of enlarged populations of peripheral blood monocytes and tissue macrophages in the spleen and the liver. Since the hyperplasia of the mononuclear phagocyte system is believed to arise as a consequence of an enhanced blood monocyte influx, we....... In conclusion, not all cell functions were altered in concert, and the previously unreported suppression of chemotactic migration might reflect a change in blood leucocyte subpopulations, deactivation in vivo or a direct suppressive effect of plasmodia induced products....

  12. Genistein inhibits human TNF-α-induced porcine endothelial cell adhesiveness for human monocytes and natural killer cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Cellular immune response is a major barrier to xenotransplantation. Human tumor necrosis factor-α (hTNF-α) possesses cross-species activity and directly amplifies the immune rejection via the upregulation of adhesion molecules on porcine endothelium. We investigated the role of protein tyrosine phosphorylation in the induction of expression of E-sclectin and vascular cell adhesion molecule-1 (VCAM-1), and the augmentation of adhesion of human peripheral blood monocytes (PBMo) and natural killer cells (PBNK), after rhTNF-α-stimulation of porcine aortic endothelial cells (PAEC) in vitro, rhTNF-α-increased adhesiveness of PAEC for both PBMo and PBNK was dose-dependently reduced by pretreatment of PAEC with the selective protein tyrosine kinase (PTK) inhibitor genistein. The inhibitory effect occurred at the early time of PAEC activation triggered by rhTNF-α, and was completely reversible. PTK activity assay indicated that genistein also suppressed rhTNF-α stimulated activation of protein tyrosine kinases (PTKs) in PAEC in a dose-dependent manner. Flow cytometric analysis showed that genistein inhibited the upregulation of E-selectin and VCAM-1 by rhTNF-α. These results suggest that PTKs may regulate the expression of E-selectin and VCAM-1 on PAEC and the adherence of PBMo and PBNK induced by rhTNF-α. Moreover, dietary genistein, used as an adhesion antagonist, may contribute to managing the cell-mediated rejection in the clinical application.

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

    Science.gov (United States)

    Kharazmi, A; Nielsen, H; Hovgaard, D; Borregaard, N; Nissen, N I

    1991-04-01

    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 undergoing GM-CSF treatment. Patients with either Hodgkin's or non-Hodgkin's lymphoma were treated with various dosages (2-16 micrograms kg-1 body weight per day for 5 days) of rhGM-CSF by intravenous or subcutaneous route. Prior to and on day 5 of rhGM-CSF treatment, neutrophil and monocyte chemotaxis and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Met-Leu-Phe and zymosan was enhanced by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence system may be enhanced by GM-CSF treatment and that this cytokine may be a useful therapeutic adjunct in compromised patients.

  14. Rhein lysinate inhibits monocyte adhesion to human umbilical vein endothelial cells by blocking p38 signaling pathway.

    Science.gov (United States)

    Lin, Yajun; Zhen, Yongzhan; Liu, Jiang; Wei, Jie; Tu, Ping; Hu, Gang

    2013-11-01

    The objective of this study was to investigate the effect of rhein lysinate (RHL) on monocyte adhesion and its mechanism. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth inhibition by drugs. The monocyte chemoattractant protein (MCP)-1 levels were assayed using MCP-1 ELISA. The expression of proteins was detected by Western blotting analysis. The results indicated that RHL inhibited monocyte adhesion in a dose- and time-dependent manner. RHL (<20 μmol/L) and lipopolysaccharide (LPS) had no effect on viability of human umbilical vein endothelial cells. Therefore, 20 μmol/L RHL was selected for this study. RHL inhibited secretion of MCP-1 induced by LPS and expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. In the meantime, both RHL and p38 inhibitor (SB203580) inhibited phosphorylation of p38 and mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2) and transcription and expression of ICAM-1 and VCAM-1. In conclusion, RHL inhibits the transcription and expression of ICAM-1 and VCAM-1 by the p38/MAPKAPK-2 signaling pathway, and the effect of RHL on transcription and expression of ICAM-1 and VCAM-1 is similar to p38 inhibitor. RHL could be a prophylactic drug for atherosclerosis.

  15. Berberine induces cell cycle arrest and apoptosis in human gastric carcinoma SNU-5 cell line

    Institute of Scientific and Technical Information of China (English)

    Jing-Pin Lin; Jai-Sing Yang; Jau-Hong Lee; Wen-Tsong Hsieh; Jing-Gung Chung

    2006-01-01

    AIM: To investigate the relationship between the inhibited growth (cytotoxic activity) of berberine and apoptotic pathway with its molecular mechanism of action.METHODS: The in vitro cytotoxic techniques were complemented by cell cycle analysis and determination of sub-G1 for apoptosis in human gastric carcinoma SNU-5 cells. Percentage of viable cells, cell cycle, and sub-G1 group (apoptosis) were examined and determined by the flow cytometric methods. The associated proteins for cell cycle arrest and apoptosis were examined by Western blotting.RESULTS: For SNU-5 cell line, the IC (50) was found to be 48 μmol/L of berberine. In SNU-5 cells treated with 25-200 μmol/L berberine, G2/M cell cycle arrest was observed which was associated with a marked increment of the expression of p53, Wee1 and CDk1 proteins and decreased cyclin B. A concentration-dependent decrease of cells in G0/G1 phase and an increase in G2/M phase were detected. In addition, apoptosis detected as sub-G0 cell population in cell cycle measurement was proved in 25-200 μmol/L berberine-treated cells by monitoring the apoptotic pathway. Apoptosis was identified by sub-G0 cell population, and upregulation of Bax, downregulation of Bcl-2, release of Ca2+, decreased the mitochondrial membrane potential and then led to the release of mitochondrial cytochrome C into the cytoplasm and caused the activation of caspase-3, and finally led to the occurrence of apoptosis.CONCLUSION: Berberine induces p53 expression and leads to the decrease of the mitochondrial membrane potential, Cytochrome C release and activation of caspase-3 for the induction of apoptosis.

  16. Pseudolaric acid B induces apoptosis, senescence, and mitotic arrest in human breast cancer MCF-7

    Institute of Scientific and Technical Information of China (English)

    Jing-hua YU; Qiao CUI; Yuan-yuan JIANG; Wei YANG; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2007-01-01

    Aim: The aim of the present study was to investigate the inhibitory effect of pseudolaric acid B (PAB) on human breast cancer MCF-7 cells. Methods: 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, morphological changes, acridine orange staining, and agarose gel electrophoresis were applied to detect apoptosis. The percentage of apoptotic and necrotic cells was calcu- lated by the lactate dehydrogenase activity-based cytotoxicity assay; senescence associated (SA)-β-galactosidase activity was detected to evaluate senescence; flow cytometric analysis of propidium iodide staining was carried out to investi- gate the distribution of cell cycle, and the protein expression was examined by Western blot analysis. Results: During apoptosis, the half maximal inhibitory concentration IC502 was 3.4 and 1.35 μmol/L at 36 and 48 h after PAB treatment, respectively. The MCF-7 cells exposed to PAB showed typical characteristics of apoptosis, including the morphological changes and DNA fragmentation. The MCF-7 cells treated with 4 μmol/L PAB for 36 h underwent apoptosis, but not necrosis. The apoptosis induced by PAB was independent of the death receptor pathway. The senescent cells became larger and flatter, and the SA-β-galactosi- dase staining was positive. PAB induced obvious mitotic arrest and it preceded apoptosis and senescence. The expressions of p21 and p53 was upregulated with PAB treatment, and cyclin B 1 was upregulated and transported from the cyto- plasm to nuclei, and sustained stable levels. Conclusion: PAB induced mitotic arrest in the MCF-7 cells and inhibited proliferation through apoptosis and senescence. The apoptosis was independent of the death receptor pathway.

  17. FastDC derived from human monocytes within 48 h effectively prime tumor antigen-specific cytotoxic T cells.

    Science.gov (United States)

    Dauer, Marc; Schad, Katharina; Herten, Jan; Junkmann, Jana; Bauer, Christian; Kiefl, Rosemarie; Endres, Stefan; Eigler, Andreas

    2005-07-01

    Previously, we have shown that dendritic cells (DCs) with full T-cell stimulatory capacity can be derived from human monocytes after 48 h of in vitro culture (FastDC). Compared to a standard 7-day protocol, this new strategy not only reduces the time span and the amount of recombinant cytokines required, but may also resemble DC development in vivo more closely. Using a melanoma antigen model, we show here that FastDC prime CTL responses against tumor antigens as effectively as standard monocyte-derived DCs (moDCs). FastDC and moDCs derived from monocytes of HLA-A2(+) donors were loaded with the melanoma-associated, HLA-A(*)0201-restricted peptide Melan-A and cocultured with autologous CD3(+) T cells. After two weekly restimulations with freshly prepared, peptide-loaded FastDC or moDCs, binding of CD8(+) T cells to fluorescently labeled MHC-I/Melan-A-peptide complexes and intracellular cytokine staining revealed that the two DC preparations had an equal capacity to prime Melan-A-specific, IFN-gamma producing CD8(+) T cells. CTLs derived from cocultures with FastDC lysed Melan-A-loaded T2 cells even more effectively than CTLs primed by moDCs. Comparative analysis also revealed that FastDC possess an equal capacity to migrate in response to the chemokine receptor CCR-7 ligand 6Ckine. Importantly, DCs can be generated with higher yield and purity using the FastDC-protocol. The reliability and efficacy of this new strategy for DC development from monocytes may facilitate clinical investigation of DC-based tumor immunotherapy.

  18. Cationic liposomal drug delivery system for specific targeting of human cd14+ monocytes in whole blood

    DEFF Research Database (Denmark)

    2013-01-01

    This invention concerns a liposome comprising lipids and at least one active ingredient, wherein at least one of the lipids is a cationic lipid; said liposome exhibiting a net positive charge at physiological conditions at which said liposome preferentially adheres to monocytes in freshly drawn b......, an infectious disease, an inflammatory disease, an autoimmune disease or allergy....

  19. 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

  20. 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.

    2012-01-01

    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 patie

  1. HIV-1 gp120 envelope glycoprotein determinants for cytokine burst in human monocytes

    Science.gov (United States)

    Coutu, Mathieu; Prévost, Jérémie; Brassard, Nathalie; Peres, Adam; Stegen, Camille; Madrenas, Joaquín; Kaufmann, Daniel E.; Finzi, Andrés

    2017-01-01

    The first step of HIV infection involves the interaction of the gp120 envelope glycoprotein to its receptor CD4, mainly expressed on CD4+ T cells. Besides its role on HIV-1 entry, the gp120 has been shown to be involved in the production of IL-1, IL-6, CCL20 and other innate response cytokines by bystander, uninfected CD4+ T cells and monocytes. However, the gp120 determinants involved in these functions are not completely understood. Whether signalling leading to cytokine production is due to CD4 or other receptors is still unclear. Enhanced chemokine receptor binding and subsequent clustering receptors may lead to cytokine production. By using a comprehensive panel of gp120 mutants, here we show that CD4 binding is mandatory for cytokine outburst in monocytes. Our data suggest that targeting monocytes in HIV-infected patients might decrease systemic inflammation and the potential tissue injury associated with the production of inflammatory cytokines. Understanding how gp120 mediates a cytokine burst in monocytes might help develop new approaches to improve the chronic inflammation that persists in these patients despite effective suppression of viremia by antiretroviral therapy. PMID:28346521

  2. Induction of oxygen free radical generation in human monocytes by lipoprotein(a)

    DEFF Research Database (Denmark)

    Riis Hansen, P; Kharazmi, A; Jauhiainen, M

    1994-01-01

    The mechanism behind the association of elevated plasma lipoprotein(a) [Lp(a)] levels with atherosclerotic disease is unknown. In the present study, Lp(a) induced generation of oxygen free radicals by monocytes from selected healthy individuals in vitro. This observation may provide a link between...

  3. Lipid-associated membrane proteins of Mycoplasma penetrans induce production of proinflammatory cytokines in human monocytic cells

    Institute of Scientific and Technical Information of China (English)

    YAN HUA ZENG; YI MOU WU; MIN JUN YU; LI ZHI TAN; ZHONG LIANG DENG; XIAO XING YOU

    2006-01-01

    The aim of this study is to explore potential pathogenicity of Mycoplasma penetrans, and to investigate whether M. penetrans lipid-associated membrane proteins (LAMPs) could induce human monocytic cell line (THP-1) to produce some proinflammatory cytokines in vitro, including interleukin-1β (IL1β), tumor necrosis factor alpha (TNF-α), and IL-8. THP-1 was stimulated with different concentrations of M. penetrans LAMPs and at different time to analyze the production of human IL-1β, TNF-α and IL-8.The protein levels of human IL-1β, TNF-α and IL-8 were measured by enzyme-linked immunoadsorbent assay (ELISA) and the mRNA levels of these proinflammatory cytokines were detected by reverse transcriptase-PCR (RT-PCR). It was demonstrated in the present study that the production of IL-1β, TNF-αand IL-8 increased in dose- and time-dependent manner after stimulation with M. penetrans LAMPs in THP-1 cells. M.penetrans LAMPs also induced the expression of IL-1β, TNF-α and IL-8 mRNA. The production of IL-1β, TNF-α and IL-8 and the expression of mRNA were down-regulated by pyrrolidine dithiocarbamate (PDTC). This study demonstrated that M. penetrans LAMPs can induce the production of proinflammatory cytokines in human monocytic cells in vitro, thus suggesting that it may be an important etiological factor.

  4. Human Adipose Derived Stem Cells Induced Cell Apoptosis and S Phase Arrest in Bladder Tumor

    Directory of Open Access Journals (Sweden)

    Xi Yu

    2015-01-01

    Full Text Available The aim of this study was to determine the effect of human adipose derived stem cells (ADSCs on the viability and apoptosis of human bladder cancer cells. EJ and T24 cells were cocultured with ADSCs or cultured with conditioned medium of ADSCs (ADSC-CM, respectively. The cell counting and colony formation assay showed ADSCs inhibited the proliferation of EJ and T24 cells. Cell viability assessment revealed that the secretions of ADSCs, in the form of conditioned medium, were able to decrease cancer cell viability. Wound-healing assay suggested ADSC-CM suppressed migration of T24 and EJ cells. Moreover, the results of the flow cytometry indicated that ADSC-CM was capable of inducing apoptosis of T24 cells and inducing S phase cell cycle arrest. Western blot revealed ADSC-CM increased the expression of cleaved caspase-3 and cleaved PARP, indicating that ADSC-CM induced apoptosis in a caspase-dependent way. PTEN/PI3K/Akt pathway and Bcl-2 family proteins were involved in the mechanism of this reaction. Our study indicated that ADSCs may provide a promising and practicable manner for bladder tumor therapy.

  5. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

    Science.gov (United States)

    Witte, Katrin; Koch, Egon; Volk, Hans-Dieter; Wolk, Kerstin; Sabat, Robert

    2015-01-01

    Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells.

  6. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

    Directory of Open Access Journals (Sweden)

    Katrin Witte

    Full Text Available Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630 on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells.

  7. Human mesenchymal stem cells shift CD8+ T cells towards a suppressive phenotype by inducing tolerogenic monocytes.

    Science.gov (United States)

    Hof-Nahor, Irit; Leshansky, Lucy; Shivtiel, Shoham; Eldor, Liron; Aberdam, Daniel; Itskovitz-Eldor, Joseph; Berrih-Aknin, Sonia

    2012-10-01

    The mechanisms underlying the immunomodulatory effects of mesenchymal stem cells (MSCs) have been investigated under extreme conditions of strong T cell activation, which induces the rapid death of activated lymphocytes. The objective of this study was to investigate these mechanisms in the absence of additional polyclonal activation. In co-cultures of peripheral mononuclear blood cells with human MSCs (hereafter referred to as hMSCs), we observed a striking decrease in the level of CD8 expression on CD8+ cells, together with decreased expression of CD28 and CD44, and impaired production of IFN-gamma and Granzyme B. This effect was specific to hMSCs, because it was not observed with several other cell lines. Downregulation of CD8 expression required CD14+ monocytes to be in direct contact with the CD8+ cells, whereas the effects of hMSCs on the CD14+ cells were essentially mediated by soluble factors. The CD14+ monocytes exhibited a tolerogenic pattern when co-cultured with hMSCs, with a clear decrease in CD80 and CD86 co-stimulatory molecules, and an increase in the inhibitory receptors ILT-3 and ILT-4. CD8+ cells that were preconditioned by MSCs had similar effects on monocytes and were able to inhibit lymphocyte proliferation. Injection of hMSCs in humanized NSG mice showed similar trends, in particular decreased levels of CD44 and CD28 in human immune cells. Our study demonstrates a new immunomodulation mechanism of action of hMSCs through the modulation of CD8+ cells towards a non-cytotoxic and/or suppressive phenotype. This mechanism of action has to be taken into account in clinical trials, where it should be beneficial in grafts and autoimmune diseases, but potentially detrimental in malignant diseases.

  8. Effect of Bacterial Lipopolysaccharide Contamination on Gutta Percha- versus Resilon-Induced Human Monocyte Cell Line Toxicity.

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    Jamshid Hadjati

    2015-04-01

    Full Text Available Cytotoxic effects of obturation materials were tested in presence and absence of endotoxin on human monocytes in vitro.Human monocytes from THP-1 cell line were cultured. Three millimeters from the tip of each Resilon and gutta percha points were cut and directly placed at the bottom of the culture wells. Cultured cells were exposed to gutta percha (groups G1 and G2 and Resilon (R1 and R2. Ten μg/ml bacterial lipopolysaccharide (LPS was added to the culture wells in groups G1 and R1. Positive control included the bacterial LPS without the root canal filling material and the negative control contained the cells in culture medium only. Viability of cells was tested in all groups after 24, 48, and 72 hours using the methylthiazolyldiphenyl-tetrazolium bromide (MTT assay for at least 3 times to obtain reproducible results. Optical density values were read and the data were analyzed using three-way ANOVA and post hoc statistical test.The results showed that cells in G2 had the lowest rate of viability at 24 hours, but the lowest rate of viable cells was recorded in G1 at 48 and 72 hours. The effect of LPS treatment was not statistically significant. Resilon groups showed cell viability values higher than those of gutta percha groups, although statistically non-significant (P=0.105. Cell viability values were lower in gutta percha than Resilon groups when LPS-treated and LPS-untreated groups were compared independently at each time point.It could be concluded that none of the tested root canal filling materials had toxic effects on cultured human monocyte cells whether in presence or absence of LPS contamination.

  9. Immature dendritic cells generated from cryopreserved human monocytes show impaired ability to respond to LPS and to induce allogeneic lymphocyte proliferation.

    Directory of Open Access Journals (Sweden)

    Guilherme Ferreira Silveira

    Full Text Available Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs, the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.

  10. High levels of WNT-5A in human glioma correlate with increased presence of tumor-associated microglia/monocytes.

    Science.gov (United States)

    Dijksterhuis, Jacomijn P; Arthofer, Elisa; Marinescu, Voichita D; Nelander, Sven; Uhlén, Mathias; Pontén, Frederik; Mulder, Jan; Schulte, Gunnar

    2015-12-10

    Malignant gliomas are among the most severe types of cancer, and the most common primary brain tumors. Treatment options are limited and the prognosis is poor. WNT-5A, a member of the WNT family of lipoglycoproteins, plays a role in oncogenesis and tumor progression in various cancers, whereas the role of WNT-5A in glioma remains obscure. Based on the role of WNT-5A as an oncogene, its potential to regulate microglia cells and the glioma-promoting capacities of microglia cells, we hypothesize that WNT-5A has a role in regulation of immune functions in glioma. We investigated WNT-5A expression by in silico analysis of the cancer genome atlas (TCGA) transcript profiling of human glioblastoma samples and immunohistochemistry experiments of human glioma tissue microarrays (TMA). Our results reveal higher WNT-5A protein levels and mRNA expression in a subgroup of gliomas (WNT-5A(high)) compared to non-malignant control brain tissue. Furthermore, we show a significant correlation between WNT-5A in the tumor and presence of major histocompatibility complex Class II-positive microglia/monocytes. Our data pinpoint a positive correlation between WNT-5A and a proinflammatory signature in glioma. We identify increased presence of microglia/monocytes as an important aspect in the inflammatory transformation suggesting a novel role for WNT-5A in human glioma.

  11. 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

    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...... are most likely to represent the inhibitory factors. The inhibitory activity of L. pneumophila sonic extract appears to be related to inhibition of killing mechanisms since uptake of Listeria was not affected by the sonicate. Our observations indicate that as Legionella infection progresses, bacterial...

  12. Role of monocytes and macrophages in experimental and human acute liver failure

    Institute of Scientific and Technical Information of China (English)

    Lucia; A; Possamai; Charalambos; Gustav; Antoniades; Quentin; M; Anstee; Alberto; Quaglia; Diego; Vergani; Mark; Thursz; Julia; Wendon

    2010-01-01

    Acute liver failure (ALF) is a devastating clinical syndrome characterised by progressive encephalopathy, coagulopathy, and circulatory dysfunction, which commonly leads to multiorgan failure and death. Central to the pathogenesis of ALF is activation of the immune system with mobilisation of cellular effectors and massive production of cytokines. As key components of the innate immune system, monocytes and macrophages are postulated to play a central role in the initiation, progression and resolution of AL...

  13. Stimulation of pro-inflammatory responses by mebendazole in human monocytic THP-1 cells through an ERK signaling pathway.

    Science.gov (United States)

    Mizuno, Katsuhiko; Toyoda, Yasuyuki; Fukami, Tatsuki; Nakajima, Miki; Yokoi, Tsuyoshi

    2011-03-01

    Oral helminthic mebendazole (MBZ) has been reported to cause liver injury with inflammatory responses. However, the underlying mechanism remains unknown. To examine the inflammatory reactions, we investigated whether MBZ and other helminthic drugs increase the release of pro-inflammatory cytokines and chemokines using human monocytic cells. The release of interleukin (IL)-8 and tumor necrosis factor (TNF) α from human monocytic THP-1 cells was significantly increased by treatment with MBZ, albendazole (ABZ), fenbendazole (FBZ), or oxibendazole (OBZ), but not by albendazole sulfoxide or praziquantel, suggesting that MBZ and structurally similar drugs can stimulate monocytes and increase the release of pro-inflammatory cytokines. MBZ also significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 in THP-1 cells. Pretreatment with the MAP kinase/ERK kinase 1/2 inhibitor U0126 significantly suppressed the increase of IL-8 and TNFα levels by MBZ, ABZ, FBZ, or OBZ treatment in THP-1 cells, but the p38 mitogen-activated protein kinase inhibitor SB203580 or JNK1/2 inhibitor SP600125 did not. These results suggested that an ERK1/2 pathway plays an important role in the release of IL-8 and TNFα in THP-1 cells treated with MBZ and structurally similar drugs. In conclusion, the release of inflammatory mediators by MBZ might be one of the mechanisms underlying immune-mediated liver injury. This in vitro method may be useful to predict adverse inflammatory reactions that lead to hepatotoxicity.

  14. Boletus edulis biologically active biopolymers induce cell cycle arrest in human colon adenocarcinoma cells.

    Science.gov (United States)

    Lemieszek, Marta Kinga; Cardoso, Claudia; Ferreira Milheiro Nunes, Fernando Hermínio; Ramos Novo Amorim de Barros, Ana Isabel; Marques, Guilhermina; Pożarowski, Piotr; Rzeski, Wojciech

    2013-04-25

    The use of biologically active compounds isolated from edible mushrooms against cancer raises global interest. Anticancer properties are mainly attributed to biopolymers including mainly polysaccharides, polysaccharopeptides, polysaccharide proteins, glycoproteins and proteins. In spite of the fact that Boletus edulis is one of the widely occurring and most consumed edible mushrooms, antitumor biopolymers isolated from it have not been exactly defined and studied so far. The present study is an attempt to extend this knowledge on molecular mechanisms of their anticancer action. The mushroom biopolymers (polysaccharides and glycoproteins) were extracted with hot water and purified by anion-exchange chromatography. The antiproliferative activity in human colon adenocarcinoma cells (LS180) was screened by means of MTT and BrdU assays. At the same time fractions' cytotoxicity was examined on the human colon epithelial cells (CCD 841 CoTr) by means of the LDH assay. Flow cytometry and Western blotting were applied to cell cycle analysis and protein expression involved in anticancer activity of the selected biopolymer fraction. In vitro studies have shown that fractions isolated from Boletus edulis were not toxic against normal colon epithelial cells and in the same concentration range elicited a very prominent antiproliferative effect in colon cancer cells. The best results were obtained in the case of the fraction designated as BE3. The tested compound inhibited cancer cell proliferation which was accompanied by cell cycle arrest in the G0/G1-phase. Growth inhibition was associated with modulation of the p16/cyclin D1/CDK4-6/pRb pathway, an aberration of which is a critical step in the development of many human cancers including colon cancer. Our results indicate that a biopolymer BE3 from Boletus edulis possesses anticancer potential and may provide a new therapeutic/preventive option in colon cancer chemoprevention.

  15. Cardiac arrest

    Science.gov (United States)

    ... Article.jsp. Accessed June 16, 2014. Myerburg RJ, Castellanos A. Approach to cardiac arrest and life-threatening ... PA: Elsevier Saunders; 2011:chap 63. Myerburg RJ, Castellanos A. Cardiac arrest and audden aardiac death. In: ...

  16. From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

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    Sebastian Weiterer

    Full Text Available Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner.The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.

  17. Effect of sesamin on apoptosis and cell cycle arrest in human breast cancer mcf-7 cells.

    Science.gov (United States)

    Siao, An-Ci; Hou, Chien-Wei; Kao, Yung-Hsi; Jeng, Kee-Ching

    2015-01-01

    Dietary prevention has been known to reduce breast cancer risk. Sesamin is one of the major components in sesame seeds and has been widely studied and proven to have anti-proliferation and anti-angiogenic effects on cancer cells. In this study, the influence of sesamin was tested in the human breast cancer MCF-7 cell line for cell viability (MTT assay) and cell cycling (flow cytometry). Results showed that sesamin dose-dependently (1, 10 and 50 μM) reduced the cell viability and increased LDH release and apoptosis (TUNEL assay). In addition, there was a significant increase of sub-G1 phase arrest in the cell cycle after sesamin treatment. Furthermore, sesamin increased the expression of apoptotic markers of Bax, caspase-3, and cell cycle control proteins, p53 and checkpoint kinase 2. Taken together, these results suggested that sesamin might be used as a dietary supplement for prevention of breast cancer by modulating apoptotic signal pathways and inhibiting tumor cell growth.

  18. Anthelmintic drug albendazole arrests human gastric cancer cells at the mitotic phase and induces apoptosis

    Science.gov (United States)

    Zhang, Xuan; Zhao, Jing; Gao, Xiangyang; Pei, Dongsheng; Gao, Chao

    2017-01-01

    As microtubules have a vital function in the cell cycle, oncologists have developed microtubule inhibitors capable of preventing uncontrolled cell division, as in the case of cancer. The anthelmintic drug albendazole (ABZ) has been demonstrated to inhibit hepatocellular, ovarian and prostate cancer cells via microtubule targeting. However, its activity against human gastric cancer (GC) cells has remained to be determined. In the present study, ABZ was used to treat GC cells (MKN-45, SGC-7901 and MKN-28). A a CCK-8 cell proliferation assay was performed to assess the effects of ABZ on cell viability and cell cycle changes were assessed using flow cytometry. SGC-7901 cells were selected for further study, and flow cytometry was employed to determine the apoptotic rate, immunofluorescence analysis was employed to show changes of the microtubule structure as well as the subcellular localization and expression levels of cyclin B1, and western blot analysis was used to identify the dynamics of microtubule assembly. The expression levels of relevant proteins, including cyclin B1 and Cdc2, the two subunits of mitosis-promoting factor as well as apoptosis-asociated proteins were also assessed by western blot analysis. The results showed that ABZ exerted its anti-cancer activity in GC cell lines by disrupting microtubule formation and function to cause mitotic arrest, which is also associated with the accumulation of cyclin B1, and consequently induces apoptosis.

  19. Effects of allitridi on cell cycle arrest of human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Min-Wen Ha; Rui Ma; Li-Ping Shun; Yue-Hua Gong; Yuan Yuan

    2005-01-01

    AIM: To determine the effect of allitridi on cell cycle of human gastric cancer (HGC) cell lines MGC803 and SGC7901 and its possible mechanism.METHODS: Trypan blue dye exclusion was used to evaluate the proliferation, inhibition of cells and damages of these cells were detected with electron microscope.Flow cytometry and cell mitotic index were used to analyze the change of cell cycle, immunohistochemistry, and RT-PCR was used to examine expression of the p21WAF1 gene.RESULTS: MGC803 cell growth was inhibited by allitridi with 24 h IC50 being 6.4 μg/mL. SGC7901 cell growth was also inhibited by allitridi with 24 h IC50 being 7.3 μg/mL.After being treated with allitridi at the concentration of 12 μg/mL for 24 h, cells were found to have direct cytotoxic effects, including broken cellular membrane, swollen and vesiculated mitochondria and rough endoplasmic reticula,and mass lipid droplet. When cells were treated with allitridi at the concentration of 3, 6, and 9 μg/mL for 24 h, the percentage of G0/G1 phase cells was decreased and that of G2/M phase cells was significantly increased (P = 0.002)compared with those in the group. When cells were treated with allitridi at the concentration of 6 μg/mL, cell mitotic index was much higher (P = 0.003) than that of control group, indicating that allitridi could cause gastric cancer cell arrest in M phase. Besides, the expression levels of p21WAF1 gene of MGC803 cells and p21WAF1 gene of SGC7901 cells were remarkably upregulated after treatment.CONCLUSION: Allitridi can cause gastric cancer cell arrest in M phase, and this may be one of the mechanisms for inhibiting cell proliferation. Effect of allitridi on cells in M phas e may be associated with the upregulation of p21WAF1 genes. This study provides experimental data for clinical use of allitridi in the treatment of gastric carcinoma.

  20. RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells.

    Directory of Open Access Journals (Sweden)

    Ryoichiro Nishibayashi

    Full Text Available Interleukin-12 (IL-12 is an important cytokine for the immunomodulatory effects of lactic acid bacteria (LAB. Using murine immune cells, we previously reported that the RNA of Enterococcus faecalis EC-12, a LAB strain exerting probiotic-like beneficial effects, is the major IL-12-inducing immunogenic component. However, it was recently revealed that bacterial RNA can be a ligand for Toll-like receptor (TLR 13, which is only expressed in mice. Because TLR13 is not expressed in humans, the immuno-stimulatory and -modulatory effects of LAB RNA in human cells should be augmented excluding TLR13 contribution. In experiment 1 of this study, the role of LAB RNA in IL-12 induction in human immune cells was studied using three LAB strains, E.faecalis EC-12, Lactobacillus gasseri JCM5344, and Bifidobacterium breve JCM1192. RNase A treatment of heat-killed LAB significantly decreased the IL-12 production of human peripheral blood mononuclear cells on stimulation, while RNase III treatment revealed virtually no effects. Further, IL-12 production against heat-killed E. faecalis EC-12 was abolished by depleting monocytes. These results demonstrated that single stranded RNA (ssRNA of LAB is a strong inducer of IL-12 production from human monocytes. In experiment 2, major receptor for ssRNA of E. faecalis EC-12 was identified using THP-1 cells, a human monocytic cell line. The type of RNA molecules of E. faecalis EC-12 responsible for IL-12 induction was also identified. IL-12 production induced by the total RNA of E. faecalis EC-12 was significantly reduced by the treatment of siRNA for TLR8 but not for TLR7. Furthermore, both 23S and 16S rRNA, but not mRNA, of E. faecalis EC-12 markedly induced IL-12 production from THP-1 cells. These results suggested that the recognition of ssRNA of E. faecalis EC-12 was mediated by TLR8 and that rRNA was the RNA molecule that exhibited IL-12-inducing ability in human cells.

  1. Induction of a type I interferon signature in normal human monocytes by gadolinium-based contrast agents: comparison of linear and macrocyclic agents.

    Science.gov (United States)

    Wermuth, P J; Jimenez, S A

    2014-01-01

    The gadolinium-based contrast agent (GdBCA) Omniscan activates human macrophages through Toll-like receptor (TLR)-4 and TLR-7 signalling. To explore the mechanisms responsible we compared the ability of linear and macrocyclic GdBCA to induce a type I interferon signature and a proinflammatory/profibrotic phenotype in normal human monocytes in vitro. Expression of genes associated with type I interferon signalling and inflammation and production of their corresponding proteins were determined. Both linear and macrocyclic GdBCA stimulated expression of multiple type I interferon-regulated genes and the expression of numerous chemokines, cytokines and growth factors in normal human peripheral blood monocytes. There was no correlation between the magnitude of the measured response and the Gd chelate used. To explore the mechanisms responsible for GdBCA induction of fibrosis in nephrogenic systemic fibrosis (NSF) in vitro, normal human dermal fibroblasts were incubated with GdBCA-treated monocyte culture supernatants and the effects on profibrotic gene expression were examined. Supernatants from monocytes exposed to all GdBCA stimulated types I and III collagen, fibronectin and α-smooth muscle actin (α-SMA) expression in normal dermal fibroblasts. The results indicate that the monocyte activation induced by GdBCA may be the initial step in the development of GdBCA associated fibrosis in NSF.

  2. Ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory chemokine ligand 2 gene in humans

    Directory of Open Access Journals (Sweden)

    Fischer Alexandra

    2012-10-01

    Full Text Available Abstract Background Coenzyme Q10 is an essential cofactor in the respiratory chain and serves in its reduced form, ubiquinol, as a potent antioxidant. Studies in vitro and in vivo provide evidence that ubiquinol reduces inflammatory processes via gene expression. Here we investigate the putative link between expression and DNA methylation of ubiquinol sensitive genes in monocytes obtained from human volunteers supplemented with 150 mg/ day ubiquinol for 14 days. Findings Ubiquinol decreases the expression of the pro-inflammatory chemokine (C-X-C motif ligand 2 gene (CXCL2 more than 10-fold. Bisulfite-/ MALDI-TOF-based analysis of regulatory regions of the CXCL2 gene identified six adjacent CpG islands which showed a 3.4-fold decrease of methylation status after ubiquinol supplementation. This effect seems to be rather gene specific, because ubiquinol reduced the expression of two other pro-inflammatory genes (PMAIP1, MMD without changing the methylation pattern of the respective gene. Conclusion In conclusion, ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory CXCL2 gene in humans. Current Controlled Trials ISRCTN26780329.

  3. Taxifolin enhances andrographolide-induced mitotic arrest and apoptosis in human prostate cancer cells via spindle assembly checkpoint activation.

    Directory of Open Access Journals (Sweden)

    Zhong Rong Zhang

    Full Text Available Andrographolide (Andro suppresses proliferation and triggers apoptosis in many types of cancer cells. Taxifolin (Taxi has been proposed to prevent cancer development similar to other dietary flavonoids. In the present study, the cytotoxic and apoptotic effects of the addition of Andro alone and Andro and Taxi together on human prostate carcinoma DU145 cells were assessed. Andro inhibited prostate cancer cell proliferation by mitotic arrest and activation of the intrinsic apoptotic pathway. Although the effect of Taxi alone on DU145 cell proliferation was not significant, the combined use of Taxi with Andro significantly potentiated the anti-proliferative effect of increased mitotic arrest and apoptosis by enhancing the cleavage of poly(ADP-ribose polymerase, and caspases-7 and -9. Andro together with Taxi enhanced microtubule polymerization in vitro, and they induced the formation of twisted and elongated spindles in the cancer cells, thus leading to mitotic arrest. In addition, we showed that depletion of MAD2, a component in the spindle assembly checkpoint (SAC, alleviated the mitotic block induced by the two compounds, suggesting that they trigger mitotic arrest by SAC activation. This study suggests that the anti-cancer activity of Andro can be significantly enhanced in combination with Taxi by disrupting microtubule dynamics and activating the SAC.

  4. Taxifolin enhances andrographolide-induced mitotic arrest and apoptosis in human prostate cancer cells via spindle assembly checkpoint activation.

    Science.gov (United States)

    Zhang, Zhong Rong; Al Zaharna, Mazen; Wong, Matthew Man-Kin; Chiu, Sung-Kay; Cheung, Hon-Yeung

    2013-01-01

    Andrographolide (Andro) suppresses proliferation and triggers apoptosis in many types of cancer cells. Taxifolin (Taxi) has been proposed to prevent cancer development similar to other dietary flavonoids. In the present study, the cytotoxic and apoptotic effects of the addition of Andro alone and Andro and Taxi together on human prostate carcinoma DU145 cells were assessed. Andro inhibited prostate cancer cell proliferation by mitotic arrest and activation of the intrinsic apoptotic pathway. Although the effect of Taxi alone on DU145 cell proliferation was not significant, the combined use of Taxi with Andro significantly potentiated the anti-proliferative effect of increased mitotic arrest and apoptosis by enhancing the cleavage of poly(ADP-ribose) polymerase, and caspases-7 and -9. Andro together with Taxi enhanced microtubule polymerization in vitro, and they induced the formation of twisted and elongated spindles in the cancer cells, thus leading to mitotic arrest. In addition, we showed that depletion of MAD2, a component in the spindle assembly checkpoint (SAC), alleviated the mitotic block induced by the two compounds, suggesting that they trigger mitotic arrest by SAC activation. This study suggests that the anti-cancer activity of Andro can be significantly enhanced in combination with Taxi by disrupting microtubule dynamics and activating the SAC.

  5. High density lipoprotein suppresses lipoprotein associated phospholipase A2 in human monocytes-derived macrophages through peroxisome proliferator-activated receptor-γ pathway

    Institute of Scientific and Technical Information of China (English)

    HAN Guan-ping; REN Jing-yi; QIN Li; SONG Jun-xian; WANG Lan; CHEN Hong

    2012-01-01

    Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) is mainly secreted by macrophages,serving as a specific marker of atherosclerotic plaque and exerting pro-atherogenic effects.It is known that high-density lipoprotein (HDL) plays an important role against atherosclerosis by inhibiting pro-inflammatory factors,however,the relationship between HDL and Lp-PLA2 remains elusive.Methods In this study,reverse transcription-polymerase chain reaction (RT-PCR),Western blotting,and a platelet-activating factor (PAF) acetylhydrolase assay were performed to determine the Lp-PLA2 mRNA level,protein expression and activity in human monocyte-derived macrophages upon HDL treatment of different concentrations and durations.To investigate the underlying mechanism of HDL-induced Lp-PLA2 action,pioglitazone,a peroxisome proliferator-activated receptor-y (PPARy) ligand,was introduced to human monocyte-derived macrophages and mRNA and protein levels of Lp-PLA2,as well as its activity,were determined.Results Lp-PLA2 mRNA levels,protein expression and activity were significantly inhibited in response to HDL treatment in a dose and time dependent manner in human monocyte-derived macrophages.Pioglitazone treatment (1-10 ng/ml) upregulated the Lp-PLA2 mRNA level,protein expression and activity in human monocyte-derived macrophages,while the effects were markedly reversed by HDL.In addition,pioglitazone resulted in a significant increase in PPARY phosphorylation in human monocyte-derived macrophages,which could be inhibited by HDL.Conclusion These findings indicate that HDL suppresses the expression and activity of Lp-PLA2 in human monocyte-derived macrophages,and the underlying mechanisms may be mediated through the PPARY pathway.

  6. The PRKAA1/AMPKα1 pathway triggers autophagy during CSF1-induced human monocyte differentiation and is a potential target in CMML.

    Science.gov (United States)

    Obba, Sandrine; Hizir, Zoheir; Boyer, Laurent; Selimoglu-Buet, Dorothée; Pfeifer, Anja; Michel, Gregory; Hamouda, Mohamed-Amine; Gonçalvès, Diogo; Cerezo, Michael; Marchetti, Sandrine; Rocchi, Stephane; Droin, Nathalie; Cluzeau, Thomas; Robert, Guillaume; Luciano, Frederic; Robaye, Bernard; Foretz, Marc; Viollet, Benoit; Legros, Laurence; Solary, Eric; Auberger, Patrick; Jacquel, Arnaud

    2015-01-01

    Autophagy is induced during differentiation of human monocytes into macrophages that is mediated by CSF1/CSF-1/M-CSF (colony stimulating factor 1 [macrophage]). However, little is known about the molecular mechanisms that link CSF1 receptor engagement to the induction of autophagy. Here we show that the CAMKK2-PRKAA1-ULK1 pathway is required for CSF1-induced autophagy and human monocyte differentiation. We reveal that this pathway links P2RY6 to the induction of autophagy, and we decipher the signaling network that links the CSF1 receptor to P2RY6-mediated autophagy and monocyte differentiation. In addition, we show that the physiological P2RY6 ligand UDP and the specific P2RY6 agonist MRS2693 can restore normal monocyte differentiation through reinduction of autophagy in primary myeloid cells from some but not all chronic myelomonocytic leukemia (CMML) patients. Collectively, our findings highlight an essential role for PRKAA1-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic interventions for CMML.

  7. Human monocytes and macrophages express NADPH oxidase 5; a potential source of reactive oxygen species in atherosclerosis.

    Science.gov (United States)

    Manea, Adrian; Manea, Simona-Adriana; Gan, Ana Maria; Constantin, Alina; Fenyo, Ioana Madalina; Raicu, Monica; Muresian, Horia; Simionescu, Maya

    2015-05-22

    Monocytes (Mon) and Mon-derived macrophages (Mac) orchestrate important oxidative and inflammatory reactions in atherosclerosis by secreting reactive oxygen species (ROS) due, in large part, to the upregulated NADPH oxidases (Nox). The Nox enzymes have been extensively investigated in human Mon and Mac. However, the expression and functional significance of the Nox5 subtypes is not known. We aimed at elucidating whether Nox5 is expressed in human Mon and Mac, and examine its potential role in atherosclerosis. Human monocytic THP-1 cell line and CD14(+) Mon were employed to search for Nox5 expression. RT-PCR, Western blot, lucigenin-enhanced chemiluminescence and dihydroethidium assays were utilized to examine Nox5 in these cells. We found that Nox5 transcription variants and proteins are constitutively expressed in THP-1 cells and primary CD14(+) Mon. Silencing of Nox5 protein expression by siRNA reduced the Ca(2+)-dependent Nox activity and the formation of ROS in Mac induced by A23187, a selective Ca(2+) ionophore. Exposure of Mac to increasing concentrations of IFNγ (5-100 ng/ml) or oxidized LDL (5-100 μg/ml) resulted in a dose-dependent increase in Nox5 protein expression and elevation in intracellular Ca(2+) concentration. Immunohistochemical staining revealed that Nox5 is present in CD68(+) Mac-rich area within human carotid artery atherosclerotic plaques. To the best of our knowledge, this is the first evidence that Nox5 is constitutively expressed in human Mon. Induction of Nox5 expression in IFNγ- and oxidized LDL-exposed Mac and the presence of Nox5 in Mac-rich atheroma are indicative of the implication of Nox5 in atherogenesis.

  8. Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood.

    Science.gov (United States)

    Meaney, Mary Pat; Nieman, David C; Henson, Dru A; Jiang, Qi; Wang, Fu-Zhang

    2016-09-12

    The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

  9. A rapid crosstalk of human gammadelta T cells and monocytes drives the acute inflammation in bacterial infections.

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    Matthias Eberl

    2009-02-01

    Full Text Available Vgamma9/Vdelta2 T cells are a minor subset of T cells in human blood and differ from other T cells by their immediate responsiveness to microbes. We previously demonstrated that the primary target for Vgamma9/Vdelta2 T cells is (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, an essential metabolite produced by a large range of pathogens. Here we wished to study the consequence of this unique responsiveness in microbial infection. The majority of peripheral Vgamma9/Vdelta2 T cells shares migration properties with circulating monocytes, which explains the presence of these two distinct blood cell types in the inflammatory infiltrate at sites of infection and suggests that they synergize in anti-microbial immune responses. Our present findings demonstrate a rapid and HMB-PP-dependent crosstalk between Vgamma9/Vdelta2 T cells and autologous monocytes that results in the immediate production of inflammatory mediators including the cytokines interleukin (IL-6, interferon (IFN-gamma, tumor necrosis factor (TNF-alpha, and oncostatin M (OSM; the chemokines CCL2, CXCL8, and CXCL10; and TNF-related apoptosis-inducing ligand (TRAIL. Moreover, under these co-culture conditions monocytes differentiate within 18 hours into inflammatory dendritic cells (DCs with antigen-presenting functions. Addition of further microbial stimuli (lipopolysaccharide, peptidoglycan induces CCR7 and enables these inflammatory DCs to trigger the generation of CD4(+ effector alphabeta T cells expressing IFN-gamma and/or IL-17. Importantly, our in vitro model replicates the responsiveness to microbes of effluent cells from peritoneal dialysis (PD patients and translates directly to episodes of acute PD-associated bacterial peritonitis, where Vgamma9/Vdelta2 T cell numbers and soluble inflammatory mediators are elevated in patients infected with HMB-PP-producing pathogens. Collectively, these findings suggest a direct link between invading pathogens, microbe

  10. HSV-1-induced chemokine expression via IFI16-dependent and IFI16-independent pathways in human monocyte-derived macrophages

    DEFF Research Database (Denmark)

    Søby, Stine; Laursen, Rune R; Østergaard, Lars Jørgen;

    2012-01-01

    ABSTRACT: BACKGROUND: Innate recognition is essential in the antiviral response against infection by herpes simplex virus (HSV). Chemokines are important for control of HSV via recruitment of natural killer cells, T lymphocytes, and antigen-presenting cells. We previously found that early HSV-1......-mediated chemokine responses are not dependent on TLR2 and TLR9 in human macrophages. Here, we investigated the role of the recently identified innate IFN-inducible DNA receptor IFI16 during HSV-1 infection in human macrophages. METHODS: Peripheral blood mononuclear cells were purified from buffy coats...... and monocytes were differentiated to macrophages. Macrophages infected with HSV-1 were analyzed using siRNA-mediated knock-down of IFI16 by real-time PCR, ELISA, and Western blotting. RESULTS: We determined that both CXCL10 and CCL3 are induced independent of HSV-1 replication. IFI16 mediates CCL3 m...

  11. Effects of dietary salt levels on monocytic cells and immune responses in healthy human subjects: a longitudinal study.

    Science.gov (United States)

    Yi, Buqing; Titze, Jens; Rykova, Marina; Feuerecker, Matthias; Vassilieva, Galina; Nichiporuk, Igor; Schelling, Gustav; Morukov, Boris; Choukèr, Alexander

    2015-07-01

    Increasing evidence indicated that excess salt consumption can impose risks on human health and a reduction in daily salt intake from the current average of approximately 12 g/d to 5-6 g/d was suggested by public health authorities. The studies on mice have revealed that sodium chloride plays a role in the modulation of the immune system and a high-salt diet can promote tissue inflammation and autoimmune disease. However, translational evidence of dietary salt on human immunity is scarce. We used an experimental approach of fixing salt intake of healthy human subjects at 12, 9, and 6 g/d for months and examined the relationship between salt-intake levels and changes in the immune system. Blood samples were taken from the end point of each salt intake period. Immune phenotype changes were monitored through peripheral leukocyte phenotype analysis. We assessed immune function changes through the characterization of cytokine profiles in response to mitogen stimulation. The results showed that subjects on the high-salt diet of 12 g/d displayed a significantly higher number of immune cell monocytes compared with the same subjects on a lower-salt diet, and correlation test revealed a strong positive association between salt-intake levels and monocyte numbers. The decrease in salt intake was accompanied by reduced production of proinflammatory cytokines interleukin (IL)-6 and IL-23, along with enhanced producing ability of anti-inflammatory cytokine IL-10. These results suggest that in healthy humans high-salt diet has a potential to bring about excessive immune response, which can be damaging to immune homeostasis, and a reduction in habitual dietary salt intake may induce potentially beneficial immune alterations.

  12. Gallic Acid Decreases Inflammatory Cytokine Secretion Through Histone Acetyltransferase/Histone Deacetylase Regulation in High Glucose-Induced Human Monocytes.

    Science.gov (United States)

    Lee, Wooje; Lee, Sang Yeol; Son, Young-Jin; Yun, Jung-Mi

    2015-07-01

    Hyperglycemia contributes to diabetes and several diabetes-related complications. Gallic acid is a polyhydroxy phenolic compound found in various natural products. In this study, we investigated the effects and mechanism of gallic acid on proinflammatory cytokine secretion in high glucose-induced human monocytes (THP-1 cells). THP-1 cells were cultured under normoglycemic or hyperglycemic conditions, in the absence or presence of gallic acid. Hyperglycemic conditions significantly induced histone acetylation, nuclear factor-κB (NF-κB) activation, and proinflammatory cytokine release from THP-1 cells, whereas gallic acid suppressed NF-κB activity and cytokine release. It also significantly reduced CREB-binding protein/p300 (CBP/p300, a NF-κB coactivator) gene expression, acetylation levels, and CBP/p300 histone acetyltransferase (HAT) activity. In addition, histone deacetylase 2 (HDAC2) expression was significantly induced. These results suggest that gallic acid inhibits hyperglycemic-induced cytokine production in monocytes through epigenetic changes involving NF-κB. Therefore, gallic acid may have potential for the treatment and prevention of diabetes and its complications.

  13. An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

    Directory of Open Access Journals (Sweden)

    Sônia Regina Nogueira Ignácio Reis

    2007-12-01

    Full Text Available An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune ethiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-alpha, IFN-alpha, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.

  14. An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation.

    Science.gov (United States)

    Reis, Sônia Regina Nogueira Ignácio; Sampaio, André Luiz Franco; Henriques, Maria das Graças Muller; Gandini, Mariana; Azeredo, Elzinandes Leal; Kubelka, Claire Fernandes

    2007-12-01

    An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune etiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-alpha, IFN-alpha, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applied for dengue fever.

  15. Long non-coding RNA-DANCR in human circulating monocytes: a potential biomarker associated with postmenopausal osteoporosis.

    Science.gov (United States)

    Tong, Xiang; Gu, Peng-cheng; Xu, San-zhong; Lin, Xiang-jin

    2015-01-01

    Osteoporosis is a common disease characterized by low bone mineral density (BMD) and low trauma fractures, mainly resulting from exceeding bone resorption by osteoclasts over bone formation by osteoblasts. Circulating monocytes are directly involved in osteoclastogenesis, and lncRNAs are believed to be involved in the osteoblast differentiation. However, no study has been conducted to identify the roles of lncRNA in circulating monocytes associated with human osteoporosis. In this study, we found significant upregulation of DANCR in the blood mononuclear cells (MNCs) from low-BMD patients with the qRT-PCR analyses. We further found that DANCR promoted the expression of IL6 and TNF-α at both mRNA level and protein level in MNCs. After deletion of DANCR with siRNAs, the levels of IL6 and TNF-α are decreased in the MNCs from low-BMD postmenopausal women. Moreover, DANCR level was correlated with IL6 and TNF-α in postmenopausal women with low BMD. Furthermore, we found that DANCR-induced IL6 and TNF-α in MNCs had bone-resorbing activity. These results indicate that DANCR is involved in the pathology of osteoporosis and may be as a biomarker for postmenopausal osteoporosis.

  16. Mitochondrial DAMPs induce endotoxin tolerance in human monocytes: an observation in patients with myocardial infarction.

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    Irene Fernández-Ruiz

    Full Text Available Monocyte exposure to mitochondrial Danger Associated Molecular Patterns (DAMPs, including mitochondrial DNA (mtDNA, induces a transient state in which these cells are refractory to further endotoxin stimulation. In this context, IRAK-M up-regulation and impaired p65 activity were observed. This phenomenon, termed endotoxin tolerance (ET, is characterized by decreased production of cytokines in response to the pro-inflammatory stimulus. We also show that monocytes isolated from patients with myocardial infarction (MI exhibited high levels of circulating mtDNA, which correlated with ET status. Moreover, a significant incidence of infection was observed in those patients with a strong tolerant phenotype. The present data extend our current understanding of the implications of endotoxin tolerance. Furthermore, our data suggest that the levels of mitochondrial antigens in plasma, such as plasma mtDNA, should be useful as a marker of increased risk of susceptibility to nosocomial infections in MI and in other pathologies involving tissue damage.

  17. Microarray analysis of human monocytes infected with Francisella tularensis identifies new targets of host response subversion.

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    Jonathan P Butchar

    Full Text Available Francisella tularensis is a gram-negative facultative bacterium that causes the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. In order to help understand the mechanisms by which this occurs, we performed Affymetrix microarray analysis on transcripts from blood monocytes infected with the virulent Type A Schu S4 strain. Results showed that expression of several host response genes were reduced such as those associated with interferon signaling, Toll-like receptor signaling, autophagy and phagocytosis. When compared to microarrays from monocytes infected with the less virulent F. tularensis subsp. novicida, we found qualitative differences and also a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes in the Schu S4 strain. Notably, the PI3K/Akt1 pathway appeared specifically down-regulated following Schu S4 infection and a concomitantly lower cytokine response was observed. This study identifies several new factors potentially important in host cell subversion by the virulent Type A F. tularensis that may serve as novel targets for drug discovery.

  18. Cucurbitacin B Causes Increased Radiation Sensitivity of Human Breast Cancer Cells via G2/M Cell Cycle Arrest

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    Suwit Duangmano

    2012-01-01

    Full Text Available Purpose. To explore the effects of cucurbitacin B on the radiation survival of human breast cancer cells and to elucidate the cellular mechanism of radiosensitization if any. Materials and Methods. Human breast carcinoma cell lines were treated with cucurbitacin B before irradiation with 0–10 Gy of C137s gamma rays. The effect of cucurbitacin B on cell-survival following irradiation was evaluated by colony-forming assay. Cell cycle distributions were investigated using flow cytometry. Real-time PCR and western blots were performed to investigate the expression of cell cycle checkpoints. Results. Cucurbitacin B inhibited breast cancer cell proliferation in a dose-dependent manner. Only MDA-MB-231 and MCF7:5C cells but not SKBR-3 cells were radiosensitized by cucurbitacin B. Flow cytometric analysis for DNA content indicated that cucurbitacin B resulted in G2/M arrest in MDA-MB-231 and MCF7:5C but not SKBR-3 cells. Moreover, Real-time PCR and western blot analysis demonstrated upregulated p21 expression before irradiation, a likely cause of the cell cycle arrest. Conclusion. Taken together, these findings suggest that cucurbitacin B causes radiosensitization of some breast cancer cells, and that cucurbitacin B induced G2/M arrest is an important mechanism. Therefore, combinations of cucurbitacin B with radiotherapy may be appropriate for experimental breast cancer treatment.

  19. Exenatide (a GLP-1 agonist) improves the antioxidative potential of in vitro cultured human monocytes/macrophages.

    Science.gov (United States)

    Bułdak, Łukasz; Łabuzek, Krzysztof; Bułdak, Rafał Jakub; Machnik, Grzegorz; Bołdys, Aleksandra; Okopień, Bogusław

    2015-09-01

    Macrophages are dominant cells in the pathogenesis of atherosclerosis. They are also a major source of reactive oxygen species (ROS). Oxidative stress, which is particularly high in subjects with diabetes, is responsible for accelerated atherosclerosis. Novel antidiabetic drugs (e.g., glucagon-like peptide-1 (GLP-1) agonists) were shown to reduce ROS level. Therefore, we conceived a study to evaluate the influence of exenatide, a GLP-1 agonist, on redox status in human monocytes/macrophages cultured in vitro, which may explain the beneficial effects of incretin-based antidiabetic treatment. Human macrophages obtained from 10 healthy volunteers were in vitro subjected to the treatment with GLP-1 agonist (exenatide) in the presence of lipopolysaccharide (LPS), antagonist of GLP-1 receptors (exendin 9-39), or protein kinase A inhibitor (H89). Afterwards, reactive oxygen species, malondialdehyde level, NADPH oxidase, and antioxidative enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase] expression was evaluated. Finally, we estimated the activity of the abovementioned enzymes in the presence of H89. According to our findings, exenatide reduced ROS and malondialdyhyde (MDA) level by decreasing the expression of ROS-generating NADPH oxidase and by increasing the expression and activities of SOD and GSH-Px. We also showed that this effect was significantly inhibited by exendin 9-39 (a GLP-1 antagonist) and blocked by H89. Exenatide improved the antioxidative potential and reduced oxidative stress in cultured human monocytes/macrophages, and this finding may be responsible for the pleiotropic effects of incretin-based therapies. This effect relied on the stimulation of GLP-1 receptor.

  20. Outer membrane vesicles from Brucella abortus promote bacterial internalization by human monocytes and modulate their innate immune response.

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    Cora N Pollak

    Full Text Available Outer membrane vesicles (OMVs released by some gram-negative bacteria have been shown to exert immunomodulatory effects that favor the establishment of the infection. The aim of the present study was to assess the interaction of OMVs from Brucella abortus with human epithelial cells (HeLa and monocytes (THP-1, and the potential immunomodulatory effects they may exert. Using confocal microscopy and flow cytometry, FITC-labeled OMVs were shown to be internalized by both cell types. Internalization was shown to be partially mediated by clathrin-mediated endocytosis. Pretreatment of THP-1 cells with Brucella OMVs inhibited some cytokine responses (TNF-α and IL-8 to E. coli LPS, Pam3Cys or flagellin (TLR4, TLR2 and TLR5 agonists, respectively. Similarly, pretreatment with Brucella OMVs inhibited the cytokine response of THP-1 cells to B. abortus infection. Treatment of THP-1 cells with OMVs during IFN-γ stimulation reduced significantly the inducing effect of this cytokine on MHC-II expression. OMVs induced a dose-dependent increase of ICAM-1 expression on THP-1 cells and an increased adhesion of these cells to human endothelial cells. The addition of OMVs to THP-1 cultures before the incubation with live B. abortus resulted in increased numbers of adhered and internalized bacteria as compared to cells not treated with OMVs. Overall, these results suggest that OMVs from B. abortus exert cellular effects that promote the internalization of these bacteria by human monocytes, but also downregulate the innate immune response of these cells to Brucella infection. These effects may favor the persistence of Brucella within host cells.

  1. Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells

    Energy Technology Data Exchange (ETDEWEB)

    Su, Miaoxian [Biology Programme (Formally Biology Dept.), School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Chung, Hau Yin, E-mail: anthonychung@cuhk.edu.hk [Biology Programme (Formally Biology Dept.), School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Li, Yaolan [Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou (China); Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drug Research, Guangzhou (China)

    2011-07-29

    Highlights: {yields} Deoxyelephantopin (ESD) inhibited cell proliferation in the human nasopharyngeal cancer CNE cells. {yields} ESD induced cell cycle arrest in S and G2/M phases via modulation of cell cycle regulatory proteins. {yields} ESD triggered apoptosis by dysfunction of mitochondria and induction of both intrinsic and extrinsic apoptotic signaling pathways. {yields} ESD also triggered Akt, ERK, and JNK signaling pathways. -- Abstract: Deoxyelephantopin (ESD), a naturally occurring sesquiterpene lactone present in the Chinese medicinal herb, Elephantopus scaber L. exerted anticancer effects on various cultured cancer cells. However, the cellular mechanisms by which it controls the development of the cancer cells are unavailable, particularly the human nasopharyngeal cancer CNE cells. In this study, we found that ESD inhibited the CNE cell proliferation. Cell cycle arrest in S and G2/M phases was also found. Western blotting analysis showed that modulation of cell cycle regulatory proteins was responsible for the ESD-induced cell cycle arrest. Besides, ESD also triggered apoptosis in CNE cells. Dysfunction in mitochondria was found to be associated with the ESD-induced apoptosis as evidenced by the loss of mitochondrial membrane potential ({Delta}{Psi}m), the translocation of cytochrome c, and the regulation of Bcl-2 family proteins. Despite the Western blotting analysis showed that both intrinsic and extrinsic apoptotic pathways (cleavage of caspases-3, -7, -8, -9, and -10) were triggered in the ESD-induced apoptosis, additional analysis also showed that the induction of apoptosis could be achieved by the caspase-independent manner. Besides, Akt, ERK and JNK pathways were found to involve in ESD-induced cell death. Overall, our findings provided the first evidence that ESD induced cell cycle arrest, and apoptosis in CNE cells. ESD could be a potential chemotherapeutic agent in the treatment of nasopharyngeal cancer (NPC).

  2. TNF and PGE2 in human monocyte-derived macrophages infected with Chlamydia trachomatis

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    E. Manor

    1993-01-01

    Full Text Available In this study levels of prostaglandin E2 (PGE2, tumour necrosis factor (TNF and interleukin-1 (IL-1 alpha in medium from monocyte derived macrophages (MdM infected with Chlamydia trachomatis (L2/434/Bu or K biovars. TNF and PGE2 were found in both cases while IL-1 alpha was not detected. Both TNF and PGE2 levels were higher in the medium of the MdM infected with K biovars. TNF reached maximum levels 24 h postinfection, and then declined, while PGE2 levels increased continuously during the infection time up to 96 h post-infection. Addition of dexamethasone inhibited production of TNF and PGE2. Inhibition of PGE2 production by indomethacin resulted in increased production of TNF, while addition of PGE2 caused partial inhibition of TNF production from infected MdM.

  3. Vitamin A induces inhibitory histone methylation modifications and down-regulates trained immunity in human monocytes

    DEFF Research Database (Denmark)

    Arts, Rob J W; Blok, Bastiaan A; van Crevel, Reinout;

    2015-01-01

    inhibited cytokine responses upon restimulation of monocytes, and this effect was exerted through increased expression of SUV39H2, a histone methyltransferase that induces the inhibitory mark H3K9me3. H3K9me3 at promoter sites of several cytokines was up-regulated by ATRA, and inhibition of SUV39H2 restored...... cytokine production. In addition to H3K9me3, the stimulatory histone mark H3K4me3 was down-regulated by ATRA at several promoter locations of cytokine genes. Therefore, we can conclude that ATRA inhibits cytokine production in models of direct stimulation or BCG-induced trained immunity...

  4. Human Invariant NKT Cells Induce IL-1β Secretion by Peripheral Blood Monocytes via a P2X7-Independent Pathway.

    Science.gov (United States)

    Felley, Laura E; Sharma, Akshat; Theisen, Erin; Romero-Masters, James C; Sauer, John-Demian; Gumperz, Jenny E

    2016-09-15

    The cytokine IL-1β plays a central role in inflammatory responses that are initiated by microbial challenges, as well as in those that are due to endogenous processes (often called sterile inflammation). IL-1β secretion that occurs independently of microbial stimulation is typically associated with the presence of endogenous alarmins, such as extracellular ATP (an indicator of cytopathic damage). In this study, we show that IL-2-activated human invariant NKT (iNKT) cells stimulate the secretion of IL-1β protein by human peripheral blood monocytes in a manner that requires neither the presence of microbial compounds nor signaling through the extracellular ATP receptor P2X7 Monocyte IL-1β production was specifically induced by iNKT cells, because similarly activated polyclonal autologous T cells did not have this effect. Secretion of IL-1β protein occurred rapidly (within 3-4 h) and required cell contact between the iNKT cells and monocytes. Similar to IL-1β production induced by TLR stimulation, the iNKT-induced pathway appeared to entail a two-step process involving NF-κB signaling and IL1B gene transcription, as well as assembly of the NLRP3 inflammasome and activation of caspase-1. However, in contrast to the classical inflammasome-mediated pathway of IL-1β production, activation of monocytes via P2X7 was dispensable for iNKT-induced IL-1β secretion, and potassium efflux was not required. Moreover, the iNKT-induced effect involved caspase-8 activity, yet it induced little monocyte death. These results suggest that IL-2-activated human iNKT cells induce monocytes to produce IL-1β through a distinctive pathway that does not require the presence of microbial danger signals or alarmins associated with cytopathic damage.

  5. Antithrombin III, but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting.

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    Kellner, Patrick; Nestler, Frank; Leimert, Anja; Bucher, Michael; Czeslick, Elke; Sablotzki, Armin; Raspè, Christoph

    2014-12-01

    In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting. Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27±7years) were pre-incubated with clinically relevant concentrations of AT-III (n=11) and C1-INH (n=12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n=6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes. This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs. Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1

  6. Glucocorticoid mediated regulation of inflammation in human monocytes is associated with depressive mood and obesity.

    Science.gov (United States)

    Cheng, Tiefu; Dimitrov, Stoyan; Pruitt, Christopher; Hong, Suzi

    2016-04-01

    Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is observed in various conditions, including depression and obesity, which are also often related. Glucocorticoid (GC) resistance and desensitization of peripheral GC receptors (GRs) are often the case in HPA dysregulation seen in depression, and GC plays a critical role in regulation of inflammation. Given the growing evidence that inflammation is a central feature of some depression cases and obesity, we aimed to investigate the immune-regulatory role of GC-GR in relation to depressive mood and obesity in 35 healthy men and women. Depressive mood and level of obesity were assessed, using Beck Depression Inventory (BDI-Ia) and body mass index (BMI), respectively. We measured plasma cortisol levels via enzyme-linked immunosorbent assay and lipopolysaccharide-stimulated intracellular tumor necrosis factor (TNF) production by monocytes, using flow cytometry. Cortisol sensitivity was determined by the difference in monocytic TNF production between the conditions of 1 and 0 μM cortisol incubation ("cortisol-mediated inflammation regulation, CoMIR"). GR vs. mineralocorticoid receptor (MR) antagonism for CoMIR was examined by using mifepristone and spironolactone. A series of multiple regression analyses were performed to investigate independent contribution of depressive mood vs. obesity after controlling for age, gender, systolic blood pressure (SBP), and plasma cortisol in predicting CoMIR. CoMIR was explained by somatic subcomponents of depressive mood (BDI-S: β=-0.499, p=0.001), or BMI (β=-0.466, pobesity and somatic depressive symptoms were associated with smaller efficacy of the blockers, which warrants further investigation. Our findings, although in a preclinical sample, signify the shared pathophysiology of immune dysregulation in depression and obesity and warrant further mechanistic investigation.

  7. Pathogenic bacteria and TNF do not induce production of macrophage migration inhibitory factor (MIF) by human monocytes.

    Science.gov (United States)

    Temple, Suzanna E L; Cheong, Karey Y; Price, Patricia; Waterer, Grant W

    2009-06-01

    Elevated serum macrophage migration inhibitory factor (MIF) is associated with severe sepsis, but it is not clear whether bacteria stimulate synthesis of MIF by blood leukocytes directly or via induction of TNF. Here we assess production of MIF mRNA and protein by blood leukocytes from healthy human subjects (n=28) following exposure to bacteria commonly associated with sepsis (Escherichia coli and Streptococcus pneumoniae). Bacteria did not increase levels of MIF mRNA or secreted protein. CD14(+) monocytes were the main cell type producing MIF before and after stimulation. Exposure of leukocytes to TNF did not induce MIF. Hence elevated levels of serum MIF observed in sepsis may not reflect MIF produced by blood leukocytes stimulated directly by bacteria or TNF.

  8. Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

    Science.gov (United States)

    Asanagi, Miki; Yamada, Shigeru; Hirata, Naoya; Itagaki, Hiroshi; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

    2016-04-01

    Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

  9. Human papillomavirus 16E6 and NFX1-123 potentiate notch signaling and differentiation without activating cellular arrest

    Energy Technology Data Exchange (ETDEWEB)

    Vliet-Gregg, Portia A.; Hamilton, Jennifer R. [Center for Global Infectious Disease Research, Seattle Children' s Research Institute, 1900 Ninth Ave., Seattle, WA 98101 (United States); Katzenellenbogen, Rachel A., E-mail: rkatzen@uw.edu [Center for Global Infectious Disease Research, Seattle Children' s Research Institute, 1900 Ninth Ave., Seattle, WA 98101 (United States); Department of Pediatrics, Division of Adolescent Medicine, University of Washington, Seattle WA (United States)

    2015-04-15

    High-risk human papillomavirus (HR HPV) oncoproteins bind host cell proteins to dysregulate and uncouple apoptosis, senescence, differentiation, and growth. These pathways are important for both the viral life cycle and cancer development. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and they collaboratively increase the growth and differentiation master regulator, Notch1. In 16E6 expressing keratinocytes (16E6 HFKs), the Notch canonical pathway genes Hes1 and Hes5 were increased with overexpression of NFX1-123, and their expression was directly linked to the activation or blockade of the Notch1 receptor. Keratinocyte differentiation genes Keratin 1 and Keratin 10 were also increased, but in contrast their upregulation was only indirectly associated with Notch1 receptor stimulation and was fully unlinked to growth arrest, increased p21{sup Waf1/CIP1}, or decreased proliferative factor Ki67. This leads to a model of 16E6, NFX1-123, and Notch1 differently regulating canonical and differentiation pathways and entirely uncoupling cellular arrest from increased differentiation. - Highlights: • 16E6 and NFX1-123 increased the Notch canonical pathway through Notch1. • 16E6 and NFX1-123 increased the differentiation pathway indirectly through Notch1. • 16E6 and NFX1-123 increased differentiation gene expression without growth arrest. • Increased NFX1-123 with 16E6 may create an ideal cellular phenotype for HPV.

  10. Epstein-Barr Virus Interferes with the Amplification of IFNα Secretion by Activating Suppressor of Cytokine Signaling 3 in Primary Human Monocytes

    Science.gov (United States)

    Michaud, François; Coulombe, François; Gaudreault, Eric; Paquet-Bouchard, Carine; Rola-Pleszczynski, Marek; Gosselin, Jean

    2010-01-01

    Background Epstein-Barr virus is recognized to cause lymphoproliferative disorders and is also associated with cancer. Evidence suggests that monocytes are likely to be involved in EBV pathogenesis, especially due to a number of cellular functions altered in EBV-infected monocytes, a process that may affect efficient host defense. Because type I interferons (IFNs) are crucial mediators of host defense against viruses, we investigated the effect of EBV infection on the IFNα pathway in primary human monocytes. Methodology/Principal Findings Infection of monocytes with EBV induced IFNα secretion but inhibited the positive feedback loop for the amplification of IFNα. We showed that EBV infection induced the expression of suppressor of cytokine signaling 3 (SOCS3) and, to a lesser extent, SOCS1, two proteins known to interfere with the amplification of IFNα secretion mediated by the JAK/STAT signal transduction pathway. EBV infection correlated with a blockage in the activation of JAK/STAT pathway members and affected the level of phosphorylated IFN regulatory factor 7 (IRF7). Depletion of SOCS3, but not SOCS1, by small interfering RNA (siRNA) abrogated the inhibitory effect of EBV on JAK/STAT pathway activation and significantly restored IFNα secretion. Finally, transfection of monocytes with the viral protein Zta caused the upregulation of SOCS3, an event that could not be recapitulated with mutated Zta. Conclusions/Significance We propose that EBV protein Zta activates SOCS3 protein as an immune escape mechanism that both suppresses optimal IFNα secretion by human monocytes and favors a state of type I IFN irresponsiveness in these cells. This immunomodulatory effect is important to better understand the aspects of the immune response to EBV. PMID:20689596

  11. Epstein-Barr virus interferes with the amplification of IFNalpha secretion by activating suppressor of cytokine signaling 3 in primary human monocytes.

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    François Michaud

    Full Text Available BACKGROUND: Epstein-Barr virus is recognized to cause lymphoproliferative disorders and is also associated with cancer. Evidence suggests that monocytes are likely to be involved in EBV pathogenesis, especially due to a number of cellular functions altered in EBV-infected monocytes, a process that may affect efficient host defense. Because type I interferons (IFNs are crucial mediators of host defense against viruses, we investigated the effect of EBV infection on the IFNalpha pathway in primary human monocytes. METHODOLOGY/PRINCIPAL FINDINGS: Infection of monocytes with EBV induced IFNalpha secretion but inhibited the positive feedback loop for the amplification of IFNalpha. We showed that EBV infection induced the expression of suppressor of cytokine signaling 3 (SOCS3 and, to a lesser extent, SOCS1, two proteins known to interfere with the amplification of IFNalpha secretion mediated by the JAK/STAT signal transduction pathway. EBV infection correlated with a blockage in the activation of JAK/STAT pathway members and affected the level of phosphorylated IFN regulatory factor 7 (IRF7. Depletion of SOCS3, but not SOCS1, by small interfering RNA (siRNA abrogated the inhibitory effect of EBV on JAK/STAT pathway activation and significantly restored IFNalpha secretion. Finally, transfection of monocytes with the viral protein Zta caused the upregulation of SOCS3, an event that could not be recapitulated with mutated Zta. CONCLUSIONS/SIGNIFICANCE: We propose that EBV protein Zta activates SOCS3 protein as an immune escape mechanism that both suppresses optimal IFNalpha secretion by human monocytes and favors a state of type I IFN irresponsiveness in these cells. This immunomodulatory effect is important to better understand the aspects of the immune response to EBV.

  12. Analysis of the human monocyte-derived macrophage transcriptome and response to lipopolysaccharide provides new insights into genetic aetiology of inflammatory bowel disease.

    Science.gov (United States)

    Baillie, J Kenneth; Arner, Erik; Daub, Carsten; De Hoon, Michiel; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Faulkner, Geoffrey J; Wells, Christine A; Rehli, Michael; Pavli, Paul; Summers, Kim M; Hume, David A

    2017-03-01

    The FANTOM5 consortium utilised cap analysis of gene expression (CAGE) to provide an unprecedented insight into transcriptional regulation in human cells and tissues. In the current study, we have used CAGE-based transcriptional profiling on an extended dense time course of the response of human monocyte-derived macrophages grown in macrophage colony-stimulating factor (CSF1) to bacterial lipopolysaccharide (LPS). We propose that this system provides a model for the differentiation and adaptation of monocytes entering the intestinal lamina propria. The response to LPS is shown to be a cascade of successive waves of transient gene expression extending over at least 48 hours, with hundreds of positive and negative regulatory loops. Promoter analysis using motif activity response analysis (MARA) identified some of the transcription factors likely to be responsible for the temporal profile of transcriptional activation. Each LPS-inducible locus was associated with multiple inducible enhancers, and in each case, transient eRNA transcription at multiple sites detected by CAGE preceded the appearance of promoter-associated transcripts. LPS-inducible long non-coding RNAs were commonly associated with clusters of inducible enhancers. We used these data to re-examine the hundreds of loci associated with susceptibility to inflammatory bowel disease (IBD) in genome-wide association studies. Loci associated with IBD were strongly and specifically (relative to rheumatoid arthritis and unrelated traits) enriched for promoters that were regulated in monocyte differentiation or activation. Amongst previously-identified IBD susceptibility loci, the vast majority contained at least one promoter that was regulated in CSF1-dependent monocyte-macrophage transitions and/or in response to LPS. On this basis, we concluded that IBD loci are strongly-enriched for monocyte-specific genes, and identified at least 134 additional candidate genes associated with IBD susceptibility from reanalysis

  13. Roles of phosphatidylinositol 3-kinase and NF-kappaB in human cytomegalovirus-mediated monocyte diapedesis and adhesion: strategy for viral persistence.

    Science.gov (United States)

    Smith, M Shane; Bivins-Smith, Elizabeth R; Tilley, A Michael; Bentz, Gretchen L; Chan, Gary; Minard, Jessica; Yurochko, Andrew D

    2007-07-01

    Infected peripheral blood monocytes are proposed to play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to tissues, a critical step in the establishment of HCMV persistence and the development of HCMV-associated diseases. We recently provided evidence for a unique strategy involved in viral dissemination: HCMV infection of primary human monocytes promotes their transendothelial migration and differentiation into proinflammatory macrophages permissive for the replication of the original input virus. To decipher the mechanism of hematogenous spread, we focused on the viral dysregulation of early cellular processes involved in transendothelial migration. Here, we present evidence that both phosphatidylinositol 3-kinase [PI(3)K] and NF-kappaB activities were crucial for the HCMV induction of monocyte motility and firm adhesion to endothelial cells. We found that the beta(1) integrins, the beta(2) integrins, intracellular adhesion molecule 1 (ICAM-1), and ICAM-3 were upregulated following HCMV infection and that they played a key role in the firm adhesion of infected monocytes to the endothelium. The viral regulation of adhesion molecule expression is complex, with PI(3)K and NF-kappaB affecting the expression of each adhesion molecule at different stages of the expression cascade. Our data demonstrate key roles for PI(3)K and NF-kappaB signaling in the HCMV-induced cellular changes in monocytes and identify the biological rationale for the activation of these pathways in infected monocytes, which together suggest a mechanism for how HCMV promotes viral spread to and persistence within host organs.

  14. Levamisole enhances immune response by affecting the activation and maturation of human monocyte-derived dendritic cells

    Science.gov (United States)

    Chen, L-Y; Lin, Y-L; Chiang, B-L

    2008-01-01

    Levamisole is a synthetic phenylimidazolthiazole that was first introduced in 1966 as an anti-helmintic agent. Current studies have been focused upon its effect on immune response and on cancer treatment. We examined the molecular mechanisms of levamisole in the activation and maturation of human monocyte-derived dendritic cells (DC) and human T cells. Treatment of DC with levamisole increased the presentation of CD80, CD86, CD83 and human leucocyte antigen D-related (HLA-DR) molecules on the cell membrane, as well as the production of interleukin (IL)-12 p40 and IL-10. Levamisole-treated human DC also enhanced T cell activation towards type 1 T helper immune response by inducing interferon-γ secretion. Neutralization with antibodies against Toll-like receptor (TLR)-2 inhibited levamisole-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-2 in signalling DC upon incubation with levamisole. The inhibition of nuclear factor-κB, extracellular signal-regulated kinases 1/2 or c-Jun N-terminal kinases pathways also prevented the effects of levamisole on DC in producing IL-12 p40 or IL-10. Taken together, levamisole could enhance immune response towards T helper 1 development through the activation of dendritic cells or T cell aspects. PMID:18005262

  15. Current Status of Monocyte Differentiation-Inducing (MDI Factors Derived from Human Fetal Membrane Chorion Cells Undergoing Apoptosis after Infl uenza Virus Infection

    Directory of Open Access Journals (Sweden)

    Noboru Uchide

    2007-01-01

    Full Text Available Influenza virus infection induces apoptosis and the expression of a set of pro-inflammatory cytokine genes, such as interleukin (IL-6, tumor necrosis factor (TNF-α, interferon (IFN-β and IFN-γ, in cultured human fetal membrane chorion cells. Monocyte differentiation-inducing (MDI activity in culture supernatants is simultaneously increased by the virus infection. The MDI activity is predominantly influenced by IL-6 molecule in culture supernatants, and partly by TNF-α and IFN-β, but not IFN-γ, molecules. The MDI factors are able to induce the mRNA expression of macrophage class A scavenger receptor (SR-A, which is one of adhesion and apoptotic cell-recognizing molecules, and gp91phox, which is a catalytic subunit of reduced nicotinamide adenine dinucleotide phosphate (NADPH oxidase enzyme complex, on monocytic cells. As a result, monocytes are initiated to differentiate into well-matured macrophages capable of adhering and producing superoxide through NADPH oxidase. The matured macrophages, obtained from human monocytic leukemia THP-1 cells by the treatment with MDI factors, phagocytose apoptotic chorion cell debris resulting from the virus infection. Subsequent to phagocytosis, an abrupt increase of superoxide production by macrophages may occur. In this article, we summarize recent knowledge about the MDI factors derived from human fetal membrane chorion cells undergoing apoptosis after influenza virus infection, and discuss their possible pathological roles during pregnancy.

  16. The CD24/P-selectin binding pathway initiates lung arrest of human A125 adenocarcinoma cells.

    Science.gov (United States)

    Friederichs, J; Zeller, Y; Hafezi-Moghadam, A; Gröne, H J; Ley, K; Altevogt, P

    2000-12-01

    Carbohydrates on tumor cells have been shown to play an important role in tumor metastasis. We demonstrated before that CD24, a Mr 35,000-60,000 mucine-type glycosylphosphatidylinositol-linked cell surface molecule, can function as ligand for P-selectin and that the sialylLex carbohydrate is essential for CD24-mediated rolling of tumor cells on P-selectin. To investigate the role of both antigens more closely, we transfected human A125 adenocarcinoma cells with CD24 and/or fucosyltransferase VII (Fuc TVII) cDNAs. Stable transfectants expressed CD24 and/or sialylLex. Biochemical analysis confirmed that in A125-CD24/FucTVII double transfectants, CD24 was modified with sialylLex. Only double transfectants showed rolling on P-selectin in vivo. When injected into mice, double transfectants arrested in the lungs, and this step was P-selectin dependent because it was strongly enhanced in lipopolysaccharide (LPS) pretreated wild-type mice but not in P-selectin knockout mice. CD24 modified by sialylLex was required on the tumor cells because the LPS-induced lung arrest was abolished by removal of CD24 from the cell surface by phosphatidylinositol-specific phospholipase C. A125-FucTVII single transfectants expressing sialylLex but not CD24 did not show P-selectin-mediated lung arrest. The sialylLex epitope is abundantly expressed on human carcinomas, and significant correlations between sialylLex expression and clinical prognosis exist. Our data suggest an important role for sialylLex-modified CD24 in the lung colonization of human tumors.

  17. 2-methoxyestradiol induces mitotic arrest, apoptosis, and synergistic cytotoxicity with arsenic trioxide in human urothelial carcinoma cells.

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    Kuan-Lin Kuo

    Full Text Available 2-Methoxyestradiol (2-ME, an endogenous derivative of 17β-estradiol, has been reported to elicit antiproliferative responses in various tumors. In this study, we investigated the effects of 2-ME on cell viability, proliferation, cell cycle, and apoptosis in human urothelial carcinoma (UC cell lines. We used two high-grade human bladder UC cell lines (NTUB1 and T24. After treatment with 2-ME, the cell viability and apoptosis were measured by MTT assay and flow cytometry (fluorescence-activated cell sorting, with annexin V-FITC staining and propidium iodide (PI labeling. DNA fragmentation was analyzed by agarose gel electrophoresis. Flow cytometry with PI labeling was used for the cell cycle analyses. The protein levels of caspase activations, poly (ADP-ribose polymerase (PARP cleavage, phospho-histone H2A.X, phospho-Bad, and cell cycle regulatory molecules were measured by Western blot. The effects of the drug combinations were analyzed using the computer software, CalcuSyn. We demonstrated that 2-ME effectively induces dose-dependent cytotoxicity and apoptosis in human UC cells after 24 h exposure. DNA fragmentation, PARP cleavage, and caspase-3, 7, 8, 9 activations can be observed with 2-ME-induced apoptosis. The decreased phospho-Bad (Ser136 and Ser155 and mitotic arrest of the cell cycle in the process of apoptosis after 2-ME treatment was remarkable. In response to mitotic arrest, the mitotic forms of cdc25C, phospho-cdc2, cyclin B1, and phospho-histone H3 (Ser10 were activated. In combination with arsenic trioxide (As2O3, 2-ME elicited synergistic cytotoxicity (combination index <1 in UC cells. We concluded that 2-ME significantly induces apoptosis through decreased phospho-Bad and arrests bladder UC cells at the mitotic phase. The synergistic antitumor effect with As2O3 provides a novel implication in clinical treatment of UC.

  18. Powerful identification of cis-regulatory SNPs in human primary monocytes using allele-specific gene expression.

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    Jonas Carlsson Almlöf

    Full Text Available A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs by genome-wide allele-specific gene expression (ASE analysis with that of traditional expression quantitative trait locus (eQTL mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers.

  19. Effect of eicosapentaenoic acid on the expression of ABCG1 gene in the human monocyte THP-1 cells.

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    Mostafa Moradi Sarabi

    2014-03-01

    Full Text Available Cardiovascular disease (CVD is the leading cause of death and disability in developed countries. Atherosclerosis is the major cause of CVD, accounting for about half of the attributed deaths. Cholesterol homeostasis is one of the most important factors in atherosclerosis. ATP-Binding cassette transporters cholesterol. Omega (ω 3 fatty acids are important ligands for regulation of ABC transporters such as ABCG1. Concern has been raised that the low absolute intakes of EPA and high ratios of ω-6 polyunsaturated fatty acids (ω-6 PUFA to EPA may predispose some individuals to CVD. Eicosapentaenoic acid (EPA is the most abundant ω3 fatty acid in the diet. The objective of this study was to evaluate the effect of different concentrations of EPA on the expression of ABCG1 gene in the human monocyte THP-1 cells. In this study, THP-1 cells were cultured in RPMI 1640 medium, THP-1 monocytes were then differentiated to macrophages with PMA (phorbol myristic acid and stimulated with 50, 75 and 100 μM of EPA for 24 h at 37°C. We examined the effects of EPA treatment on the expression of ABCG1 gene using Quantitative Real time RT-PCR (qRT-PCR. Our results, indicate that ABCG1 mRNA expression was significantly reduced by 50, 75 and 100 μM EPA fatty acid treatments as compared to the control cells (р = 0.009, р < 0.001 and р = 0.002, respectively. These results suggest that polyunsaturated fatty acids (PUFAs such as EPA have an effect on the cholesterol homeostasis in macrophages, and they can change the expression of ABCG1 gene. It seems that EPA has different effects on gene expression and lipid metabolism.

  20. Effect of eicosapentaenoic acid on the expression of ABCG1 gene in the human monocyte THP-1 cells.

    Science.gov (United States)

    Moradi Sarabi, Mostafa; Doosti, Mahmood; Einollahi, Nahid; Hesami, Soroush Shahryar; Dashti, Nasrin

    2014-01-01

    Cardiovascular disease (CVD) is the leading cause of death and disability in developed countries. Atherosclerosis is the major cause of CVD, accounting for about half of the attributed deaths. Cholesterol homeostasis is one of the most important factors in atherosclerosis. ATP-Binding cassette transporters cholesterol. Omega (ω) 3 fatty acids are important ligands for regulation of ABC transporters such as ABCG1. Concern has been raised that the low absolute intakes of EPA and high ratios of ω-6 polyunsaturated fatty acids (ω-6 PUFA) to EPA may predispose some individuals to CVD. Eicosapentaenoic acid (EPA) is the most abundant ω3 fatty acid in the diet. The objective of this study was to evaluate the effect of different concentrations of EPA on the expression of ABCG1 gene in the human monocyte THP-1 cells. In this study, THP-1 cells were cultured in RPMI 1640 medium, THP-1 monocytes were then differentiated to macrophages with PMA (phorbol myristic acid) and stimulated with 50, 75 and 100 μM of EPA for 24 h at 37°C. We examined the effects of EPA treatment on the expression of ABCG1 gene using Quantitative Real time RT-PCR (qRT-PCR). Our results, indicate that ABCG1 mRNA expression was significantly reduced by 50, 75 and 100 μM EPA fatty acid treatments as compared to the control cells (р = 0.009, р < 0.001 and р = 0.002, respectively). These results suggest that polyunsaturated fatty acids (PUFAs) such as EPA have an effect on the cholesterol homeostasis in macrophages, and they can change the expression of ABCG1 gene. It seems that EPA has different effects on gene expression and lipid metabolism.

  1. Alcohol and cannabinoids differentially affect HIV infection and function of human monocyte-derived dendritic cells (MDDC

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    Marisela eAgudelo

    2015-12-01

    Full Text Available During human immunodeficiency virus (HIV infection, alcohol has been known to induce inflammation while cannabinoids have been shown to have an anti-inflammatory role. For instance cannabinoids have been shown to reduce susceptibility to HIV-1 infection and attenuate HIV replication in macrophages. Recently, we demonstrated that alcohol induces cannabinoid receptors and regulates cytokine production by monocyte-derived dendritic cells (MDDC. However, the ability of alcohol and cannabinoids to alter MDDC function during HIV infection has not been clearly elucidated yet. In order to study the potential impact of alcohol and cannabinoids on differentiated MDDC infected with HIV, monocytes were cultured for 7 days with GM-CSF and IL-4, differentiated MDDC were infected with HIV-1Ba-L and treated with EtOH (0.1 and 0.2%, THC (5 and 10 uM, or JWH-015 (5 and 10 uM for 4-7 days. HIV infection of MDDC was confirmed by p24 and Long Terminal Repeats (LTR estimation. MDDC endocytosis assay and cytokine array profiles were measured to investigate the effects of HIV and substances of abuse on MDDC function. Our results show the HIV+EtOH treated MDDC had the highest levels of p24 production and expression when compared with the HIV positive controls and the cannabinoid treated cells. Although both cannabinoids, THC and JWH-015 had lower levels of p24 production and expression, the HIV+JWH-015 treated MDDC had the lowest levels of p24 when compared to the HIV+THC treated cells. In addition, MDDC endocytic function and cytokine production were also differentially altered after alcohol and cannabinoid treatments. Our results show a differential effect of alcohol and cannabinoids, which may provide insights into the divergent inflammatory role of alcohol and cannabinoids to modulate MDDC function in the context of HIV infection.

  2. Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma

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    Sander Bekeschus

    2016-01-01

    Full Text Available In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α, differentiation (retinoic acid signaling and interferon inducible factors, and cell growth (Yin Yang 1. Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1 and of the neutrophil attractant chemokine interleukin-8 (IL-8. Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.

  3. Monocyte procoagulant activity and plasminogen activator. Role in human renal allograft rejection

    Energy Technology Data Exchange (ETDEWEB)

    Cole, E.H.; Cardella, C.J.; Schulman, J.; Levy, G.A.

    1985-10-01

    Currently the mechanism of renal allograft rejection is not well understood. This study was designed to determine whether induction of monocyte procoagulant activity (MCPA) is important in the pathogenesis of renal allograft rejection. The MPCA assay was performed utilizing a one stage clotting assay both in normal and in factor-VII-deficient plasma. There was no increase in spontaneous MPCA in 20 patients with endstage renal failure and in 10 patients following abdominal or orthopedic operation, as compared with 20 normal controls. MPCA was assessed daily in 18 patients who had received renal allografts. Rejection episodes (RE) were predicted on the basis of persistent elevation in MPCA as compared with pretransplant levels. Rejection was diagnosed clinically and treated on the basis of standard criteria. Treated RE were compared with those predicted by elevated MPCA, and 3 patients were assessed as having no RE by MPCA and by standard criteria. In 8 RE, MPCA correlated temporally with RE (same day) when compared with standard criteria. In 12 RE, MPCA was predictive of rejection preceding standard criteria by at least 24 hr. There were 7 false-positive predictions on the basis of MPCA; however, there was only 1 false negative. MPCA was shown to be a prothrombinase by its dependence only on prothrombin and fibrinogen for full activity. MPCA may be important in the pathogenesis of allograft rejection, and additionally it may be a useful adjunct in the clinical management of this disease.

  4. In vitro Catecholamine Exposure Produces Variable Effects on the B7 Costimulatory Pathway in Human Monocytic Cells

    Science.gov (United States)

    Salicru, A. N.; Crucian, B.; Sams, Clarence; Actor, J. K.; Marshall, G. D., Jr.

    2006-01-01

    Catecholamines have been associated with immunomodulation of the adaptive immune system towards a Th2 response in vitro. We therefore examined the role of in vitro epinephrine (EPI) and norepinephrine (NE) exposure on the B7 costimulatory expression of antigen presenting cells (APC) from human monocytic cell lines and human peripheral blood mononuclear cells (PBMC). THP1 monocytic cells and CD14+ cells from normal human PBMC were stimulated with lipopolysaccharide (LPS) and incubated with physiologic stress levels (10(exp -6) - 10(exp -8)M) of EPI or NE for 24 hours. Cells were subsequently stained with CD80 FITC, CD86 PE, and CD14 PC5 antibodies and analyzed by flow cytometry for changes in fluorescence and mean fluorescence intensity (MFI). Exposure of THP1 to EPI in vitro at concentrations of 10(exp -6), 10(exp -7) and 10(exp -8)M significantly decreased mean CD80 from 42 plus or minus 0.7% to 11 plus or minus 0.44%, 19.1 plus or minus 2.0%, and 30.7 plus or minus 2.1% expression, respectively (p less than 0.01). In addition, CD86 expression increased with EPI at 10(exp -6), 10(exp -7) and 10(exp -8) M from 9.2 plus or minus 0.52% to 41 plus or minus 3.8%, 26.4 plus or minus 1.9%, and 15.74 plus or minus 1.8% expression, respectively (p less than 0.01). Similar results for mean CD80 and CD86 percent expression were observed for CD14+ cells from PBMC with a sample size of N = 6 and for NE when substituted for EPI. The data show that in vitro exposure to catecholamines significantly decreases %CD86 expression and significantly increases %CD86 expression in THP1 cells and human CD14+ APC. Previous studies have suggested an association between increased CD86 expression and TH2 activity. Thus, these data suggest that immunomodulation by catecholamines results in part by the variable effects of the B7 costimulatory pathway in APC.

  5. HIV-1 infection of in vitro cultured human monocytes: early events and influence of anti HIV-1 antibodies

    DEFF Research Database (Denmark)

    Arendrup, M; Olofsson, S; Nielsen, Jens Ole;

    1994-01-01

    To characterize the role of the humoral immune response on HIV-1 infection of monocytes and macrophages (M phi s) we examined the susceptibility of in vitro cultured monocyte/M phi s to various HIV-1 isolates and the influence of heterologous and particularly autologous anti HIV-1 sera on this in...

  6. Antiproliferative effect of rapamycin on human T-cell leukemia cell line Jurkat by cell cycle arrest and telomerase inhibition

    Institute of Scientific and Technical Information of China (English)

    Yan-min ZHAO; Qian ZHOU; Yun XU; Xiao-yu LAI; He HUANG

    2008-01-01

    Aim:To examine the ability of rapamycin to suppress growth and regulate telomerase activity in the human T-cell leukemia cell line Jurkat. Methods:Cell proliferation was assessed after exposure to rapamycin by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were determined by flow cytometry. The proteins important for cell cycle progres-sion and Akt/mammalian target of rapamycin signaling cascade were assessed by Western blotting. Telomerase activity was quantified by telomeric repeat amplication protocol assay. The human telomerase reverse transcriptase (hTERT) mRNA levels were determined by semi-quantitative RT-PCR. Results:Rapamycin inhibited the proliferation of Jurkat, induced G1 phase arrest, unregulated the pro-tein level of p21 as well as p27, and downregulated cyclinD3, phospho-p70s6k, and phospho-s6, but had no effect on apoptosis. Treatment with rapamycin reduced telomerase activity, and reduced hTERT mRNA and protein expression. Conclusion:Rapamycin displayed a potent antileukemic effect in the human T-cell leukemia cell line by inhibition of cell proliferation through G1 cell cycle arrest and also through the suppression of telomerase activity, suggesting that rapamycin may have potential clinical implications in the treatment of some leukemias.

  7. Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Valeria de Turris

    Full Text Available Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

  8. Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

    Science.gov (United States)

    de Turris, Valeria; Teloni, Raffaela; Chiani, Paola; Bromuro, Carla; Mariotti, Sabrina; Pardini, Manuela; Nisini, Roberto; Torosantucci, Antonella; Gagliardi, Maria Cristina

    2015-01-01

    Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

  9. Inhibition of HIV-1 replication in human monocyte-derived macrophages by parasite Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Guadalupe Andreani

    Full Text Available BACKGROUND: Cells of monocyte/macrophage lineage are one of the major targets of HIV-1 infection and serve as reservoirs for viral persistence in vivo. These cells are also the target of the protozoa Trypanosoma cruzi, the causative agent of Chagas disease, being one of the most important endemic protozoonoses in Latin America. It has been demonstrated in vitro that co-infection with other pathogens can modulate HIV replication. However, no studies at cellular level have suggested an interaction between T. cruzi and HIV-1 to date. METHODOLOGY/PRINCIPAL FINDINGS: By using a fully replicative wild-type virus, our study showed that T. cruzi inhibits HIV-1 antigen production by nearly 100% (p99% being stronger than HIV-T. cruzi (approximately 90% for BaL and approximately 85% for VSV-G infection. In MDM with established HIV-1 infection, T. cruzi significantly inhibited luciferate activity (p<0.01. By quantifying R-U5 and U5-gag transcripts by real time PCR, our study showed the expression of both transcripts significantly diminished in the presence of trypomastigotes (p<0.05. Thus, T. cruzi inhibits viral post-integration steps, early post-entry steps and entry into MDM. Trypomastigotes also caused a approximately 60-70% decrease of surface CCR5 expression on MDM. Multiplication of T. cruzi inside the MDM does not seem to be required for inhibiting HIV-1 replication since soluble factors secreted by trypomastigotes have shown similar effects. Moreover, the major parasite antigen cruzipain, which is secreted by the trypomastigote form, was able to inhibit viral production in MDM over 90% (p<0.01. CONCLUSIONS/SIGNIFICANCE: Our study showed that T. cruzi inhibits HIV-1 replication at several replication stages in macrophages, a major cell target for both pathogens.

  10. Phagocytosis of haemozoin (malarial pigment enhances metalloproteinase-9 activity in human adherent monocytes: Role of IL-1beta and 15-HETE

    Directory of Open Access Journals (Sweden)

    Giribaldi Giuliana

    2008-08-01

    Full Text Available Abstract Background It has been shown previously that human monocytes fed with haemozoin (HZ or trophozoite-parasitized RBCs displayed increased matrix metalloproteinase-9 (MMP-9 enzyme activity and protein/mRNA expression and increased TNF production, and showed higher matrix invasion ability. The present study utilized the same experimental model to analyse the effect of phagocytosis of: HZ, delipidized HZ, beta-haematin (lipid-free synthetic HZ and trophozoites on production of IL-1beta and MMP-9 activity and expression. The second aim was to find out which component of HZ was responsible for the effects. Methods Native HZ freshly isolated from Plasmodium falciparum (Palo Alto strain, Mycoplasma-free, delipidized HZ, beta-haematin (lipid-free synthetic HZ, trophozoites and control meals such as opsonized non-parasitized RBCs and inert latex particles, were fed to human monocytes. The production of IL-1beta by differently fed monocytes, in presence or absence of specific MMP-9 inhibitor or anti-hIL-1beta antibodies, was quantified in supernatants by ELISA. Expression of IL-1beta was analysed by quantitative real-time RT-PCR. MMP-9 activity and protein expression were quantified by gelatin zymography and Western blotting. Results Monocytes fed with HZ or trophozoite-parasitized RBCs generated increased amounts of IL-1beta and enhanced enzyme activity (in cell supernatants and protein/mRNA expression (in cell lysates of monocyte MMP-9. The latter appears to be causally related to enhanced IL-1beta production, as enhancement of both expression and enzyme activity were abrogated by anti-hIL-1beta Abs. Upregulation of IL-1beta and MMP-9 were absent in monocytes fed with beta-haematin or delipidized HZ, indicating a role for HZ-attached or HZ-generated lipid components. 15-HETE (15(S,R-hydroxy-6,8,11,13-eicosatetraenoic acid a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis was identified as one mediator

  11. Daily Arrests

    Data.gov (United States)

    Montgomery County of Maryland — This dataset provides the public with arrest information from the Montgomery County Central Processing Unit (CPU) systems. The data presented is derived from every...

  12. Hispolon from Phellinus linteus induces G0/G1 cell cycle arrest and apoptosis in NB4 human leukaemia cells.

    Science.gov (United States)

    Chen, Yi-Chuan; Chang, Heng-Yuan; Deng, Jeng-Shyan; Chen, Jian-Jung; Huang, Shyh-Shyun; Lin, I-Hsin; Kuo, Wan-Lin; Chao, Wei; Huang, Guan-Jhong

    2013-01-01

    Hispolon (a phenolic compound isolated from Phellinus linteus) has been shown to possess strong antioxidant, anti-inflammatory, anticancer, and antidiabetic properties. In this study, we investigated the antiproliferative effect of hispolon on human hepatocellular carcinoma NB4 cells using the MTT assay, DNA fragmentation, DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) staining, and flow cytometric analysis. Hispolon inhibited the cellular growth of NB4 cells in a dose-dependent manner through the induction of cell cycle arrest at G0/G1 phase measured using flow cytometric analysis and apoptotic cell death, as demonstrated by DNA laddering. Exposure of NB4 cells to hispolon-induced apoptosis-related protein expressions, such as the cleavage form of caspase 3, caspase 8, caspase 9, poly (ADP ribose) polymerase, and the proapoptotic Bax protein. Western blot analysis showed that the protein levels of extrinsic apoptotic proteins (Fas and FasL), intrinsic related proteins (cytochrome c), and the ratio of Bax/Bcl-2 were increased in NB4 cells after hispolon treatment. Hispolon-induced G0/G1-phase arrest was associated with a marked decrease in the protein expression of p53, cyclins D1, and cyclins E, and cyclin-dependent kinases (CDKs) 2, and 4, with concomitant induction of p21waf1/Cip1 and p27Kip1. We conclude that hispolon induces both of extrinsic and intrinsic apoptotic pathways in NB4 human leukemia cells in vitro.

  13. Jatamanvaltrate P induces cell cycle arrest, apoptosis and autophagy in human breast cancer cells in vitro and in vivo.

    Science.gov (United States)

    Yang, Bo; Zhu, Rui; Tian, Shasha; Wang, Yiqi; Lou, Siyue; Zhao, Huajun

    2017-03-10

    Jatamanvaltrate P is a novel iridoid ester isolated from Valeriana jatamansi Jones, a traditional medicine used to treat nervous disorders. In this study, we found that Jatamanvaltrate P possessed notable antitumor properties and therefore evaluated its anticancer effects against human breast cancer cells in vitro and in vivo. Jatamanvaltrate P inhibited the growth and proliferation of MCF-7 and triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-453 and MDA-MB-468) in a concentration-dependent manner, while displayed relatively low cytotoxicity to human breast epithelial cells (MCF-10A). Treatment with Jatamanvaltrate P induced G2/M-phase arrest in TNBC and G0/G1-phase arrest in MCF-7 cells. Further study of the molecular mechanisms of this cytotoxic compound demonstrated that Jatamanvaltrate P enhanced cleavage of PARP and caspases, while decreased the expression levels of cell cycle-related Cyclin B1, Cyclin D1 and Cdc-2. It also activated autophagy, as indicated by the triggered autophagosome formation and increased LC3-II levels. Autophagy inhibition by 3-MA co-treatment undermined Jatamanvaltrate P-induced cell death. Finally, Jatamanvaltrate P exhibited a potential antitumor effect in MDA-MB-231 xenografts without apparent toxicity. These results suggest that Jatamanvaltrate P is a potential therapeutic agent for breast cancer, providing a basis for development of the compound as a novel chemotherapeutic agent.

  14. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Bin; Cai, Yingyue; Li, Yongshu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Jingjing [College of Life Science, Hubei Normal University, Huangshi 435002, Hubei (China); Liu, Kaiyu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Yi, E-mail: johnli2668@hotmail.com [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Bioengineering Department, Wuhan Bioengineering Institute, Wuhan 430415, Hubei (China); Yang, Yongbo, E-mail: yongboyang@mail.ccnu.edu.cn [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China)

    2013-05-25

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization.

  15. Immunomodulatory effects of adult Haemonchus contortus excretory/secretory products on human monocyte-derived dendritic cells.

    Science.gov (United States)

    Rehman, Z U; Knight, J S; Koolaard, J; Simpson, H V; Pernthaner, A

    2015-12-01

    The levels of expression of surface molecules and release of cytokines and chemokines of human monocyte-derived dendritic cells were determined after their exposure to adult H. contortus excretory/secretory (ES) products or a combination of ES products and bacterial lipopolysaccharide (LPS). Worm products provoked a weak response and only partial maturation of the dendritic cells, consistent with the hyporesponsiveness and more tolerogenic immune environment present in parasitized animals and humans. Co-stimulation with LPS demonstrated that H. contortus secretions, like those of other helminths, contain immunomodulators capable of reducing some aspects of the strong T(H)1/T(H)2 response evoked by bacterial LPS. There were significant reductions in the release of some cytokine/chemokines by LPS-stimulated mdDCs and a trend (although not significant at P < 0.05) for reduced expression levels of CD40, CD80 and HLA-DR. A prominent feature was the variability in responses of dendritic cells from the four donors, even on different days in repeat experiments, suggesting that generalized conclusions may be difficult to make, except in genetically related animals. Such observations may therefore be applicable only to restricted populations. In addition, previous exposure to parasites in a target population for immunomodulatory therapy may be an important factor in assessing the likelihood of adverse reactions or failures in the treatment to worm therapy.

  16. Correlation between Endotoxin Tolerance in Human Monocyte Leukemia Cell Line THP-1 with Glucocorticoid Receptor-α

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Human monocyte leukemia cell line THP-1 was stimulated with lipopolysaccharide (LPS) to simulate the sepsis model and the expression of human glucocorticoid receptor-α (GR-α) mRNA in montocytes with endotoxin tolerance was investigated. THP-1 cells were cultured in serum-free medium, randomly divided into groups A, B, C, D and E, and stimulated with 0, 10, 10,100, 0 ng/mL LPS for 24 h followed with 100, 100, 10, 100, 0 ng/mL LPS for another 24 h respectively. The expression of GR-α mRNA was detected by semi-quantitative reverse transcriptional polymerase chain reaction. Tumor necrosis factor-α (TNF-α) was determined by enzyme linked immunosorbent assay (ELISA). The results showed that the A values of GR-α/β-actin in groups A,B, C, D and E was 0. 607±0. 006, 0. 368±0. 005, 0. 484±0. 008, 0. 509±0. 004 and 0. 564± 0. 014 respectively with the difference being significant among the groups (P<0. 05). The GR-α mRNA expression was negatively correlated with the TNF-α expression (P<0.01). It was concluded that the down-regulation of the expression of GR-α mRNA in endotoxin tolerance THP-1 cells might play an important role in the development of endotoxin tolerance in THP-1 cells.

  17. Low CCR7-mediated migration of human monocyte derived dendritic cells in response to human respiratory syncytial virus and human metapneumovirus.

    Directory of Open Access Journals (Sweden)

    Cyril Le Nouën

    2011-06-01

    Full Text Available Human respiratory syncytial virus (HRSV and, to a lesser extent, human metapneumovirus (HMPV and human parainfluenza virus type 3 (HPIV3, can re-infect symptomatically throughout life without significant antigenic change, suggestive of incomplete or short-lived immunity. In contrast, re-infection by influenza A virus (IAV largely depends on antigenic change, suggestive of more complete immunity. Antigen presentation by dendritic cells (DC is critical in initiating the adaptive immune response. Antigen uptake by DC induces maturational changes that include decreased expression of the chemokine receptors CCR1, CCR2, and CCR5 that maintain DC residence in peripheral tissues, and increased expression of CCR7 that mediates the migration of antigen-bearing DC to lymphatic tissue. We stimulated human monocyte-derived DC (MDDC with virus and found that, in contrast to HPIV3 and IAV, HMPV and HRSV did not efficiently decrease CCR1, 2, and 5 expression, and did not efficiently increase CCR7 expression. Consistent with the differences in CCR7 mRNA and protein expression, MDDC stimulated with HRSV or HMPV migrated less efficiently to the CCR7 ligand CCL19 than did IAV-stimulated MDDC. Using GFP-expressing recombinant virus, we showed that the subpopulation of MDDC that was robustly infected with HRSV was particularly inefficient in chemokine receptor modulation. HMPV- or HRSV-stimulated MDDC responded to secondary stimulation with bacterial lipopolysaccharide or with a cocktail of proinflammatory cytokines by increasing CCR7 and decreasing CCR1, 2 and 5 expression, and by more efficient migration to CCL19, suggesting that HMPV and HRSV suboptimally stimulate rather than irreversibly inhibit MDDC migration. This also suggests that the low concentration of proinflammatory cytokines released from HRSV- and HMPV-stimulated MDDC is partly responsible for the low CCR7-mediated migration. We propose that inefficient migration of HRSV- and HMPV-stimulated DC to

  18. Chemoresistance of human monocyte-derived dendritic cells is regulated by IL-17A.

    Directory of Open Access Journals (Sweden)

    Selma Olsson Åkefeldt

    Full Text Available Dendritic cells initiate adaptive immune responses, leading either to control cancer by effector T cells or to exacerbate cancer by regulatory T cells that inhibit IFN-γ-mediated Th1-type response. Dendritic cells can also induce Th17-type immunity, mediated by IL-17A. However, the controversial role of this cytokine in cancer requires further investigations. We generated dendritic cells from peripheral blood monocytes to investigate lifespan, phenotype and chemoresistance of dendritic cells, treated with IL-17A with or without IFN-γ. Studying the expression of Bcl-2 family members, we demonstrated that dendritic cells constitutively express one pro-survival Bcl-2 member: MCL1. Immature dendritic cells were CD40(lowHLADR(low CD1a(+ MCL1(+, did not express CD14, CD68 or BCL2A1, and displayed a short 2-day lifespan. IL-17A-treated DC exhibited a semi-mature (CD40(high HLADR(low pre-M2 (CCL22(+ CD206(+ CD163(+ IL1RN(+ IL-10(- CXCL10(- IL-12(- mixed (CD1a(+ CD14+ CD68(+ macrophage-dendritic cell phenotype. They efficiently exerted mannose receptor-mediated endocytosis and did not produce superoxide anions, in the absence of TLR engagement. Interestingly, IL-17A promoted a long-term survival of dendritic cells, beyond 12 days, that correlated to BCL2A1 induction, a pro-survival Bcl-2 family member. BCL2A1 transcription was activated by NF-κB, downstream of IL-17A transduction. Thus, immature dendritic cells only express MCL1, whereas IL-17A-treated dendritic cells concomitantly expressed two pro-survival Bcl-2 family members: MCL1 and BCL2A1. These latter developed chemoresistance to 11 of the 17 chemotherapy agents tested. However, high doses of either vinblastine or cytarabine decreased MCL1 expression and induced dendritic cell death. When IL-17A is produced in vivo, administration of anti-IL-17A biotherapy may impair dendritic cell survival by targeting BCL2A1 expression. Consequently, depending on the effector or regulatory role of dendritic

  19. Ganoderma lucidum polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function

    Directory of Open Access Journals (Sweden)

    Lau Yu

    2008-07-01

    Full Text Available Abstract Background Previous studies demonstrated Ganoderma lucidum polysaccharides (GL-PS, a form of bioactive β-glucan can stimulate the maturation of monocyte-derived dendritic cells (DC. The question of how leukemic cells especially in monocytic lineage respond to GL-PS stimuli remains unclear. Results In this study, we used in vitro culture model with leukemic monocytic cell-lines THP-1 and U937 as monocytic effectors cells for proliferation responses and DCs induction. We treated the THP-1 and U937 cells with purified GL-PS (100 μg/mL or GL-PS with GM-CSF/IL-4. GL-PS alone induced proliferative response on both THP-1 and U937 cells but only THP-1 transformed into typical DC morphology when stimulated with GL-PS plus GM-CSF/IL-4. The transformed THP-1 DCs had significant increase expression of HLA-DR, CD40, CD80 and CD86 though not as high as the extent of normal monocyte-derived DCs. They had similar antigen-uptake ability as the normal monocyte-derived DCs positive control. However, their potency in inducing allogeneic T cell proliferation was also less than that of normal monocyte-derived DCs. Conclusion Our findings suggested that GL-PS could induce selected monocytic leukemic cell differentiation into DCs with immuno-stimulatory function. The possible clinical impact of using this commonly used medicinal mushroom in patients with monocytic leukemia (AML-M4 and M5 deserved further investigation.

  20. GM-CSF and IL-3 Modulate Human Monocyte TNF-α Production and Renewal in In Vitro Models of Trained Immunity.

    Science.gov (United States)

    Borriello, Francesco; Iannone, Raffaella; Di Somma, Sarah; Loffredo, Stefania; Scamardella, Eloise; Galdiero, Maria Rosaria; Varricchi, Gilda; Granata, Francescopaolo; Portella, Giuseppe; Marone, Gianni

    2016-01-01

    GM-CSF and IL-3 are hematopoietic cytokines that also modulate the effector functions of several immune cell subsets. In particular, GM-CSF and IL-3 exert a significant control on monocyte and macrophage effector functions, as assessed in experimental models of inflammatory and autoimmune diseases and also in human studies. Here, we sought to investigate the mechanisms and the extent to which GM-CSF and IL-3 modulate the pro-inflammatory, LPS-mediated, activation of human CD14(+) monocytes taking into account the new concept of trained immunity (i.e., the priming stimulus modulates the response to subsequent stimuli mainly by inducing chromatin remodeling and increased transcription at relevant genetic loci). We demonstrate that GM-CSF and IL-3 priming enhances TNF-α production upon subsequent LPS stimulation (short-term model of trained immunity) in a p38- and SIRT2-dependent manner without increasing TNF primary transcript levels (a more direct measure of transcription), thus supporting a posttranscriptional regulation of TNF-α in primed monocytes. GM-CSF and IL-3 priming followed by 6 days of resting also results in increased TNF-α production upon LPS stimulation (long-term model of trained immunity). In this case, however, GM-CSF and IL-3 priming induces a c-Myc-dependent monocyte renewal and increase in cell number that is in turn responsible for heightened TNF-α production. Overall, our results provide insights to understand the biology of monocytes in health and disease conditions in which the hematopoietic cytokines GM-CSF and IL-3 play a role and also extend our knowledge of the cellular and molecular mechanisms of trained immunity.

  1. GM-CSF and IL-3 Modulate Human Monocyte TNF-α Production and Renewal in In Vitro Models of Trained Immunity

    Science.gov (United States)

    Borriello, Francesco; Iannone, Raffaella; Di Somma, Sarah; Loffredo, Stefania; Scamardella, Eloise; Galdiero, Maria Rosaria; Varricchi, Gilda; Granata, Francescopaolo; Portella, Giuseppe; Marone, Gianni

    2017-01-01

    GM-CSF and IL-3 are hematopoietic cytokines that also modulate the effector functions of several immune cell subsets. In particular, GM-CSF and IL-3 exert a significant control on monocyte and macrophage effector functions, as assessed in experimental models of inflammatory and autoimmune diseases and also in human studies. Here, we sought to investigate the mechanisms and the extent to which GM-CSF and IL-3 modulate the pro-inflammatory, LPS-mediated, activation of human CD14+ monocytes taking into account the new concept of trained immunity (i.e., the priming stimulus modulates the response to subsequent stimuli mainly by inducing chromatin remodeling and increased transcription at relevant genetic loci). We demonstrate that GM-CSF and IL-3 priming enhances TNF-α production upon subsequent LPS stimulation (short-term model of trained immunity) in a p38- and SIRT2-dependent manner without increasing TNF primary transcript levels (a more direct measure of transcription), thus supporting a posttranscriptional regulation of TNF-α in primed monocytes. GM-CSF and IL-3 priming followed by 6 days of resting also results in increased TNF-α production upon LPS stimulation (long-term model of trained immunity). In this case, however, GM-CSF and IL-3 priming induces a c-Myc-dependent monocyte renewal and increase in cell number that is in turn responsible for heightened TNF-α production. Overall, our results provide insights to understand the biology of monocytes in health and disease conditions in which the hematopoietic cytokines GM-CSF and IL-3 play a role and also extend our knowledge of the cellular and molecular mechanisms of trained immunity. PMID:28138327

  2. HIV-1 infection of in vitro cultured human monocytes: early events and influence of anti HIV-1 antibodies

    DEFF Research Database (Denmark)

    Arendrup, M; Olofsson, S; Nielsen, Jens Ole;

    1994-01-01

    on this infection. Depending on the period of in vitro cultivation and the virus isolate used different patterns of susceptibility were detected. One week old monocyte/M phi s were highly susceptible to HIV-1 infection, in contrast to monocyte/M phi s cultured 4 weeks. The infection by virus isolated immediately...... to CD4 and that post binding events may be common to the infection of lymphocytes. Anti HIV-1 sera showed neutralizing activity against heterologous and even autologous escape virus. This finding, together with the observation that monocytes and M phi s are infected in vivo, suggests that protection...

  3. Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

    Energy Technology Data Exchange (ETDEWEB)

    Byrne, Ann; McLaren, Rajashree P.; Mason, Paul; Chai, Lilly; Dufault, Michael R.; Huang, Yinyin; Liang, Beirong; Gans, Joseph D.; Zhang, Mindy; Carter, Kara; Gladysheva, Tatiana B.; Teicher, Beverly A.; Biemann, Hans-Peter N.; Booker, Michael; Goldberg, Mark A.; Klinger, Katherine W.; Lillie, James [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Madden, Stephen L., E-mail: steve.madden@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Jiang, Yide, E-mail: yide.jiang@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States)

    2010-01-15

    The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

  4. Subcellular proteomic analysis of host-pathogen interactions using human monocytes exposed to Yersinia pestis and Yersinia pseudotuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, C G; Gonzales, A D; Choi, M W; Chromy, B A; Fitch, J P; McCutchen-Maloney, S L

    2004-05-20

    Yersinia pestis, the etiological agent of plague, is of concern to human health both from an infectious disease and a civilian biodefense perspective. While Y. pestis and Y. pseudotuberculosis share more than 90% DNA homology, they have significantly different clinical manifestations. Plague is often fatal if untreated, yet Y. pseudotuberculosis causes severe intestinal distress and is rarely fatal. A better understanding of host response to these closely related pathogens may help explain the different mechanisms of virulence and pathogenesis that result in such different clinical outcomes. The aim of this study was to characterize host protein expression changes in human monocyte-like U937 cells after exposure to Y. pestis and Y. pseudotuberculosis. In order to gain global proteomic coverage of host response, proteins from cytoplasmic, nuclear and membrane fractions of host cells were studied by 2-dimensional differential gel electrophoresis (2-D DIGE) and relative protein expression differences were quantitated. Differentially expressed proteins, with at least 1.5 fold expression changes and p values of 0.01 or less, were identified by MALDI-MS or LC/MS/MS. With these criteria, differential expression was detected in 16 human proteins after Y. pestis exposure and 13 human proteins after Y. pseudotuberculosis exposure, of which only two of the differentially expressed proteins identified were shared between the two exposures. Proteins identified in this study are reported to be involved in a wide spectrum of cellular functions and host defense mechanisms including apoptosis, cytoskeletal rearrangement, protein synthesis and degradation, DNA replication and transcription, metabolism, protein folding, and cell signaling. Notably, the differential expression patterns observed can distinguish the two pathogen exposures from each other and from unexposed host cells. The functions of the differentially expressed proteins identified provide insight on the different

  5. Human XCR1+ Dendritic Cells Derived In Vitro from CD34+ Progenitors Closely Resemble Blood Dendritic Cells, Including Their Adjuvant Responsiveness, Contrary to Monocyte-Derived Dendritic Cells

    OpenAIRE

    S. Balan; Ollion, V.; Colletti, N.; Chelbi, R.; Montanana-Sanchis, F.; LIU, H.; Vu Manh, T.-P.; Sanchez, C.; Savoret, J.; Perrot, I.; Doffin, A.-C.; Fossum, E.; Bechlian, D.; Chabannon, C.; Bogen, B

    2014-01-01

    Human monocyte-derived dendritic cell (MoDC) have been used in the clinic with moderately encouraging results. Mouse XCR1+ DC excel at cross-presentation, can be targeted in vivo to induce protective immunity, and share characteristics with XCR1+ human DC. Assessment of the immunoactivation potential of XCR1+ human DC is hindered by their paucity in vivo and by their lack of a well-defined in vitro counterpart. We report in this study a protocol generating both XCR1+ and XCR1− human DC in CD3...

  6. Overexpression of cyclin L2 induces apoptosis and cell-cycle arrest in human lung cancer cells

    Institute of Scientific and Technical Information of China (English)

    LI Hong-li; WANG Tong-shan; LI Xiao-yu; LI Nan; HUANG Ding-zhi; CHEN Qi; BA Yi

    2007-01-01

    Background Uncontrolled cell division is one of the hallmarks of tumor growth. Researches have been focused on numerous molecules involved in this process. Cyclins are critical regulatory proteins of cell cycle progression and/or transcription. The present study aimed to investigate the anti-proliferative effect of cyclin L2, and to define its growth regulatory mechanisms using human lung adenocarcinoma cell line A549.Methods Human cyclin L2 was transfected into human lung adenocarcinoma cells (A549 cell), and was expressed in a mammalian expression vector pcDNA3.1. The effects and mechanisms of the cyclin L2 in cell growth, cell cycle analysis and apoptosis were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry or Western blot, respectively.Results Overexpression of cyclin L2 inhibited the growth of A549 cells. Cell cycle analysis in cells transfected with pCCNL2 revealed an increment in proportion in G0/G1 phase ((68.07 ± 4.2)%) in contrast to (60.39 ± 2.82)% of the cells transfected with mock vector. Apoptosis occurred in (7.25 ± 0.98)% cells transfected with pCCNL2, as compared with (1.25 ± 0.21)% of the mock vector control group. Cyclin L2-induced-G0/G1 arrest and apoptosis involved upregulation of caspase-3 and downregulation of Bcl-2 and survivin.Conclusion The results indicate that overexpression of cyclin L2 protein may promote efficient growth inhibition of human lung adenocarcinoma cells by inducing G0/G1 cell cycle arrest and apoptosis.

  7. Age-dependent alterations of monocyte subsets and monocyte-related chemokine pathways in healthy adults

    Directory of Open Access Journals (Sweden)

    Trautwein Christian

    2010-06-01

    Full Text Available Abstract Background Recent experimental approaches have unraveled essential migratory and functional differences of monocyte subpopulations in mice. In order to possibly translate these findings into human physiology and pathophysiology, human monocyte subsets need to be carefully revisited in health and disease. In analogy to murine studies, we hypothesized that human monocyte subsets dynamically change during ageing, potentially influencing their functionality and contributing to immunosenescence. Results Circulating monocyte subsets, surface marker and chemokine receptor expression were analyzed in 181 healthy volunteers (median age 42, range 18-88. Unlike the unaffected total leukocyte or total monocyte counts, non-classical CD14+CD16+ monocytes significantly increased with age, but displayed reduced HLA-DR and CX3CR1 surface expression in the elderly. Classical CD14++CD16- monocyte counts did not vary dependent on age. Serum MCP-1 (CCL2, but not MIP1α (CCL3, MIP1β (CCL4 or fractalkine (CX3CL1 concentrations increased with age. Monocyte-derived macrophages from old or young individuals did not differ with respect to cytokine release in vitro at steady state or upon LPS stimulation. Conclusions Our study demonstrates dynamic changes of circulating monocytes during ageing in humans. The expansion of the non-classical CD14+CD16+ subtype, alterations of surface protein and chemokine receptor expression as well as circulating monocyte-related chemokines possibly contribute to the preserved functionality of the monocyte pool throughout adulthood.

  8. Zinc oxide nanoparticles-induced epigenetic change and G2/M arrest are associated with apoptosis in human epidermal keratinocytes

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    Gao F

    2016-08-01

    Full Text Available Fei Gao, Ningjie Ma, Hong Zhou, Qing Wang, Hao Zhang, Pu Wang, Haoli Hou, Huan Wen, Lijia Li State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of China Abstract: As an engineered nanomaterial, zinc oxide nanoparticles (ZnO NPs are used frequently in biological applications and can make contact with human skin. Here, we systematically investigated the effects of ZnO NPs on non-tumorigenic human epidermal keratinocytes, which were used as a test model for this in vitro study, at the epigenetic and molecular levels. Our results showed that ZnO NPs induced cell cycle arrest at the G2/M checkpoint before the viability of human epidermal keratinocytes was reduced, which was associated with the chromatin changes at the epigenetic level, including increased methylation of histone H3K9 and decreased acetylation of histone H4K5 accompanied by chromatin condensation at 24 hours. The mRNA expression of the methyltransferase genes G9a and GLP was also increased upon treatment with ZnO NPs, and the acetyltransferase genes GCN5, P300, and CBP were downregulated. Reactive oxygen species were found to be more abundant after treatment with ZnO NPs for 6 hours, and DNA damage was observed at 24 hours. Transmission electron microscopy and flow cytometry confirmed that ZnO NPs were absorbed into the cell when they were added to the medium. Apoptotic human epidermal keratinocytes were detected, and the expression of the proapoptotic genes Bax, Noxa, and Puma increased significantly, while the expression of the antiapoptotic gene Bcl-xl decreased 24 hours after exposure to ZnO NPs. These findings suggest that the ZnO NPs induced cell cycle arrest at G2/M, which was associated with epigenetic changes and accompanied by p53-Bax mitochondrial pathway-mediated apoptosis. Keywords: ZnO nanoparticle, cell cycle G2/M arrest, histone modification, p53-Bax mitochondrial apoptosis pathway, reactive oxygen species

  9. Insoluble β-glucan from the cell wall of Candida albicans induces immune responses of human THP-1 monocytes through Dectin-1

    Institute of Scientific and Technical Information of China (English)

    LI Min; LIU Ze-hu; CHEN Qing; ZHOU Wu-qing; YU Mei-wen; L(U) Gui-xia; L(U) Xue-lian; SHEN Yong-nian; LIU Wei-da; WU Shao-xi

    2009-01-01

    Background β-glucan is the major structure component of Candida albicans (C. albicans) cell wall. It has been demonstrated that Dectin-1 as the principal C-type lectin pattern-recognition receptor (PRR) can recognize fungal β-glucan and induce immune responses. In this study, we sought to clarify whether insoluble β-glucan from the cell wall of C. albicans (CalG) could induce immune responses in human THP-1 monocytes (a human acute monocytic leukemia cell line) and to determine the underlying mechanisms.Methods Human THP-1 monocytes were challenged with CalG in vitro. The mRNA expression of Dectin-1, Toll-like receptors (TLR2), proinflammatory cytokine (TNF-α) and chemokine (IL-8) was assayed by real-time reverse transcription polymerase chain reaction (RT-PCR). The secretion of TNF-α and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA). H2O2 release was determined by microplate fluorescent assay. Western blotting was used to analyze IkB-α phosphorylation and degradation.Results Exposure of THP-1 monocytes to CalG led to increased gene expression and secretion of TNF-α and IL-8.CalG induced H2O2 release in a time-dependent manner. CalG hydrolyzed with zymolyase failed to induce gene expression and secretion of TNF-α, IL-8 and H2O2 release. CalG up-regulated the mRNA of Dectin-1, whereas the mRNA level of TLR2 was not altered. THP-1 monocytes challenged with CalG resulted in the activation of NF-KB in a time-dependent manner. Dectin-1 inhibitor laminarin blocked the CalG-induced production of TNF-α and H2O2 in THP-1 monocytes, but no such effect was observed in pretreatment with anti-TLR2 neutralizing antibody and the LPS inhibitor (polymyxin B).Conclusion CalG may play a role in activation of immune responses in human THP-1 cells through Dectin-1, not TLR2.This work was supported by a grant from National Natural ScienceFoundation of China (No. 30671893).

  10. Integrating genome-wide genetic variations and monocyte expression data reveals trans-regulated gene modules in humans.

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    Maxime Rotival

    2011-12-01

    Full Text Available One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns-independent component analysis-to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739, previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1 is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178, which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644 was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the

  11. Interaction of E-cadherin and PTEN regulates morphogenesis and growth arrest in human mammary epithelial cells

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    Fournier, Marcia V.; Fata, Jimmie E.; Martin, Katherine J.; Yaswen, Paul; Bissell, Mina J.

    2009-06-03

    PTEN is a dual function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two non-malignant human mammary epithelial cell lines (HMECs) that form polarized, growth-arrested structures (acini) when cultured in 3-dimensional laminin-rich extracellular matrix gels (3D lrECM). As acini begin to form, PTEN accumulates in both the cytoplasm, and at cell-cell contacts where it colocalizes with E-cadherin/{beta}-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in SKBR3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in 3D lrECM indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus appears to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells.

  12. Selective COX-2 inhibitor, NS-398, suppresses cellular proliferation in human hepatocellular carcinoma cell lines via cell cycle arrest

    Institute of Scientific and Technical Information of China (English)

    Ji Yeon Baek; Wonhee Hur; Jin Sang Wang; Si Hyun Bae; Seung Kew Yoon

    2007-01-01

    AIM: To investigate the growth inhibitory mechanism of NS-398, a selective cyclooxygenase-2 (COX-2) inhibitor,in two hepatocellular carcinoma (HCC) cell lines (HepG2and Huh7).METHODS: HepG2 and Huh7 cells were treated with NS-398. Its effects on cell viability, cell proliferation,cell cycles, and gene expression were respectively evaluated by water-soluble tetrazolium salt (WST-1)assay, 4'-6-diamidino-2-phenylindole (DAPI) staining,flow cytometer analysis, and Western blotting,with dimethyl sulfoxide (DMSO) as positive control.RESULTS: NS-398 showed dose- and time-dependent growth-inhibitory effects on the two cell lines.Proliferating cell nuclear antigen (PCNA) expressions in HepG2 and Huh7 cells, particularly in Huh7 cells were inhibited in a time- and dose-independent manner.NS-398 caused cell cycle arrest in the G1 phase with cell accumulation in the sub-G1 phase in HepG2 and Huh7cell lines. No evidence of apoptosis was observed in two cell lines.CONCLUSION: NS-398 reduces cell proliferation by inducing cell cycle arrest in HepG2 and Huh7 cell lines,and COX-2 inhibitors may have potent chemoprevention effects on human hepatocellular carcinoma.

  13. Honokiol, a chemopreventive agent against skin cancer, induces cell cycle arrest and apoptosis in human epidermoid A431 cells.

    Science.gov (United States)

    Chilampalli, Chandeshwari; Guillermo, Ruth; Kaushik, Radhey S; Young, Alan; Chandrasekher, Gudiseva; Fahmy, Hesham; Dwivedi, Chandradhar

    2011-11-01

    Honokiol is a plant lignan isolated from bark and seed cones of Magnolia officinalis. Recent studies from our laboratory indicated that honokiol pretreatment decreased ultraviolet B-induced skin cancer development in SKH-1 mice. The aim of the present investigation was to study the effects of honokiol on human epidermoid squamous carcinoma A431 cells and to elucidate possible mechanisms involved in preventing skin cancer. A431 cells were pretreated with different concentrations of honokiol for a specific time period and investigated for effects on apoptosis and cell cycle analysis. Treatment with honokiol significantly decreased cell viability and cell proliferation in a concentration- and time-dependent manner. Honokiol pretreatment at 50 μmol/L concentration induced G0/G1 cell cycle arrest significantly (P Cdk4 and Cdk6 proteins and up-regulated the expression of Cdk's inhibitor proteins p21 and p27. Pretreatment of A431 cells with honokiol leads to induction of apoptosis and DNA fragmentation. These findings indicate that honokiol provides its effects in squamous carcinoma cells by inducing cell cycle arrest at G0/G1 phase and apoptosis.

  14. Characterization of HIV-1 Infection and Innate Sensing in Different Types of Primary Human Monocyte-Derived Macrophages

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    Elisabeth A. Diget

    2013-01-01

    Full Text Available Macrophages play an important role in human immunodeficiency virus (HIV pathogenesis and contribute to establishment of a viral reservoir responsible for continuous virus production and virus transmission to T cells. In this study, we investigated the differences between various monocyte-derived macrophages (MDMs generated through different differentiation protocols and evaluated different cellular, immunological, and virological properties. We found that elevated and persistent HIV-1 pWT/BaL replication could be obtained only in MDMs grown in RPMI containing macrophage colony-stimulating factor (M-CSF. Interestingly, this MDM type was also most responsive to toll-like receptor stimulation. By contrast, all MDM types were activated to a comparable extent by intracellular DNA, and the macrophage serum-free medium-(Mac-SFM-differentiated MDMs responded strongly to membrane fusion through expression of CXCL10. Finally, we found that HIV infection of RPMI/M-CSF-differentiated MDMs induced low-grade expression of two interferon-stimulated genes in some donors. In conclusion, our study demonstrates that the differentiation protocol used greatly influences the ability of MDMs to activate innate immune reactions and support HIV-1 replication. Paradoxically, the data show that the MDMs with the strongest innate immune response were also the most permissive for HIV-1 replication.

  15. Retinoic acid regulates CD1d gene expression at the transcriptional level in human and rodent monocytic cells.

    Science.gov (United States)

    Chen, Qiuyan; Ross, A Catharine

    2007-04-01

    CD1d belongs to a group of nonclassical antigen-presenting molecules that present glycolipid antigens and thereby activate natural killer T (NKT) cells, a subset of bifunctional T cells. Little is known so far regarding the expression and physiologic regulation of CD1d. Here we show that all-trans-retinoic acid (RA), the active metabolite of vitamin A, rapidly (1 hr after treatment) increases CD1d mRNA in human and rodent monocytic cells at a physiologic dose (10 nM). The induction is RA specific and RA receptor (RAR) dependent-RA and an RARalpha agonist, Am580, both had a pronounced positive effect, whereas the addition of RARalpha antagonist partially blocked the increase in CD1d mRNA induced by RA and Am580. The induction was also completely blocked by the presence of actinomycin D. A putative RA-response element was identified in the distal 5' flanking region of the CD1d gene, which binds nuclear retinoid receptors and was responsive to RA in both gel mobility shift assay and transient transfection assay in THP-1 cells. These results further confirmed the transcriptional regulation of RA in CD1d gene expression. Moreover, RA significantly increased alpha-galactosylceramide-induced spleen cell proliferation. These studies together provide evidence for a previously unknown mechanism of CD1d gene expression regulation by RA and suggest that RA is a significant modulator of NKT cell activation.

  16. Human monocyte heat shock protein 72 responses to acute hypoxic exercise after 3 days of exercise heat acclimation.

    Science.gov (United States)

    Lee, Ben J; Mackenzie, Richard W A; Cox, Valerie; James, Rob S; Thake, Charles D

    2015-01-01

    The aim of this study was to determine whether short-term heat acclimation (STHA) could confer increased cellular tolerance to acute hypoxic exercise in humans as determined via monocyte HSP72 (mHSP72) expression. Sixteen males were separated into two matched groups. The STHA group completed 3 days of exercise heat acclimation; 60 minutes cycling at 50% V̇O2peak in 40°C 20% relative humidity (RH). The control group (CON) completed 3 days of exercise training in 20°C, 40% RH. Each group completed a hypoxic stress test (HST) one week before and 48 hours following the final day of CON or STHA. Percentage changes in HSP72 concentrations were similar between STHA and CON following HST1 (P = 0.97). STHA induced an increase in basal HSP72 (P = 0.03) with no change observed in CON (P = 0.218). Basal mHSP72 remained elevated before HST2 for the STHA group (P 0.05). Percent change in mHSP72 was lower after HST2 in STHA compared to CON (P = 0.02). The mHSP72 response to hypoxic exercise was attenuated following 3 days of heat acclimation. This is indicative of improved tolerance and ability to cope with the hypoxic insult, potentially mediated in part by increased basal reserves of HSP72.

  17. In vitro detection of contact allergens: development of an optimized protocol using human peripheral blood monocyte-derived dendritic cells.

    Science.gov (United States)

    Reuter, Hendrik; Spieker, Jochem; Gerlach, Silke; Engels, Ursula; Pape, Wolfgang; Kolbe, Ludger; Schmucker, Robert; Wenck, Horst; Diembeck, Walter; Wittern, Klaus-Peter; Reisinger, Kerstin; Schepky, Andreas G

    2011-02-01

    Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive will introduce a testing ban for cosmetic ingredients after 2013. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. To solve the issues associated with the use of primary cells, the culture and exposure conditions (cytokine concentrations, incubation time, readout, pooled vs. single donors and cytotoxicity) were re-assessed and optimized. Here we propose a stable and reproducible protocol based on PBMDCs. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in an integrated testing strategy.

  18. Genomic Profiling of a Human Leukemic Monocytic Cell-Line (THP-1 Exposed to Alpha Particle Radiation

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    Vinita Chauhan

    2012-01-01

    Full Text Available This study examined alpha (α- particle radiation effects on global changes in gene expression in human leukemic monocytic cells (THP-1 for the purposes of mining for candidate biomarkers that could be used for the development of a biological assessment tool. THP-1 cells were exposed to α-particle radiation at a dose range of 0 to 1.5 Gy. Twenty-four hours and three days after exposure gene expression was monitored using microarray technology. A total of 16 genes were dose responsive and classified as early onset due to their expression 24 h after exposure. Forty-eight transcripts were dose responsive and classified as late-onset as they were expressed 72 h after exposure. Among these genes, 6 genes were time and dose responsive and validated further using alternate technology. These transcripts were upregulated and associated with biological processes related to immune function, organelle stability and cell signalling/communication. This panel of genes merits further validation to determine if they are strong candidate biomarkers indicative of α-particle exposure.

  19. Large-scale monocyte enrichment coupled with a closed culture system for the generation of human dendritic cells.

    Science.gov (United States)

    Pullarkat, Vinod; Lau, Roy; Lee, Sun-Min; Bender, James G; Weber, Jeffrey S

    2002-09-15

    Conventional methods for generating monocyte-derived dendritic cells (DC) for clinical trials utilize the property of plastic adherence to select monocytes from leukapheresis samples. This method is labor-intensive and has the potential for contamination at various steps. We evaluated a large-scale monocyte enrichment procedure using a cell selector (Isolex 300i(R)) followed by culture in a sterile bag system (Stericell(R)) for generation of DC. DC generated in tissue culture flasks after monocyte selection by plastic adherence were compared to those generated in Stericell(R) bags after monocyte enrichment by negative selection with the Isolex(R) 300i. DC were matured with lipopolysaccharide and pulsed with a peptide derived from the melanoma antigen gp100. Peptide-pulsed DC cultured by the two techniques were evaluated for phenotype, viability, ability to induce allogeneic and peptide-specific autologous proliferative responses as well as peptide-specific cytotoxic T-cell responses. The mean monocyte yield from leukapheresis collections was 17+/-2.4%, which increased to 52+/-11% after Isolex(R) selection. The DC yield of plated mononuclear cells from flasks or bags was 2.7+/-0.96% and 4.84+/-2.65%, respectively. DC cultured by both methods expressed high levels of CD86, CD80, CD40, CD83, CD44, CD11c and CD58, and was comparable in their ability to induce allogeneic and peptide-specific autologous proliferative responses as well as gp100 peptide-specific cytotoxic T-cell responses. These results indicate that potent monocyte-derived DC can be generated in a closed culture bag system after monocyte enrichment by immunomagnetic negative selection. Due to the closed nature of the enrichment and culture systems, the potential for contamination is minimized. This protocol is well suited for culturing large numbers of DC for clinical immunotherapy trials.

  20. Parthenolide Induces Apoptosis and Cell Cycle Arrest of Human 5637 Bladder Cancer Cells In Vitro

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    Guang Cheng

    2011-08-01

    Full Text Available Parthenolide, the principal component of sesquiterpene lactones present in medical plants such as feverfew (Tanacetum parthenium, has been reported to have anti-tumor activity. In this study, we evaluated the therapeutic potential of parthenolide against bladder cancer and its mechanism of action. Treatment of bladder cancer cells with parthenolide resulted in a significant decrease in cell viability. Parthenolide induced apoptosis through the modulation of Bcl-2 family proteins and poly (ADP-ribose polymerase degradation. Treatment with parthenolide led to G1 phase cell cycle arrest in 5637 cells by modulation of cyclin D1 and phosphorylated cyclin-dependent kinase 2. Parthenolide also inhibited the invasive ability of bladder cancer cells. These findings suggest that parthenolide could be a novel therapeutic agent for treatment of bladder cancer.

  1. Induction of Apoptosis and Cell Cycle Arrest in Human Colorectal Carcinoma by Litchi Seed Extract

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    Chih-Ping Hsu

    2012-01-01

    Full Text Available The Litchi (Litchi chinensis fruit products possess rich amounts of flavanoids and proanthocyanidins. Its pericarp has been shown to inhibit breast and liver cancer cell growth. However, the anticolorectal cancer effect of Litchi seed extract has not yet been reported. In this study, the effects of polyphenol-rich Litchi seed ethanol extract (LCSP on the proliferation, cell cycle, and apoptosis of two colorectal cancer cell lines Colo320DM and SW480 were examined. The results demonstrated that LCSP significantly induced apoptotic cell death in a dose-dependent manner and arrested cell cycle in G2/M in colorectal carcinoma cells. LCSP also suppressed cyclins and elevated the Bax : Bcl-2 ratio and caspase 3 activity. This study provides in vitro evidence that LCSP serves as a potential chemopreventive agent for colorectal cancer.

  2. Omega-3 Polyunsaturated Fatty Acids Trigger Cell Cycle Arrest and Induce Apoptosis in Human Neuroblastoma LA-N-1 Cells

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    Wai Wing So

    2015-08-01

    Full Text Available Omega-3 (n-3 fatty acids are dietary long-chain fatty acids with an array of health benefits. Previous research has demonstrated the growth-inhibitory effect of n-3 fatty acids on different cancer cell lines in vitro, yet their anti-tumor effects and underlying action mechanisms on human neuroblastoma LA-N-1 cells have not yet been reported. In this study, we showed that docosahexaenoic acid (DHA and eicosapentaenoic acid (EPA exhibited time- and concentration-dependent anti-proliferative effect on the human neuroblastoma LA-N-1 cells, but had minimal cytotoxicity on the normal or non-tumorigenic cells, as measured by MTT reduction assay. Mechanistic studies indicated that DHA and EPA triggered G0/G1 cell cycle arrest in LA-N-1 cells, as detected by flow cytometry, which was accompanied by a decrease in the expression of CDK2 and cyclin E proteins. Moreover, DHA and EPA could also induce apoptosis in LA-N-1 cells as revealed by an increase in DNA fragmentation, phosphatidylserine externalization and mitochondrial membrane depolarization. Up-regulation of Bax, activated caspase-3 and caspase-9 proteins, and down-regulation of Bcl-XL protein, might account for the occurrence of apoptotic events. Collectively, our results suggest that the growth-inhibitory effect of DHA and EPA on LA-N-1 cells might be mediated, at least in part, via triggering of cell cycle arrest and apoptosis. Therefore, DHA and EPA are potential anti-cancer agents which might be used for the adjuvant therapy or combination therapy with the conventional anti-cancer drugs for the treatment of some forms of human neuroblastoma with minimal toxicity.

  3. Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages.

    Science.gov (United States)

    Mattana, Antonella; Sanna, Manuela; Cano, Antonella; Delogu, Giuseppe; Erre, Giuseppe; Roberts, Craig W; Henriquez, Fiona L; Fiori, Pier Luigi; Cappuccinelli, Piero

    2016-10-01

    Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response.

  4. Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages

    Science.gov (United States)

    Sanna, Manuela; Cano, Antonella; Delogu, Giuseppe; Erre, Giuseppe; Roberts, Craig W.; Henriquez, Fiona L.; Fiori, Pier Luigi; Cappuccinelli, Piero

    2016-01-01

    Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response. PMID:27481240

  5. Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters In Vivo during Interaction of the Pathogenic Fungus Aspergillus fumigatus with Human Monocytes

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    Mohebbi, Sara; Erfurth, Florian; Hennersdorf, Philipp; Brakhage, Axel A.; Saluz, Hans Peter

    2016-01-01

    Hyperspectral imaging (HSI) is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus Aspergillus fumigatus conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised A. fumigatus conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of A. fumigatus is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell. PMID:27727286

  6. Differential Activation of Human Monocyte-Derived and Plasmacytoid Dendritic Cells by West Nile Virus Generated in Different Host Cells▿

    Science.gov (United States)

    Silva, Maria Carlan; Guerrero-Plata, Antonieta; Gilfoy, Felicia D.; Garofalo, Roberto P.; Mason, Peter W.

    2007-01-01

    Dendritic cells (DCs) play a central role in innate immunity and antiviral responses. In this study, we investigated the production of alpha interferon (IFN-α) and inducible chemokines by human monocyte-derived dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) infected with West Nile virus (WNV), an emergent pathogen whose infection can lead to severe cases of encephalitis in the elderly, children, and immunocompromised individuals. Our experiments demonstrated that WNV grown in mammalian cells (WNVVero) was a potent inducer of IFN-α secretion in pDCs and, to a lesser degree, in mDCs. The ability of WNVVero to induce IFN-α in pDCs did not require viral replication and was prevented by the treatment of cells with bafilomycin A1 and chloroquine, suggesting that it was dependent on endosomal Toll-like receptor recognition. On the other hand, IFN-α production in mDCs required viral replication and was associated with the nuclear translocation of IRF3 and viral antigen expression. Strikingly, pDCs failed to produce IFN-α when stimulated with WNV grown in mosquito cells (WNVC7/10), while mDCs responded similarly to WNVVero or WNVC7/10. Moreover, the IFN-dependent chemokine IP-10 was produced in substantial amounts by pDCs in response to WNVVero but not WNVC7/10, while interleukin-8 was produced in greater amounts by mDCs infected with WNVC7/10 than in those infected with WNVVero. These findings suggest that cell-specific mechanisms of WNV recognition leading to the production of type I IFN and inflammatory chemokines by DCs may contribute to both the innate immune response and disease pathogenesis in human infections. PMID:17913823

  7. How is mRNA expression predictive for protein expression? A correlation study on human circulating monocytes

    Institute of Scientific and Technical Information of China (English)

    Yanfang Guo; Yuan Chen; Hui Jiang; Lijun Tan; Jingyun Xie; Xuezhen Zhu; Songping Liang; Hongwen Deng; Peng Xiao; Shufeng Lei; Feiyan Deng; Gary Guishan Xiao; Yaozhong Liu; Xiangding Chen; Liming Li; Shan Wu

    2008-01-01

    A key assumption in studying mRNA expression is that it is informative in the prediction of protein expression. However,only limited studies have explored the mRNA-protein expression correlation in yeast or human tissues and the results have been relatively inconsistent. We carried out correlation analyses on mRNA-protein expressions in freshly isolated human circulating monocytes from 30 unrelated women. The expressed proteins for 71 genes were quantified and identified by 2-D electrophoresis coupled with mass spectrometry. The corresponding mRNA expressions were quantified by Affymetrix gene chips. Significant correlation (r=0.235, P<0.0001) was observed for the whole dataset including all studied genes and all samples. The correlations varied in different biological categories of gene ontology. For example, the highest correlation was achieved for genes of the extracellular region in terms of cellular component (r=0.643, P<0.0001) and the lowest correlation was obtained for genes of regulation (r=0.099, P=0.213) in terms of biological process. In the genome, half of the samples showed significant positive correlation for the 71 genes and significant correlation was found between the average mRNA and the average protein expression levels in all samples (r=0.296, P<0.01). However, at the study group level, only five studied genes had significant positive correlation across all the samples. Our results showed an overall positive correlation between mRNA and protein expression levels.However, the moderate and varied correlations suggest that mRNA expression might be sometimes useful, but certainly far from perfect, in predicting protein expression levels.

  8. Analysis of the human monocyte-derived macrophage transcriptome and response to lipopolysaccharide provides new insights into genetic aetiology of inflammatory bowel disease

    Science.gov (United States)

    Arner, Erik; De Hoon, Michiel; Carninci, Piero; Hayashizaki, Yoshihide; Pavli, Paul; Summers, Kim M.; Hume, David A.

    2017-01-01

    The FANTOM5 consortium utilised cap analysis of gene expression (CAGE) to provide an unprecedented insight into transcriptional regulation in human cells and tissues. In the current study, we have used CAGE-based transcriptional profiling on an extended dense time course of the response of human monocyte-derived macrophages grown in macrophage colony-stimulating factor (CSF1) to bacterial lipopolysaccharide (LPS). We propose that this system provides a model for the differentiation and adaptation of monocytes entering the intestinal lamina propria. The response to LPS is shown to be a cascade of successive waves of transient gene expression extending over at least 48 hours, with hundreds of positive and negative regulatory loops. Promoter analysis using motif activity response analysis (MARA) identified some of the transcription factors likely to be responsible for the temporal profile of transcriptional activation. Each LPS-inducible locus was associated with multiple inducible enhancers, and in each case, transient eRNA transcription at multiple sites detected by CAGE preceded the appearance of promoter-associated transcripts. LPS-inducible long non-coding RNAs were commonly associated with clusters of inducible enhancers. We used these data to re-examine the hundreds of loci associated with susceptibility to inflammatory bowel disease (IBD) in genome-wide association studies. Loci associated with IBD were strongly and specifically (relative to rheumatoid arthritis and unrelated traits) enriched for promoters that were regulated in monocyte differentiation or activation. Amongst previously-identified IBD susceptibility loci, the vast majority contained at least one promoter that was regulated in CSF1-dependent monocyte-macrophage transitions and/or in response to LPS. On this basis, we concluded that IBD loci are strongly-enriched for monocyte-specific genes, and identified at least 134 additional candidate genes associated with IBD susceptibility from reanalysis

  9. International news coverage of human trafficking arrests and prosecutions: a content analysis

    OpenAIRE

    Denton, Erin

    2009-01-01

    Trafficking in human beings is a growing phenomenon with an expanding body of literature. However, a gap is evident in this body of literature: original research focusing on specific incidents of human trafficking. At this time, the human trafficking literature is permeated with discussions of the sex trade, sexual exploitation, organized crime, global socioeconomic problems and human rights. While these issues are all pertinent to the human trafficking debate, the literature requires maturat...

  10. Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development.

    Science.gov (United States)

    Nogueira, D; Ron-El, R; Friedler, S; Schachter, M; Raziel, A; Cortvrindt, R; Smitz, J

    2006-01-01

    Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6-12 mm) were retrieved 34-36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

  11. Induction of cell cycle arrest, DNA damage, and apoptosis by nimbolide in human renal cell carcinoma cells.

    Science.gov (United States)

    Hsieh, Yi-Hsien; Lee, Chien-Hsing; Chen, Hsiao-Yun; Hsieh, Shu-Ching; Lin, Chia-Liang; Tsai, Jen-Pi

    2015-09-01

    Nimbolide is a tetranortriterpenoid isolated from the leaves and flowers of Azadirachta indica which has been shown to exhibit anticancer, antioxidant, anti-inflammatory, and anti-invasive properties in a variety of cancer cells. However, the anti-tumor effect on human renal cell carcinoma (RCC) cells is unknown. In this study, we found that nimbolide treatment had a cytotoxic effect on 786-O and A-498 RCC cells in a dose-dependent manner. According to flow cytometric analysis, nimbolide treatment resulted in G2/M arrest in 786-O and A-498 cells accompanied with an increase in the phosphorylation status of p53, cdc2, cdc25c, and decreased expressions of cyclin A, cyclin B, cdc2, and cdc25c. Nimbolide also caused DNA damage in a dose-dependent manner as determined by comet assay and measurement of γ-H2AX. In addition, apoptotic cells were observed in an Annexin V-FITC/propidium iodide double-stained assay. The activities of caspase-3, -9, and poly ADP-ribose polymerase (PARP) were increased, and the expression of pro-caspase-8 was decreased in nimbolide-treated 786-O and A-498 cells. Western blot analysis revealed that the levels of intrinsic-related apoptotic proteins Bax and extrinsic-related proteins (DR5, CHOP) were significantly increased in nimbolide-treated 786-O and A-498 cells. In addition, the expressions of Bcl-2 and Mcl-1 were decreased in 786-O and A-498 cells after nimbolide treatment. We conclude that nimbolide can inhibit the growth of human RCC cells by inducing G2/M phase arrest by modulating cell cycle-related proteins and cell apoptosis by regulating intrinsic and extrinsic caspase signaling pathways. Nimbolide may be a promising therapeutic strategy for the treatment of RCC.

  12. Mycobacterium tuberculosis-infected human monocytes down-regulate microglial MMP-2 secretion in CNS tuberculosis via TNFα, NFκB, p38 and caspase 8 dependent pathways

    Directory of Open Access Journals (Sweden)

    Elkington Paul T

    2011-05-01

    Full Text Available Abstract Tuberculosis (TB of the central nervous system (CNS is a deadly disease characterized by extensive tissue destruction, driven by molecules such as Matrix Metalloproteinase-2 (MMP-2 which targets CNS-specific substrates. In a simplified cellular model of CNS TB, we demonstrated that conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb, but not direct infection, unexpectedly down-regulates constitutive microglial MMP-2 gene expression and secretion by 72.8% at 24 hours, sustained up to 96 hours (P M.tb-infected monocyte-dependent networks paradoxically involves the pro-inflammatory mediators TNF-α, p38 MAP kinase and NFκB in addition to a novel caspase 8-dependent pathway.

  13. Comparative assessment of the recognition of domain-specific CD163 monoclonal antibodies in human monocytes explains wide discrepancy in reported levels of cellular surface CD163 expression

    DEFF Research Database (Denmark)

    Maniecki, Maciej Bogdan; Etzerodt, Anders; Moestrup, Søren Kragh;

    2011-01-01

    continue to exhibit great discrepancy in the measured percentage of CD163-expressing blood monocytes in healthy individuals. In this study we sought to clarify this inconsistency in reported levels of CD163 surface expression by a detailed analysis of a panel of CD163 antibodies used in previous studies...... 1 (MAC2-158), domain 4 (R-20), domain 7 (GHI/61), and domain 9 (RM3/1). The CD163 monoclonal antibodies were characterized in binding and endocytosis experiments in human macrophages and CD163-transfected Flp-In CHO cells. Calcium-dependent ligand binding was assessed using surface plasmon resonance......-terminal part of CD163, remote from the membrane surface. Moreover, the proportion of CD163 positive monocytes observed was highly dependent on free calcium. GHI/61 did not exhibit CD163 binding in the presence of calcium as measured by surface plasmon resonance, which was in agreement with the concordant loss...

  14. A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

    Science.gov (United States)

    Kawasaki, Kengo; Muroyama, Koutarou; Yamamoto, Norio; Murosaki, Shinji

    2015-01-01

    The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

  15. Candida albicans Yeast and Germ Tube Forms Interfere Differently with Human Monocyte Differentiation into Dendritic Cells: a Novel Dimorphism-Dependent Mechanism To Escape the Host's Immune Response

    Science.gov (United States)

    Torosantucci, Antonella; Romagnoli, Giulia; Chiani, Paola; Stringaro, Annarita; Crateri, Pasqualina; Mariotti, Sabrina; Teloni, Raffaela; Arancia, Giuseppe; Cassone, Antonio; Nisini, Roberto

    2004-01-01

    The ability of Candida albicans to convert from the yeast (Y) form to mycelial forms through germ tube (GT) formation is considered a key feature of the transition of the organism from commensalism to virulence. We show here that human monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) after phagocytosis of Y forms did not differentiate into dendritic cells (DCs); they retained CD14, did not acquire CD1a, and were unable to express the maturation markers CD83 and CCR7. Moreover, they did not produce IL-12p70 but secreted IL-10. In addition, they spontaneously expressed high levels of tumor necrosis factor alpha (TNF-α), IL-6, and IL-8 mRNA transcripts and were able to induce proliferation of alloreactive memory but not naïve T lymphocytes. Conversely, monocytes that had phagocytosed GT forms differentiated into mature CD83+ and CCR7+ DCs; however, there was no up-regulation of CD40, CD80, and major histocompatibility complex class II, irrespective of lipopolysaccharide (LPS) treatment. In addition, these cells were unable to produce IL-12 even after LPS stimulation, but they were not functionally exhausted, as shown by their capacity to express TNF-α and IL-8 mRNA transcripts. These cells were able to prime naïve T cells but not to induce their functional polarization into effector cells. These data indicate that phagocytosis of Y and GT forms has profound and distinct effects on the differentiation pathway of monocytes. Thus, the differentiation of human monocytes into DCs appears to be tunable and exploitable by C. albicans to elude immune surveillance. PMID:14742527

  16. Differential interleukin-10 (IL-10) and IL-23 production by human blood monocytes and dendritic cells in response to commensal enteric bacteria.

    Science.gov (United States)

    Manuzak, Jennifer; Dillon, Stephanie; Wilson, Cara

    2012-08-01

    Human peripheral blood contains antigen-presenting cells (APC), including dendritic cells (DC) and monocytes, that may encounter microbes that have translocated from the intestine to the periphery in disease states like HIV-1 infection and inflammatory bowel disease. We investigated the response of DC and monocytes in peripheral blood mononuclear cells (PBMC) to a panel of representative commensal enteric bacteria, including Escherichia coli, Enterococcus sp., and Bacteroides fragilis. All three bacteria induced significant upregulation of the maturation and activation markers CD40 and CD83 on myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC). However, only mDC produced cytokines, including interleukin-10 (IL-10), IL-12p40/70, and tumor necrosis factor alpha (TNF-α), in response to bacterial stimulation. Cytokine profiles in whole PBMC differed depending on the stimulating bacterial species: B. fragilis induced production of IL-23, IL-12p70, and IL-10, whereas E. coli and Enterococcus induced an IL-10-predominant response. mDC and monocyte depletion experiments indicated that these cell types differentially produced IL-10 and IL-23 in response to E. coli and B. fragilis. Bacteroides thetaiotaomicron did not induce levels of IL-23 similar to those of B. fragilis, suggesting that B. fragilis may have unique proinflammatory properties among Bacteroides species. The addition of recombinant human IL-10 to PBMC cultures stimulated with commensal bacteria abrogated the IL-23 response, whereas blocking IL-10 significantly enhanced IL-23 production, suggesting that IL-10 controls the levels of IL-23 produced. These results indicate that blood mDC and monocytes respond differentially to innate stimulation with whole commensal bacteria and that IL-10 may play a role in controlling the proinflammatory response to translocated microbes.

  17. Messenger RNA electroporation of human monocytes, followed by rapid in vitro differentiation, leads to highly stimulatory antigen-loaded mature dendritic cells.

    Science.gov (United States)

    Ponsaerts, Peter; Van den Bosch, Glenn; Cools, Nathalie; Van Driessche, Ann; Nijs, Griet; Lenjou, Marc; Lardon, Filip; Van Broeckhoven, Christine; Van Bockstaele, Dirk R; Berneman, Zwi N; Van Tendeloo, Viggo F I

    2002-08-15

    Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83(+) DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.

  18. Screening of Compounds Toxicity against Human Monocytic cell line-THP-1 by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Pick Neora

    2004-01-01

    Full Text Available The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death.

  19. Acute stress reduces wound-induced activation of microbicidal potential of ex vivo isolated human monocyte-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Ulrike Kuebler

    Full Text Available BACKGROUND: Psychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM, and (b that these reductions are modulated by stress hormone release. METHODS: Fourty-one healthy men (mean age 35 ± 13 years were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker before assessing HMDM microbicidal potential. RESULTS: Compared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p's <.05. Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14-44.72. Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001. This effect was blocked by prior incubation with phentolamine. CONCLUSIONS: Our results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing.

  20. In vitro interactions of Candida parapsilosis wild type and lipase deficient mutants with human monocyte derived dendritic cells

    Directory of Open Access Journals (Sweden)

    Vágvölgyi Csaba

    2011-05-01

    Full Text Available Abstract Background Candida parapsilosis typically is a commensal of human skin. However, when host immune defense is compromised or the normal microflora balance is disrupted, C. parapsilosis transforms itself into an opportunistic pathogen. Candida-derived lipase has been identified as potential virulence factor. Even though cellular components of the innate immune response, such as dendritic cells, represent the first line of defense against invading pathogens, little is known about the interaction of these cells with invading C. parapsilosis. Thus, the aim of our study was to assess the function of dendritic cells in fighting C. parapsilosis and to determine the role that C. parapsilosis-derived lipase plays in the interaction with dendritic cells. Results Monocyte-derived immature and mature dendritic cells (iDCs and mDCs, respectively co-cultured with live wild type or lipase deficient C. parapsilosis strains were studied to determine the phagocytic capacity and killing efficiency of host cells. We determined that both iDCs and mDCs efficiently phagocytosed and killed C. parapsilosis, furthermore our results show that the phagocytic and fungicidal activities of both iDCs and mDCs are more potent for lipase deficient compared to wild type yeast cells. In addition, the lipase deficient C. parapsilosis cells induce higher gene expression and protein secretion of proinflammatory cytokines and chemokines in both DC types relative to the effect of co-culture with wild type yeast cells. Conclusions Our results show that DCs are activated by exposure to C. parapsilosis, as shown by increased phagocytosis, killing and proinflammatory protein secretion. Moreover, these data strongly suggest that C. parapsilosis derived lipase has a protective role during yeast:DC interactions, since lipase production in wt yeast cells decreased the phagocytic capacity and killing efficiency of host cells and downregulated the expression of host effector molecules.

  1. Activation of the aryl hydrocarbon receptor affects activation and function of human monocyte-derived dendritic cells.

    Science.gov (United States)

    Wang, C; Ye, Z; Kijlstra, A; Zhou, Y; Yang, P

    2014-08-01

    Aryl hydrocarbon receptor (AhR) is well known for mediating the toxic effects of dioxin-containing pollutants, but has also been shown to be involved in the natural regulation of the immune response. In this study, we investigated the effect of AhR activation by its endogenous ligands 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the differentiation, maturation and function of monocyte-derived DCs in Behçet's disease (BD) patients. In this study, we showed that AhR activation by FICZ and ITE down-regulated the expression of co-stimulatory molecules including human leucocyte antigen D-related (HLA-DR), CD80 and CD86, while it had no effect on the expression of CD83 and CD40 on DCs derived from BD patients and normal controls. Lipopolysaccharide (LPS)-treated dendritic cells (DCs) from active BD patients showed a higher level of interleukin (IL)-1β, IL-6, IL-23 and tumour necrosis factor (TNF)-α production. FICZ or ITE significantly inhibited the production of IL-1β, IL-6, IL-23 and TNF-α, but induced IL-10 production by DCs derived from active BD patients and normal controls. FICZ or ITE-treated DCs significantly inhibited the T helper type 17 (Th17) and Th1 cell response. Activation of AhR either by FICZ or ITE inhibits DC differentiation, maturation and function. Further studies are needed to investigate whether manipulation of the AhR pathway may be used to treat BD or other autoimmune diseases.

  2. Effect of Allitridi on Inducing Mitotic Arrest in Human Gastric Cell Line SGC-7901 and Its Possible Mechanism

    Institute of Scientific and Technical Information of China (English)

    CHEN Tie-jun; HA Min-wen; GONG Yue-hua; XU Qian; YUAN Yuan

    2008-01-01

    Objective:To learn the effect of allitridi on inducing mitotic arrest in human gastric cell line SGC-7901 and its possible mechanisms.Methods:We treated SGC-7901 cells with ailitridi,and observed the proliferation inhibitory rate with MTT colometric assay,changes of cell cycle using flow cytometry and Switzerland-Giemsa's staining,and morphologic changes of the microtubule structure and location changes of cyclin B1 expression using immunofluorescence and confocal laser scanning microscope.Furthermore,the expression of cyclin B1 was analyzed quantitatively using Leica confocal software.Results:SGC-7901 cells were inhibited after exposure tO allitridi and the IC50 was 7.2μg/ml for 24 h,20μg/ml for 72 h.When the cells were treated with allitridi at concentrations of 3,6,and 9μg/ml for 24 h respectively,there was a declining tendency in the percentage of G0/G1 cell but an increasing tendency in G2/M cell in the allitridi treated group compared with that of control (P<0.01).When cells were treated allitridi at concentration of 6 μg/ml for 24 h,its mitotic index was much higher(P<0.01) than that of control,suggesting that allitridi caused arrest of gastric cancer cells in M phase.The cells were treated with allitridi became more shrunken and nepheioid,in which the microtubule networks disappeared,while the control cell exhibited an intact microtubule network.Contrasting with normal existence mainly in the cytoplasm,the cyclin B1 was expressed more significantly and concentrated in the nucleus after exposure tO allitridi.Fluorescence intensity of cyclin B1 protein in cells treated with allitridi was much more higher than that of control(P<0.001).Conclusion:Allitridi can induce arrest of SGC-7901 cells in M phase,probably through enhancing microtubule depolymerization by elevating the expression of cyclin B1.

  3. Involvement of MINK, a Ste20 Family Kinase, in Ras Oncogene-Induced Growth Arrest in Human Ovarian Surface Epithelial Cells

    NARCIS (Netherlands)

    Nicke, B.; Bastien, J.; Khanna, S.J.; Warne, P.H.; Cowling, V.; Cook, S.J.; Peters, G.; Delpuech, O.; Schulze, A.; Berns, K.; Mullenders, J.; Beijersbergen, R.L.; Bernards, R.A.; Ganesan, T.S.; Downward, J.; Hancock, D.C.

    2005-01-01

    The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kino

  4. Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction

    Directory of Open Access Journals (Sweden)

    Ou Chern-Han

    2007-11-01

    Full Text Available Abstract Background Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-α. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells. Results Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach. Conclusion Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

  5. Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-DR on peripheral blood monocytes during mild and severe dengue fever.

    Science.gov (United States)

    Azeredo, Elzinandes L; Neves-Souza, Patrícia C; Alvarenga, Allan R; Reis, Sônia R N I; Torrentes-Carvalho, Amanda; Zagne, Sonia-Maris O; Nogueira, Rita M R; Oliveira-Pinto, Luzia M; Kubelka, Claire F

    2010-06-01

    Dengue fever (DF), a public health problem in tropical countries, may present severe clinical manifestations as result of increased vascular permeability and coagulation disorders. Dengue virus (DENV), detected in peripheral monocytes during acute disease and in in vitro infection, leads to cytokine production, indicating that virus-target cell interactions are relevant to pathogenesis. Here we investigated the in vitro and in vivo activation of human peripheral monocytes after DENV infection. The numbers of CD14(+) monocytes expressing the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) were significantly increased during acute DF. A reduced number of CD14(+) human leucocyte antigen (HLA)-DR(+) monocytes was observed in patients with severe dengue when compared to those with mild dengue and controls; CD14(+) monocytes expressing toll-like receptor (TLR)2 and TLR4 were increased in peripheral blood from dengue patients with mild disease, but in vitro DENV-2 infection up-regulated only TLR2. Increased numbers of CD14(+) CD16(+) activated monocytes were found after in vitro and in vivo DENV-2 infection. The CD14(high) CD16(+) monocyte subset was significantly expanded in mild dengue, but not in severe dengue. Increased plasma levels of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin (IL)-18 in dengue patients were inversely associated with CD14(high) CD16(+), indicating that these cells might be involved in controlling exacerbated inflammatory responses, probably by IL-10 production. We showed here, for the first time, phenotypic changes on peripheral monocytes that were characteristic of cell activation. A sequential monocyte-activation model is proposed in which DENV infection triggers TLR2/4 expression and inflammatory cytokine production, leading eventually to haemorrhagic manifestations, thrombocytopenia, coagulation disorders, plasmatic leakage and shock development, but may also produce factors that act in

  6. Sargaquinoic Acid Inhibits TNF-α-Induced NF-κB Signaling, Thereby Contributing to Decreased Monocyte Adhesion to Human Umbilical Vein Endothelial Cells (HUVECs).

    Science.gov (United States)

    Gwon, Wi-Gyeong; Lee, Bonggi; Joung, Eun-Ji; Choi, Min-Woo; Yoon, Nayoung; Shin, Taisun; Oh, Chul-Woong; Kim, Hyeung-Rak

    2015-10-21

    Sargaquinoic acid (SQA) has been known for its antioxidant and anti-inflammatory properties. This study investigated the effects of SQA isolated from Sargassum serratifolium on the inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). SQA decreased the expression of cell adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as chemotactic cytokines such as interleukin-8 and monocyte chemoattractant protein-1 in TNF-α-treated HUVECs. As a result, SQA prevented monocyte adhesion to TNF-α-induced adhesion. SQA also inhibited TNF-α-induced nuclear factor kappa B (NF-κB) translocation into the nucleus by preventing proteolytic degradation of inhibitor κB-α. Overall, SQA protects against TNF-α-induced vascular inflammation through inhibition of the NF-κB pathway in HUVECs. These data suggest that SQA may be used as a therapeutic agent for vascular inflammatory diseases such as atherosclerosis.

  7. Human monocyte-derived dendritic cells expressing both chemotactic cytokines IL-8, MCP-1, RANTES and their receptors,and their selective migration to these chemokines

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To characterize the mRNA expression of CXC chemokine IL-8, CC chemokine monocyte chemothractant protein-1 (MCP-1) and regulated on activation,normal T cell expressed and secreted (RANTES), and a newly defined DC chemokine DC- CK1 as well as the expression of IL-8 receptor, MCP-1 receptor and RANTES receptor in human monocyte derived dendritic cells (MoDCs).The migratory responsiveness of MoDC to IL-8, MCP-1 and RANTES was alsso studied. Methods In vitro generated MoDCs were obtained by differentiating monocytes in the presence of GM-CSF and IL-4 for 5 days. The time course of RNA expression was analyzed by RT-PCR and migratoly ability was assessed by a micromultiwell chemotaxis chamber assay. Results IL-8, MCP-1, RANTES and their corres ponding receptors were consistently expressed in MoDCs. DC-CK-1 expression was detectable efter 48 hours of differentiation. MoDC selectively migrated in response to MCP-1 and RANTES but not to IL-8 though transcripts of IL-8 receptor were present. Conclusion Because the capacity of dendritic cells to initiate immune responses depends on their specialized migratory and tissue homing properties, the expression of chemokines and their receptors along with the migratory responsiveness to chemokines of MoDC in our study suggests a potential role of chemokines in the interaction between dendritic cells and T cells and the induction of immune responses.

  8. A unique anti-CD115 monoclonal antibody which inhibits osteolysis and skews human monocyte differentiation from M2-polarized macrophages toward dendritic cells.

    Science.gov (United States)

    Haegel, Hélène; Thioudellet, Christine; Hallet, Rémy; Geist, Michel; Menguy, Thierry; Le Pogam, Fabrice; Marchand, Jean-Baptiste; Toh, Myew-Ling; Duong, Vanessa; Calcei, Alexandre; Settelen, Nathalie; Preville, Xavier; Hennequi, Marie; Grellier, Benoit; Ancian, Philippe; Rissanen, Jukka; Clayette, Pascal; Guillen, Christine; Rooke, Ronald; Bonnefoy, Jean-Yves

    2013-01-01

    Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163(+)CD64(+) M2-polarized suppressor macrophages, skewing their differentiation toward CD14(-)CD1a(+) dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.

  9. Integrated microRNA-mRNA-analysis of human monocyte derived macrophages upon Mycobacterium avium subsp. hominissuis infection.

    Directory of Open Access Journals (Sweden)

    Jutta Sharbati

    Full Text Available BACKGROUND: Many efforts have been made to understand basal mechanisms of mycobacterial infections. Macrophages are the first line of host immune defence to encounter and eradicate mycobacteria. Pathogenic species have evolved different mechanisms to evade host response, e.g. by influencing macrophage apoptotic pathways. However, the underlying molecular regulation is not fully understood. A new layer of eukaryotic regulation of gene expression is constituted by microRNAs. Therefore, we present a comprehensive study for identification of these key regulators and their targets in the context of host macrophage response to mycobacterial infections. METHODOLOGY/PRINCIPAL FINDINGS: We performed microRNA as well as mRNA expression analysis of human monocyte derived macrophages infected with several Mycobacterium avium hominissuis strains by means of microarrays as well as quantitative reverse transcription PCR (qRT-PCR. The data revealed the ability of all strains to inhibit apoptosis by transcriptional regulation of BCL2 family members. Accordingly, at 48 h after infection macrophages infected with all M. avium strains showed significantly decreased caspase 3 and 7 activities compared to the controls. Expression of let-7e, miR-29a and miR-886-5p were increased in response to mycobacterial infection at 48 h. The integrated analysis of microRNA and mRNA expression as well as target prediction pointed out regulative networks identifying caspase 3 and 7 as potential targets of let-7e and miR-29a, respectively. Consecutive reporter assays verified the regulation of caspase 3 and 7 by these microRNAs. CONCLUSIONS/SIGNIFICANCE: We show for the first time that mycobacterial infection of human macrophages causes a specific microRNA response. We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs. This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response

  10. Resibufogenin Induces G1-Phase Arrest through the Proteasomal Degradation of Cyclin D1 in Human Malignant Tumor Cells.

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    Masami Ichikawa

    Full Text Available Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3β (GSK-3β induces the proteasomal degradation of cyclin D1. The addition of GSK-3β inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3β.

  11. The adenovirus E4orf4 protein induces growth arrest and mitotic catastrophe in H1299 human lung carcinoma cells.

    Science.gov (United States)

    Li, S; Szymborski, A; Miron, M-J; Marcellus, R; Binda, O; Lavoie, J N; Branton, P E

    2009-01-22

    The human adenovirus E4orf4 protein, when expressed alone, induces p53-independent death in a wide range of cancer cells. Earlier studies by our groups suggested that although in some cases cell death can be associated with some hallmarks of apoptosis, it is not always affected by caspase inhibitors. Thus it is unlikely that E4orf4-induced cell death occurs uniquely through apoptosis. In the present studies using H1299 human lung carcinoma cells as a model system we found that death is induced in the absence of activation of any of the caspases tested, accumulation of reactive oxygen species, or release of cytochrome c from mitochondria. E4orf4 caused a substantial change in cell morphology, including vigorous membrane blebbing, multiple nuclei in many cells and increased cell volume. Most of these characteristics are not typical of apoptosis, but they are of necrosis. FACS analysis and western blotting for cell cycle markers showed that E4orf4-expressing cells became arrested in G(2)/M and also accumulated high levels of cyclin E. The presence of significant numbers of tetraploid and polyploid cells and some cells with micronuclei suggested that E4orf4 appears to induce death in these cells through a process resulting from mitotic catastrophe.

  12. Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation.

    Directory of Open Access Journals (Sweden)

    Natalia Bailon-Moscoso

    Full Text Available Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL, a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment.

  13. Leaf Extracts of Calocedrus formosana (Florin Induce G2/M Cell Cycle Arrest and Apoptosis in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sheau-Yun Yuan

    2011-01-01

    Full Text Available Calocedrus formosana (Florin bark acetone/ethylacetate extracts are known to exert an antitumor effect on some human cancer cell lines, but the mechanism is yet to be defined. The aim of this study was to determine the effects of Florin leaf methanol extracts on the growth and apoptosis of human bladder cancer cell lines. MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay showed that the growth of these bladder cancer cells was potently inhibited by the Florin leaf extracts. The cell cycle of these extract-treated cells (TCCSUP cells was arrested at the G2/M phase as determined by flow cytometry. Western blot analysis revealed the increases of cyclin B1 and Cdc2 kinase levels, alone with the decrease of phosphorylated Cdc2 kinase, after treating these cells with the extracts. An immunofluorescence assessment of β-tubulin showed decreased levels of polymerized tubulin in treated cells. However, the proteolytic cleavage of poly ADP-ribose polymerase and the activation of caspase-3/-8/-9 were all increased upon treatments of extracts. The concurrent increase of Bax and decrease of Bcl-2 levels indicated that the extracts could induce apoptosis in these treated cells. Taken together, these results suggest that the Florin leaf extracts may be an effective antibladder cancer agent.

  14. Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation

    Science.gov (United States)

    Bailon-Moscoso, Natalia; González-Arévalo, Gabriela; Velásquez-Rojas, Gabriela; Malagon, Omar; Vidari, Giovanni; Zentella-Dehesa, Alejandro; Ratovitski, Edward A.; Ostrosky-Wegman, Patricia

    2015-01-01

    Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites) may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL), a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment. PMID:26309132

  15. Induction of G1 Cell Cycle Arrest in Human Glioma Cells by Salinomycin Through Triggering ROS-Mediated DNA Damage In Vitro and In Vivo.

    Science.gov (United States)

    Zhao, Shi-Jun; Wang, Xian-Jun; Wu, Qing-Jian; Liu, Chao; Li, Da-Wei; Fu, Xiao-Ting; Zhang, Hui-Fang; Shao, Lu-Rong; Sun, Jing-Yi; Sun, Bao-Liang; Zhai, Jing; Fan, Cun-Dong

    2016-12-19

    Chemotherapy has always been one of the most effective ways in combating human glioma. However, the high metastatic potential and resistance toward standard chemotherapy severely hindered the chemotherapy outcomes. Hence, searching effective chemotherapy drugs and clarifying its mechanism are of great significance. Salinomycin an antibiotic shows novel anticancer potential against several human tumors, including human glioma, but its mechanism against human glioma cells has not been fully elucidated. In the present study, we demonstrated that salinomycin treatment time- and dose-dependently inhibited U251 and U87 cells growth. Mechanically, salinomycin-induced cell growth inhibition against human glioma was mainly achieved by induction of G1-phase arrest via triggering reactive oxide species (ROS)-mediated DNA damage, as convinced by the activation of histone, p53, p21 and p27. Furthermore, inhibition of ROS accumulation effectively attenuated salinomycin-induced DNA damage and G1 cell cycle arrest, and eventually reversed salinomycin-induced cytotoxicity. Importantly, salinomycin treatment also significantly inhibited the U251 tumor xenograft growth in vivo through triggering DNA damage-mediated cell cycle arrest with involvement of inhibiting cell proliferation and angiogenesis. The results above validated the potential of salinomycin-based chemotherapy against human glioma.

  16. Unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not promote human monocyte differentiation toward alternative macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Bouhlel, Mohamed Amine [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Brozek, John [Genfit, Loos (France); Derudas, Bruno [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Zawadzki, Christophe; Jude, Brigitte [Inserm ERI-9 and Equipe d' Accueil 2693, IFR114, Universite de Lille, Lille (France); Staels, Bart, E-mail: bart.staels@pasteur-lille.fr [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Chinetti-Gbaguidi, Giulia [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France)

    2009-08-28

    Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPAR{gamma} promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPAR{beta}/{delta} in this process has been reported only in mice and no data are available for PPAR{alpha}. Here, we show that in contrast to PPAR{gamma}, expression of PPAR{alpha} and PPAR{beta}/{delta} overall does not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPAR{alpha} and PPAR{beta}/{delta} do not appear to modulate the alternative differentiation of human macrophages.

  17. Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Li-Wen [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hsieh, Bau-Shan; Cheng, Hsiao-Ling [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hu, Yu-Chen [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Chang, Wen-Tsan [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Division of Hepatobiliarypancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan (China); Chang, Kee-Lung, E-mail: Chang.KeeLung@msa.hinet.net [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China)

    2012-01-15

    Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis. Highlights: ► Arecoline has potential to prevent against basal cell carcinoma tumorigenesis. ► It has more effectiveness on BCC as compared with a human keratinocyte cell line. ► Mechanisms involved including reducing tumor cells’ survival factor IL-6, ► Decreasing Cdc25C phosphatase, enhancing tumor suppressor factor p53, ► Eliciting G2/M

  18. Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study

    Science.gov (United States)

    2014-01-01

    Background Cordyceps cicadae is a medicinal fungus that is often used for treating cancer. However, the anticancer mechanisms of C. cicadae are largely unknown. This study aims to investigate the anticancer mechanisms of C. cicadae against hepatocellular carcinoma cells in vitro using a proteomic approach. Methods Human hepatocellular carcinoma MHCC97H cells were treated with a water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) for 48 h and harvested for cell viability assays. The significant differences in protein expression between control and C. cicadae-treated cells were analyzed by two-dimensional gel-based proteomics coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Flow cytometry analysis was employed to investigate the cell cycle and cell death. The anticancer molecular mechanism was analyzed by whole proteome mapping. Results The water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) inhibited the growth of MHCC97H cells in a dose-dependent manner via G2/M phase cell cycle arrest with no evidence of apoptosis. Among the identified proteins with upregulated expression were dynactin subunit 2, N-myc downstream-regulated gene 1, heat shock protein beta-1, alpha-enolase isoform 1, phosphatidylinositol transfer protein, and WD repeat-containing protein 1. Meanwhile, the proteins with downregulated expression were 14-3-3 gamma, BUB3, microtubule-associated protein RP/EB family member 1, thioredoxin-like protein, chloride intracellular channel protein 1, ectonucleoside triphosphate diphosphohydrolase 5, xaa-Pro dipeptidase, enoyl-CoA delta isomerase 1, protein-disulfide isomerase-related chaperone Erp29, hnRNP 2H9B, peroxiredoxin 1, WD-40 repeat protein, and serine/threonine kinase receptor-associated protein. Conclusion The water extract of C. cicadae reduced the growth of human hepatocellular carcinoma MHCC97H cells via G2/M cell cycle arrest. PMID:24872842

  19. Monocytes-derived macrophages mediated stable expression of human brain-derived neurotrophic factor, a novel therapeutic strategy for neuroAIDS.

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    Jing Tong

    Full Text Available HIV-1 associated dementia remains a significant public health burden. Clinical and experimental research has shown that reduced levels of brain-derived neurotrophic factor (BDNF may be a risk factor for neurological complications associated with HIV-1 infection. We are actively testing genetically modified macrophages for their possible use as the cell-based gene delivery vehicle for the central nervous system (CNS. It can be an advantage to use the natural homing/migratory properties of monocyte-derived macrophages to deliver potentially neuroprotective BDNF into the CNS, as a non-invasive manner. Lentiviral-mediated gene transfer of human (hBDNF plasmid was constructed and characterized. Defective lentiviral stocks were generated by transient transfection of 293T cells with lentiviral transfer plasmid together with packaging and envelope plasmids. High titer lentiviral vector stocks were harvested and used to transduce human neuronal cell lines, primary cultures of human peripheral mononocyte-derived macrophages (hMDM and murine myeloid monocyte-derived macrophages (mMDM. These transduced cells were tested for hBDNF expression, stability, and neuroprotective activity. The GenomeLab GeXP Genetic Analysis System was used to evaluate transduced cells for any adverse effects by assessing gene profiles of 24 reference genes. High titer vectors were prepared for efficient transduction of neuronal cell lines, hMDM, and mMDM. Stable secretion of high levels of hBDNF was detected in supernatants of transduced cells using western blot and ELISA. The conditioned media containing hBDNF were shown to be protective to neuronal and monocytic cell lines from TNF-α and HIV-1 Tat mediated cytotoxicity. Lentiviral vector-mediated gene transduction of hMDM and mMDM resulted in high-level, stable expression of the neuroprotective factorBDNF in vitro. These findings form the basis for future research on the potential use of BDNF as a novel therapy for neuroAIDS.

  20. Monocytes-derived macrophages mediated stable expression of human brain-derived neurotrophic factor, a novel therapeutic strategy for neuroAIDS.

    Science.gov (United States)

    Tong, Jing; Buch, Shilpa; Yao, Honghong; Wu, Chengxiang; Tong, Hsin-I; Wang, Youwei; Lu, Yuanan

    2014-01-01

    HIV-1 associated dementia remains a significant public health burden. Clinical and experimental research has shown that reduced levels of brain-derived neurotrophic factor (BDNF) may be a risk factor for neurological complications associated with HIV-1 infection. We are actively testing genetically modified macrophages for their possible use as the cell-based gene delivery vehicle for the central nervous system (CNS). It can be an advantage to use the natural homing/migratory properties of monocyte-derived macrophages to deliver potentially neuroprotective BDNF into the CNS, as a non-invasive manner. Lentiviral-mediated gene transfer of human (h)BDNF plasmid was constructed and characterized. Defective lentiviral stocks were generated by transient transfection of 293T cells with lentiviral transfer plasmid together with packaging and envelope plasmids. High titer lentiviral vector stocks were harvested and used to transduce human neuronal cell lines, primary cultures of human peripheral mononocyte-derived macrophages (hMDM) and murine myeloid monocyte-derived macrophages (mMDM). These transduced cells were tested for hBDNF expression, stability, and neuroprotective activity. The GenomeLab GeXP Genetic Analysis System was used to evaluate transduced cells for any adverse effects by assessing gene profiles of 24 reference genes. High titer vectors were prepared for efficient transduction of neuronal cell lines, hMDM, and mMDM. Stable secretion of high levels of hBDNF was detected in supernatants of transduced cells using western blot and ELISA. The conditioned media containing hBDNF were shown to be protective to neuronal and monocytic cell lines from TNF-α and HIV-1 Tat mediated cytotoxicity. Lentiviral vector-mediated gene transduction of hMDM and mMDM resulted in high-level, stable expression of the neuroprotective factorBDNF in vitro. These findings form the basis for future research on the potential use of BDNF as a novel therapy for neuroAIDS.

  1. Mesenchymal stem cells derived from human exfoliated deciduous teeth (SHEDs induce immune modulatory profile in monocyte-derived dendritic cells.

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    Fernando de Sá Silva

    Full Text Available BACKGROUND: Mesenchymal stem cells have prominent immune modulatory properties, which may have clinical applications; however their major source, bone marrow, is of limited availability. On the other hand, mesenchymal stem cells derived from human exfoliated deciduous teeth (SHEDs are readily accessible, but their immune regulatory properties have not been completely investigated. This study was designed, therefore, to evaluate the SHEDs influence on DCs differentiation, maturation, ability to activate T cells and to expand CD4(+Foxp3(+ T cells. METHODOLOGY/PRINCIPAL FINDINGS: The experiments were based in cellular co-culture during differentiation and maturation of monocyte derived-DCs (moDCs, with, or not, presence of SHEDs. After co-culture with SHEDs, (moDCs presented lower expression of BDCA-1 and CD11c, in comparison to DC cultivated without SHEDs. CD40, CD80, CD83 and CD86 levels were also decreased in mature DCs (mDCs after co-cultivation with SHEDs. To assess the ability of SHEDs-exposed moDCs to modulate T cell responses, the former were separated from SHEDs, and co-cultured with peripheral blood lymphocytes. After 5 days, the proliferation of CD4(+ and CD8(+ T cells was evaluated and found to be lower than that induced by moDCs cultivated without SHEDs. In addition, an increase in the proportion of CD4(+Foxp3(+IL-10(+ T cells was observed among cells stimulated by mature moDCs that were previously cultivated with SHEDs. Soluble factors released during co-cultures also showed a reduction in the pro-inflammatory cytokines (IL-2, TNF-α and IFN-γ, and an increase in the anti-inflammatory molecule IL-10. CONCLUSION/SIGNIFICANCE: This study shows that SHEDs induce an immune regulatory phenotype in moDCs cells, evidenced by changes in maturation and differentiation rates, inhibition of lymphocyte stimulation and ability to expand CD4(+Foxp3(+ T cells. Further characterization and validation of this phenomenon could support the use of SHEDs

  2. Platelet-derived growth factor (PDGF-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation

    Directory of Open Access Journals (Sweden)

    Bethel-Brown Crystal

    2012-12-01

    Full Text Available Abstract Chemokine (C-C motif ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1 is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND. The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS via the disrupted blood-brain barrier (BBB. We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK1/2, c-Jun N-terminal kinase (JNK and p38 mitogen-activated protein (MAP kinases and phosphatidylinositol 3-kinase (PI3K/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB. Chromatin immunoprecipitation (ChIP assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs, an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R blocker. PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte

  3. The specific role of pRb in p16 (INK4A) -mediated arrest of normal and malignant human breast cells.

    Science.gov (United States)

    Bazarov, Alexey V; Lee, Won Jae; Bazarov, Irina; Bosire, Moses; Hines, William C; Stankovich, Basha; Chicas, Agustin; Lowe, Scott W; Yaswen, Paul

    2012-03-01

    RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G 1 was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.

  4. Growth arrest and apoptosis of human hepatocellular carcinoma cells induced by hexamethylene bisacetamide

    Institute of Scientific and Technical Information of China (English)

    Gao-Liang Ouyang; Qiu-Feng Cai; Min Liu; Rui-Chuan Chen; Zhi Huang; Rui-Sheng Jiang; Fu Chen; Shui-Gen Hong; Shi-Deng Bao

    2004-01-01

    AIM: To investigate the cellular effects of hybrid polar compound hexamethylene bisacetamide (HMBA) on the growth and apoptosis of human hepatocellular carcinoma cells and to provide the molecular mechanism for potential application of HMBA in the treatment of liver cancer.METHODS: Effects of HMBA on the growth of human hepatocellular carcinoma SMMC-7721 cells were assayed by MTT chronometry. Apoptosis induced by HMBA was detected by phase-contrast microscopy, flow cytometry,propidium iodide staining and immunocytochemical analysis.RESULTS: The growth of SMMC-7721 cells was significantly inhibited by HMBA, and the growth inhibitory rate was 51.1%, 62.6%, 68.7% and 73.9% respectively after treatment with 5.0, 7.5, 10.0 and 12.5 mmol/L of HMBA.In the cells treated with 10 mmol/L of HMBA for 72 h, the population of cells at sub-G1 phase significantly increased,and the apoptotic bodies and condensed nuclei were detected. Moreover, treatment of SMMC-7721 cells with 10 mmol/L of HMBA down-regulated the expression of Bcl2 anti-apoptotic protein, while slightly up-regulated the level of pro-apoptotic protein Bax.CONCLUSION: Treatment with 10.0 mmol/L of HMBA can significantly inhibit the growth and induce apoptosis of human hepatocellular carcinoma SMMC-7721 cells by decreasing the ratio of Bcl-2 to Bax.

  5. 1,25-Dihydroxyvitamin D3 inhibits cytokine production by human blood monocytes at the post-transcriptional level

    DEFF Research Database (Denmark)

    Müller, K; Haahr, P M; Diamant, M

    1992-01-01

    (TNF-alpha), produced by the antigen presenting cells. In the present study we examined the effect of 1,25-(OH)2D3 on the production of these cytokines, as well as superoxide generation by freshly isolated mononuclear cells and partially purified monocytes. The immediate precursor of 1,25(OH)2D3, 25-OH...... D3, and the synthetic analogue MC 903 ('Calcipotriol') were examined in parallel. 1,25-(OH)2D3 dose-dependently inhibited the production of IL-alpha, IL-6 and TNF-alpha by Escherichia coli lipopolysaccharide (LPS)-stimulated monocytes, without affecting superoxide production. MC 903 had comparable......1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits lymphocyte proliferation and production of antibodies and lymphokines such as interleukin (IL)-2 and interferon gamma. These lymphocyte functions are dependent upon cytokines, including IL-1 alpha, IL-1 beta, IL-6 and tumour necrosis factor alpha...

  6. Schisandrin B inhibits the proliferation of human lung adenocarcinoma A549 cells by inducing cycle arrest and apoptosis.

    Science.gov (United States)

    Lv, Xue-Jiao; Zhao, Li-Jing; Hao, Yu-Qiu; Su, Zhen-Zhong; Li, Jun-Yao; Du, Yan-Wei; Zhang, Jie

    2015-01-01

    Lung cancer is the leading cause of cancer death in the world. Schizandrin B (Sch B) is one of the main dibenzocyclooctadiene lignans present in the fruit of Schisandra chinensis (Schisandraceae). Sch B has multiple functions against cancer. The aim of this study was to determine the effect of Sch B on the proliferation, cell cycling, apoptosis and invasion of lung adenocarcinoma A549 cells by MTT, flow cytometry, wound healing and transwell invasion assays. Treatment with Sch B inhibited the proliferation of A549 cells in a dose-dependent manner. Sch B induced cell cycle arrest at G0/G1 phase by down-regulating the expression of cyclin D1, cyclin-dependent kinase (CDK)4, and CDK6, but up-regulating p53 and p21 expression in A549 cells. Furthermore, Sch B triggered A549 cell apoptosis by increasing Bax, cleaved caspase-3, 9, Cyto C, but decreasing Bcl-2 and PCNA expression. In addition, Sch B inhibited the invasion and migration of A549 cells by down-regulating the expressions of HIF-1, VEGF, MMP-9 and MMP-2. Therefore, Sch B has potent anti-tumor activity and may be a promising traditional Chinese medicine for human lung carcinoma.

  7. Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells.

    Science.gov (United States)

    Ko, Hyeonseok; Kim, Young-Joo; Amor, Evangeline C; Lee, Jong Wha; Kim, Han-Cheon; Kim, Hee Ju; Yang, Hyun Ok

    2011-09-01

    Dimethyl cardamonin (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone; DMC) is a naturally occurring chalcone, and it is the major compound isolated from the leaves of Syzygium samarangense (Blume) Merr. & L.M. Perry (Myrtaceae). Experiments were conducted to determine the effects of DMC on cell proliferation, cell-cycle distribution, and programmed cell death in cultures of human colorectal carcinoma HCT116 and LOVO cells. Results showed that DMC inhibited HCT116 and LOVO cell proliferation and induced G(2) /M cell cycle arrest, which was associated with the conversion of microtubule associated protein light chain 3 (LC3)-I-LC3-II, an autophagosome marker, and the incorporation of monodansylcadaverine (MDC), a marker for the acidic compartment of autolysosomes or acidic vesicular organelles. The treatment of HCT116 and LOVO cells using a combination of DMC with an autophagy inhibitor, such as 3-methyladenine (3-MA), beclin 1 siRNA, or atg5 siRNA, suppressed the effect of DMC-mediated anti-proliferation. These results imply that DMC can suppress colorectal carcinoma HCT116 and LOVO cell proliferation through a G(2) /M phase cell-cycle delay, and can induce autophagy, the hallmark of Type II programmed cell death (PCD). Taken together, our results suggest that DMC may be an effective chemotherapeutic agent for HCT116 and LOVO colorectal carcinoma cells.

  8. Lectin from Agaricus Bisporus Suppresses Akt Phosphorylation and Arrests Cell Cycle Progression in Primary Human Retinal Pigment Epithelial Cells

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    Y. H. Cheung

    2011-05-01

    Full Text Available Anomalous retinal pigment epithelial (RPE cells have been implicated in the development of retinal diseases. Lectin from the edible mushroom Agaricus bisporus (ABL was found to inhibit growth of RPE cells. To elucidate the mechanism through which ABL inhibits RPE cell proliferation, we investigated the changes in cell proliferation-related signaling pathways and cell cycle distribution patterns. Primary human RPE cells were grown with or without the lectin (ABL supplement (20ug or 90ug/ml for three days. Phosphorylation statuses of Akt, Jnk and p38 as well as p53 expression level were investigated by Western blotting. Cellular distributions in various cell cycle phases were investigated using flow cytometry. After ABL treatment (90ug/ml, Akt was found to be hypo-phosphorylated while the expression levels of p53, phosphorylated-Jnk and phosphorylated-p38 were not altered. The amount of cells present at S phase was reduced. Our results showed that ABL hypo-phosphorylated Akt and this observation is in line with the finding that ABL could attenuate cell proliferation. As the level of p53 was not significantly altered by ABL, this suggested that the mechanism in which ABL arrested cell proliferation was independent of Akt-mediated MDM2 activation but was possibly mediated by altering G1 to S phase transition.

  9. Exposure of Human Lung Cancer Cells to 8-Chloro-Adenosine Induces G2/M Arrest and Mitotic Catastrophe

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    Hong-Yu Zhang

    2004-11-01

    Full Text Available 8-Chloro-adenosine (8-CI-Ado is a potent chemotherapeutic agent whose cytotoxicity in a variety of tumor cell lines has been widely investigated. However, the molecular mechanisms are uncertain. In this study, we found that exposure of human lung cancer cell lines A549 (p53-wt and H1299 (p53-depleted to 8-CI-Ado induced cell arrest in the G2/M phase, which was accompanied by accumulation of binucleated and polymorphonucleated cells resulting from aberrant mitosis and failed cytokinesis. Western blotting showed the loss of phosphorylated forms of Cdc2 and Cdc25C that allowed progression into mitosis. Furthermore, the increase in Ser10-phosphorylated histone H3-positive cells revealed by fluorescence-activated cell sorting suggested that the agent-targeted cells were able to exit the G2 phase and enter the M phase. Immunocytochemistry showed that microtubule and microfilament arrays were changed in exposed cells, indicating that the dynamic instability of microtubules and microfilaments was lost, which may correlate with mitotic dividing failure. Aberrant mitosis resulted in mitotic catastrophe followed by varying degrees of apoptosis, depending on the cell lines. Thus, 8-CI-Ado appears to exert its cytotoxicity toward cells in culture by inducing mitotic catastrophe.

  10. Molecular mechanisms underlying mangiferin-induced apoptosis and cell cycle arrest in A549 human lung carcinoma cells.

    Science.gov (United States)

    Shi, Wei; Deng, Jiagang; Tong, Rongsheng; Yang, Yong; He, Xia; Lv, Jianzhen; Wang, Hailian; Deng, Shaoping; Qi, Ping; Zhang, Dingding; Wang, Yi

    2016-04-01

    Mangiferin, which is a C‑glucosylxanthone (1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside) purified from plant sources, has recently gained attention due to its various biological activities. The present study aimed to determine the apoptotic effects of mangiferin on A549 human lung adenocarcinoma cells. In vitro studies demonstrated that mangiferin exerted growth‑inhibitory and apoptosis-inducing effects against A549 cells. In addition, mangiferin exhibited anti-tumor properties in A549 xenograft mice in vivo. Mangiferin triggered G2/M phase cell cycle arrest via downregulating the cyclin-dependent kinase 1-cyclin B1 signaling pathway, and induced apoptotic cell death by inhibiting the protein kinase C-nuclear factor-κB pathway. In addition, mangiferin was able to enhance the antiproliferative effects of cisplatin on A549 cells, thus indicating the potential for a combined therapy. Notably, mangiferin exerted anticancer effects in vivo, where it was able to markedly decrease the volume and weight of subcutaneous tumor mass, and expand the lifespan of xenograft mice. The present study clarified the molecular mechanisms underlying mangiferin-induced antitumor activities, and suggested that mangiferin may be considered a potential antineoplastic drug for the future treatment of cancer.

  11. Tocotrienol-Rich Fraction Prevents Cell Cycle Arrest and Elongates Telomere Length in Senescent Human Diploid Fibroblasts

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    Suzana Makpol

    2011-01-01

    Full Text Available This study determined the molecular mechanisms of tocotrienol-rich fraction (TRF in preventing cellular senescence of human diploid fibroblasts (HDFs. Primary culture of HDFs at various passages were incubated with 0.5 mg/mL TRF for 24 h. Telomere shortening with decreased telomerase activity was observed in senescent HDFs while the levels of damaged DNA and number of cells in G0/G1 phase were increased and S phase cells were decreased. Incubation with TRF reversed the morphology of senescent HDFs to resemble that of young cells with decreased activity of SA-β-gal, damaged DNA, and cells in G0/G1 phase while cells in the S phase were increased. Elongated telomere length and restoration of telomerase activity were observed in TRF-treated senescent HDFs. These findings confirmed the ability of tocotrienol-rich fraction in preventing HDFs cellular ageing by restoring telomere length and telomerase activity, reducing damaged DNA, and reversing cell cycle arrest associated with senescence.

  12. Identification of biological markers of liver X receptor (LXR activation at the cell surface of human monocytes.

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    Cédric Rébé

    Full Text Available BACKGROUND: Liver X receptor (LXR α and LXR β (NR1H3 and NR1H2 are oxysterol-activated nuclear receptors involved in the control of major metabolic pathways such as cholesterol homeostasis, lipogenesis, inflammation and innate immunity. Synthetic LXR agonists are currently under development and could find applications in various fields such as cardiovascular diseases, cancer, diabetes and neurodegenerative diseases. The clinical development of LXR agonists requires the identification of biological markers for pharmacodynamic studies. In this context, monocytes represent an attractive target to monitor LXR activation. They are easily accessible cells present in peripheral blood; they express LXR α and β and respond to LXR agonist stimulation in vitro. The aim of our study was to identify cell surface markers of LXR agonists on monocytes. For this, we focused on clusters of differentiation (CD markers because they are well characterized and accessible cell surface molecules allowing easy immuno-phenotyping. METHODOLOGY/PRINCIPAL FINDINGS: By using microarray analysis of monocytes treated or not with an LXR agonist in vitro, we selected three CD, i.e. CD82, CD226, CD244 for further analysis by real time PCR and flow cytometry. The three CD were up-regulated by LXR agonist treatment in vitro in a time- and dose- dependent manner and this induction was LXR specific as assessed by a SiRNA or LXR antagonist strategy. By using flow cytometry, we could demonstrate that the expression of these molecules at the cell surface of monocytes was significantly increased after LXR agonist treatment. CONCLUSIONS/SIGNIFICANCE: We have identified three new cell surface markers that could be useful to monitor LXR activation. Future studies will be required to confirm the biological and diagnostic significance of the markers.

  13. Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to uropathogenic Escherichia coli.

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    Benjamin L Duell

    Full Text Available Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10 in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

  14. Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to uropathogenic Escherichia coli.

    Science.gov (United States)

    Duell, Benjamin L; Carey, Alison J; Dando, Samantha J; Schembri, Mark A; Ulett, Glen C

    2013-01-01

    Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC) being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10) in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

  15. Aortic endothelial cells regulate proliferation of human monocytes in vitro via a mechanism synergistic with macrophage colony-stimulating factor. Convergence at the cyclin E/p27(Kip1) regulatory checkpoint.

    Science.gov (United States)

    Antonov, A S; Munn, D H; Kolodgie, F D; Virmani, R; Gerrity, R G

    1997-06-15

    Monocyte-derived macrophages (Mphis) are pivotal participants in the pathogenesis of atherosclerosis. Evidence from both animal and human plaques indicates that local proliferation may contribute to accumulation of lesion Mphis, and the major Mphi growth factor, macrophage colony stimulating factor (MCSF), is present in atherosclerotic plaques. However, most in vitro studies have failed to demonstrate that human monocytes/Mphis possess significant proliferative capacity. We now report that, although human monocytes cultured in isolation showed only limited MCSF-induced proliferation, monocytes cocultured with aortic endothelial cells at identical MCSF concentrations underwent enhanced (up to 40-fold) and prolonged (21 d) proliferation. In contrast with monocytes in isolation, this was optimal at low seeding densities, required endothelial cell contact, and could not be reproduced by coculture with smooth muscle cells. Intimal Mphi isolated from human aortas likewise showed endothelial cell contact-dependent, MCSF-induced proliferation. Consistent with a two-signal mechanism governing Mphi proliferation, the cell cycle regulatory protein, cyclin E, was rapidly upregulated by endothelial cell contact in an MCSFindependent fashion, but MCSF was required for successful downregulation of the cell cycle inhibitory protein p27(Kip1) before cell cycling. Thus endothelial cells and MCSF differentially and synergistically regulate two Mphi genes critical for progression through the cell cycle.

  16. Monocyte Subpopulations in Angiogenesis

    Science.gov (United States)

    Dalton, Heather J.; Armaiz-Pena, Guillermo; Gonzalez-Villasana, Vianey; Lopez-Berestein, Gabriel; Bar-Eli, Menashe; Sood, Anil K.

    2014-01-01

    Growing understanding of the role of the tumor microenvironment in angiogenesis has brought monocyte-derived cells into focus. Monocyte subpopulations are an increasingly attractive therapeutic target in many pathologic states, including cancer. Before monocyte-directed therapies can be fully harnessed for clinical use, understanding of monocyte-driven angiogenesis in tissue development and homeostasis, as well as malignancy, is required. Here, we provide an overview of the mechanisms by which monocytic subpopulations contribute to angiogenesis in tissue and tumor development, highlight gaps in our existing knowledge, and discuss opportunities to exploit these cells for clinical benefit. PMID:24556724

  17. Down-regulation of AP-4 inhibits proliferation, induces cell cycle arrest and promotes apoptosis in human gastric cancer cells.

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    Xinghua Liu

    Full Text Available BACKGROUND: AP-4 belongs to the basic helix-loop-helix leucine-zipper subgroup; it controls target gene expression, regulates growth, development and cell apoptosis and has been implicated in tumorigenesis. Our previous studies indicated that AP-4 was frequently overexpressed in gastric cancers and may be associated with the poor prognosis. The purpose of this study is to examine whether silencing of AP-4 can alter biological characteristics of gastric cancer cells. METHODS: Two specific siRNAs targeting AP-4 were designed, synthesized, and transfected into gastric cancer cell lines and human normal mucosa cells. AP-4 expression was measured with real-time quantitative PCR and Western blot. Cell proliferation and chemo-sensitivity were detected by CCK-8 assay. Cell cycle assay and apoptosis assay were performed by flow cytometer, and relative expression of cell cycle regulators were detected by real-time quantitative PCR and Western blot, expression of the factors involved in the apoptosis pathway were examined in mRNA and protein level. RESULTS: The expression of AP-4 was silenced by the siRNAs transfection and the effects of AP-4 knockdown lasted 24 to 96 hrs. The siRNA-mediated silencing of AP-4 suppressed the cellular proliferation, induced apoptosis and sensitized cancer cells to anticancer drugs. In addition, the expression level of p21, p53 and Caspase-9 were increased when AP-4 was knockdown, but the expression of cyclin D1, Bcl-2 and Bcl-x(L was inhibited. It didn't induce cell cycle arrest when AP-4 was knockdown in p53 defect gastric cancer cell line Kato-III. CONCLUSIONS: These results illustrated that gene silencing of AP-4 can efficiently inhibited cell proliferation, triggered apoptosis and sensitized cancer cells to anticancer drugs in vitro, suggesting that AP-4 siRNAs mediated silencing has a potential value in the treatment of human gastric cancer.

  18. Peroxisome proliferator-activated receptor γ ligands induce cell cycle arrest and apoptosis in human renal carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Feng-guang YANG; Zhi-wen ZHANG; Dian-qi XIN; Chang-jin SHI; Jie-ping WU; Ying-lu GUO; You-fei GUAN

    2005-01-01

    Aim: To study the effect of peroxisome proliferator-actived receptor γ (PPARγ)ligands on cell proliferation and apoptosis in human renal carcinoma cell lines.Methods: The expression of PPARγ was investigated by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry.The effect of thiazolidinedione (TZD) PPARγ ligands on growth of renal cell carcinoma (RCC) cells was measured by MTT assay and flow cytometric analysis. Cell death ELISA, Hoechst 33342 fluorescent staining and DNA ladder assay were used to observe the effects of PPARγ ligands on apoptosis. Regulatory proteins of cell cycle and apoptosis were detected by Western blot analysis. Results:PPARγ was expressed at much higher levels in renal tumors than in the normal kidney (2.16±0.85 vs 0.90±0.73; P<0.01 ). TZD PPARγ ligands inhibited RCC cell growth in a dose-dependent manner with IC50 values of 7.08 μmol/L and 11.32 μmol/L for pioglitazone, and 5.71 μmol/L and 8.38 μmol/L for troglitazone in 786-O and A498 cells, respectively. Cell cycle analysis showed a G0/G1 arrest in human RCC cells following 24-h exposure to TZD. Analysis of cell cycle regulatory proteins revealed that TZD decreased the protein levels of proliferating cell nuclear antigen, pRb, cyclin D1, and Cdk4 but increased the levels of p21 and p27 in a timedependent manner. Furthermore, high doses of TZD induced massive apoptosis in renal cancer cells, with increased Bax expression and decreased Bcl-2 expression.Conclusion: TZD PPARγ ligands showed potent inhibitory effect on proliferation,and could induce apoptosis in RCC cells. These results suggest that ligands for PPARγ have potential antitumor effects on renal carcinoma cells.

  19. Sinomenine influences capacity for invasion and migration in activated human monocytic THP-1 cells by inhibiting the expression of MMP-2, MMP-9, and CD147

    Institute of Scientific and Technical Information of China (English)

    Yang-qiong OU; Li-hua CHEN; Xue-jun LI; Zhi-bin LIN; Wei-dong LI

    2009-01-01

    Aim: The aim of this study was to investigate the mechanism of the effects of Sinomenine (SIN) on the invasion and migration ability of activated human monocytic THP-1 cells (A-THP-1). Sinomenine is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum.Methods: Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-ac-etate (PMA). Cells were treated with different concentrations of SIN. The invasion and migration ability of cells was tested by in vitro transwell assays. The levels of CD147 and MMPs were evaluated by flow cytometric analysis and zymographic analysis, respectively. The mRNA expression of CD147, MMP-2, and MMP-9 was measured by RT-PCR. Results: The invasion and migration ability of A-THP-1 cells was significantly inhibited by SIN in a concentration-depen-dent fashion; at the same time, the levels of CD147, MMP-2, and MMP-9 were markedly down-regulated. This inhibitory effect was most notable at concentrations of 0.25 mmol/L and 1.00 mmol/L (P<0.01). Conclusion: A possible mechanism of the inhibitory effect of SIN on cell invasion and migration ability is repression of the expression of MMP-2 and MMP-9, which strongly correlates with the inhibition of CD147 activity.

  20. Expression of dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin on dendritic cells generated from human peripheral blood monocytes

    Institute of Scientific and Technical Information of China (English)

    Jun Li; Zhi-Hua Feng; Guang-Yu Li; Dan-Lei Mou; Qing-He Nie

    2006-01-01

    AIM: To generate dendritic cells (DCs) from human peripheral blood and to detect the expression of dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN; CD209) for the further study of DC-SIGN in hepatitis C virus (HCV) transmission.METHODS: Peripheral blood monocytes were isolated from blood of healthy individuals by Ficoll-Hypaque sedimentation and cultured in complete medium containing rhGM-CSF and rhIL-4. Cells were cultured for seven days, with cytokine addition every two days to obtain immature DCs. Characteristics of the cultured cells were observed under light and scanning microscope, and the expression of DC-SIGN was detected by immunofluorescence staining.RESULTS: After seven-day culture, a large number of cells with typical characteristics of DCs appeared. Their characteristics were observed under light and scanning electron microscope. These cells had a variety of cell shapes such as those of bipolar elongate cells, elaborate stellate cells and DCs. DC-SIGN was detected by immunofluorescence staining and its expression level on cultivated dendritic cells was high.CONCLUSION: DCs with a high expression of DC-SIGN can be generated from human peripheral blood monocytes in complete medium containing rhGM-CSF and rhIL-4.

  1. Tailored control and optimisation of the number of phosphonic acid termini on phosphorus-containing dendrimers for the ex-vivo activation of human monocytes.

    Science.gov (United States)

    Rolland, Olivier; Griffe, Laurent; Poupot, Mary; Maraval, Alexandrine; Ouali, Armelle; Coppel, Yannick; Fournié, Jean-Jacques; Bacquet, Gérard; Turrin, Cédric-Olivier; Caminade, Anne-Marie; Majoral, Jean-Pierre; Poupot, Rémy

    2008-01-01

    The syntheses of a series of phosphonic acid-capped dendrimers is described. This collection is based on a unique set of dendritic structural parameters-cyclo(triphosphazene) core, benzylhydrazone branches and phosphonic acid surface-and was designed to study the influence of phosphonate (phosphonic acid) surface loading towards the activation of human monocytes ex vivo. Starting from the versatile hexachloro-cyclo(triphosphazene) N(3)P(3)Cl(6), six first-generation dendrimers were obtained, bearing one to six full branches, that lead to 4, 8, 12, 16, 20 and 24 phosphonate termini, respectively. The surface loading was also explored at the limit of dense packing by means of a first-generation dendrimer having a cyclo(tetraphosphazene) core and bearing 32 termini, and with a first-generation dendrimer based on a AB(2)/CD(5) growing pattern and bearing 60 termini. Human monocyte activation by these dendrimers confirms the requirement of the whole dendritic structure for bioactivity and identifies the dendrimer bearing four branches, thus 16 phosphonate termini, as the most bioactive.

  2. Growth factors induce monocyte binding to vascular smooth muscle cells: implications for monocyte retention in atherosclerosis.

    Science.gov (United States)

    Cai, Qiangjun; Lanting, Linda; Natarajan, Rama

    2004-09-01

    Adhesive interactions between monocytes and vascular smooth muscle cells (VSMC) may contribute to subendothelial monocyte-macrophage retention in atherosclerosis. We investigated the effects of angiotensin II (ANG II) and platelet-derived growth factor (PDGF)-BB on VSMC-monocyte interactions. Treatment of human aortic VSMC (HVSMC) with ANG II or PDGF-BB significantly increased binding to human monocytic THP-1 cells and to peripheral blood monocytes. This was inhibited by antibodies to monocyte beta(1)- and beta(2)-integrins. The binding was also attenuated by blocking VSMC arachidonic acid (AA) metabolism by inhibitors of 12/15-lipoxygenase (12/15-LO) or cyclooxygenase-2 (COX-2). Conversely, binding was enhanced by overexpression of 12/15-LO or COX-2. Direct treatment of HVSMC with AA or its metabolites also increased binding. Furthermore, VSMC derived from 12/15-LO knockout mice displayed reduced binding to mouse monocytic cells relative to genetic control mice. Using specific signal transduction inhibitors, we demonstrated the involvement of Src, phosphoinositide 3-kinase, and MAPKs in ANG II- or PDGF-BB-induced binding. Interestingly, after coculture with HVSMC, THP-1 cell surface expression of the scavenger receptor CD36 was increased. These results show for the first time that growth factors may play additional roles in atherosclerosis by increasing monocyte binding to VSMC via AA metabolism and key signaling pathways. This can lead to monocyte subendothelial retention, CD36 expression, and foam cell formation.

  3. Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes

    Directory of Open Access Journals (Sweden)

    Sharbati Soroush

    2012-01-01

    Full Text Available Abstract Background Small interfering and non-coding RNAs regulate gene expression across all kingdoms of life. MicroRNAs constitute an important group of metazoan small RNAs regulating development but also disease. Accordingly, in functional genomics microRNA expression analysis sheds more and more light on the dynamic regulation of gene expression in various cellular processes. Results We have developed custom RT-qPCR arrays allowing for accurate quantification of 31 small RNAs in triplicate using a 96 well format. In parallel, we provide accurate normalisation of microRNA expression data based on the quantification of 5 reference snRNAs. We have successfully employed such arrays to study microRNA regulation during human monocyte differentiation as well as Salmonella infection. Besides well-known protagonists such as miR-146 or miR-155, we identified the up-regulation of miR-21, miR-222, miR-23b, miR-24, miR-27a as well as miR-29 upon monocyte differentiation or infection, respectively. Conclusions The provided protocol for RT-qPCR arrays enables straight-forward microRNA expression analysis. It is fully automatable, compliant with the MIQE guidelines and can be completed in only 1 day. The application of these arrays revealed microRNAs that may mediate monocyte host defence mechanisms by regulating the TGF-β signalling upon Salmonella infection. The introduced arrays are furthermore suited for customised quantification of any class of small non-coding RNAs as exemplified by snRNAs and thus provide a versatile tool for ubiquitous applications.

  4. Modulation of TREM2 by CD33: a protein QTL study integrates Alzheimer loci in human monocytes

    Science.gov (United States)

    Chan, Gail; White, Charles C.; Winn, Phoebe A.; Cimpean, Maria; Replogle, Joseph M.; Glick, Laura R.; Cuerdon, Nicole E.; Ryan, Katie J.; Johnson, Keith A.; Schneider, Julie A.; Bennett, David A.; Chibnik, Lori B.; Sperling, Reisa A.; Bradshaw, Elizabeth M.; De Jager, Philip L.

    2015-01-01

    Here, we report results from a protein quantitative trait analysis in monocytes from 226 individuals to evaluate cross-talk between Alzheimer loci. We find that the NME8 locus influences PTK2B and that the CD33 risk allele leads to greater TREM2 expression. Further, we observe (1) a decreased TREM1/TREM2 ratio with a TREM1 risk allele, (2) decreased TREM2 expression with CD33 suppression, and (3) elevated cortical TREM2 mRNA expression with amyloid pathology. PMID:26414614

  5. Notch1 signaling inhibits growth of human hepatocellular carcinoma through induction of cell cycle arrest and apoptosis.

    Science.gov (United States)

    Qi, Runzi; An, Huazhang; Yu, Yizhi; Zhang, Minghui; Liu, Shuxun; Xu, Hongmei; Guo, Zhenghong; Cheng, Tao; Cao, Xuetao

    2003-12-01

    Notch signaling plays a critical role in maintaining the balance between cell proliferation, differentiation, and apoptosis; hence, perturbed Notch signaling may contribute to tumorigenesis. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in Africa and Asia. The mechanisms that orchestrate the multiple oncogenic insults required for initiation and progression of HCC are not clear. We constitutively overexpressed active Notch1 in human HCC to explore the effects of Notch1 signaling on HCC cell growth and to investigate the underlying molecular mechanisms. We show here that overexpression of Notch1 was able to inhibit the growth of HCC cells in vitro and in vivo. Biochemical analysis revealed the involvement of cell cycle regulated proteins in Notch1-mediated G(0)/G(1) arrest of HCC cells. Compared with green fluorescent protein (GFP) control, transient transfection of Notch1 ICN decreased expression of cyclin A (3.5-fold), cyclin D1 (2-fold), cyclin E (4.5-fold), CDK2 (2.8-fold), and the phosphorylated form of retinoblastoma protein (3-fold). Up-regulation of p21(waf/cip1) protein expression was observed in SMMC7721-ICN cells stably expressing active Notch1 but not in SMMC7721-GFP cells, which only express GFP. Furthermore, a 12-fold increase in p53 expression and an increase (4.8-fold) in Jun-NH(2)-terminal kinase activation were induced in SMMC7721-ICN cells compared with SMMC7721-GFP cells. In contrast, expression of the antiapoptotic Bcl-2 protein could not be detected in SMMC7721-ICN cells. These findings suggest that Notch1 signaling may participate in the development of HCC cells, affecting multiple pathways that control both cell proliferation and apoptosis.

  6. RRR-α-tocopheryl succinate inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

    Institute of Scientific and Technical Information of China (English)

    Kun Wu; Yan Zhao; Bai-He Liu; Yao Li; Fang Liu; Jian Guo; Wei-Ping Yu

    2002-01-01

    AIM: To investigate the effects of growth inhibition ofhuman gastric cancer SGC-7901 cell with RRR-α-tocopherylsuccinate (VES), a derivative of natural Vitamin E, viainducing apoptosis and DNA synthesis arrest.METHODS: Human gastric cancer SGC-7901 cells wereregularly incubated in the presence of VES at 5, 10 and20mg@ L 1(VES was dissolved in absolute ethanol anddiluted in RPMI 1640 complete condition mediacorrespondingly to a final concentration of VES and 1mL@L-1 ethanol), succinic acid and ethanol equivalents asvehicle (VEH) control andcondition media only asuntreated (UT) control. Trypan blue dye exclusionanalysis and MTT assay were applied to detect the cellproliferation. 37kBq of tritiated thymidine was added tocells and [3H] TdR uptake was measured to observe DNAsynthesis. Apoptotic morphology was observed byelectron microscopy and DAPI staining. Flow cytometryand terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) assay were performed to detectVES-triggered apoptosis.RESULTS: VES inhibited SGC-7901 cell growth in a dose-dependent manner. The growth curve showed suppressionby 24.7%, 49.2% and 68.7% following 24h of VEStreatment at 5, 10 and 20 mg@L 1, respectively, similar tothe findings from MTT assay. DNA synthesis wasevidently reduced by 35%, 45% and 98% after 24h VEStreatment at 20 mg@ L-1 and 48h at 10 and 20 mg@ L 1,respectively. VES induced SGC-7901 cells to undergoapoptosis with typically apoptotic characteristics,including morphological changes of chromatincondensation, chromatin crescent formation/margination,nucleus fragmentation and apoptotic body formation,typical apoptotic sub-G1 peak by flow cytometry andincrease of apoptotic cells by TUNEL assay in which 90%of cells underwent apoptosis after 48h of VES treatment at20 mcg@L-1.CONCLUSION: VES can inhibit human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesisarrest. Inhibition of SGC-7901 cell growth by VES is dose-and time

  7. Reduction of Monocyte Chemoattractant Protein-1 and Interleukin-8 Levels by Ticlopidine in TNF-α Stimulated Human Umbilical Vein Endothelial Cells

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    Chaur-Jong Hu

    2009-01-01

    Full Text Available Atherosclerosis and its associated complications represent major causes of morbidity and mortality in the industrialized or Western countries. Monocyte chemoattractant protein-1 (MCP-1 is critical for the initiating and developing of atherosclerotic lesions. Interleukin-8 (IL-8, a CXC chemokine, stimulates neutrophil chemotaxis. Ticlopidine is one of the antiplatelet drugs used to prevent thrombus formation relevant to the pathophysiology of atherothrombosis. In this study, we found that ticlopidine dose-dependently decreased the mRNA and protein levels of TNF-α-stimulated MCP-1, IL-8, and vascular cell adhesion molecule-1 (VCAM-1 in human umbilical vein endothelial cells (HUVECs. Ticlopidine declined U937 cells adhesion and chemotaxis as compared to TNF-α stimulated alone. Furthermore, the inhibitory effects were neither due to decreased HUVEC viability, nor through NF-kB inhibition. These results suggest that ticlopidine decreased TNF-α induced MCP-1, IL-8, and VCAM-1 levels in HUVECs, and monocyte adhesion. Therefore, the data provide additional therapeutic machinery of ticlopidine in treatment and prevention of atherosclerosis.

  8. The natural flavonoid apigenin suppresses Th1- and Th2-related chemokine production by human monocyte THP-1 cells through mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Huang, Ching-Hua; Kuo, Po-Lin; Hsu, Ya-Ling; Chang, Tai-Tsung; Tseng, Hsing-I; Chu, Yu-Te; Kuo, Chang-Hung; Chen, Huan-Nan; Hung, Chih-Hsing

    2010-04-01

    Dietary flavonoids have various biological functions, and there is increasing evidence that reduced prevalence and severity of allergic reactions are associated with the intake of flavonoids. Among natural flavonoids, apigenin is a potent anti-inflammatory agent. However, the mechanisms of apigenin's effect remain uncertain. Monocyte-derived chemokine (MDC) plays a pivotal role in recruiting T-helper (Th) 2 cells in the allergic inflammation process. In the late phase of allergic inflammation, the Th1 chemokine interferon-inducible protein 10 (IP-10) has also been found in elevated levels in the bronchial alveolar fluid of asthmatic children. We used human THP-1 monocyte cells, pretreated with or without apigenin, prior to lipopolysaccharide stimulation. By means of enzyme-linked immunosorbent assay, we found that apigenin inhibited production of both MDC and IP-10 by THP-1 cells and that the suppressive effect of apigenin was not reversed by the estrogen receptor antagonist ICI182780. The p65 phosphorylation of nuclear factor kappaB remained unaffected, but the phosphorylation of p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase mitogen-activated protein kinase pathways were all blocked. We found that inhibition of c-raf phosphorylation might be the target of apigenin's anti-inflammation property.

  9. Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells

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    Royere Dominique

    2006-03-01

    Full Text Available Abstract Background Zygote arrest 1 (ZAR1 is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. Methods Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. Results We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. Conclusion Our data suggest that in addition to its role in early embryo

  10. HIV-1 inhibits phagocytosis and inflammatory cytokine responses of human monocyte-derived macrophages to P. falciparum infected erythrocytes.

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    Louise E Ludlow

    Full Text Available HIV-1 infection increases the risk and severity of malaria by poorly defined mechanisms. We investigated the effect of HIV-1(Ba-L infection of monocyte-derived macrophages (MDM on phagocytosis of opsonised P. falciparum infected erythrocytes (IE and subsequent proinflammatory cytokine secretion. Compared to mock-infected MDM, HIV-1 infection significantly inhibited phagocytosis of IE (median (IQR (10 (0-28 versus (34 (27-108; IE internalised/100 MDM; p = 0.001 and decreased secretion of IL-6 (1,116 (352-3,387 versus 1,552 (889-6,331; pg/mL; p = 0.0078 and IL-1β (16 (7-21 versus 33 (27-65; pg/mL; p = 0.0078. Thus inadequate phagocytosis and cytokine production may contribute to impaired control of malaria in HIV-1 infected individuals.

  11. Nitric oxide is the key mediator of death induced by fisetin in human acute monocytic leukemia cells.

    Science.gov (United States)

    Ash, Dipankar; Subramanian, Manikandan; Surolia, Avadhesha; Shaha, Chandrima

    2015-01-01

    Nitric oxide (NO) has been shown to be effective in cancer chemoprevention and therefore drugs that help generate NO would be preferable for combination chemotherapy or solo use. This study shows a new evidence of NO as a mediator of acute leukemia cell death induced by fisetin, a promising chemotherapeutic agent. Fisetin was able to kill THP-1 cells in vivo resulting in tumor shrinkage in the mouse xenograft model. Death induction in vitro was mediated by an increase in NO resulting in double strand DNA breaks and the activation of both the extrinsic and the intrinsic apoptotic pathways. Double strand DNA breaks could be reduced if NO inhibitor was present during fisetin treatment. Fisetin also inhibited the downstream components of the mTORC1 pathway through downregulation of levels of p70 S6 kinase and inducing hypo-phosphorylation of S6 Ri P kinase, eIF4B and eEF2K. NO inhibition restored phosphorylation of downstream effectors of mTORC1 and rescued cells from death. Fisetin induced Ca(2+) entry through L-type Ca(2+) channels and abrogation of Ca(2+) influx reduced caspase activation and cell death. NO increase and increased Ca(2+) were independent phenomenon. It was inferred that apoptotic death of acute monocytic leukemia cells was induced by fisetin through increased generation of NO and elevated Ca(2+) entry activating the caspase dependent apoptotic pathways. Therefore, manipulation of NO production could be viewed as a potential strategy to increase efficacy of chemotherapy in acute monocytic leukemia.

  12. Impairment of in vitro generation of monocyte-derived human dendritic cells by inactivated human immunodeficiency virus-1: Involvement of type I interferon produced from plasmacytoid dendritc cells.

    Science.gov (United States)

    Kodama, Akira; Tanaka, Reiko; Zhang, Li Feng; Adachi, Tetsuya; Saito, Mineki; Ansari, Aftab A; Tanaka, Yuetsu

    2010-06-01

    In an attempt to simplify the protocol of DC generation in vitro, studies conducted herein show that functional DCs could be generated from bulk peripheral blood mononuclear cells (PBMCs) in media containing GM-CSF and IL-4. Interestingly, when PBMCs, but not purified monocytes, were exposed to either CCR5- or CXCR4-tropic inactivated HIV-1 isolates (iHIV-1) at the initiation of the culture, DC yields were significantly reduced in a dose-dependent manner because of monocyte apoptosis. Similar impairment of DC generation was noted using type I IFNs and poly IC not only in cultures of PBMCs but also using highly enriched monocytes. This effect was reversed by antihuman type I IFN receptor, but not by anti-FasL, anti-TRAIL, anti-TNF, or a mixture of these antibodies. iHIV-1-exposed PBMCs, but not monocytes, produced high levels of IFN-alpha but not IFN-beta. PBMCs depleted of CD123(+) plasmacytoid DCs produced low levels of IFN-alpha and were resistant to iHIV-1-mediated DC impairment. Interestingly, exogenously added TNF reversed the impairment by iHIV-1 in the PBMC cultures. In conclusion, the present results indicate that iHIV-1 impairs the in vitro generation of functional DCs from PBMCs through the induction of IFN-alpha from plasmacytoid DCs in a CD4-dependent fashion in the absence of TNF.

  13. PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme.

    Science.gov (United States)

    Tselepis, A D; Dentan, C; Karabina, S A; Chapman, M J; Ninio, E

    1995-10-01

    In human plasma, platelet activating factor (PAF)-degrading acetylhydrolase (acetylhydrolase) is principally transported in association with LDLs and HDLs; this enzyme hydrolyzes PAF and short-chain forms of oxidized phosphatidylcholine, transforming them into lyso-PAF and lysophosphatidylcholine, respectively. We have examined the distribution, catalytic characteristics, and transfer of acetylhydrolase activity among plasma lipoprotein subspecies separated by isopycnic density gradient ultracentrifugation; the possibility that the plasma enzyme may be partially derived from adherent monocytes has also been evaluated. In normolipidemic subjects with Lp(a) levels VHDL-1; d = 1.156 to 1.179 g/mL), representing 23.9 +/- 1.7% and 20.6 +/- 3.2%, respectively, of total plasma activity. The apparent Km values for PAF of the enzyme associated with such lipoproteins were 89.7 +/- 23.4 and 34.8 +/- 4.5 mumol/L for LDL-5 and VHDL-1, respectively: indeed, the Km value for LDL-5 was some 10-fold higher than that of the light LDL-1, LDL-2, and LDL-3 subspecies, whereas the Km of VHDL-1 was some twofold greater than those of the HDL-2 and HDL-3 subspecies. Furthermore, when expressed on the basis of unit plasma volume, the Vmax of the acetylhydrolase associated with LDL-5 was some 150-fold greater than that in LDL-1 (d = 1.019 to 1.023 g/mL). No significant differences in the pH dependence of enzyme activity or in sensitivity to protease inactivation, sulfydryl reagents, the serine protease inhibitor Pefabloc, or the PAF antagonist CV 3988 could be detected between apo B-containing and apo A-I-containing lipoprotein particle subspecies. Incubation of LDL-1 (Km = 8.4 +/- 2.6 mumol/L) and LDL-2 (d = 1.023 to 1.029 g/mL; Km = 8.4 +/- 3.3 mumol/L) subspecies with LDL-5, in which acetylhydrolase had been inactivated by pretreatment with Pefabloc, demonstrated preferential transfer of acetylhydrolase to LDL-5. Acetylhydrolase transferred to LDL-5 from the light LDL subspecies exhibited

  14. Induction of G1 arrest and apoptosis by schisandrin C isolated from Schizandra chinensis Baill in human leukemia U937 cells.

    Science.gov (United States)

    Park, Cheol; Choi, Young-Whan; Hyun, Sook Kyung; Kwon, Hyun Ju; Hwang, Hye Jin; Kim, Gi-Young; Choi, Byung Tae; Kim, Byung-Woo; Choi, Il-Whan; Moon, Sung-Kwon; Kim, Wun-Jae; Choi, Yung Hyun

    2009-10-01

    We isolated two phytochemical lignans, schisandrin and schisandrin C, from Schizandra chinensis Baill and investigated their anti-cancer effects in human leukemia U937 cells. Schisandrin C inhibited cell growth in a dose-dependent manner, which was associated with the induction of G1 arrest of the cell cycle and apoptosis; schisandrin did not inhibit growth. Schisandrin C induced G1 arrest was correlated with down-regulation of cyclin D1, cyclin E, cyclin-dependent kinase (Cdk) 4 and E2Fs expression, inhibition of phosphorylation of retinoblastoma protein (pRB), and up-regulation of the Cdk inhibitor p21(WAF1/CIP1). In addition, schisandrin C-induced apoptosis was associated with down-regulation of expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, proteolytic activation of caspase-3 and -9, and a concomitant degradation of poly(ADP-ribose) polymerase (PARP). Furthermore, schisandrin C-induced apoptosis was significantly inhibited by a caspase-3 specific inhibitor z-DEVD-fmk, indicating an important role for caspase-3 in the schisandrin C mechanism. In summary, growth inhibition by schisandrin C is related to cell cycle arrest at G1 and induction of caspase-3-dependent apoptosis in U937 cells; these findings suggest that schisandrin C may be a useful chemotherapeutic agent.

  15. Aqueous extract of Curcuma aromatica induces apoptosis and G2/M arrest in human colon carcinoma LS-174-T cells independent of p53.

    Science.gov (United States)

    Hu, Bing; Shen, Ke-Ping; An, Hong-Mei; Wu, Yang; Du, Qin

    2011-02-01

    Curcuma aromatica is a common Chinese herb for treating diseases with blood stasis and has been regarded as an anticancer herb in modern clinical practice. However, the anticancer effects and related molecular mechanisms of Curcuma aromatica remain unclear. In the present study, human colon carcinoma LS-174-T cell line with wild-type p53 was used as a model cell to evaluate the anticancer effects of aqueous extract of Curcuma aromatica (AECA). AECA inhibits LS-174-T cell proliferation in a dose- and time-dependent manner and colony formation in a dose-dependent manner. AECA treatment induces apoptosis accompanied by caspase-8, -9, and -3 activation in LS-174-T cells. Moreover, blocking the activities of these caspases with a specific inhibitor significantly protected LS-174-T cells from AECA-induced apoptosis. AECA treatment also induces G2/M phase arrest in LS-174-T cells. Expression of p53 was unchanged after AECA treatment; specific silence of p53 did not influence AECA-induced apoptosis and G2/M phase arrest. Further, the expression of cyclin B1 and CDK1 was reduced by AECA. This study suggests that AECA might be effective as an antiproliferative herb for colon carcinoma, the antitumor activity of AECA may involve both extrinsic and intrinsic apoptosis, and AECA induces G2/M phase arrest via downregulation of cyclin B1 and CDK1 and without the participation of p53.

  16. Human Herpesvirus-6 U14 Induces Cell-Cycle Arrest in G2/M Phase by Associating with a Cellular Protein, EDD.

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    Junko Mori

    Full Text Available The human herpesvirus-6 (HHV-6 infection induces cell-cycle arrest. In this study, we found that the HHV-6-encoded U14 protein induced cell-cycle arrest at G2/M phase via an association with the cellular protein EDD, a mediator of DNA-damage signal transduction. In the early phase of HHV-6 infection, U14 colocalized with EDD dots in the nucleus, and similar colocalization was also observed in cells transfected with a U14 expression vector. When the carboxyl-terminal region of U14 was deleted, no association of U14 and EDD was observed, and the percentage of cells in G2/M decreased relative to that in cells expressing wild-type U14, indicating that the C-terminal region of U14 and the U14-EDD association are critical for the cell-cycle arrest induced by U14. These results indicate that U14 is a G2/M checkpoint regulator encoded by HHV-6.

  17. The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells

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    Wu Xinjiang [Institute of Indoor and Environmental Toxicology, Faculty of Medicine, Justus-Liebig-University of Giessen, Aulweg 123, D-35385 Giessen (Germany); Kassie, Fekadu [Institute of Indoor and Environmental Toxicology, Faculty of Medicine, Justus-Liebig-University of Giessen, Aulweg 123, D-35385 Giessen (Germany); Mersch-Sundermann, Volker [Institute of Indoor and Environmental Toxicology, Faculty of Medicine, Justus-Liebig-University of Giessen, Aulweg 123, D-35385 Giessen (Germany)]. E-mail: Volker.mersch-sundermann@uniklinikum-giessen.de

    2005-11-11

    Diallyl disulfide (DADS), an oil soluble constituent of garlic (Allium sativum), has been reported to cause antimutagentic and anticarcinogenic effects in vitro and in vivo by modulating phases I and II enzyme activities. In recent years, several studies suggested that the chemopreventive effects of DADS can also be attributed to induction of cell cycle arrest and apoptosis in cancer cells. In the present study, we reported that DADS-induced cell cycle arrest at G2/M and apoptosis in human A549 lung cancer cells in a time- and dose-dependent manner. Additionally, a significant increase of intracellular reactive oxygen species (ROS) was induced in A549 cells less than 0.5 h after DADS treatment, indicating that ROS may be an early event in DADS-modulated apoptosis. Treatment of A549 cells with N-acetyl cysteine (NAC) completely abrogated DADS-induced cell cycle arrest and apoptosis. The result indicated that oxidative stress modulates cell proliferation and cell death induced by DADS.

  18. In vitro evidence for the protective role of Sida rhomboidea. Roxb extract against LDL oxidation and oxidized LDL-induced apoptosis in human monocyte-derived macrophages.

    Science.gov (United States)

    Thounaojam, Menaka C; Jadeja, Ravirajsinh N; Devkar, Ranjisinh V; Ramachandran, A V

    2011-06-01

    The present study was undertaken to evaluate protective role of S. rhomboidea. Roxb (SR) leaf extract against in vitro low-density lipoprotein (LDL) oxidation and oxidized LDL (Ox-LDL) induced macrophage apoptosis. Copper and cell-mediated LDL oxidation, Ox-LDL-induced peroxyl radical generation, mitochondrial activity, and apoptosis in human monocyte-derived macrophages (HMDMs) were assessed in presence of SR extract. Results clearly indicated that SR was capable of reducing LDL oxidation and formation of intermediary oxidation products. Also, SR successfully attenuated peroxyl radical formation, mitochondrial dysfunction, nuclear condensation, and apoptosis in Ox-LDL-exposed HMDMs. This scientific report is the first detailed investigation that establishes anti-atherosclerotic potential of SR extract.

  19. Inhibitive effect of 3-bromopyruvic acid on human breast cancer MCF-7 cells involves cell cycle arrest and apoptotic induction

    Institute of Scientific and Technical Information of China (English)

    LIU Xiao-hong; ZHENG Xue-fang; WANG Yong-li

    2009-01-01

    Background Breast cancer is one of the most common malignancies in women and is highly resistant to chemotherapy. Due to its high tumour selectivity, 3-bromopyruvic acid (3-BrPA), a well-known inhibitor of energy metabolism has been proposed as a specific anticancer agent. The present study determined the effect of 3-BrPA on proliferation, cell cycle and apoptosis in the human breast cancer MCF-7 cell line and other antitumour mechanisms. Methods MCF-7 cells were treated with various concentrations of 3-BrPA for 1-4 days, and cell growth was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Marked morphological changes in MCF-7 cells after treatment with 3-BrPA were observed using transmission electron microscopy. The distributions of the cell cycle and apoptosis were analyzed by flow cytometry. Immunohistochemistry was used to indicate the changes in the expression of Bcl-2, c-Myc, and mutant p53. Results 3-BrPA (25 μg/ml) significantly inhibited the proliferation of MCF-7 cells in a time-dependent manner. The MCF-7 cells exposed to 3-BrPA showed the typical morphological characteristics of apoptosis, including karyopycnosis, nuclear condensation and oversize cytoplasmic particles. In addition, flow cytometric assay also showed more apoptotic cells after 3-BrPA stimulation. The cells at the GO and G1 phases were dramatically decreased while cells at the S and G2/M phases were increased in response to 3-BrPA treatment after 48 hours. Furthermore, 3-BrPA stimulation decreased the expressions of Bcl-2, c-Myc and mutant p53, which were strongly associated with the programmed cell death signal transduction pathway. Conclusion 3-BrPA inhibits proliferation, induces S phase and G2/M phase arrest, and promotes apoptosis in MCF-7 cells, which processes might be mediated by the downregulation of the expressions of Bcl-2, c-Myc and mutant p53.

  20. Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    V. Vucic

    2006-02-01

    Full Text Available Gamma-irradiation (gamma-IR is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD, specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001 antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control to 49% (IR cells, with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

  1. 5-(2-Carboxyethenyl) isatin derivative induces G{sub 2}/M cell cycle arrest and apoptosis in human leukemia K562 cells

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    Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin; Yang, Yao; Song, Bin-Bin; Guo, Qian-Nan [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Fan, Zhen-Chuan [Key Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Ministry of Education, Tianjin 300457 (China); Obesita and Algaegen LLC, College Station, TX 77845 (United States); Zhang, Yong-Min [Université Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moléculaire UMR CNRS 8232, 4 Place Jussieu, 75005 Paris (France); Teng, Yu-Ou, E-mail: tyo201485@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Yu, Peng, E-mail: yupeng@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China)

    2014-08-08

    Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G{sub 2}/M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC{sub 50}) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G{sub 2}/M phase and accumulated subsequently in the sub-G{sub 1} phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G{sub 2}/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.

  2. Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Vucic V.

    2006-01-01

    Full Text Available Gamma-irradiation (gamma-IR is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD, specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001 antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control to 49% (IR cells, with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

  3. Involvement of the p38 MAPK signaling pathway in S-phase cell-cycle arrest induced by Furazolidone in human hepatoma G2 cells.

    Science.gov (United States)

    Sun, Yu; Tang, Shusheng; Jin, Xi; Zhang, Chaoming; Zhao, Wenxia; Xiao, Xilong

    2013-12-01

    Given the previously described essential role for the p38 mitogen-activation protein kinase (p38 MAPK) signaling pathway in human hepatoma G2 cells (HepG2), we undertook the present study to investigate the role of the p38 MAPK signaling pathway in cell-cycle arrest induced by Furazolidone (FZD). The aim of this study was to determine the effects of FZD on HepG2 cells by activating and inhibiting the p38 MAPK signaling pathway. The cell cycle and proliferation of HepG2 cells treated with FZD were detected by flow cytometry and MTT assay in the presence or absence of p38 MAPK inhibitors (SB203580), respectively. Cyclin D1, cyclin D3 and CDK6 were detected by quantitative real-time PCR and western blot analysis. Our data showed that p38 MAPK became phosphorylated after stimulation with FZD. Activation of p38 MAPK could arise S-phase cell-cycle arrest and suppress cell proliferation. Simultaneously, inhibition of the p38 MAPK signaling pathway significantly prevented S-phase cell-cycle arrest, increased the percentage of cell viability and decreased the expression of cyclin D1, cyclin D3 and CDK6. These results demonstrated that FZD arose S-phase cell-cycle arrest via activating the p38 MAPK signaling pathway in HepG2 cells. Cyclin D1, cyclin D3 and CDK6 are target genes functioning at the downstream of p38 MAPK in HepG2 cells induced by FZD.

  4. Both common and specialty mushrooms inhibit adhesion molecule expression and in vitro binding of monocytes to human aortic endothelial cells in a pro-inflammatory environment

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    Martin Keith R

    2010-07-01

    Full Text Available Abstract Background Cardiovascular disease (CVD is a leading cause of mortality in the United States as well as globally. Epidemiological studies show that regular fruit and vegetable consumption reduces CVD risk, in part, due to antioxidant activity and immunomodulation since oxidative stress and inflammation are features of atherogenesis. Accumulating evidence also shows that dietary fungi, viz., mushrooms, can protect against chronic disease by altering inflammatory environments such as those associated with CVD although most research has focused on specialty mushrooms. In this study, we tested the ability of both common and specialty mushrooms to inhibit cellular processes associated with CVD. Methods Human aortic endothelial cells (HAEC were incubated overnight with control media with dimethylsulfoxide (DMSO vehicle (1% v/v or containing DMSO extracts of whole dehydrated mushrooms (0.1 mg/mL, which included Agaricus bisporus (white button and crimini, Lentinula edodes (shiitake, Pleurotus ostreatus (oyster, and Grifola frondosa (maitake. Monolayers were subsequently washed and incubated with medium alone or containing the pro-inflammatory cytokine IL-1β (5 ng/mL for 6 h to upregulate pro-atherosclerotic adhesion molecules (AM. AM expression was assayed by ELISA and binding of U937 human monocytes pre-loaded with fluorescent dye was determined. Results White button mushrooms consistently reduced (p Conclusion These data provide evidence that dietary mushrooms can inhibit cellular processes such as adhesion molecule expression and ultimate binding of monocytes to the endothelium under pro-inflammatory conditions, which are associated with CVD. As a result, these findings support the notion that dietary mushrooms can be protective against CVD.

  5. Knockdown of p54nrb inhibits migration, invasion and TNF-α release of human acute monocytic leukemia THP1 cells.

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    Zhang, Xiujuan; Wu, Changli; Xiong, Wei; Chen, Chunling; Li, Rong; Zhou, Guangji

    2016-06-01

    54 kDa nuclear RNA- and DNA-binding protein (p54nrb) which is also called non-POU domain-containing octamer-binding protein (NONO) is known to be multifunctional involved in many nuclear processes. It was shown that p54nrb/NONO was closely related to the occurrence of erythroleukemia. Whether p54nrb/NONO plays a role in progress of human acute monocytic leukemia remains unknown. In the present study, we examined the effects of p54nrb/NONO silencing on the biological characteristics of human acute monocytic leukemia THP1 cells. The results showed that p54nrb was strongly expressed in THP1 cells, and knockdown of p54nrb slightly promoted proliferation and strongly inhibited motility and invasion of THP1 cells. Moreover, knockdown of p54nrb strongly decreased the release of TNF-α from THP1 cells by inhibiting certain process of TNF-α secretion, specially for the release of TNF-α induced by lipopolysaccharide (LPS). Notably, the infection of negative control shRNA-containing lentiviruses promoted the migration and the release of TNF-α induced by LPS in THP1 cells. All the above results demonstrated that p54nrb slightly inhibited THP1 cell proliferation, but significantly promoted migration, invasion and release of TNF-α induced by LPS in THP1 cells. The present study indicates that p54nrb is a powerful molecule involved in the regulation of cell motility and promotes the migration and invasion of THP1 cells, and it is more likely to be involved in the release of inflammatory mediators and the motility of inflammatory cells.

  6. A novel class I histone deacetylase inhibitor, I-7ab, induces apoptosis and arrests cell cycle progression in human colorectal cancer cells.

    Science.gov (United States)

    Yang, Liyan; Liang, Qiannan; Shen, Ke; Ma, Li; An, Na; Deng, Weiping; Fei, Zhewei; Liu, Jianwen

    2015-04-01

    Epigenetic mutations are closely associated with human diseases, especially cancers. Among them, dysregulations of histone deacetylases (HDACs) are commonly observed in human cancers. Recent years, HDAC inhibitors have been identified as promising anticancer agents; several HDAC inhibitors have been applied in clinical practice. In this study, we synthesized a novel N-hydroxyacrylamide-derived HDAC inhibitor, I-7ab, and examined its antitumor activity. Our investigations demonstrated that I-7ab exerted cytotoxicity toward and inhibited the growth of human cancer cell lines at micromolar concentrations. Among tested cells, HCT116 was the most sensitive one to the treatment of I-7ab. However, I-7ab displayed far less cytotoxicity in human normal cells. In HCT116 cells, I-7ab inhibited the expression of class I HDACs, especially that of HDAC3, and suppressed EGFR signaling pathway. With respect to the cytotoxic effect of I-7ab, it induced apoptosis via increasing the Bax/Bcl-2 ratio and suppressing the translocation of NF-κB. Other than inducing apoptosis, I-7ab inhibited the expression of cyclin B1 and thereby arrests cell cycle progression at G2/M phase. Further analyses revealed potential role of p53 and p21 in I-7ab-induced apoptosis and cell cycle arrest. According to our findings, I-7ab may serve as a lead compound for potential antitumor drugs.

  7. Arrest functions of the MIF ligand/receptor axes in atherogenesis

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    Sabine eTillmann

    2013-05-01

    Full Text Available Macrophage migration inhibitory factor (MIF has been defined as an important chemokine-like function (CLF chemokine with an essential role in monocyte recruitment and arrest. Adhesion of monocytes to the vessel wall and their transendothelial migration are critical in atherogenesis and many other inflammatory diseases. Chemokines carefully control all steps of the monocyte recruitment process. Those chemokines specialized in controlling arrest are typically immobilized on the endothelial surface, mediating the arrest of rolling monocytes by chemokine receptor-triggered pathways. The chemokine receptor CXCR2 functions as an important arrest receptor on monocytes. An arrest function has been revealed for the bona fide CXCR2 ligands CXCL1 and CXCL8, but genetic studies also suggested that additional arrest chemokines are likely to be involved in atherogenic leukocyte recruitment. While CXCR2 is known to interact with numerous CXC chemokine ligands, the CLF-chemokine MIF, which structurally does not belong to the CXC chemokine sub-family, was surprisingly identified as a non-cognate ligand of CXCR2, responsible for critical arrest functions during the atherogenic process. MIF was originally identified as macrophage migration inhibitory factor, but is now known as a potent inflammatory cytokine with chemokine-like functions including chemotaxis and leukocyte arrest. This review will cover the mechanisms underlying these functions, including MIF’s effects on LFA1 integrin activity and signal transduction, and will discuss the structural similarities between MIF and the bona fide CXCR2 ligand CXCL8 while emphasizing the structural differences. As MIF also interacts with CXCR4, a chemokine receptor implicated in CXCL12-elicited lymphocyte arrest, the arrest potential of the MIF/CXCR4 axis will also be scrutinized as well as the recently identified role of pericyte MIF in attracting leukocytes exiting through venules as part of the pericyte 'motility

  8. Overexpression of the promyelocytic leukemia gene suppresses growth of human bladder cancer cells by inducing G1 cell cycle arrest and apoptosis

    Institute of Scientific and Technical Information of China (English)

    HE Dalin 贺大林; NAN Xunyi 南勋义; Chang Kun-Song; WANG Yafeng 王亚峰; Chung Leland W.K.

    2003-01-01

    Objectives To examine the anti-oncogenic effects of promyelocytic leukemia (PML) on bladder cancer and to explore its molecular mechanisms of growth suppression.Methods Wild-type PML was transfected into bladder cancer cells (5637 cell) and expressed in a replication-deficient adenovirus-mediated gene delivery system and introduced into human bladder cancer cells (5637 cell) in vitro and in vivo. The effect and mechanisms of the PML gene in cell growth, clonogenicity, and tumorigenicity of bladder cancer cells were studied using in vitro and in vivo growth assays, soft agar colony-forming assay, cell cycle analysis, apoptosis assay and in vivo tumorigenicity assay.Results Overexpression of PML in 5637 cells significantly reduced their growth rate and clonogenicity on soft agar. PML suppressed bladder cancer cell growth by inducing G1 cell cycle arrest and apoptosis. Adenovirus-mediated PML (Ad-PML) significantly suppressed the tumorigenicity and growth of bladder cancer cells. Intratumoral injection of Ad-PML into tumors induced by 5637 cells dramatically suppressed their growth. Conclusions The results indicated that overexpression of PML protein may promote efficient growth inhibition of human bladder cancer cells by inducing G1 cell cycle arrest and apoptosis, and adenovirus-mediated PML (Ad-PML) expression efficiently suppresses human bladder cancer growth.

  9. Hinokitiol inhibits cell growth through induction of S-phase arrest and apoptosis in human colon cancer cells and suppresses tumor growth in a mouse xenograft experiment.

    Science.gov (United States)

    Lee, Youn-Sun; Choi, Kyeong-Mi; Kim, Wonkyun; Jeon, Young-Soo; Lee, Yong-Moon; Hong, Jin-Tae; Yun, Yeo-Pyo; Yoo, Hwan-Soo

    2013-12-27

    Hinokitiol (1), a tropolone-related natural compound, induces apoptosis and has anti-inflammatory, antioxidant, and antitumor activities. In this study, the inhibitory effects of 1 were investigated on human colon cancer cell growth and tumor formation of xenograft mice. HCT-116 and SW-620 cells derived from human colon cancers were found to be similarly susceptible to 1, with IC50 values of 4.5 and 4.4 μM, respectively. Compound 1 induced S-phase arrest in the cell cycle progression and decreased the expression levels of cyclin A, cyclin E, and Cdk2. Conversely, 1 increased the expression of p21, a Cdk inhibitor. Compound 1 decreased Bcl-2 expression and increased the expression of Bax, and cleaved caspase-9 and -3. The effect of 1 on tumor formation when administered orally was evaluated in male BALB/c-nude mice implanted intradermally separately with HCT-116 and SW-620 cells. Tumor volumes and tumor weights in the mice treated with 1 (100 mg/kg) were decreased in both cases. These results suggest that the suppression of tumor formation by compound 1 in human colon cancer may occur through cell cycle arrest and apoptosis.

  10. Cis and trans acting factors involved in human cytomegalovirus experimental and natural latent infection of CD14 (+ monocytes and CD34 (+ cells.

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    Cyprian C Rossetto

    Full Text Available The parameters involved in human cytomegalovirus (HCMV latent infection in CD14 (+ and CD34 (+ cells remain poorly identified. Using next generation sequencing we deduced the transcriptome of HCMV latently infected CD14 (+ and CD34 (+ cells in experimental as well as natural latency settings. The gene expression profile from natural infection in HCMV seropositive donors closely matched experimental latency models, and included two long non-coding RNAs (lncRNAs, RNA4.9 and RNA2.7 as well as the mRNAs encoding replication factors UL84 and UL44. Chromatin immunoprecipitation assays on experimentally infected CD14 (+ monocytes followed by next generation sequencing (ChIP-Seq were employed to demonstrate both UL84 and UL44 proteins interacted with the latent viral genome and overlapped at 5 of the 8 loci identified. RNA4.9 interacts with components of the polycomb repression complex (PRC as well as with the MIE promoter region where the enrichment of the repressive H3K27me3 mark suggests that this lncRNA represses transcription. Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE, which identifies nucleosome-depleted viral DNA, was used to confirm that latent mRNAs were associated with actively transcribed, FAIRE analysis also showed that the terminal repeat (TR region of the latent viral genome is depleted of nucleosomes suggesting that this region may contain an element mediating viral genome maintenance. ChIP assays show that the viral TR region interacts with factors associated with the pre replication complex and a plasmid subclone containing the HCMV TR element persisted in latently infected CD14 (+ monocytes, strongly suggesting that the TR region mediates viral chromosome maintenance.

  11. Surface modification of biomaterials based on high-molecular polylactic acid and their effect on inflammatory reactions of primary human monocyte-derived macrophages: perspective for personalized therapy.

    Science.gov (United States)

    Stankevich, Ksenia S; Gudima, Alexandru; Filimonov, Victor D; Klüter, Harald; Mamontova, Evgeniya M; Tverdokhlebov, Sergei I; Kzhyshkowska, Julia

    2015-06-01

    Polylactic acid (PLA) based implants can cause inflammatory complications. Macrophages are key innate immune cells that control inflammation. To provide higher biocompatibility of PLA-based implants with local innate immune cells their surface properties have to be improved. In our study surface modification technique for high-molecular PLA (MW=1,646,600g/mol) based biomaterials was originally developed and successfully applied. Optimal modification conditions were determined. Treatment of PLA films with toluene/ethanol=3/7 mixture for 10min with subsequent exposure in 0.001M brilliant green dye (BGD) solution allows to entrap approximately 10(-9)mol/cm(2) model biomolecules. The modified PLA film surface was characterized by optical microscopy, SERS, FT-IR, UV and TG/DTA/DSC analysis. Tensile strain of modified films was determined as well. The effect of PLA films modified with BGD on the inflammatory reactions of primary human monocyte-derived macrophages was investigated. We developed in vitro test-system by differentiating primary monocyte-derived macrophages on a coating material. Type 1 and type 2 inflammatory cytokines (TNFα, CCL18) secretion and histological biomarkers (CD206, stabilin-1) expression were analyzed by ELISA and confocal microscopy respectively. BGD-modified materials have improved thermal stability and good mechanical properties. However, BGD modifications induced additional donor-specific inflammatory reactions and suppressed tolerogenic phenotype of macrophages. Therefore, our test-system successfully demonstrated specific immunomodulatory effects of original and modified PLA-based biomaterials, and can be further applied for the examination of improved coatings for implants and identification of patient-specific reactions to implants.

  12. Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells

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    Stefanie Hoyer

    2015-01-01

    Full Text Available For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs. However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs’ immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1, and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

  13. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

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    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  14. Mechanisms of modulation of cytokine release by human cord blood monocytes exposed to high concentrations of caffeine

    Science.gov (United States)

    Chavez-Valdez, Raul; Ahlawat, Rajni; Wills-Karp, Marsha; Gauda, Estelle B.

    2016-01-01

    Background Serum caffeine concentrations >20µg/mL (100 µM) in infants treated for apnea of prematurity increases TNF-α and decreases IL-10, change that perhaps is linked to co-morbidities. We hypothesize that this pro-inflammatory cytokine profile may be linked to differential binding of caffeine to adenosine receptor subtypes (AR), inhibition of phosphodiesterases (PDEs), and modulation of toll-like receptors (TLR). Methods LPS-activated cord blood monocytes (CBM) from 19 infants were exposed to caffeine (0 to 200 µM) with or without previous exposure to A1R, A3R, or PDE IV antagonists to determine changes in dose-response curves. Cytokines levels (ELISA), intracellular cAMP accumulation (EIA) and TLR gene expression (real time qRT PCR) were measured. Results Caffeine at ≤100µM decreased TNF-α levels (~25%, p=0.01) and cAMP. All caffeine concentrations decreased IL-10 levels (17 to 35%, p<0.01). A1R, A3R and PDE blockades decreased TNF-α (31%, 21%, and 88%, p≤0.01), but not IL-10. Caffeine further decreased TNF-α following A3R and PDE blockades. Caffeine concentrations directly correlated to TLR4 gene expression (r=0.84; p<0.001). Conclusion Neither A3R, nor PDE blockades are involved in caffeine’s modulation of cytokine release by CBM at any concentration. Besides A1R blockade, caffeine’s up-regulation of TLR4 may promote inflammation at high concentrations. PMID:26982450

  15. KU004 induces G1 cell cycle arrest in human breast cancer SKBR-3 cells by modulating PI3K/Akt pathway.

    Science.gov (United States)

    Fu, Jing; Tian, Chongchong; Xing, Mengtao; Wang, Xinzhi; Guo, Hongli; Sun, Lixin; Sun, Lan; Jiang, Zhenzhou; Zhang, Luyong

    2014-06-01

    KU004 is a newly synthesized compound which has been demonstrated possessing potent anti-cancer activities through targeting the highly-expressed protein HER2 on the surface of the cells. In this study, we investigated the potential roles of KU004 in the induced-cell cycle arrest in human breast cancer SK-BR-3 cells. KU004 could not only inhibit the proliferation of SK-BR-3 in a concentration-dependent manner but also induce G1 phase arrest in SK-BR-3 cells. The western blot results showed KU004 decreased the expression of cyclin D, CDK-4, p-Rb708/780, and up-regulated the p21. In order to verify whether KU004 takes the anti-tumor effect thought the regulation of PI3K/Akt pathway, we used western blot to detect the expression of protein Akt, Her2, p-Akt and p-Her2. Our results shown that after KU004 treatment, the amount of p-Akt and p-Her2 decreased but the total amount of Akt and Her2 remained unchanged. In conclusion, these results provide a framework for further exploration of KU004 as a novel chemotherapeutic for human breast tumors by modulating PI3K/Akt pathway.

  16. Antrodia camphorata induces G(1) cell-cycle arrest in human premyelocytic leukemia (HL-60) cells and suppresses tumor growth in athymic nude mice.

    Science.gov (United States)

    Yang, Hsin-Ling; Kumar, K J Senthil; Kuo, Ya-Ting; Chang, Hebron C; Liao, Jiunn-Wang; Hsu, Li-Sung; Hseu, You-Cheng

    2014-09-01

    Antrodia camphorata is a well-known medicinal mushroom in Taiwan. The broth from a fermented culture of Antrodia camphorata (AC) has been shown to induce apoptosis in cultured human premyelocytic leukemia (HL-60) cells. In the present study, we examined the effects of AC on cell cycle arrest in vitro in HL-60 cells and on tumor regression in vivo using an athymic nude mouse model. We found that AC (20-80 μg mL(-1)) treatment significantly induced G1 cell-cycle arrest in HL-60 cells by reducing the levels of cyclin D1, CDK4, cyclin E, CDK2, cyclin A, and phosphorylation of retinoblastoma protein (p-Rb). Moreover, AC treatment led to significantly increased protein expression levels of CDK inhibitors, including p21(WAF1) and p15(NIK4B). Additionally, AC treatment markedly induced intracellular ROS generation and mitochondrial dysfunction in HL-60 cells. Furthermore, the in vivo study results revealed that AC treatment was effective in terms of delaying the tumor incidence in nude mice that had been inoculated with HL-60 cells as well as in reducing the tumor burden. Histological analysis confirmed that AC treatment significantly modulated the xenografted tumor progression as demonstrated by a reduction in mitotic cells. Our data strongly suggest that Antrodia camphorata could be an anti-cancer agent for human leukemia.

  17. Source and Purity of Dengue-Viral Preparations Impact Requirement for Enhancing Antibody to Induce Elevated IL-1β Secretion: A Primary Human Monocyte Model.

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    Justin B Callaway

    Full Text Available Dengue virus is a major global health threat and can lead to life-threatening hemorrhagic complications due to immune activation and cytokine production. Cross-reactive antibodies to an earlier dengue virus infection are a recognized risk factor for severe disease. These antibodies bind heterologous dengue serotypes and enhance infection into Fc-receptor-bearing cells, a process known as antibody-dependent enhancement of infection. One crucial cytokine seen elevated in severe dengue patients is IL-1β, a potent inflammatory cytokine matured by the inflammasome. We used a highly-physiologic system by studying antibody-dependent enhancement of IL-1β in primary human monocytes with anti-dengue human monoclonal antibodies isolated from patients. Antibody-enhancement increased viral replication in primary human monocytes inoculated with supernatant harvested from Vero cells infected with dengue virus serotype 2 (DENV-2 16681. Surprisingly, IL-1β secretion induced by infectious supernatant harvested from two independent Vero cell lines was not enhanced by antibody. Secretion of multiple other inflammatory cytokines was also independent of antibody signaling. However, IL-1β secretion did require NLRP3 and caspase-1 activity. Immunodepletion of dengue virions from the infectious supernatant confirmed that virus was not the main IL-1β-inducing agent, suggesting that a supernatant component(s not associated with the virion induced IL-1β production. We excluded RNA, DNA, contaminating LPS, viral NS1 protein, complement, and cytokines. In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction. Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion. This study indicates that Vero cells infected with DENV-2 16681 may produce inflammatory components

  18. Source and Purity of Dengue-Viral Preparations Impact Requirement for Enhancing Antibody to Induce Elevated IL-1β Secretion: A Primary Human Monocyte Model.

    Science.gov (United States)

    Callaway, Justin B; Smith, Scott A; Widman, Douglas G; McKinnon, Karen P; Scholle, Frank; Sempowski, Gregory D; Dittmer, Dirk P; Crowe, James E; de Silva, Aravinda M; Ting, Jenny P-Y

    2015-01-01

    Dengue virus is a major global health threat and can lead to life-threatening hemorrhagic complications due to immune activation and cytokine production. Cross-reactive antibodies to an earlier dengue virus infection are a recognized risk factor for severe disease. These antibodies bind heterologous dengue serotypes and enhance infection into Fc-receptor-bearing cells, a process known as antibody-dependent enhancement of infection. One crucial cytokine seen elevated in severe dengue patients is IL-1β, a potent inflammatory cytokine matured by the inflammasome. We used a highly-physiologic system by studying antibody-dependent enhancement of IL-1β in primary human monocytes with anti-dengue human monoclonal antibodies isolated from patients. Antibody-enhancement increased viral replication in primary human monocytes inoculated with supernatant harvested from Vero cells infected with dengue virus serotype 2 (DENV-2) 16681. Surprisingly, IL-1β secretion induced by infectious supernatant harvested from two independent Vero cell lines was not enhanced by antibody. Secretion of multiple other inflammatory cytokines was also independent of antibody signaling. However, IL-1β secretion did require NLRP3 and caspase-1 activity. Immunodepletion of dengue virions from the infectious supernatant confirmed that virus was not the main IL-1β-inducing agent, suggesting that a supernatant component(s) not associated with the virion induced IL-1β production. We excluded RNA, DNA, contaminating LPS, viral NS1 protein, complement, and cytokines. In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction. Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion. This study indicates that Vero cells infected with DENV-2 16681 may produce inflammatory components during dengue virus

  19. HIV-1 Latency in Monocytes/Macrophages

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    Amit Kumar

    2014-04-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 targets CD4+ T cells and cells of the monocyte/macrophage lineage. HIV pathogenesis is characterized by the depletion of T lymphocytes and by the presence of a population of cells in which latency has been established called the HIV-1 reservoir. Highly active antiretroviral therapy (HAART has significantly improved the life of HIV-1 infected patients. However, complete eradication of HIV-1 from infected individuals is not possible without targeting latent sources of infection. HIV-1 establishes latent infection in resting CD4+ T cells and findings indicate that latency can also be established in the cells of monocyte/macrophage lineage. Monocyte/macrophage lineage includes among others, monocytes, macrophages and brain resident macrophages. These cells are relatively more resistant to apoptosis induced by HIV-1, thus are important stable hideouts of the virus. Much effort has been made in the direction of eliminating HIV-1 resting CD4+ T-cell reservoirs. However, it is impossible to achieve a cure for HIV-1 without considering these neglected latent reservoirs, the cells of monocyte/macrophage lineage. In this review we will describe our current understanding of the mechanism of latency in monocyte/macrophage lineage and how such cells can be specifically eliminated from the infected host.

  20. Monocytic HLA DR antigens in schizophrenic patients.

    Science.gov (United States)

    Krause, Daniela; Wagner, Jenny; Matz, Judith; Weidinger, Elif; Obermeier, Michael; Riedel, Michael; Gruber, Rudolf; Schwarz, Markus; Mueller, Norbert

    2012-01-01

    A genetic association of specific human leukocyte antigens (HLA) DR genes and schizophrenia has recently been shown. These HLA play a fundamental role in the control of immune responses. Furthermore infectious agents have been proposed to be involved in the pathogenesis of schizophrenia. In this study we investigated the rate of HLA DR positive monocytes in schizophrenic patients compared to controls with a special focus on the adaption to in vitro stimulation with toll-like receptor ligands. Patients with schizophrenia and matched controls were included. For each individual, we evaluated the rate of HLA DR positive monocytes (either incubated at 37 °C or after stimulation with lipopolysaccharide or Poly I:C). We found a significantly higher percentage of schizophrenic patients with elevated HLA DR positive cells (p=0.045) as compared to controls. The adjustment rate from baseline levels of monocytic HLA DR positive cells to stimulation with Poly I:C was significantly lower in schizophrenic patients (p=0.038). The increased monocytic HLA DR in schizophrenic patients and the maladjustment of their monocytic HLA DR levels to an infectious stimulus might be a sign for a disturbed monocytic immune balance in schizophrenic individuals.

  1. Human XCR1+ dendritic cells derived in vitro from CD34+ progenitors closely resemble blood dendritic cells, including their adjuvant responsiveness, contrary to monocyte-derived dendritic cells.

    Science.gov (United States)

    Balan, Sreekumar; Ollion, Vincent; Colletti, Nicholas; Chelbi, Rabie; Montanana-Sanchis, Frédéric; Liu, Hong; Vu Manh, Thien-Phong; Sanchez, Cindy; Savoret, Juliette; Perrot, Ivan; Doffin, Anne-Claire; Fossum, Even; Bechlian, Didier; Chabannon, Christian; Bogen, Bjarne; Asselin-Paturel, Carine; Shaw, Michael; Soos, Timothy; Caux, Christophe; Valladeau-Guilemond, Jenny; Dalod, Marc

    2014-08-15

    Human monocyte-derived dendritic cell (MoDC) have been used in the clinic with moderately encouraging results. Mouse XCR1(+) DC excel at cross-presentation, can be targeted in vivo to induce protective immunity, and share characteristics with XCR1(+) human DC. Assessment of the immunoactivation potential of XCR1(+) human DC is hindered by their paucity in vivo and by their lack of a well-defined in vitro counterpart. We report in this study a protocol generating both XCR1(+) and XCR1(-) human DC in CD34(+) progenitor cultures (CD34-DC). Gene expression profiling, phenotypic characterization, and functional studies demonstrated that XCR1(-) CD34-DC are similar to canonical MoDC, whereas XCR1(+) CD34-DC resemble XCR1(+) blood DC (bDC). XCR1(+) DC were strongly activated by polyinosinic-polycytidylic acid but not LPS, and conversely for MoDC. XCR1(+) DC and MoDC expressed strikingly different patterns of molecules involved in inflammation and in cross-talk with NK or T cells. XCR1(+) CD34-DC but not MoDC efficiently cross-presented a cell-associated Ag upon stimulation by polyinosinic-polycytidylic acid or R848, likewise to what was reported for XCR1(+) bDC. Hence, it is feasible to generate high numbers of bona fide XCR1(+) human DC in vitro as a model to decipher the functions of XCR1(+) bDC and as a potential source of XCR1(+) DC for clinical use.

  2. Wear particles from studded tires and granite pavement induce pro-inflammatory alterations in human monocyte-derived macrophages: a proteomic study.

    Science.gov (United States)

    Karlsson, Helen; Lindbom, John; Ghafouri, Bijar; Lindahl, Mats; Tagesson, Christer; Gustafsson, Mats; Ljungman, Anders G

    2011-01-14

    Airborne particulate matter is considered to be one of the environmental contributors to the mortality in cancer, respiratory, and cardiovascular diseases. For future preventive actions, it is of major concern to investigate the toxicity of defined groups of airborne particles and to clarify their pathways in biological tissues. To expand the knowledge beyond general inflammatory markers, this study examined the toxicoproteomic effects on human monocyte derived macrophages after exposure to wear particles generated from the interface of studded tires and a granite-containing pavement. As comparison, the effect of endotoxin was also investigated. The macrophage proteome was separated using two-dimensional gel electrophoresis. Detected proteins were quantified, and selected proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Among analyzed proteins, seven were significantly decreased and three were increased by exposure to wear particles as compared to unexposed control cells. Endotoxin exposure resulted in significant changes in the expression of six proteins: four decreased and two increased. For example, macrophage capping protein was significantly increased after wear particle exposure only, whereas calgizzarin and galectin-3 were increased by both wear particle and endotoxin exposure. Overall, proteins associated with inflammatory response were increased and proteins involved in cellular functions such as redox balance, anti-inflammatory response, and glycolysis were decreased. Investigating the effects of characterized wear particles on human macrophages with a toxicoproteomic approach has shown to be useful in the search for more detailed information about specific pathways and possible biological markers.

  3. Malarial pigment haemozoin, IFN-gamma, TNF-alpha, IL-1beta and LPS do not stimulate expression of inducible nitric oxide synthase and production of nitric oxide in immuno-purified human monocytes

    Directory of Open Access Journals (Sweden)

    Ceretto Monica

    2007-06-01

    Full Text Available Abstract Background Enhanced production of nitric oxide (NO following upmodulation of the inducible isoform of NO synthase (iNOS by haemozoin (HZ, inflammatory cytokines and LPS may provide protection against Plasmodium falciparum malaria by killing hepatic and blood forms of parasites and inhibiting the cytoadherence of parasitized erythrocytes (RBC to endothelial cells. Monocytes and macrophages are considered to contribute importantly to protective upregulation of iNOS and production of NO. Data obtained with murine phagocytes fed with human HZ and synthetic HZ (sHZ indicate that supplemental treatment of those cells with IFN-gamma elicited significant increases in protein and mRNA expression of iNOS and NO production, providing a potential mechanism linking HZ phagocytosis and increased production of NO. Purpose of this study was to analyse the effect of P. falciparum HZ and sHZ supplemental to treatment with IFN-gamma and/or a stimulatory cytokine-LPS mix on iNOS protein and mRNA expression in immuno-purified human monocytes. Methods Adherent immunopurified human monocytes (purity >85%, and murine phagocytic cell lines RAW 264.7, N11 and ANA1 were fed or not with P. falciparum HZ or sHZ and treated or not with IFN-gamma or a stimulatory cytokine-LPS mix. Production of NO was quantified in supernatants, iNOS protein and mRNA expression were measured after immunoprecipitation and Western blotting and quantitative RT-PCT, respectively. Results Phagocytosis of HZ/sHZ by human monocytes did not increase iNOS protein and mRNA expression and NO production either after stimulation by IFN-gamma or the cytokine-LPS mix. By contrast, in HZ/sHZ-laden murine macrophages, identical treatment with IFN-gamma and the cytokine-LPS mix elicited significant increases in protein and mRNA expression of iNOS and NOS metabolites production, in agreement with literature data. Conclusion Results indicate that human monocytes fed or not with HZ/sHZ were constantly

  4. Effects of Cigarette Smoke Condensate on Oxidative Stress, Apoptotic Cell Death, and HIV Replication in Human Monocytic Cells.

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    Pss Rao

    Full Text Available While cigarette smoking is prevalent amongst HIV-infected patients, the effects of cigarette smoke constituents in cells of myeloid lineage are poorly known. Recently, we have shown that nicotine induces oxidative stress through cytochrome P450 (CYP 2A6-mediated pathway in U937 monocytic cells. The present study was designed to examine the effect of cigarette smoke condensate (CSC, which contains majority of tobacco constituents, on oxidative stress, cytotoxicity, expression of CYP1A1, and/or HIV-1 replication in HIV-infected (U1 and uninfected U937 cells. The effects of CSC on induction of CYP1 enzymes in HIV-infected primary macrophages were also analyzed. The results showed that the CSC-mediated increase in production of reactive oxygen species (ROS in U937 cells is dose- and time-dependent. Moreover, CSC treatment was found to induce cytotoxicity in U937 cells through the apoptotic pathway via activation of caspase-3. Importantly, pretreatment with vitamin C blocked the CSC-mediated production of ROS and induction of caspase-3 activity. In U1 cells, acute treatment of CSC increased ROS production at 6H (>2-fold and both ROS (>2 fold and HIV-1 replication (>3-fold after chronic treatment. The CSC mediated effects were associated with robust induction in the expression of CYP1A1 mRNA upon acute CSC treatment of U937 and U1 cells (>20-fold, and upon chronic CSC treatment to U1 cells (>30-fold. In addition, the CYP1A1 induction in U937 cells was mediated through the aromatic hydrocarbon receptor pathway. Lastly, CSC, which is known to increase viral replication in primary macrophages, was also found to induce CYP1 enzymes in HIV-infected primary macrophages. While mRNA levels of both CYP1A1 and CYP1B1 were elevated following CSC treatment, only CYP1B1 protein levels were increased in HIV-infected primary macrophages. In conclusion, these results suggest a possible association between oxidative stress, CYP1 expression, and viral replication in

  5. Human monocyte-derived dendritic cells from leukoreduction system chambers after plateletpheresis are functional in an in vitro co-culture assay with intestinal epithelial cells.

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    Tiscornia, Inés; Sánchez-Martins, Viviana; Hernández, Ana; Bollati-Fogolín, Mariela

    2012-10-31

    The dendritic cells (DC) found in the intestine are involved both in the maintenance of tolerance towards commensal microbiota, and in the generation of protective immune responses against pathogens, thus contributing to gut immune homeostasis. There is an increasing interest in the use of lactic acid bacteria (LAB) as probiotics; among their beneficial effects we highlight the modulation of the immune system which is one of their fundamental properties. As these effects are strain-dependent, it is important to have in vitro systems that include DC and intestinal epithelial cells (IEC), which are crucial for intestinal homeostasis, to identify candidates by means of bacterial screening. Obtaining enough human cells, necessary to simultaneously test several bacteria, is a major challenge for researchers. In this study we analyzed the usefulness of the cellular fraction retained in leukoreduction system chambers following plateletpheresis (PP) as a source of DC. We compared the capacity of peripheral blood mononuclear cells (PBMC) from buffy coats (BC) or PP to generate DC using a short differentiation protocol. The functionality of the DC obtained was analyzed in co-cultures together with intestinal epithelial HT-29 cells, stimulating with LPS alone or with two LAB commonly used in the food industry, Streptococcus thermophilus and Lactobacillus delbrueckii. DC surface markers CD86, HLA-DR and cytokine production were measured. The behavior of DC derived from PP was similar to the behavior observed for DC derived from BC. When we tested the response of DC to bacteria, we found significant differences in cytokine secretion, especially for IL-10, suggesting that the system has the ability to discriminate LAB with different immunomodulatory properties. We also found that DC derived from both sources displayed a similar ability to phagocyte bacteria. In conclusion, we hereby propose a modification of the two-day protocol for obtaining human DC previously described, using

  6. Hypoxia modifies the transcriptome of primary human monocytes: modulation of novel immune-related genes and identification of CC-chemokine ligand 20 as a new hypoxia-inducible gene.

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    Bosco, Maria Carla; Puppo, Maura; Santangelo, Clara; Anfosso, Luca; Pfeffer, Ulrich; Fardin, Paolo; Battaglia, Florinda; Varesio, Luigi

    2006-08-01

    Peripheral blood monocytes migrate to and accumulate in hypoxic areas of inflammatory and tumor lesions. To characterize the molecular bases underlying monocyte functions within a hypoxic microenvironment, we investigated the transcriptional profile induced by hypoxia in primary human monocytes using high-density oligonucleotide microarrays. Profound changes in the gene expression pattern were detected following 16 h exposure to 1% O(2), with 536 and 677 sequences showing at least a 1.5-fold increase and decrease, respectively. Validation of this analysis was provided by quantitative RT-PCR confirmation of expression differences of selected genes. Among modulated genes, 74 were known hypoxia-responsive genes, whereas the majority were new genes whose responsiveness to hypoxia had not been previously described. The hypoxic transcriptome was characterized by the modulation of a significant cluster of genes with immunological relevance. These included scavenger receptors (CD163, STAB1, C1qR1, MSR1, MARCO, TLR7), immunoregulatory, costimulatory, and adhesion molecules (CD32, CD64, CD69, CD89, CMRF-35H, ITGB5, LAIR1, LIR9), chemokines/cytokines and receptors (CCL23, CCL15, CCL8, CCR1, CCR2, RDC1, IL-23A, IL-6ST). Furthermore, we provided conclusive evidence of hypoxic induction of CCL20, a chemoattractant for immature dendritic cells, activated/memory T lymphocytes, and naive B cells. CCL20 mRNA up-regulation was paralleled by increased protein expression and secretion. This study represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into monocyte functional behavior within ischemic/hypoxic tissues. CCL20 up-regulation by hypoxia may constitute an important mechanism to promote recruitment of specific leukocyte subsets at pathological sites and may have implications for the pathogenesis of chronic inflammatory diseases.

  7. Interaction between M-CSF and IL-10 on productions of IL-12 and IL-18 and expressions of CD14, CD23, and CD64 by human monocytes

    Institute of Scientific and Technical Information of China (English)

    Xiao-hui JI; Ting YAO; Jun-chuan QIN; Shu-kui WANG; Hui-juan WANG; Kun YAO

    2004-01-01

    AIM: To Study the interaction of macrophage colony-stimulating factor (M-CSF) and interleukin-10 (IL-10) in productions of IL-12 and IL-18 and expressions of CD14, CD23, and CD64 by human monocytes. METHODS:Purified adherent human monocytes were cultured with M-CSF or IL-10 alone, or with M-CSF+IL-10 and 2-3d later, the culture supernatants and cells were separated and collected. IL-12P40 and IL-18 levels in the supernatants were determined by ELISA and the percentages of CD14, CD23, and CD64 positive cells were examined by flow cytometry. RESULTS: (1) IL-10 decreased M-CSF-induced IL-18 levels, while M-CSF further reduced IL-12P40 level in the culture supernatants of IL-10-treated monocytes; (2) IL-10 alone had no effect on the percentage of CD14-positive cells, but further increased the percentage of CD14-positive cells induced by M-CSF; M-CSF alone had no effect on the percentage of CD64-positive cells, but further increased the percentage of CD64-positive cells induced by IL-10; (3) IL-10 decreased the percentage of CD23-positive cells induced by M-CSF.CONCLUSION: Between M-CSF and IL-10, there were antagonistic effects on inducing IL-18 and CD23 expressions by monocytes; there were also synergistic effects on inhibiting IL-12P40 production and inducing CD 14 and CD64 expressions by monocytes.

  8. Human parvovirus B19 DNA replication induces a DNA damage response that is dispensable for cell cycle arrest at phase G2/M.

    Science.gov (United States)

    Lou, Sai; Luo, Yong; Cheng, Fang; Huang, Qinfeng; Shen, Weiran; Kleiboeker, Steve; Tisdale, John F; Liu, Zhengwen; Qiu, Jianming

    2012-10-01

    Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G(2)/M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G(2)/M significantly, during B19V infection.

  9. Sesamin inhibits bacterial formylpeptide-induced inflammatory responses in a murine air-pouch model and in THP-1 human monocytes.

    Science.gov (United States)

    Cui, Youhong; Hou, Xinwei; Chen, Juan; Xie, Lianying; Yang, Lang; Le, Yingying

    2010-02-01

    The reaction of human leukocytes to chemoattractants is an important component of the host immune response and also plays a crucial role in the development of inflammation. Sesamin has been shown to inhibit lipid peroxidation and regulate cytokine production. In this study, we examined the effect of sesamin on inflammatory responses elicited by the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF) in vitro and in vivo and explored the mechanisms involved. fMLF is recognized by a human G protein-coupled receptor formyl peptide receptor (FPR) on phagocytic leukocytes. Sesamin at concentrations between 12.5 and 50 micromol/L inhibited fMLF-induced chemotaxis of human monocyte cell line THP-1 differentiated with dibutyryl cyclic AMP (P sesamin inhibited FPR-transfected rat basophilic leukemia cell [epitope-tagged human FPR (ETFR) cell] migration toward fMLF (P sesamin (12 mgkg(-1)d(-1) for 2 d) suppressed leukocyte infiltration into the air pouch induced by fMLF [(62.89 +/- 7.93) x 10(4) vs. (19.67 +/- 4.43) x 10(4) cells/air pouch; n = 9; P sesamin inhibited fMLF-induced ERK1/2 phosphorylation in a dose-dependent manner but did not affect fMLF-induced Ca(2+) flux. Electrophoretic mobility shift assay showed that pretreatment of THP-1 cells with sesamin dose dependently inhibited fMLF-induced nuclear factor-kappaB (NF-kappaB) activation. These results suggest that sesamin inhibits leukocyte activation by fMLF through ERK1/2- and NF-kappaB-related signaling pathways and thus is a potential compound for the management of inflammatory diseases.

  10. Technical advance: Generation of human pDC equivalents from primary monocytes using Flt3-L and their functional validation under hypoxia.

    Science.gov (United States)

    Sekar, Divya; Brüne, Bernhard; Weigert, Andreas

    2010-08-01

    The division of labor between DC subsets is evolutionarily well-defined. mDC are efficient in antigen presentation, whereas pDC act as rheostats of the immune system. They activate NK cells, cause bystander activation of mDC, and interact with T cells to induce tolerance. This ambiguity positions pDC at the center of inflammatory diseases, such as cancer, arthritis, and autoimmune diseases. The ability to generate human mDC ex vivo made it possible to engineer them to suit therapy needs. Unfortunately, a similar, easily accessible system to generate human pDC is not available. We describe a method to generate human pDC equivalents ex vivo, termed mo-pDC from peripheral blood monocytes using Flt3-L. mo-pDC showed a characteristic pDC profile, such as high CD123 and BDCA4, but low CD86 and TLR4 surface expression and a low capacity to induce autologous lymphocyte proliferation and to phagocytose apoptotic debris in comparison with mDC. Interestingly, mo-pDC up-regulated the pDC lineage-determining transcription factor E2-2 as well as expression of BDCA2, which is under the transcriptional control of E2-2 but not its inhibitor ID2, during differentiation. mo-pDC produced high levels of IFN-alpha when pretreated overnight with TNF-alpha. Under hypoxia, E2-2 was down-regulated, and ID2 was induced in mo-pDC, whereas surface expression of MHCI, CD86, and BDCA2 was decreased. Furthermore, mo-pDC produced high levels of inflammatory cytokines when differentiated under hypoxia compared with normoxia. Hence, mo-pDC can be used to study differentiation and functions of human pDC under microenvironmental stimuli.

  11. Coleusin factor exerts cytotoxic activity by inducing G0/G1 cell cycle arrest and apoptosis in human gastric cancer BGC-823 cells.

    Science.gov (United States)

    Sun, Bo; Geng, Shuo; Huang, Xiaojia; Zhu, Jin; Liu, Shu; Zhang, Yajing; Ye, Jian; Li, Yongjin; Wang, Jingze

    2011-02-01

    Coleusin factor (CF), a kind of diterpenoids, is isolated and purified from the root of a Chinese tropical plant Coleus forskohlii by our laboratory. Our previous studies have demonstrated that CF significantly inhibits growth in some kinds of cancer cell lines. Here, we found that CF remarkably inhibited growth in human gastric cancer BGC-823 cells by decreasing cell proliferation, inducing G(0)/G(1) cell cycle arrest and apoptosis. CF also decreased the mitochondrial membrane potential in BGC-823 cells. Immunoblotting analysis revealed that CF significantly decreased the expressions of cyclinD1, Bcl-2, and Bcl-x(L), increased the expressions of cytosol cytochrome c, p53, p21, and Rb. In addition, CF significantly increased the expressions and activities of caspase-3 and -9. More importantly, CF potently inhibited the growth of BGC-823 cells xenografted in athymic nude mice with negligible body weight loss and damage towards the spleen. These results indicate that CF exerts a cytotoxic effect on BGC-823 cells by inducing cell cycle arrest and apoptosis.

  12. MiR-107 and MiR-185 can induce cell cycle arrest in human non small cell lung cancer cell lines.

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    Yukari Takahashi

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are short single stranded noncoding RNAs that suppress gene expression through either translational repression or degradation of target mRNAs. The annealing between messenger RNAs and 5' seed region of miRNAs is believed to be essential for the specific suppression of target gene expression. One miRNA can have several hundred different targets in a cell. Rapidly accumulating evidence suggests that many miRNAs are involved in cell cycle regulation and consequentially play critical roles in carcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Introduction of synthetic miR-107 or miR-185 suppressed growth of the human non-small cell lung cancer cell lines. Flow cytometry analysis revealed these miRNAs induce a G1 cell cycle arrest in H1299 cells and the suppression of cell cycle progression is stronger than that by Let-7 miRNA. By the gene expression analyses with oligonucleotide microarrays, we find hundreds of genes are affected by transfection of these miRNAs. Using miRNA-target prediction analyses and the array data, we listed up a set of likely targets of miR-107 and miR-185 for G1 cell cycle arrest and validate a subset of them using real-time RT-PCR and immunoblotting for CDK6. CONCLUSIONS/SIGNIFICANCE: We identified new cell cycle regulating miRNAs, miR-107 and miR-185, localized in frequently altered chromosomal regions in human lung cancers. Especially for miR-107, a large number of down-regulated genes are annotated with the gene ontology term 'cell cycle'. Our results suggest that these miRNAs may contribute to regulate cell cycle in human malignant tumors.

  13. Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in a human lymphoblastic leukemia cell line

    Directory of Open Access Journals (Sweden)

    Rahman HS

    2014-01-01

    Full Text Available Heshu Sulaiman Rahman,1–3 Abdullah Rasedee,1,2 Ahmad Bustamam Abdul,2,4 Nazariah Allaudin Zeenathul,1,2 Hemn Hassan Othman,1,3 Swee Keong Yeap,2 Chee Wun How,2 Wan Abd Ghani Wan Nor Hafiza4,51Faculty of Veterinary Medicine, 2Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia; 3Faculty of Veterinary Medicine, University of Sulaimanyah, Sulaimanyah City, Kurdistan Region, Northern Iraq; 4Faculty of Medicine and Health Science, Universiti Putra Malaysia, Selangor, Malaysia; 5College of Medical Laboratory Technology, Institute for Medical Research, Kuala Lumpur, MalaysiaAbstract: This investigation evaluated the antileukemia properties of a zerumbone (ZER-loaded nanostructured lipid carrier (NLC prepared by hot high-pressure homogenization techniques in an acute human lymphoblastic leukemia (Jurkat cell line in vitro. The apoptogenic effect of the ZER-NLC on Jurkat cells was determined by fluorescent and electron microscopy, Annexin V-fluorescein isothiocyanate, Tdt-mediated dUTP nick-end labeling assay, cell cycle analysis, and caspase activity. An MTT (3-(4,5-dimethylthiazol-2-yl-2,5 diphenyltetrazolium bromide assay showed that ZER-NLC did not have adverse effects on normal human peripheral blood mononuclear cells. ZER-NLC arrested the Jurkat cells at G2/M phase with inactivation of cyclin B1 protein. The study also showed that the antiproliferative effect of ZER-NLC on Jurkat cells is through the intrinsic apoptotic pathway via activation of caspase-3 and caspase-9, release of cytochrome c from the mitochondria into the cytosol, and subsequent cleavage of poly (adenosine diphosphate-ribose polymerase (PARP. These findings show that the ZER-NLC is a potentially useful treatment for acute lymphoblastic leukemia in humans.Keywords: zerumbone-loaded nanostructured lipid carrier, cell cycle arrest, apoptosis, mitochondrial pathway

  14. Cytotoxicity and Proliferation Studies with Arsenic in Established Human Cell Lines: Keratinocytes, Melanocytes, Dendritic Cells, Dermal Fibroblasts, Microvascular Endothelial Cells, Monocytes and T-Cells

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    Hari H. P. Cohly

    2003-01-01

    Full Text Available Abstract: Based on the hypothesis that arsenic exposure results in toxicity and mitogenecity, this study examined the dose-response of arsenic in established human cell lines of keratinocytes (HaCaT, melanocytes (1675, dendritic cells (THP-1/A23187, dermal fibroblasts (CRL1904, microvascular endothelial cells (HMEC, monocytes (THP-1, and T cells (Jurkat. Cytotoxicity was determined by incubating THP-1, THP-1+ A23187 and JKT cells in RPMI 1640, 1675 in Vitacell, HMEC in EBM, and dermal fibroblasts and HaCaT in DMEM with 10% fetal bovine serum, 1% streptomycin and penicillin for 72 hrs in 96-well microtiter plates, at 37oC in a 5% CO2 incubator with different concentrations of arsenic using fluorescein diacetate (FDA. Cell proliferation in 96-well plates was determined in cultured cells starved by prior incubation for 24 hrs in 1% FBS and exposed for 72 hours, using the 96 cell titer proliferation solution (Promega assay. Cytotoxicity assays yielded LD50s of 9 μg/mL for HaCaT, 1.5 μg/mL for CRL 1675, 1.5 μg/mL for dendritic cells, 37 μg/mL for dermal fibroblasts, 0.48 μg/mL for HMEC, 50 μg/mL for THP-1 cells and 50 μg/mL for JKT-T cells. The peak proliferation was observed at 6 μg/mL for HaCaT and THP-1 cells, 0.19 μg/mL for CRL 1675, dendritic cells, and HMEC, and 1.5 μg/mL for dermal fibroblasts and Jurkat T cells. These results show that arsenic is toxic at high doses to keratinocytes, fibroblasts, monocytes and T cells, and toxic at lower doses to melanocytes, microvascular endothelial cells and dendritic cells. Proliferation studies showed sub-lethal doses of arsenic to be mitogenic.

  15. Essential involvement of cross-talk between IFN-gamma and TNF-alpha in CXCL10 production in human THP-1 monocytes.

    Science.gov (United States)

    Qi, Xu-Feng; Kim, Dong-Heui; Yoon, Yang-Suk; Jin, Dan; Huang, Xue-Zhu; Li, Jian-Hong; Deung, Young-Kun; Lee, Kyu-Jae

    2009-09-01

    Interferon (IFN)-gamma-induced protein 10 (IP-10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN-gamma depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN-gamma and TNF-alpha have not been adequately elucidated in human monocytes. In this study, we showed that TNF-alpha had more potential than IFN-gamma to induce CXCL10 production in THP-1 monocytes. Furthermore, IFN-gamma synergistically enhanced the production of CXCL10 in parallel with the activation of NF-kappaB in TNF-alpha-stimulated THP-1 cells. Blockage of STAT1 or NF-kappaB suppressed CXCL10 production. JAKs inhibitors suppressed IFN-gamma plus TNF-alpha-induced production of CXCL10 in parallel with activation of STAT1 and NF-kappaB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF-kappaB, but not that of STAT1. IFN-gamma-induced phosphorylation of JAK1 and JAK2, whereas TNF-alpha induced phosphorylation of ERK1/2. Interestingly, IFN-gamma alone had no effect on phosphorylation and degradation of IkappaB-alpha, whereas it significantly promoted TNF-alpha-induced phosphorylation and degradation of IkappaB-alpha. These results suggest that TNF-alpha induces CXCL10 production by activating NF-kappaB through ERK and that IFN-gamma induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF-alpha-induced NF-kappaB may be the primary pathway contributing to CXCL10 production in THP-1 cells. IFN-gamma potentiates TNF-alpha-induced CXCL10 production in THP-1 cells by increasing the activation of STAT1 and NF-kappaB through JAK1 and JAK2.

  16. Dietary oleic and palmitic acids modulate the ratio of triacylglycerols to cholesterol in postprandial triacylglycerol-rich lipoproteins in men and cell viability and cycling in human monocytes.

    Science.gov (United States)

    López, Sergio; Bermúdez, Beatriz; Pacheco, Yolanda M; López-Lluch, Guillermo; Moreda, Wenceslao; Villar, José; Abia, Rocío; Muriana, Francisco J G

    2007-09-01

    The postprandial metabolism of dietary fats produces triacylglycerol (TG)-rich lipoproteins (TRL) that could interact with circulating cells. We investigated whether the ratios of oleic:palmitic acid and monounsaturated fatty acids (MUFA):SFA in the diet affect the ratio of TG:cholesterol (CHOL) in postprandial TRL of healthy men. The ability of postprandial TRL at 3 h (early postprandial period) and 5 h (late postprandial period) to affect cell viability and cycle in the THP-1 human monocytic cell line was also determined. In a randomized, crossover experiment, 14 healthy volunteers (Caucasian men) ate meals enriched (50 g/m(2) body surface area) in refined olive oil, high-palmitic sunflower oil, butter, and a mixture of vegetable and fish oils, which had ratios of oleic:palmitic acid (MUFA:SFA) of 6.83 (5.43), 2.36 (2.42), 0.82 (0.48), and 13.81 (7.08), respectively. The ratio of TG:CHOL in postprandial TRL was inversely correlated (r = -0.89 to -0.99) with the ratio of oleic:palmitic acid and with the MUFA:SFA ratio in the dietary fats (P the cell cycle in THP-1 cells.

  17. Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells

    DEFF Research Database (Denmark)

    Lund, L R; Rønne, E; Roldan, A L;

    1991-01-01

    We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-fold...... enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments...... show a time-dependent increase in the u-PAR gene transcription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNA is paralleled by a time-dependent increase in u-PAR protein as detected by cross-linking studies with radiolabeled ligand. We conclude that PMA stimulates...

  18. Different Transcriptional Profiles of Human Monocyte-Derived Dendritic Cells Infected with Distinct Strains of Mycobacterium tuberculosis and Mycobacterium bovis Bacillus Calmette-Guérin

    Directory of Open Access Journals (Sweden)

    Nunzia Sanarico

    2011-01-01

    Full Text Available In order to analyze dendritic cells (DCs activation following infection with different mycobacterial strains, we studied the expression profiles of 165 genes of human monocyte-derived DCs infected with H37Rv, a virulent Mycobacterium tuberculosis (MTB laboratory strain, CMT97, a clinical MTB isolate, Mycobacterium bovis bacillus Calmette-Guérin (BCG, Aventis Pasteur, and BCG Japan, both employed as vaccine against tuberculosis. The analysis of the gene expression reveals that, despite a set of genes similarly modulated, DCs response resulted strain dependent. In particular, H37Rv significantly upregulated EBI3 expression compared with BCG Japan, while it was the only strain that failed to release a significant IL-10 amount. Of note, BCG Japan showed a marked increase in CCR7 and TNF-α expression regarding both MTB strains and it resulted the only strain failing in exponential intracellular growth. Our results suggest that DCs display the ability to elicit a tailored strain-specific immune response.

  19. Inhibitory effects of Zataria multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in glucose-stimulated human monocyte.

    Science.gov (United States)

    Kavoosi, Gholamreza; Teixeira da Silva, Jaime A

    2012-09-01

    The inhibitory effects of Zataria multiflora essential oil on nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) production were examined in human monocytes cultured in the presence of 20mM glucose. Z. multiflora essential oil was extracted by water-distillation and then analyzed by GC-MS. Carvacrol (29.2%), thymol (25.4%), p-cymene (11.2%), linalool (9.6%) and γ-terpinene (8%) were the main components detected in the essential oil. Cells cultured in the presence of 20mM glucose showed an increase in NO and H(2)O(2) production as well as NO synthase (NOS) and NADH oxidase (NOX) activities compared to cells cultured in the presence of 5mM glucose. Pretreatment with Z. multiflora essential oil, carvacrol and thymol reduced NO and H(2)O(2) production as well as NOS and NOX activities in those cells cultured in the presence of 20mM glucose. However, p-cymene, linalool and γ-terpinene did not show any such activities. Accordingly, it was concluded that Z. multiflora can reduce oxidative stress and can be used in the therapy of oxidative damage accompanying hyperglycemia and some inflammatory conditions.

  20. Biphasic influence of PGE2 on the resorption activity of osteoclast-like cells derived from human peripheral blood monocytes and mouse RAW264.7 cells.

    Science.gov (United States)

    Lutter, Anne-Helen; Hempel, Ute; Anderer, Ursula; Dieter, Peter

    2016-08-01

    Osteoclasts are large bone-resorbing cells of hematopoietic origin. Their main function is to dissolve the inorganic component hydroxyapatite and to degrade the organic bone matrix. Prostaglandin E2 (PGE2) indirectly affects osteoclasts by stimulating osteoblasts to release factors that influence osteoclast activity. The direct effect of PGE2 on osteoclasts is still controversial. To study the influence of PGE2 on osteoclast activity, human peripheral blood monocytes (hPBMC) and mouse RAW264.7 cells were cultured on osteoblast-derived extracellular matrix. hPBMC and RAW264.7 cells were differentiated by the addition of macrophage colony-stimulation factor and receptor activator of NFκB ligand and treated with PGE2 before and after differentiation induction. The pit area, an indicator of resorption activity, and the activity of tartrate-resistant acid phosphatase were dose-dependently inhibited when PGE2 was present ab initio, whereas the resorption activity remained unchanged when the cells were exposed to PGE2 from day 4 of culture. These results lead to the conclusion that PGE2 treatment inhibits only the differentiation of precursor osteoclasts whereas differentiated osteoclasts are not affected.

  1. A matrix of cholesterol crystals, but not cholesterol alone, primes human monocytes/macrophages for excessive endotoxin-induced production of tumor necrosis factor-alpha. Role in atherosclerotic inflammation?

    DEFF Research Database (Denmark)

    Bendtzen, Klaus; Christensen, Ole; Nielsen, Claus Henrik

    2014-01-01

    When exposed to small amounts of bacterial endotoxin, matrices of cholesterol crystals, but not cholesterol itself, primed human monocytes/macrophages to a highly augmented (>10-fold) production of inflammatory tumor necrosis factor-α. Priming also sensitized the cells, as 10- to 100-fold lower...... suggest that cholesterol matrix formation may play a pathogenic role in atherosclerotic inflammation, and they indicate a mechanism by which bacteria and/or bacterial products may play a role in processes leading to arteriosclerosis....

  2. Anticancer activity of chemically prepared shrimp low molecular weight chitin evaluation with the human monocyte leukaemia cell line, THP-1.

    Science.gov (United States)

    Salah, R; Michaud, P; Mati, F; Harrat, Z; Lounici, H; Abdi, N; Drouiche, N; Mameri, N

    2013-01-01

    In the present study, anticancer activities of chitin, chitosan and low molecular weight chitin were evaluated using a human tumour cell line, THP-1. A molecular weight-activity relationship and an electrostatic interaction-activity relationship were determined. The cytotoxic effects of chitin and derivatives were also evaluated using a normal human foetal lung fibroblastic cell line, MRC-5 and the specific cytotoxicity of chitin and derivatives to tumour cell lines was demonstrated. The high antitumour effect of low molecular weight of chitin was established.

  3. Curcumin inhibits the proliferation of a human colorectal cancer cell line Caco-2 partially by both apoptosis and G2/M cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Yohko Fujimoto

    2014-06-01

    Full Text Available The aim of this study was to assess the possible roles of the phytochemical compounds, curcumin, quercetin and resveratrol in the proliferation of human colorectal cancer cell line Caco-2. All three phytochemical compounds inhibited Caco-2 cell proliferation, with curcumin being more effective than quercetin and resveratrol. Investigations concerning DNA fragmentation in the nucleus, Bax and Bcl-2 mRNA expression levels, and caspase-3/7 activity indicated that curcumin induced apoptosis in Caco-2 cells through an increase in the Bax/Bcl-2 ratio and activation of caspase-3/7. Furthermore, the analysis of flow-cytometry showed that curcumin caused an arrest of G2/M phase in Caco-2 cells. These results suggest that curcumin suppresses Caco-2 proliferation partially via activation of the mitochondrial apoptotic pathway and cell cycle retardation.

  4. Ferrocenyl and dicobalt hexacarbonyl chromones--new organometallics inducing oxidative stress and arresting human cancer cells in G2/M phase.

    Science.gov (United States)

    Kowalski, Konrad; Hikisz, Paweł; Szczupak, Łukasz; Therrien, Bruno; Koceva-Chyła, Aneta

    2014-06-23

    The straightforward syntheses of four new ferrocenyl and dicobalt hexacarbonyl chromones are presented. The redox behavior of the novel metallo-chromones has been examined by cyclic voltammetry (CV), revealing a reversible behavior of the ferrocenyl groups, while the dicobalt hexacarbonyl derivatives show irreversible oxidation. The anticancer activity of the products has been evaluated against hepatocellular carcinoma (Hep G2), ER+ (MCF-7) and ER- (MDA-MB-231) breast adenocarcinoma, and leukemic (CCRF-CEM) human cancer cell lines. The mechanism of action for the most active complexes has been investigated and it seems to involve oxidative stress and apoptosis induction. Moreover, the results show that the investigated metallo-chromones generate damage to DNA and arrest the cell cycle in G2/M phase.

  5. The Putative Metal Coordination Motif in the Endonuclease Domain of Human Parvovirus B19 NS1 Is Critical for NS1 Induced S Phase Arrest and DNA Damage

    Directory of Open Access Journals (Sweden)

    Violetta Kivovich, Leona Gilbert, Matti Vuento, Stanley J. Naides

    2012-01-01

    Full Text Available The non-structural proteins (NS of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV, minute virus of mice (MVM and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2 cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.

  6. The putative metal coordination motif in the endonuclease domain of human Parvovirus B19 NS1 is critical for NS1 induced S phase arrest and DNA damage.

    Science.gov (United States)

    Kivovich, Violetta; Gilbert, Leona; Vuento, Matti; Naides, Stanley J

    2012-01-01

    The non-structural proteins (NS) of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV), minute virus of mice (MVM) and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2) cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.

  7. Oridonin nanosuspension was more effective than free oridonin on G2/M cell cycle arrest and apoptosis in the human pancreatic cancer PANC-1 cell line

    Directory of Open Access Journals (Sweden)

    Qi XL

    2012-04-01

    Full Text Available Xiaoli Qi1, Dianrui Zhang2, Xia Xu1, Feifei Feng2, Guijie Ren1, Qianqian Chu1, Qiang Zhang3, Keli Tian11Department of Biochemistry and Molecular Biology, Shandong University School of Medicine, Jinan, 2Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan, 3State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, People's Republic of ChinaAbstract: Oridonin, a diterpenoid isolated from Rabdosia rubescencs, has been reported to have antitumor effects. However, low solubility has limited its clinical applications. Preparation of drugs in the form of nanosuspensions is an extensively utilized protocol. In this study, we investigated the anticancer activity of oridonin and oridonin nanosuspension on human pancreatic carcinoma PANC-1 cells. 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay was performed to investigate the effect of oridonin on cell growth. Propidium iodide and Hoechst 33342 staining were used to detect morphologic changes. The percentage of apoptosis and cell cycle progression was determined by flow cytometric method staining with propidium iodide. Annexin V-fluorescein isothiocyanate (FITC/PI staining was used to evaluate cell apoptosis by flow cytometry. Caspase-3 activity was measured by spectrophotometry. The apoptotic and cell cycle protein expression were determined by Western blot analysis. Both oridonin and oridonin nanosuspension induced apoptosis and G2/M phase cell cycle arrest, and the latter had a more significant cytotoxic effect. The ratio of Bcl-2/Bax protein expression was decreased and caspase-3 activity was stimulated. The expression of cyclin B1 and p-cdc2 (T161 was suppressed. Our results showed that oridonin nanosuspension was more effective than free oridonin on G2/M cell cycle arrest and apoptosis in the human pancreatic cancer PANC-1 cell line.Keywords: cyclin B1, cdc2, caspase-3, Bcl-2, Bax

  8. Sinomenine prevents metastasis of human osteosarcoma cells via S phase arrest and suppression of tumor-related neovascularization and osteolysis through the CXCR4-STAT3 pathway.

    Science.gov (United States)

    Xie, Tao; Ren, Hai-Yong; Lin, Hai-Qing; Mao, Jin-Ping; Zhu, Ting; Wang, Sheng-Dong; Ye, Zhao-Ming

    2016-05-01

    Osteosarcoma is the most common primary malignant tumor of the bone. The long-term survivals continue to be unsatisfactory for patients with metastatic and recurrent disease. Metastasis is still a severe challenge in osteosarcoma treatment. Sinomenine, an alkaloid from traditional Chinese medicine, has been proved to possess potent antitumor and anti-invasion effect on various cancers. However, the effect of sinomenine on human osteosarcoma and the underlying mechanisms remains unknown. We report here that sinomenine inhibited proliferation by inducing S phase arrest and suppressing the clone formation. Significant inhibitory effects were found in invasion and metastasis in osteosarcoma, but little cytotoxicity was observed in tested concentrations. Exposure to sinomenine resulted in suppression of invasion and migration in osteosarcoma cells as well as tube formation ability in the human umbilical vein endothelial cells (HUVEC) and U2OS cells. Furthermore, it demonstrated that CXCR4 played a key role contributing to invasion in osteosarcoma which is considered to be a core target site in sinomenine treatment. Sinomenine inhibited invasion by suppressing CXCR4 and STAT3 phosphorylation then downregulating the expression of MMP-2, MMP-9, RANKL, VEGF downstream. In addition, then RANKL-mediated bone destruction stimulated by osteoclastogenesis and VEGF-related neovascularization were restrained. Importantly, in vivo, sinomenine suppressed proliferation, osteoclastogenesis and bone destruction. Through these various comprehensive means, sinomenine inhibits metastasis in osteosarcoma. Taken together, our results revealed that sinomenine caused S phase arrest, inhibited invasion and metastasis via suppressing the CXCR4-STAT3 pathway and then osteoclastogenesis-mediated bone destruction and neovascularization in osteosarcoma. Sinomenine is therefore a promising adjuvant agent for metastasis control in osteosarcoma.

  9. Notch3 overexpression causes arrest of cell cycle progression by inducing Cdh1 expression in human breast cancer cells.

    Science.gov (United States)

    Chen, Chun-Fa; Dou, Xiao-Wei; Liang, Yuan-Ke; Lin, Hao-Yu; Bai, Jing-Wen; Zhang, Xi-Xun; Wei, Xiao-Long; Li, Yao-Chen; Zhang, Guo-Jun

    2016-01-01

    Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27(Kip) accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27(Kip) at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27(Kip) accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment.

  10. Neferine, an alkaloid from lotus seed embryo, inhibits human lung cancer cell growth by MAPK activation and cell cycle arrest.

    Science.gov (United States)

    Poornima, Paramasivan; Weng, Ching Feng; Padma, Viswanadha Vijaya

    2014-01-01

    Neferine is the major bisbenzylisoquinoline alkaloid isolated from the seed embryo of a traditional medicinal plant Nelumbo nucifera (Lotus). Epidemiological studies have revealed the therapeutic potential of lotus seed embryo. Although several mechanisms have been proposed, a clear anticancer action mechanism of neferine on lung cancer cells is still not known. Lung cancer is the most common cause of cancer death in the world, and the patients with advanced stage of nonsmall lung cancer require adjunct chemotherapy after surgical resection for the eradication of cancer cells. In this study, the effects of neferine were evaluated and characterized in A549 cells. Neferine induced apoptosis in a dose-dependent manner with the hypergeneration of reactive oxygen species, activation of MAPKs, lipid peroxidation, depletion of cellular antioxidant pool, loss of mitochondrial membrane potential, and intracellular calcium accumulation. Furthermore, neferine treatment leads to the inhibition of nuclear factor kappaB and Bcl2, upregulation of Bax and Bad, release of cytochrome C, activation of caspase cascade, and DNA fragmentation. In addition, neferine could induce p53 and its effector protein p21 and downregulation of cell cycle regulatory protein cyclin D1 thereby inducing G1 cell cycle arrest. These results suggest a novel function of neferine as an apoptosis inducer in lung cancer cells.

  11. DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by Ruta graveolens in human colon cancer cells.

    Science.gov (United States)

    Arora, Shagun; Tandon, Simran

    2015-01-01

    In the present study, we investigated the anti-cancer effect of various potencies of Ruta graveolens (Ruta) on COLO-205 cell line, as evidenced by cytotoxicity, migration, clonogenecity, morphological and biochemical changes and modification in the levels of genes associated with apoptosis and cell cycle. On treatment of COLO-205 cells maximal effects were seen with mother tincture (MT) and 30C potencies, wherein decrease in cell viability along with reduced clonogenecity and migration capabilities were noted. In addition morphological and biochemical alterations such as nuclear changes (fragmented nuclei with condensed chromatin) and DNA ladder-like pattern (increased amount of fragmented DNA) in COLO-205 cells indicating apoptotic related cell death were seen. The expression of apoptosis and cell-cycle related regulatory genes assessed by reverse transcriptase-PCR revealed an up-regulation of caspase 9, caspase-3, Bax, p21 and p27 expression and down-regulation of Bcl-2 expression in treated cells. The mode of cell death was suggestive of intrinsic apoptotic pathway along with cell cycle arrest at the G2/M of the cell cycle. Our findings indicate that phytochemicals present in Ruta showed potential for natural therapeutic product development for colon carcinoma.

  12. Silica Nanoparticles Sensitize Human Multiple Myeloma Cells to Snake (Walterinnesia aegyptia Venom-Induced Apoptosis and Growth Arrest

    Directory of Open Access Journals (Sweden)

    Douaa Sayed

    2012-01-01

    Full Text Available Background. Multiple myeloma (MM, an almost incurable disease, is the second most common blood cancer. Initial chemotherapeutic treatment could be successful; however, resistance development urges the use of higher toxic doses accompanied by hematopoietic stem cell transplantation. The establishment of more effective treatments that can overcome or circumvent chemoresistance has become a priority. We recently demonstrated that venom extracted from Walterinnesia aegyptia (WEV either alone or in combination with silica nanoparticles (WEV+NPs mediated the growth arrest and apoptosis of prostate cancer cells. In the present study, we evaluated the impact of WEV alone and WEV+NP on proliferation and apoptosis of MM cells. Methods. The impacts of WEV alone and WEV+NP were monitored in MM cells from 70 diagnosed patients. The influences of WEV and WEV+NP were assessed with flow cytometry analysis. Results. WEV alone and WEV+NP decreased the viability of MM cells. Using a CFSE proliferation assay, we found that WEV+NP strongly inhibited MM cell proliferation. Furthermore, analysis of the cell cycle using the propidium iodide (PI staining method indicated that WEV+NP strongly altered the cell cycle of MM cells and enhanced the induction of apoptosis. Conclusions. Our data reveal the biological effects of WEV and WEV+NP on MM cells that enable these compounds to function as effective treatments for MM.

  13. Dicentrine Analogue-Induced G2/M Arrest and Apoptosis through Inhibition of Topoisomerase II Activity in Human Cancer Cells.

    Science.gov (United States)

    Lin, Huei-Fang; Huang, Huey-Lan; Liao, Jyh-Fei; Shen, Chien-Chang; Huang, Ray-Ling

    2015-07-01

    Lindera megaphylla has been traditionally used as an antineoplastic and wound healing remedy. We previously demonstrated the antitumor effects of D-dicentrine, a natural aporphine alkaloid from the root of L. megaphylla. To generate analogues, series of phenanthrene alkaloids from D-dicentrine were synthesized by degradation with ethyl chloroformate in pyridine, base hydrolysis, and N-alkylation. In this study, we demonstrated that one of the synthesized D-dicentrine analogues (here after designated as analogue 1) exhibited more potent cytotoxic effects than D-dicentrine in colon adenocarcinoma, hepatoma, leukemia, and epidermoid carcinoma cells. We performed cell cycle and apoptotic analysis by flow cytometry, an apoptotic DNA detection ELISA assay, and topoisomerase II activity by the kinetoplast DNA concatenation assay for studying their cytotoxic mechanisms. We found that both D-dicentrine and analogue 1 induced apoptosis and G2/M arrest in HL-60 leukemia cells. The percentage of apoptotic cells induced by analogue 1 was 4.5-fold higher than that induced by D-dicentrine as evident from measuring the amount of histone-bound DNA fragments. Moreover, we found that analogue 1 was 28-fold more potent than D-dicentrine for inhibition of topoisomerase II activity by the kinetoplast DNA concatenation assay. Our findings indicate that D-dicentrine analogue 1 is very promising as a potential antitumor agent for future study.

  14. Fatal Monocytic Ehrlichiosis in Woman, Mexico, 2013

    Science.gov (United States)

    Sosa-Gutierrez, Carolina G.; Solorzano-Santos, Fortino; Walker, David H.; Torres, Javier; Serrano, Carlos A.

    2016-01-01

    Human monocytic ehrlichiosis is a febrile illness caused by Ehrlichia chaffeensis, an intracellular bacterium transmitted by ticks. In Mexico, a case of E. chaffeensis infection in an immunocompetent 31-year-old woman without recognized tick bite was fatal. This diagnosis should be considered for patients with fever, leukopenia, thrombocytopenia, and elevated liver enzyme levels. PMID:27088220

  15. The soluble recombinant Neisseria meningitidis adhesin NadA(Δ351-405) stimulates human monocytes by binding to extracellular Hsp90.

    Science.gov (United States)

    Cecchini, Paola; Tavano, Regina; Polverino de Laureto, Patrizia; Franzoso, Susanna; Mazzon, Cristina; Montanari, Paolo; Papini, Emanuele

    2011-01-01

    The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadA(Δ351-405,) devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadA(Δ351-405) cellular effects in monocytes. We show that NadA(Δ351-405) (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadA(Δ351-405) cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadA(Δ351-405) /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadA(Δ351-405) and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadA(Δ351-405) determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadA(Δ351-405) alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by

  16. Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Yong-Cheng [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Su, Nan [Department of Quality Detection and Management, Henan University of Animal Husbandry and Economy, Zhengzhou, Henan (China); Shi, Xiao-Jing; Zhao, Wen; Ke, Yu [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China); Zi, Xiaolin [Department of Urology, University of California, Irvine, Orange, CA (United States); Department of Pharmacology, University of California, Irvine, Orange, CA (United States); Department of Pharmaceutical Sciences, University of California, Irvine, Orange, CA (United States); Zhao, Ning-Min; Qin, Yu-Hua; Zhao, Hong-Wei [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Liu, Hong-Min, E-mail: liuhm@zzu.edu.cn [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China)

    2015-01-15

    Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore, Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.

  17. Monocyte functions in diabetes mellitus

    DEFF Research Database (Denmark)

    Geisler, C; Almdal, T; Bennedsen, J

    1982-01-01

    The aim of this study was to investigate the functions of monocytes obtained from 14 patients with diabetes mellitus (DM) compared with those of monocytes from healthy individuals. It was found that the total number of circulating monocytes in the 14 diabetic patients was lower than that from...... for the elucidation of concomitant infections in diabetic patients are discussed....

  18. Interaction of angiogenically stimulated intermediate CD163+ monocytes/macrophages with soft hydrophobic poly(n-butyl acrylate) networks with elastic moduli matched to that of human arteries.

    Science.gov (United States)

    Mayer, Anke; Kratz, Karl; Hiebl, Bernhard; Lendlein, Andreas; Jung, Friedrich

    2012-03-01

    The cell population of peripheral blood monocytes/macrophages (MO) is heterogeneous: The majority of the MO are CD14++ CD16- and named "classical" (= MO1). Furthermore, two other subpopulations were described: CD14++ CD16+ ("intermediate" = MO2) and CD14+ CD16++ ("non-classical" = MO3). It is reported that MO2 possess anti-inflammatory properties and express the MO lineage marker CD163. On a hydrophilic neutrally charged acrylamide-based hydrogel human intermediate (CD14++ CD16+ ), angiogenically stimulated CD163++ monocytes/macrophages (aMO2) maintained a proangiogenic and noninflammatory status for at least 14 days. Here, we explored whether this aMO2 subset adhered to hydrophobic poly(n-butyl acrylate) networks (cPnBA) and also remained in its proangiogenic and noninflammatory status. Because substrate elasticity can impact adherence, morphology, and function of cells, cPnBAs with different Young's modulus (250 and 1100 kPa) were investigated, whereby their elasticity was tailored by variation of the cross-linker content and matched to the elasticity of human arteries. The cPnBAs exhibited similar surface properties (e.g., surface roughness), which were maintained after ethylene oxide sterilization and exposure in serum-free cell culture medium for 18 h at 37°C. aMO2 were seeded on cPnBA samples (1.7 × 10(5) cells/1.33 cm(2) ) in Dulbecco's modified Eagle medium (DMEM high glucose) supplemented with vascular endothelial growth factor 165 (VEGF-A(165) , 10 ng/mL) and fetal calf serum (10 vol%) for 3 and 72 h. On both polymeric samples (n = 3 each), the numbers of adherent cells per unit area were significantly higher (P glass, 3 h: 6 ± 3 cells/mm(2) , 72 h: 130 ± 83 cells/mm(2) ) and showed a typically spread morphology. The mRNA expression profile of the aMO2 was not influenced by the substrate elasticity. In the supernatant of aMO2 on cPnBA0250, significantly less VEGF-A(165) product was found than expected based on

  19. Honokiol arrests cell cycle, induces apoptosis, and potentiates the cytotoxic effect of gemcitabine in human pancreatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Sumit Arora

    Full Text Available Survival rates for patients with pancreatic cancer are extremely poor due to its asymptomatic progression to advanced and metastatic stage for which current therapies remain largely ineffective. Therefore, novel therapeutic agents and treatment approaches are desired to improve the clinical outcome. In this study, we determined the effects of honokiol, a biologically active constituent of oriental medicinal herb Magnolia officinalis/grandiflora, on two pancreatic cancer cell lines, MiaPaCa and Panc1, alone and in combination with the standard chemotherapeutic drug, gemcitabine. Honokiol exerted growth inhibitory effects on both the pancreatic cancer cell lines by causing cell cycle arrest at G₁ phase and induction of apoptosis. At the molecular level, honokiol markedly decreased the expression of cyclins (D1 and E and cyclin-dependent kinases (Cdk2 and Cdk4, and caused an increase in Cdk inhibitors, p21 and p27. Furthermore, honokiol treatment led to augmentation of Bax/Bcl-2 and Bax/Bcl-xL ratios to favor apoptosis in pancreatic cancer cells. These changes were accompanied by enhanced cytoplasmic accumulation of NF-κB with a concomitant decrease in nuclear fraction and reduced transcriptional activity of NF-κB responsive promoter. This was associated with decreased phosphorylation of inhibitor of kappa B alpha (IκB-α causing its stabilization and thus increased cellular levels. Importantly, honokiol also potentiated the cytotoxic effects of gemcitabine, in part, by restricting the gemcitabine-induced nuclear accumulation of NF-κB in the treated pancreatic cancer cell lines. Altogether, these findings demonstrate, for the first time, the growth inhibitory effects of honokiol in pancreatic cancer and indicate its potential usefulness as a novel natural agent in prevention and therapy.

  20. SCYL1-BP1 affects cell cycle arrest in human hepatocellular carcinoma cells via Cyclin F and RRM2.

    Science.gov (United States)

    Wang, Yang; Zhi, Qiaoming; Ye, Qin; Zhou, Chengyuan; Zhang, Lei; Yan, Wei; Wu, Qun; Zhang, Di; Li, Pu; Huo, Keke

    2016-01-01

    The cell cycle is regulated via important biological mechanisms. Controlled expression of cell cycle regulatory proteins is crucial to maintain cell cycle progression. However, unbalanced protein expression leads to many diseases, such as cancer. Previous research suggests that SCYL1-BP1 function might be related to cell cycle progression and SCYL1-BP1 dysfunction to diseases through undefined mechanisms. In this research, an unbiased yeast two-hybrid screen was used to find protein(s) with potential biological relevance to SCYL1-BP1 function, and a novel interaction was recognized between SCYL1-BP1 and Cyclin F. This interaction was chosen as a paradigm to study SCYL1-BP1 function in cell cycle progression and its possible role in tumorigenesis. We found that SCYL1-BP1 binds to Cyclin F both in vivo and in vitro. SCYL1-BP1 overexpression promoted expression of the CCNF gene and simultaneously delayed Cyclin F protein degradation. SCYL1-BP1 knockdown reduced the expression of endogenous Cyclin F. It was also demonstrated in functional assays that SCYL1-BP1 overexpression induces G2/M arrest in cultured liver cells. Furthermore, SCYL1-BP1 sustained RRM2 protein expression by reducing its ubiquitination. Thus, we propose that SCYL1- BP1 affects the cell cycle through increasing steady state levels of Cyclin F and RRM2 proteins, thus constituting a dual regulatory circuit. This study provides a possible mechanism for SCYL1-BP1-mediated cell cycle regulation and related diseases.

  1. Down-regulation of the chemokine receptor CCR5 by activation of chemotactic formyl peptide receptor in human monocytes.

    Science.gov (United States)

    Shen, W; Li, B; Wetzel, M A; Rogers, T J; Henderson, E E; Su, S B; Gong, W; Le, Y; Sargeant, R; Dimitrov, D S; Oppenheim, J J; Wang, J M

    2000-10-15

    Interactions between cell surface receptors are important regulatory elements in the complex host responses to infections. In this study, it is shown that a classic chemotactic factor, the bacterial chemotactic peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), rapidly induced a protein-kinase-C-mediated serine phosphorylation and down-regulation of the chemokine receptor CCR5, which serves as a major human immunodeficiency virus (HIV)-1 coreceptor. The fMLF binding to its receptor, formyl peptide receptor (FPR), resulted in significant attenuation of cell responses to CCR5 ligands and in inhibition of HIV-1-envelope-glycoprotein-mediated fusion and infection of cells expressing CD4, CCR5, and FPR. The finding that the expression and function of CCR5 can be regulated by peptides that use an unrelated receptor may provide a novel approach to the design of anti-inflamatory and antiretroviral agents. (Blood. 2000;96:2887-2894)

  2. 4β-Hydroxywithanolide E from Physalis peruviana (golden berry inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

    Directory of Open Access Journals (Sweden)

    Guo Zong-Lun

    2010-02-01

    Full Text Available Abstract Background The crude extract of the fruit bearing plant, Physalis peruviana (golden berry, demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Methods Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299 using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE assay was used to evaluate the DNA damage due to the drug. Results It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p p 50 of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 μg/mL for 24 h. Conclusions In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

  3. The novel anthraquinone derivative IMP1338 induces death of human cancer cells by p53-independent S and G2/M cell cycle arrest.

    Science.gov (United States)

    Choi, Hyun Kyung; Ryu, Hwani; Son, A-Rang; Seo, Bitna; Hwang, Sang-Gu; Song, Jie-Young; Ahn, Jiyeon

    2016-04-01

    To identify novel small molecules that induce selective cancer cell death, we screened a chemical library containing 1040 compounds in HT29 colon cancer and CCD18-Co normal colon cells, using a phenotypic cell-based viability assay system with the Cell Counting Kit-8 (CCK-8). We discovered a novel anthraquinone derivative, N-(4-[{(9,10-dioxo-9,10-dihydro-1-anthracenyl)sulfonyl}amino]phenyl)-N-methylacetamide (IMP1338), which was cytotoxic against the human colon cancer cells tested. The MTT cell viability assay showed that treatment with IMP1338 selectively inhibited HCT116, HCT116 p53(-/-), HT29, and A549 cancer cell proliferation compared to that of Beas2B normal epithelial cells. To elucidate the cellular mechanism underlying the cytotoxicity of IMP1338, we examined the effect of IMP1338 on the cell cycle distribution and death of cancer cells. IMP1338 treatment significantly arrested the cell cycle at S and G2/M phases by DNA damage and led to apoptotic cell death, which was determined using FACS analysis with Annexin V/PI double staining. Furthermore, IMP1338 increased caspase-3 cleavage in wild-type p53, p53 knockout HCT116, and HT29 cells as determined using immunoblotting. In addition, IMP1338 markedly induced the phosphorylation of histone H2AX and Chk1 in both cell lines while the combination of 5-fluorouracil (5-FU) and radiation inhibited the viability of HCT116, HCT116 p53(-/-), and HT29 cells compared to 5-FU or radiation alone. Our findings indicated that IMP1338 induced p53-independent cell death through S and G2/M phase arrest as well as DNA damage. These results provide a basis for future investigations assessing the promising anticancer properties of IMP1338.

  4. The pleiotropic effects of fisetin and hesperetin on human acute promyelocytic leukemia cells are mediated through apoptosis, cell cycle arrest, and alterations in signaling networks.

    Science.gov (United States)

    Adan, Aysun; Baran, Yusuf

    2015-11-01

    Fisetin and hesperetin, flavonoids from various plants, have several pharmaceutical activities including antioxidative, anti-inflammatory, and anticancer effects. However, studies elucidating the role and the mechanism(s) of action of fisetin and hesperetin in acute promyelocytic leukemia are absent. In this study, we investigated the mechanism of the antiproliferative and apoptotic actions exerted by fisetin and hesperetin on human HL60 acute promyelocytic leukemia cells. The viability of HL60 cells was evaluated using the MTT assay, apoptosis by annexin V/propidium iodide (PI) staining and cell cycle distribution using flow cytometry, and changes in caspase-3 enzyme activity and mitochondrial transmembrane potential. Moreover, we performed whole-genome microarray gene expression analysis to reveal genes affected by fisetin and hesperetin that can be important for developing of future targeted therapy. Based on data obtained from microarray analysis, we also described biological networks modulated after fisetin and hesperetin treatment by KEGG and IPA analysis. Fisetin and hesperetin treatment showed a concentration- and time-dependent inhibition of proliferation and induced G2/M arrest for both agents and G0/G1 arrest for hesperetin at only the highest concentrations. There was a disruption of mitochondrial membrane potential together with increased caspase-3 activity. Furthermore, fisetin- and hesperetin-triggered apoptosis was confirmed by annexin V/PI analysis. The microarray gene profiling analysis revealed some important biological pathways including mitogen-activated protein kinases (MAPK) and inhibitor of DNA binding (ID) signaling pathways altered by fisetin and hesperetin treatment as well as gave a list of genes modulated ≥2-fold involved in cell proliferation, cell division, and apoptosis. Altogether, data suggested that fisetin and hesperetin have anticancer properties and deserve further investigation.

  5. Histamine Regulates Actin Cytoskeleton in Human Toll-like Receptor 4-activated Monocyte-derived Dendritic Cells Tuning CD4+ T Lymphocyte Response.

    Science.gov (United States)

    Aldinucci, Alessandra; Bonechi, Elena; Manuelli, Cinzia; Nosi, Daniele; Masini, Emanuela; Passani, Maria Beatrice; Ballerini, Clara

    2016-07-08

    Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1-4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance.

  6. Effect of extracts of poly(ether imide) microparticles on cytotoxicity, ROS generation and proinflammatory effects on human monocytic (THP-1) cells.

    Science.gov (United States)

    Kumar, Reddi K; Basu, Sayantani; Lemke, Horst-Dieter; Jankowski, Joachim; Kratz, Karl; Lendlein, Andreas; Tetali, Sarada D

    2016-01-01

    Current haemodialysis techniques are not capable to remove efficiently low molecular weight hydrophobic uremic toxins from the blood of patients suffering from chronic renal failure. With respect to the hydrophobic characteristics and the high level of protein binding of these uremic toxins, hydrophobic adsorber materials might be an alternative to remove these substances from the plasma of the chronic kidney disease (CKD) patients. Here nanoporous microparticles prepared from poly(ether imide) (PEI) with an average diameter of 90 ± 30 μm and a porosity around 88 ± 2% prepared by a spraying/coagulation process are considered as candidate adsorber materials. A prerequisite for the clinical application of such particles is their biocompatibility, which can be examined i.e. indirectly in cell culture experiments with the particles' extracts. In this work we studied the effects of aqueous extracts of PEI microparticles on the viability of THP-1 cells, a human leukemia monocytic cell line, as well as their macrophage differentiation, reactive oxygen species (ROS) generation and inflammation.A high cell viability of around 99 ± 18% and 99 ± 5% was observed when THP-1 cells were cultured in the presence of aqueous extracts of the PEI microparticles in medium A and medium B respectively. The obtained microscopic data suggested that PEI particle extracts have no significant effect on cell death, oxidative stress or differentiation to macrophages. It was further found that the investigated proinflammatory markers in THP-1 cells were not up-regulated. These results are promising with regard to the biocompatibility of PEI microparticles and in a next step the hemocompatibility of the microparticles will be examined.

  7. Production of proinflammatory cytokines in the human THP-1 monocyte cell line following induction by Tp0751,a recombinant protein of Treponema pallidum

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The tissue destruction characteristic of syphilis infection may be caused by inflammation due to Treponema pallidum and the ensuing immune responses to the pathogen.T.pallidum membrane proteins are thought to be potent inducers of inflammation during the early stages of infection.However,the actual membrane proteins that induce inflammatory cytokine production are not known,nor are the molecular mechanisms responsible for triggering and sustaining the inflammatory cascades.In the present study,Tp0751 recombinant protein from T.pallidum was found to induce the production of proinflammatory cytokines,including TNF-α,IL-1βand IL-6,in a THP-1 human monocyte cell line.The signal transduction pathways involved in the production of these cytokines were then further investigated.No inhibition of TNF-a,IL-1β,or IL-6 production was observed following treatment with the SAPK/JNK specific inhibitor SP600125 or with an ERK inhibitor PD98059.By contrast,anti-TLR2 mAb,anti-CD14 mAb,and the p38 inhibitor SB203580 significantly inhibited the production of all three cytokines.In addition,pyrrolidine dithiocarbamate (PDTC),a specific inhibitor of NF-κB,profoundly inhibited the production of these cytokines.Tp0751 treatment strongly activated NF-κB,as revealed by Western blotting.However,NF-κB translocation was significantly inhibited by treatment with PDTC.These results indicated that TLR2,CD14,MAPKs/p38,and NF-κB might be implicated in the inflammatory reaction caused by T.pallidum infection.

  8. Osteoblastic and osteoclastic differentiation of human mesenchymal stem cells and monocytes in a miniaturized three-dimensional culture with mineral granules.

    Science.gov (United States)

    Gamblin, Anne Laure; Renaud, Audrey; Charrier, Céline; Hulin, Philippe; Louarn, Guy; Heymann, Dominique; Trichet, Valérie; Layrolle, Pierre

    2014-12-01

    The pathologies of the skeleton have a significant socioeconomic impact on our population. Although therapies have improved the treatment of osteosarcoma and osteoporosis, their efficacy still remains limited. In this context, we developed a miniaturized 3-D culture model of bone cells on calcium phosphate ceramics. Human bone marrow mesenchymal stem cells (MSCs) were three-dimensionally cultured on particles of biphasic calcium phosphate (BCP, 125-200μm) in osteogenic media. The MSCs seeded on the BCP particles adhered and proliferated, producing abundant collagenous extracellular matrix (ECM). Light and confocal laser scanning microscopy showed that the MSCs created bridges between the BCP particles and formed a 3-D structure. Energy dispersive X-ray analysis in a scanning electron microscope confirmed the mineralization of the collagen matrix. The 96-well sized bone constructs were tested by immunohistology and transcription analysis, proving cell differentiation. Both techniques corroborated the osteoblastic differentiation with high production of bone sialoprotein and osteocalcin. Peripheral blood CD14-positive monocytes (MOs) were pre-differentiated into osteoclasts prior to seeding on the 3-D constructs. Multinucleated and tartrate-resistant acid phosphatase-positive cells were also identified at the surface of the 3-D constructs after 90days of culture. In addition, cell viability within these constructs was measured by flow cytometry. In summary, we have developed a miniaturized 3-D culture of bone cell precursors with osteoblasts and osteoclasts. This 3-D culture may make it possible to test the effects of new drugs for bone healing, osteoporosis and osteosarcomas, in more appropriate cell-cell and cell-matrix interactions than conventional 2-D cultures.

  9. Lipopolysaccharide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate mediators in the sequence of inflammatory and antiinflammatory responses of human monocytes.

    Science.gov (United States)

    Brattig, N W; Rathjens, U; Ernst, M; Geisinger, F; Renz, A; Tischendorf, F W

    2000-08-01

    The majority of Onchocerca volvulus-infected persons show signs of cellular anergy, and long-time survival of adult and larval parasites in subcutaneous tissue is observed. The mechanisms leading to immunological hyporesponsiveness are poorly understood. Monocytes/macrophages represent a link between the innate and acquired immune system and are candidate cells to promote inflammatory and antiinflammatory processes. In the present study we have shown that products of microfilarial (O. volvulus) and adult (O. volvulus and O. ochengi) parasites affect monocytes in vitro. An early production of TNF-alpha by exposed monocytes was followed by the production of IL-10 and a reduced expression of HLA-DR and the costimulatory molecules B7-1 and B7-2, while other adhesion receptors remained unaffected. Downregulation of the functional membrane receptors failed to occur after treatment of the cells with anti-IL-10 antibodies. The engagement of CD14, a dominant membrane receptor on monocytes and major binding protein for lipopolysaccharides, was indicated by partial blocking of monocyte modulation by neutralizing antibodies to CD14 and by the antagonistic lipid A analog compound 406. Lipopolysaccharide-like molecules were detected in sterile products of O. volvulus stages which could originate from Wolbachia bacteria related to Gram-negative Rickettsiales, known to be abundant in the hypodermis and the female reproductive organs of O. volvulus. The present results indicate that the monocyte/macrophage may be a major target cell for immunomodulatory parasite-derived and intraparasitic, bacteria-derived molecules, thereby contributing to the host's cellular hyporesponsiveness.

  10. A novel,rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+monocytes within 48 hours of in vitro culture

    Institute of Scientific and Technical Information of China (English)

    Xin Guan; Ji-Run Peng; Lan Yuan; Hui Wang; Yu-Hua Wei; Xi-Sheng Leng

    2004-01-01

    AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factorα (TNFα), interleukin1β (IL-1β), interleukin-6 (IL-6)and prostaglandin E2 (PGE2).One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h ofin vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocytederived dendritic cells within 48 h ofin vitro culture (FastDC).METHODS: HCCLMl3 cells were cultured in RPMI 1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/Lheat inactivated human AB serum, 2 mmol/L L-glutamine,100 μg/mL gentamicin, 1000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFα(1000 U/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL) and PGE2(1μg/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/L polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritomas. The FastDCs and novel dendritomas were immunostained with antiCD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence

  11. Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells

    Institute of Scientific and Technical Information of China (English)

    Sinikka Latvala; Taija E Pietil(a); Ville Veckman; Riina A Kekkonen; Soile Tynkkynen; Riitta Korpela; Ilkka Julkunen

    2008-01-01

    MM: To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyLe-derived dendritic cells (moDCs).METHODS: Cytokine production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis (FACS),respectively.The kinetics of mRNA expression of cytokine genes was determined by Northern blotting.The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors.RESULTS: All studied bacteria induced the maturation of moDCs in a dose-dependent manner.More detailed analysis with S.thermophilus THS,B.breve Bb99,and L.lactis subsp,cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class II and CD86 as efficiently as pathogenic bacteria.However,these bacteria differed in their ability to induce moDC cytokine gene expression.S.therrnophilus induced the expression of pro-inflammatory (TNF-a,IL-12,IL-6,and CCL20)and Th1 type (IL-12 and IFN-y) cytokines,while B.breve and L.lactis were also potent inducers of antiinflammatory IL-10.Mitogen-activated protein kinase (MAPK) p38,phosphatidylinositol 3 (PI3) kinase,and nuclear factor-kappa B (NF-κB) signaling pathways were shown to be involved in bacteria-induced cytokine production.CONCLUSION: Our results indicate that potentially probiotic bacteria are able to induce moDC maturation,but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another.

  12. HIV-1-infected monocytes and monocyte-derived macrophages are impaired in their ability to produce superoxide radicals.

    Science.gov (United States)

    Howell, A L; Groveman, D S; Wallace, P K; Fanger, M W

    1997-01-01

    Monocytes and monocyte-derived macrophages play a key role in immune defense against pathogenic organisms. Superoxide anion production is a key mechanism by which phagocytes kill pathogens. We sought to determine whether human immunodeficiency virus-infected monocytes and monocyte-derived macrophages are compromised in their ability to produce the superoxide anion following stimulation with phorbol myristate acetate (PMA) or after cross-linking the type I Fc receptor for IgG (Fc gamma RI). Fc gamma RI was cross-linked by the binding of monoclonal antibody 197, which reacts with an epitope of Fc gamma RI via its Fc region. Monocytes and monocyte-derived macrophages obtained from seronegative donors were infected in vitro with human immunodeficiency virus-1JR-FL and used in effector assays that measured superoxide anion production by the reduction of nitroblue tetrazolium. Reduced nitroblue tetrazolium was measured spectrophotometrically and by microscopy in which the percentage of cells containing intracellular deposits of the dye was assessed. By spectrophotometric measurement, we found that human immunodeficiency virus-infected monocytes and monocyte-derived macrophages produced less superoxide anion following either phorbol myristate acetate stimulation or Fc gamma RI cross-linking than uninfected cells from the same donor. Using microscopy we saw no difference in the percentage of infected and uninfected macrophages containing intracellular deposits of nitroblue tetrazolium suggesting that human immunodeficiency virus-infected macrophages produce less superoxide anion on a per cell basis than uninfected macrophages. Activation of human immunodeficiency virus-infected monocytes with interferon-gamma for 72 h prior to stimulation with phorbol myristate acetate or monoclonal antibody 197 increased their ability to reduce nitroblue tetrazolium. These findings suggest that impairment in the production of reactive oxygen intermediates may, in some cases, contribute to

  13. Pivotal Role for CD16+ Monocytes in Immune Surveillance of the Central Nervous System.

    Science.gov (United States)

    Waschbisch, Anne; Schröder, Sina; Schraudner, Dana; Sammet, Laura; Weksler, Babette; Melms, Arthur; Pfeifenbring, Sabine; Stadelmann, Christine; Schwab, Stefan; Linker, Ralf A

    2016-02-15

    Monocytes represent a heterogeneous population of primary immune effector cells. At least three different subsets can be distinguished based on expression of the low-affinity FcγRIII: CD14(++)CD16 -: classical monocytes, CD14(++)CD16(+) intermediate monocytes, and CD14(+)CD16 ++: non-classical monocytes. Whereas CD16 -: classical monocytes are considered key players in multiple sclerosis (MS), little is known on CD16(+) monocytes and how they contribute to the disease. In this study, we examined the frequency and phenotype of monocyte subpopulations in peripheral blood, cerebrospinal fluid (CSF), and brain biopsy material derived from MS patients and controls. Furthermore, we addressed a possible monocyte dysfunction in MS and analyzed migratory properties of monocyte subsets using human brain microvascular endothelial cells. Our ex vivo studies demonstrated that CD16(+) monocyte subpopulations are functional but numerically reduced in the peripheral blood of MS patients. CD16(+) monocytes with an intermediate-like phenotype were found to be enriched in CSF and dominated the CSF monocyte population under noninflammatory conditions. In contrast, an inversed CD16(+) to CD16 -: CSF monocyte ratio was observed in MS patients with relapsing-remitting disease. Newly infiltrating, hematogenous CD16(+) monocytes were detected in a perivascular location within active MS lesions, and CD16(+) monocytes facilitated CD4(+) T cell trafficking in a blood -: brain barrier model. Our findings support an important role of CD16(+) monocytes in the steady-state immune surveillance of the CNS and suggest that CD16(+) monocytes shift to sites of inflammation and contribute to the breakdown of the blood-brain barrier in CNS autoimmune diseases.

  14. Securinine from Phyllanthus glaucus Induces Cell Cycle Arrest and Apoptosis in Human Cervical Cancer HeLa Cells

    Science.gov (United States)

    Krauze-Baranowska, Mirosława; Ochocka, J. Renata

    2016-01-01

    Background The Securinega-type alkaloids occur in plants belonging to Euphorbiaceae family. One of the most widely distributed alkaloid of this group is securinine, which was identified next to allosecurinine in Phyllanthus glaucus (leafflower). Recently, some Securinega-type alkaloids have paid attention to its antiproliferative potency towards different cancer cells. However, the cytotoxic properties of allosecurinine have not yet been evaluated. Methods The cytotoxicity of the extract, alkaloid fraction obtained from P. glaucus, isolated securinine and allosecurinine against HeLa cells was evaluated by real-time xCELLigence system and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and 7-amino-actinomycin (7-AAD) staining and confirmed with fluorescent Hoechst 33342 dye. The assessment of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, the level of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), caspase-3/7 activity and cell cycle analysis were measured by flow cytometry. The enzymatic activity of caspase-9 was assessed by a luminometric assay. The expression of apoptosis associated genes was analyzed by real-time PCR. Results The experimental data revealed that securinine and the alkaloid fraction were significantly potent on HeLa cells growth inhibition with IC50 values of 7.02 ± 0.52 μg/ml (32.3 μM) and 25.46 ± 1.79 μg/ml, respectively. The activity of allosecurinine and Phyllanthus extract were much lower. Furthermore, our study showed that the most active securinine induced apoptosis in a dose-dependent manner in the tested cells, increased the percentage of ROS positive cells and depolarized cells as well as stimulated the activity of ERK1/2, caspase-9 and -3/7. Securinine also induced cell cycle arrest in S phase. Real-time PCR analysis showed high expression of TNFRSF genes in the cells stimulated with securinine. Conclusions Securinine

  15. 1-Oxoeudesm-11(13)-eno-12,8a-lactone induces G2/M arrest and apoptosis of human glioblastoma cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Shan-shan LIU; Yan-feng WANG; Li-sha MA; Bei-bei ZHENG; Lin LI; Wei-dong XiE; Xia LI

    2013-01-01

    Aim: To investigate the effects of 1-oxoeudesm-11(13)eno-12,8a-lactone (OEL),a novel eudesmane-type sesquiterpene isolated from Aster himalaicus,on the cell cycle and apoptosis in human glioblastoma cells in vitro.Methods: Human malignant glioblastoma cell lines U87 and A172 were used.The cytotoxicity of OEL was examined using the MTT assay.Cell apoptosis was assessed with DAPI staining and flow cytometry.DNA damage was determined by measuring the phosphorylation of H2AX using immunofluorescence staining and Western blotting.Cell cycle profiles were measured with flow cytometry.The mRNA expression of p53 and p21Waf1/Cip1 was investigated using real-time PCR.The protein expression of γ-H2AX,caspase-9,caspase-3,p53,p21Waf1/Cip1,cyclin B1,and cdc2 was analyzed with Western blotting.Results: Treatment of the malignant glioblastoma cells with OEL inhibited the cell growth in dose-and time-dependent manners (thevalues of IC50 at 48 and 72 h were 29.5 and 16.99 μmol/L,respectively,in U87 cells; 7.2 and 9.5 μmol/L,respectively,in A172 cells).OEL (10-30μmol/L) induced apoptosis and G2/M phase arrest in both U87 and A172 cells.OEL induced the phosphorylation of cdc2,a G2/M phase cyclin-dependent kinase,and decreased the expression of cyclin B1 required for progression through the G2/M phase in U87 cells.The compound remarkably increased the phosphorylation of H2AX in U87 cells.Moreover,OEL increased the mRNA and protein levels of p53 and its target gene p21waf1/cip1 in U87 cells.The compound also induced p53 phosphorylation.Pretreatment withPFT-α,a specific inhibitor of p53 transcriptional activity,could partially reverse the inhibition of OEL on the viability of U87 and A172 cells.Conclusion: OEL suppresses the growth of human glioblastoma cells in vitro via inducing DNA damage,p53-mediated cell cycle arrest and apoptosis,thus warrants further studies as a lead compound of anti-glioblastoma drug.

  16. Honokiol, a potential therapeutic agent, induces cell cycle arrest and program cell death in vitro and in vivo in human thyroid cancer cells.

    Science.gov (United States)

    Lu, Chieh-Hsiang; Chen, Shu-Hsin; Chang, Yi-Sheng; Liu, Yi-Wen; Wu, Jin-Yi; Lim, Yun-Ping; Yu, Hui-I; Lee, Ying-Ray

    2017-01-01

    Thyroid cancer is the most common endocrine malignancy, the global incidence rate of which is rapidly rising. Surgery and radioiodine therapies are common and effective treatments only for nonmetastasized primary tumors. Therefore, effective treatment modalities are imperative for patients with radioiodine-resistant thyroid cancer. Honokiol, a biophenolic compound derived from Magnolia spp., has been shown have diverse biological and pharmacological activities, including anti-inflammatory, antioxidative, antiangiogenic, and anticancer properties. In the present study, three human thyroid cancer cell lines, namely anaplastic, follicular, and poorly differentiated thyroid cancer cells, were used to evaluate the chemotherapeutic activity of honokiol. Cell viability, cell cycle, apoptosis, and autophagy induction were determined through flow cytometry and western blot analysis. We found that honokiol treatment can suppress cell growth, induce cell cycle arrest, and enhance the induction of caspase-dependent apoptosis and autophagy in cancer cells. Moreover, honokiol treatment modulated signaling pathways including Akt/mTOR, ERK, JNK, and p38 in the studied cells. In addition, the antitumorigenic activity of honokiol was also confirmed in vitro and in vivo. Our data provide evidence that honokiol has a unique application in chemotherapy for human thyroid cancers.

  17. Alpinia pricei Rhizome Extracts Induce Cell Cycle Arrest in Human Squamous Carcinoma KB Cells and Suppress Tumor Growth in Nude Mice

    Directory of Open Access Journals (Sweden)

    You-Cheng Hseu

    2011-01-01

    Full Text Available Alpinia pricei has been shown to induce apoptosis in human squamous carcinoma (KB cells. In this study, we report the effectiveness of the ethanol (70% extracts of A. pricei rhizome (AP extracts in terms of tumor regression as determined using both in vitro cell culture and in vivo athymic nude mice models of KB cells. We found that the AP extract (25–200 μg/mL treatment decreased the proliferation of KB cells by arresting progression through the G2/M phase of the cell cycle. This cell cycle blockade was associated with reductions in cyclin A and B1, Cdc2, and Cdc25C, and increased p21/WAF1, Wee1, p53 and phospho-p53 (p-p53 in a dose- and time-dependent manner. Moreover, we found that AP extract treatment decreased metalloproteinase-9 (MMP-9 and urokinase plasminogen activator (u-PA expression, while expression of their endogenous inhibitors, tissue inhibitor of MMP-1 (TIMP-1 and plasminogen activator inhibitor-1 (PAI-1, were increased in KB cells. Furthermore, AP extract treatment effectively delayed tumor incidence in nude mice inoculated with KB cells and reduced the tumor burden. AP extract treatment also induced apoptotic DNA fragmentation, as detected by in situ TUNEL staining. Thus, A. pricei may possess antitumor activity in human squamous carcinoma (KB cells.

  18. Acetylsalicylic acid-induced oxidative stress, cell cycle arrest, apoptosis and mitochondrial dysfunction in human hepatoma HepG2 cells.

    Science.gov (United States)

    Raza, Haider; John, Annie; Benedict, Sheela

    2011-10-01

    It is widely accepted that non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, reduce the risk of cancer. The anti-cancer and anti-inflammatory effects of NSAIDs are associated with the inhibition of prostaglandin synthesis and cyclooxygenase-2 activity. Several other mechanisms which contribute to the anti-cancer effect of these drugs in different cancer models both in vivo and in vitro are also presumed to be involved. The precise molecular mechanism, however, is still not clear. We investigated, therefore, the effects of acetylsalicylic acid (ASA, aspirin) on multiple cellular and functional targets, including mitochondrial bioenergetics, using human hepatoma HepG2 cancer cells in culture. Our results demonstrate that ASA induced G0/G1 cell cycle arrest and apoptosis in HepG2 cells. ASA increased the production of reactive oxygen species, reduced the cellular glutathione (GSH) pool and inhibited the activities of the mitochondrial respiratory enzyme complexes, NADH-ubiquinone oxidoreductase (complex I), cytochrome c oxidase (complex IV) and the mitochondrial matrix enzyme, aconitase. Apoptosis was triggered by alteration in mitochondrial permeability transition, inhibition of ATP synthesis, decreased expression of the anti-apoptotic protein Bcl-2, release of cytochrome c and activation of pro-apoptotic caspase-3 and the DNA repairing enzyme, poly (-ADP-ribose) polymerase (PARP). These findings strongly suggest that ASA-induced toxicity in human hepatoma HepG2 cells is mediated by increased metabolic and oxidative stress, accompanied by mitochondrial dysfunction which result in apoptosis.

  19. Fucoidan induces G1 arrest of the cell cycle in EJ human bladder cancer cells through down-regulation of pRB phosphorylation

    Directory of Open Access Journals (Sweden)

    Hye Young Park

    2015-06-01

    Full Text Available AbstractFucoidan, a sulfated polysaccharide found in marine algae and brown seaweeds, has been shown to inhibit the in vitro growth of human cancer cells. This study was conducted in cultured human bladder cancer EJ cells to elucidate the possible mechanisms by which fucoidan exerts its anti-proliferative activity, which until now has remained poorly understood. Fucoidan treatment of EJ cells resulted in dose-dependent inhibition of cell growth and induced apoptotic cell death. Flow cytometric analysis revealed that fucoidan led to G1 arrest in cell cycle progression. It was associated with down-regulation of cyclin D1, cyclin E, and cyclin-dependent-kinases (Cdks in a concentration-dependent manner, without any change in Cdk inhibitors, such as p21 and p27. Furthermore, dephosphorylation of retinoblastoma protein (pRB by this compound was associated with enhanced binding of pRB wit