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Sample records for human granulocyte macrophage

  1. CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Kasinrerk, W; Baumruker, T; Majdic, O; Knapp, W; Stockinger, H

    1993-01-15

    In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome...... of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system....

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

    Directory of Open Access Journals (Sweden)

    S Montagnani

    2009-12-01

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

  4. In vivo effect of human granulocyte-macrophage colony-stimulating factor on megakaryocytopoiesis

    International Nuclear Information System (INIS)

    Aglietta, M.; Monzeglio, C.; Sanavio, F.; Apra, F.; Morelli, S.; Stacchini, A.; Piacibello, W.; Bussolino, F.; Bagnara, G.; Zauli, G.

    1991-01-01

    The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on megakaryocytopoiesis and platelet production was investigated in patients with normal hematopoiesis. Three findings indicated that GM-CSF plays a role in megakaryocytopoiesis. During treatment with GM-CSF (recombinant mammalian, glycosylated; Sandoz/Schering-Plough, 5.5 micrograms protein/kg/d, subcutaneously for 3 days) the percentage of megakaryocyte progenitors (megakaryocyte colony forming unit [CFU-Mk]) in S phase (evaluated by the suicide technique with high 3H-Tdr doses) increased from 31% +/- 16% to 88% +/- 11%; and the maturation profile of megakaryocytes was modified, with a relative increase in more immature stage I-III forms. Moreover, by autoradiography (after incubation of marrow cells with 125I-labeled GM-CSF) specific GM-CSF receptors were detectable on megakaryocytes. Nevertheless, the proliferative stimulus induced on the progenitors was not accompanied by enhanced platelet production (by contrast with the marked granulomonocytosis). It may be suggested that other cytokines are involved in the regulation of the intermediate and terminal stages of megakaryocytopoiesis in vivo and that their intervention is an essential prerequisite to turn the GM-CSF-induced proliferative stimulus into enhanced platelet production

  5. Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells

    International Nuclear Information System (INIS)

    Moonen, P.; Mermod, J.J.; Ernst, J.F.; Hirschi, M.; DeLamarter, J.F.

    1987-01-01

    Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coli. The E. coli produced-protein-lacking any carbohydrate- had by far the highest specific activity observed for the recombinant hGM-CSFs

  6. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    International Nuclear Information System (INIS)

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F.

    1990-01-01

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of 125 I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB- 125 I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein

  7. Combined application of alginate dressing and human granulocyte-macrophage colony stimulating factor promotes healing in refractory chronic skin ulcers.

    Science.gov (United States)

    Huang, Guobao; Sun, Tangqing; Zhang, Lei; Wu, Qiuhe; Zhang, Keyan; Tian, Qingfen; Huo, Ran

    2014-06-01

    The aim of the present study was to evaluate the clinical therapeutic effect of the combined application of alginate and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on the healing of refractory chronic skin ulcers. A single center, three arm, randomized study was performed at Jinan Central Hospital (Jinan, Shandong, China). A total of 60 patients with refractory chronic skin ulcers, which persisted for >1 month, were enrolled and randomly assigned into one of the following three groups: alginate dressing/rhGM-CSF group (group A), rhGM-CSF only group (group B) and conventional (vaseline dressing) group (group C). The wound area rate was measured, granulation and color were observed and pain was evaluated. The data were summarized and statistical analysis was performed. The results demonstrated that group A exhibited a significantly faster wound healing rate and lower pain score compared with the other groups (PCSF for the treatment of refractory chronic skin ulcers demonstrated significant advantages. It promoted the growth of granulation tissue, accelerated re-epithelialization and also effectively reduced wound pain, and thus improved the quality of life for the patient. This suggests that the combined application of alginate and rhGM-CSF may be an effective therapeutic strategy for the clinical treatment of refractory chronic skin ulcers.

  8. Nuclear proteins interacting with the promoter region of the human granulocyte/macrophage colony-stimulating factor gene

    International Nuclear Information System (INIS)

    Shannon, M.F.; Gamble, J.R.; Vadas, M.A.

    1988-01-01

    The gene for human granulocyte/macrophage colony-stimulating factor (GM-CSF) is expressed in a tissue-specific as well as an activation-dependent manner. The interaction of nuclear proteins with the promoter region of the GM-CSF gene that is likely to be responsible for this pattern of GM-CSF expression was investigated. The authors show that nuclear proteins interact with DNA fragments from the GM-CSF promoter in a cell-specific manner. A region spanning two cytokine-specific sequences, cytokine 1 (CK-1, 5', GAGATTCCAC 3') and cytokine 2 (CK-2, 5' TCAGGTA 3') bound two nuclear proteins from GM-CSF-expressing cells in gel retardation assays. NF-GMb was inducible with phorbol 12-myristate 13-acetate and accompanied induction of GM-CSF message. NF-GMb was absent in cell lines not producing GM-CSF, some of which had other distinct binding proteins. NF-GMa and NF-GMb eluted from a heparin-Sepharose column at 0.3 and 0.6 M KCl, respectively. They hypothesize that the sequences CK-1 and CK-2 bind specific proteins and regulate GM-CSF transcription

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

    Science.gov (United States)

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

    2015-07-01

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

  10. Characterization and molecular features of the cell surface receptor for human granulocyte-macrophage colony-stimulating factor

    International Nuclear Information System (INIS)

    Chiba, S.; Tojo, A.; Kitamura, T.; Urabe, A.; Miyazono, K.; Takaku, F.

    1990-01-01

    The receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the surfaces of normal and leukemic myeloid cells were characterized using 125I-labeled bacterially synthesized GM-CSF. The binding was rapid, specific, time dependent, and saturable. Scatchard analysis of the 125I-GM-CSF binding to peripheral blood neutrophils indicated the presence of a single class of binding site (Kd = 99 +/- 21 pM; 2,304 +/- 953 sites/cell). However, for peripheral blood monocytes and two GM-CSF-responsive myeloid cell lines (U-937 and TF-1), the Scatchard plots were biphasic curvilinear, which were best fit by curves derived from two binding site model: one with high affinity (Kd1 = 10-40 pM) and the other with low affinity (Kd2 = 0.9-2.0 nM). For U-937 cells, the number of high-affinity receptors was 1,058 +/- 402 sites/cell and that of low-affinity receptors was estimated to be 10,834 +/- 2,396 sites/cell. Cross-linking studies yielded three major bands with molecular masses of 150 kDa, 115 kDa, and 95 kDa, which were displaced by an excess amount of unlabeled GM-CSF, suggesting 135-kDa, 100-kDa, and 80-kDa species for the individual components of the human GM-CSF receptor. These bands comigrated for different cell types including peripheral blood neutrophils, U-937 cells and TF-1 cells. In experiments using U-937 cells, only the latter two bands appeared to be labeled in a dose-dependent manner in a low-affinity state. These results suggest that the human GM-CSF receptor possibly forms a multichain complex

  11. Differential expression of the human thymosin-β4 gene in lymphocytes, macrophages, and granulocytes

    International Nuclear Information System (INIS)

    Gondo, H.; Kudo, J.; White, J.W.; Barr, C.; Selvanayagam, P.; Saunders, G.F.

    1987-01-01

    A cDNA clone encoding human thymosin-β 4 was isolated from a cDNA library prepared from peripheral blood leukocytes of a patient with acute lymphocytic leukemia. This clone contained the entire coding sequence of 43 amino acid residues of thymosin-β 4 and had an initiation codon and two termination codons. The amino acid and nucleotide sequences in the coding region were well conserved between rat and human. No signal peptide was found in the deduced protein sequence. Human thymosin-β 4 mRNA, approximately 830 nucleotides in length, was about 30 nucleotides larger than rat thymosin-β 4 mRNA. Expression of the human thymosin-β 4 gene in various primary myeloid and lymphoid malignant cells and in a few human hemopoietic cell lines was studied. Northern blot analyses of different neoplastic B lymphocytes revealed that steady state levels of thymosin-β 4 mRNA varied as a function of differentiation stage. Thymosin-β 4 mRNA levels were decreased in myeloma cells as are class II human leukocyte antigen, Fc receptor, and complement receptor, suggesting a relationship between thymosin-β 4 and the immune response. Treatment of THP-1 cells, a human monocytic cell line, with recombinant human interferon-γ reduced the levels of thymosin-β 4 mRNA. The pattern of thymosin-β 4 gene expression suggests that it may play a fundamental role in the host defense mechanism

  12. Effect of human granulocyte macrophage-colony stimulating factor on differentiation and apoptosis of the human osteosarcoma cell line SaOS-2

    Directory of Open Access Journals (Sweden)

    L Postiglione

    2009-06-01

    Full Text Available We investigated the effects of human granulocyte macrophage- colony stimulating factor (GM-CSF on the relation between differentiation and apoptosis in SaOS-2 cells, an osteoblast-like cell line. To determine the relationship between these cellular processes, SaOS-2 cells were treated in vitro for 1, 7 and 14 days with 200 ng/mL GM-CSF and compared with untreated cells. Five nM insulin-like growth factor (IGF-I and 30 nM okadaic acid were used as negative and positive controls of apoptosis, respectively. Effects on cell differentiation were determined by ECM (extracellular matrix mineralization, morphology of some typical mature osteoblast differentiation markers, such as osteopontin and sialoprotein II (BSP-II, and production of bone ECM components such as collagen I. The results showed that treatment with GM-CSF caused cell differentiation accompanied by increased production of osteopontin and BSP-II, together with increased ECM deposition and mineralization. Flow cytometric analysis of annexin V and propidium iodide incorporation showed that GM-CSF up-regulated apoptotic cell death of SaOS-2 cells after 14 days of culture in contrast to okadaic acid, which stimulated SaOS-2 apoptosis only during the early period of culture. Endonucleolytic cleavage of genomic DNA, detected by “laddering analysis”, confirmed these data. The results suggest that GM-CSF induces osteoblastic differentiation and long-term apoptotic cell death of the SaOS-2 human osteosarcoma cell line, which in turn suggests a possible in vivo physiological role for GM-CSF on human osteoblast cells.

  13. Interleukin-6 production by human monocytes treated with granulocyte-macrophage colony-stimulating factor in the presence of lipopolysaccharide of oral microorganisms.

    Science.gov (United States)

    Baqui, A A; Meiller, T F; Chon, J J; Turng, B F; Falkler, W A

    1998-06-01

    This study focused on the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide of the putative periodontal pathogens Porphyromonas gingivalis or Fusobacterium nucleatum on IL-6 production by THP-1 cells (a human monocytic cell line). Resting THP-1 cells were alternatively treated with GM-CSF (50 IU/ml) and lipopolysaccharide of P. gingivalis or F. nucleatum, in varying concentrations for varying time periods. IL-6 production in supernatant fluids of treated cells was evaluated by an enzyme-linked immunosorbent assay (ELISA) and a reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate gene expression. Untreated THP-1 cells did not produce IL-6 as determined by ELISA. RT-PCR also failed to detect IL-6 mRNA in untreated THP-1 cells, indicating that IL-6 was not constitutively produced. After stimulation of THP-1 cells with lipopolysaccharide of F. nucleatum or P. gingivalis, IL-6 was produced, peaking at 4 h (200-300 pg/ml) and thereafter sharply declining by 8 h. When GM-CSF was added together with lipopolysaccharide of P. gingivalis or F. nucleatum, there was a synergistic quantitative increase in production of IL-6 as measured by ELISA as compared with lipopolysaccharide alone. IL-6 mRNA was detected by RT-PCR, 15 min after stimulation with lipopolysaccharide of either P. gingivalis or F. nucleatum. GM-CSF supplementation with lipopolysaccharide of P. gingivalis shortened the transcription of IL-6 mRNA to 5 min, a shift which was not observed with lipopolysaccharide of F. nucleatum, possibly indicating a different mechanism of initiation of transcription. Production of IL-6 by GM-CSF-treated THP-1 cells in the presence of lipopolysaccharide of oral microorganisms may provide a model for studying the role of macrophages in acute and chronic periodontal diseases, including the clinical periodontal exacerbation as observed in chemotherapy patients receiving GM-CSF for bone marrow recovery.

  14. Enhanced interleukin-8 production in THP-1 human monocytic cells by lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

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    Baqui, A A; Meiller, T F; Falkler, W A

    1999-10-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-8 (IL-8) plays an important role in macrophage mediated inflammatory processes including exacerbation of periodontal diseases, one of the most common complications in GM-CSF receiving cancer patients. The effect of GM-CSF supplementation on IL-8 production was investigated in a human monocyte cell line THP-1, stimulated with lipopolysaccharide extracted from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. Resting THP-1 cells were treated with lipopolysaccharide (1 microgram/ml) of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) for varying time periods. The production of IL-8 in THP-1 cells was measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). A very low level of the cytokine IL-8 was produced constitutive in THP-1 cells. Starting from 8 h of treatment and afterwards GM-CSF alone significantly increased IL-8 production in THP-1 cells. Lipopolysaccharide (1 microgram/ml) extracts from either F. nucleatum or P. gingivalis amplified IL-8 production 500-800 times in comparison to resting THP-1 cells. When lipopolysaccharide of F. nucleatum or P. gingivalis was supplemented with 50 IU/ml of GM-CSF, there was a statistically significant enhanced production of IL-8 by THP-1 cells after 1 day to 7 days of treatment as compared with lipopolysaccharide treatment alone. GM-CSF (50 IU/ml) also significantly increased IL-8 production from 2-7 days of treatment of THP-1 cells when supplemented with a positive control, phorbol-12-myristate-13 acetate (PMA), as compared to PMA treatment alone. These investigations using the in vitro THP-1 human monocyte cell model indicate that there may be an increase in the response on a cellular level to oral endotoxin following GM-CSF therapy as evidenced by enhanced production of the tissue-reactive inflammatory cytokine, IL-8.

  15. Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Tran, Anh-Minh; Nguyen, Thanh-Thao; Nguyen, Cong-Thuan; Huynh-Thi, Xuan-Mai; Nguyen, Cao-Tri; Trinh, Minh-Thuong; Tran, Linh-Thuoc; Cartwright, Stephanie P; Bill, Roslyn M; Tran-Van, Hieu

    2017-04-04

    Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.

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

    DEFF Research Database (Denmark)

    Agerholm, Inge; Loft, Anne; Hald, Finn

    2010-01-01

    -vitro culture of human embryos in the presence of 2 ng/ml GM-CSF resulted in 34.8% (8/23) uniformly normal embryos. Culture without 2 ng/ml GM-CSF resulted in 33.3% (9/27) uniformly normal embryos. A trend towards a higher number of TQE in the test group was observed; however, due to lack of TQE in the control...... women donating 86 oocytes. The primary endpoint was to investigate the chromosomal constitution of human embryos (fluorescence in-situ hybridization analysis for chromosomes 13, 16, 18, 21, 22, X and Y) cultured with or without GM-CSF. The secondary endpoints were number of top-quality embryos (TQE......) and number of normally developed embryos evaluated morphologically on day 3. The cytogenetic analyses demonstrated non-inferiority and therefore the chromosomal constitution of human embryos cultured in vitro in the presence of 2 ng/ml GM-CSF was no worse than the control group cultured without GM-CSF. In...

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

    DEFF Research Database (Denmark)

    Agerholm, Inge; Loft, Anne; Hald, Finn

    2010-01-01

    women donating 86 oocytes. The primary endpoint was to investigate the chromosomal constitution of human embryos (fluorescence in-situ hybridization analysis for chromosomes 13, 16, 18, 21, 22, X and Y) cultured with or without GM-CSF. The secondary endpoints were number of top-quality embryos (TQE......) and number of normally developed embryos evaluated morphologically on day 3. The cytogenetic analyses demonstrated non-inferiority and therefore the chromosomal constitution of human embryos cultured in vitro in the presence of 2 ng/ml GM-CSF was no worse than the control group cultured without GM-CSF. In......-vitro culture of human embryos in the presence of 2 ng/ml GM-CSF resulted in 34.8% (8/23) uniformly normal embryos. Culture without 2 ng/ml GM-CSF resulted in 33.3% (9/27) uniformly normal embryos. A trend towards a higher number of TQE in the test group was observed; however, due to lack of TQE in the control...

  18. Granulocyte-Macrophage Colony-Stimulating Factor Amplification of Interleukin-1β and Tumor Necrosis Factor Alpha Production in THP-1 Human Monocytic Cells Stimulated with Lipopolysaccharide of Oral Microorganisms

    OpenAIRE

    Baqui, A. A. M. A.; Meiller, Timothy F.; Chon, Jennifer J.; Turng, Been-Foo; Falkler, William A.

    1998-01-01

    Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1β and TNF-α production following GM-CSF suppl...

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    -induced CD34(+) progenitor chemotaxis. These chemotactic attracted CD34(+) progenitors are colony-forming units-granulocyte-macrophage. gamma IP-10 and Mig also induced GM-CSF-stimulated CD34(+) progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 m......Ab blocked these functions of gammaIP-10 and Mig but not of chemokine stromal cell-derived factor 1 alpha. gamma IP-10-induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF-stimulated CD34(+) progenitors. Moreover, gamma IP-10 and Mig...... stimulated CXCR3 redistribution and cellular polarization in GM-CSF-stimulated CD34(+) progenitors. These results indicate that CXCR3-gamma IP-10 and CXCR3-Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment...

  20. Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

    Science.gov (United States)

    Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

    2015-04-01

    Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. The role of granulocyte macrophage-colony stimulating factor in gastrointestinal immunity to salmonellosis.

    Science.gov (United States)

    Coon, C; Beagley, K W; Bao, S

    2009-08-01

    Human Salmonella infection, in particular, typhoid fever is a highly infectious disease that remains a major public health problem causing significant morbidity and mortality. The outcome of these infections depends on the host's immune response, particularly the actions of granulocytes and macrophages. Using a mouse model of human typhoid fever, with Salmonella typhimurium infection of wild type and granulocyte macrophage-colony stimulating factor (GM-CSF) knock out mice we show a delay in the onset of immune-mediated tissue damage in the spleens and livers of GM-CSF(-/-) mice. Furthermore, GM-CSF(-/-) mice have a prolonged sequestration of S. typhimurium in affected tissues despite an increased production of F4/80+ effector cells. Moreover in the absence of GM-CSF, a decrease in pro-inflammatory cytokine expression of tumor necrosis factor-alpha, interleukin-12 (IL-12) and IL-18 was found, which may alter the host's immune response to infection. GM-CSF appears to play an important role in the pathogenesis of Salmonellosis, and may contribute significantly to the development of protective gastrointestinal mucosal immune responses against oral pathogens.

  2. Activation of human gingival epithelial cells by cell-surface components of black-pigmented bacteria: augmentation of production of interleukin-8, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor and expression of intercellular adhesion molecule 1.

    Science.gov (United States)

    Sugiyama, A; Uehara, A; Iki, K; Matsushita, K; Nakamura, R; Ogawa, T; Sugawara, S; Takada, H

    2002-01-01

    Black-pigmented anaerobic bacteria, such as Porphyromonas gingivalis and Prevotella intermedia, are amongst the predominant bacteria in periodontal pockets and have been implicated in periodontal diseases. To elucidate the roles of gingival keratinocytes, which are the first cells encountered by oral bacteria in periodontal diseases, human gingival keratinocytes in primary culture were stimulated with cell-surface components of P gingivalis and Pr. intermedia. A glycoprotein fraction from Pr. intermedia (PGP) clearly augmented the release of interleukin-8, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, as determined by enzyme-linked immunosorbent assay. This PGP also induced expression of intercellular adhesion molecule-1 (ICAM-1), as determined by flow cytometry. The augmentation of mRNA expression for these molecules was also confirmed by reverse transcription PCR. In contrast, lipopolysaccharide (LPS) from Pr. intermedia and Escherichia coli was completely inactive in these assays. LPS fraction and purified fimbriae from P gingivalis exhibited weak activities. Cytokine production and ICAM-1 expression by gingival keratinocytes might cause accumulation and activation of neutrophils in the epithelium and, therefore, may be involved in the initiation and development of inflammation in periodontal tissues.

  3. An antigen shared by human granulocytes, monocytes, marrow granulocyte precursors and leukemic blasts.

    Science.gov (United States)

    Shumak, K H; Rachkewich, R A

    1983-01-01

    An antibody to human granulocytes was raised in rabbits by immunization with granulocytes pretreated with rabbit antibody to contaminating antigens. The antibody reacted not only with granulocytes but also with monocytes and bone marrow granulocyte precursors including colony-forming units in culture (CFU-C). In tests with leukemic cells, the antibody reacted with blasts from most (8 of 9) patients with acute myelomonoblastic leukemia and from some patients with acute myeloblastic leukemia, morphologically undifferentiated acute leukemia and chronic myelogenous leukemia in blast crisis. The antibody did not react with blasts from patients with acute lymphoblastic leukemia nor with leukemic cells from patients with chronic lymphocytic leukemia.

  4. (TH) diazepam binding to human granulocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bond, P.A.; Cundall, R.L.; Rolfe, B.

    1985-07-08

    (TH)-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These (TH)-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14 M). Binding of (TH) diazepam at 0 is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and K/sub D/ of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. 11 references, 3 figures, 1 table.

  5. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    International Nuclear Information System (INIS)

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

    1990-01-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

  6. Carp macrophages and neutrophilic granulocytes secrete an interleukin-1-like factor.

    NARCIS (Netherlands)

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

    1995-01-01

    Carp, Cyprinus carpio L, macrophages and neutrophilic granulocytes obtained from pronephros were cultured. Supernatant was harvested after 48 h and tested for interleukin-1 (IL-1) bioactivity. A concentration-dependent stimulation of proliferation was found of carp Ig− lymphocytes as well as of the

  7. Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

    Science.gov (United States)

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

    2015-06-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

    Science.gov (United States)

    Laricchia-Robbio, L; Liedberg, B; Platou-Vikinge, T; Rovero, P; Beffy, P; Revoltella, R P

    1996-10-01

    An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct

  9. [Macrophages in human semen].

    Science.gov (United States)

    Bouvet, Beatriz Reina; Brufman, Adriana Silvia; Paparella, Cecilia Vicenta; Feldman, Rodolfo Nestor; Gatti, Vanda Nora; Solis, Edita Amalia

    2003-11-01

    To investigate the presence of macrophages in human semen samples and the function they carry out in the seminal fluid. Their presence was studied in relation to spermatic morphology, percentage of spermatozoids with native DNA, and presence of antispermatic antibodies. The work was performed with semen samples from 31 unfertile males from 63 couples in which the "female factor" was ruled out as the cause of infertility. Sperm study according to WHO (1992) was carried out in all samples, in addition to: DNA study with acridine orange as fluorocrom, macrophage concentration by neutral red in a Neubauer camera, and detection of antispermatic antibodies with a mixed agglutination test (TAC II) (validated with Mar Screen-Fertility technologies). Sperm morphology was evaluated by Papanicolaou test. 19/31 selected sperm samples (61.3%) showed increased concentration of macrophages, 13 of them (41.9%) with denaturalized DNA, and 8 (25.8%) abnormal morphology. Six samples showed increased macrophage concentration and predominance of native DNA, whereas 11 samples showed increased macrophages and abnormal morphology. Among 18 (58.1%) samples showing antispermatic antibodies 14 (77.7%) had an increased concentration of macrophages. Statistical analysis resulted in a high correlation between macrophage concentration and increased percentage of spermatozoids with denaturalized DNA (p < 0.05). An increased concentration of macrophages is associated with the presence of antispermatic antibodies (p < 0.05). There was not evidence of significant association between concentration of macrophages and percentage of morphologically normal spermatozoids (p < 0.05). We can conclude that macrophages are present in human semen and participate in immunovigilance contributing to improve the seminal quality.

  10. Granulocyte-macrophage colony-stimulating factor amplification of interleukin-1beta and tumor necrosis factor alpha production in THP-1 human monocytic cells stimulated with lipopolysaccharide of oral microorganisms.

    Science.gov (United States)

    Baqui, A A; Meiller, T F; Chon, J J; Turng, B F; Falkler, W A

    1998-05-01

    Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1beta and TNF-alpha production following GM-CSF supplementation with lipopolysaccharide (LPS) from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. LPS of P. gingivalis or F. nucleatum was prepared by a phenol-water extraction method and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determination of total protein and endotoxin contents. Resting THP-1 cells were treated with LPS of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) by using different concentrations for various time periods. Production of IL-1beta and TNF-alpha in THP-1 cells was measured by solid-phase enzyme-linked immunosorbent assay. Reverse transcription (RT)-PCR was used to evaluate the gene expression of resting and treated THP-1 cells. IL-1beta was not detected in untreated THP-1 cells. IL-1beta production was, however, stimulated sharply at 4 h. GM-CSF amplified IL-1beta production in THP-1 cells treated with LPS from both oral anaerobes. No IL-1beta-specific mRNA transcript was detected in untreated THP-1 cells. However, IL-1beta mRNA was detected by RT-PCR 2 h after stimulation of THP-1 cells with LPS from both organisms. GM-CSF did not shorten the IL-1beta transcriptional activation time. GM-CSF plus F. nucleatum or P. gingivalis LPS activated THP-1 cells to produce a 1.6-fold increase in TNF-alpha production at 4 h over LPS stimulation alone. These investigations with the in vitro THP-1 model indicate that there may be an increase in the cellular immune response to oral

  11. Development and characterization of antiserum to murine granulocyte-macrophage colony-stimulating factor

    International Nuclear Information System (INIS)

    Mochizuki, D.Y.; Eisenman, J.R.; Conlon, P.J.; Park, L.S.; Urdal, D.L.

    1986-01-01

    The expression in yeast of a cDNA clone encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has made possible the purification of large quantities of this recombinant protein. Rabbits immunized with pure recombinant GM-CSF generated antibodies that were shown to be specific for both recombinant GM-CSF and GM-CSF isolated from natural sources. Other lymphokines, including IL 1β, IL 2, IL 3, and recombinant human GM-CSF did not react with the antiserum. The antiserum together with recombinant GM-CSF that had been radiolabeled with 125 I to high specific activity, formed the foundation for a rapid, sensitive, and quantitative radioimmunoassay specific for murine GM-CSF. Furthermore, the antiserum was found to inhibit the biologic activities of GM-CSF as measured in both a bone marrow proliferation assay and a colony assay, and thus should prove to be a useful reagent for dissecting the complex growth factor activities involved in murine hematopoiesis

  12. Efficacy and safety of granulocyte, monocyte/macrophage adsorptive in pediatric ulcerative colitis

    DEFF Research Database (Denmark)

    Ruuska, Tarja; Küster, Peter; Grahnquist, Lena

    2016-01-01

    AIM: To investigate efficacy and safety for granulocyte, monocyte apheresis in a population of pediatric patients with ulcerative colitis. METHODS: The ADAPT study was a prospective, open-label, multicenter study in pediatric patients with moderate, active ulcerative colitis with pediatric...... ulcerative colitis activity index (PUCAI) of 35-64. Patients received one weekly apheresis with Adacolumn(®) granulocyte, monocyte/macrophage adsorptive (GMA) apheresis over 5 consecutive weeks, optionally followed by up to 3 additional apheresis treatments over 3 consecutive weeks. The primary endpoint...... mg daily on average from Baseline to week 12. CONCLUSION: Adacolumn(®) GMA apheresis treatment was effective in pediatric patients with moderate active Ulcerative Colitis. No new safety signals were reported. The present data contribute to considering GMA apheresis as a therapeutic option...

  13. Recurrent spleen enlargement during cyclic granulocyte-macrophage colony-stimulating factor therapy for myelodysplastic syndrome

    International Nuclear Information System (INIS)

    Delmer, A.; Karmochkine, M.; Cadiou, M.; Gerhartz, H.; Zittoun, R.

    1990-01-01

    A 65-year-old woman with refractory anemia with excess of blasts received sequential courses of granulocyte-macrophage colony-stimulating factor therapy (GM-CSF) and low-dose cytosine arabinoside. Each course of GM-CSF induced a rapid and tremendous increase in leukocyte count as well as in spleen size, 111-indium chloride scanning suggested a myeloid metaplasia of the spleen. This observation suggests that in some patients the granulopoietic response to the myeloid growth factor stimulation may be predominant in the spleen

  14. Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis)

    KAUST Repository

    Sugumar, Thennarasu; Ganesan, Pugalenthi; Harishankar, Murugesan; Dhinakar Raj, Gopal

    2013-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface. © 2013 John Wiley & Sons Ltd.

  15. Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis)

    KAUST Repository

    Sugumar, Thennarasu

    2013-06-25

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface. © 2013 John Wiley & Sons Ltd.

  16. A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization

    DEFF Research Database (Denmark)

    Ziebe, Søren; Loft, Anne; Povlsen, Betina B

    2013-01-01

    To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR).......To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR)....

  17. Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to Pseudomonas aeruginosa infection.

    Directory of Open Access Journals (Sweden)

    Sonali Singh

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF, on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1 and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human

  18. Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to Pseudomonas aeruginosa infection.

    Science.gov (United States)

    Singh, Sonali; Barr, Helen; Liu, Yi-Chia; Robins, Adrian; Heeb, Stephan; Williams, Paul; Fogarty, Andrew; Cámara, Miguel; Martínez-Pomares, Luisa

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages

  19. A Randomized Case-Controlled Study of Recombinant Human Granulocyte Colony Stimulating Factor for the Treatment of Sepsis in Preterm Neutropenic Infants

    OpenAIRE

    Aktaş, Doğukan; Demirel, Bilge; Gürsoy, Tuğba; Ovalı, Fahri

    2015-01-01

    To investigate the efficacy and safety of recombinant human granulocyte colony-stimulating factor, recombinant human granulocyte-macrophage colony-stimulating factor (rhG-CSF) to treat sepsis in neutropenic preterm infants. Methods: Fifty-six neutropenic preterm infants with suspected or culture-proven sepsis hospitalized in Zeynep Kamil Maternity and Children's Educational and Training Hospital, Kozyatağı/Istanbul, Turkey between January 2008 and January 2010 were enrolled. Patients were ...

  20. Increased production of granulocyte-macrophage colony-stimulating factor in Crohn's disease--a possible target for infliximab treatment

    DEFF Research Database (Denmark)

    Agnholt, Jørgen; Kelsen, Jens; Brandsborg, Birgitte

    2004-01-01

    The presence of neutrophils among epithelial cells is one of the major features of the inflammation in Crohn's disease, and has been used to indicate disease activity. The survival of neutrophils outside the blood vessels is limited and their longevity is influenced by granulocyte-macrophage colo...

  1. Promotion of Tumor Invasion by Cooperation of Granulocytes and Macrophages Activated by Anti-tumor Antibodies

    Directory of Open Access Journals (Sweden)

    Emilio Barbera-Guillem

    1999-11-01

    Full Text Available We investigated the potential role of anti-tumor antibodies and tumor antigens in the formation of immune complexes which promote matrix degradation and angiogenesis. B-cell deficient or B-cell depleted mice showed a reduction in tumor invasion and metastasis. In vitro invasion assays and in vivo models of metastasis showed that anti-sTn antibodies and sTn tumor antigens form complexes which induce granulocytes and macrophages together to mediate tumor invasion and metastasis by processes including extracellular matrix degradation and angiogenesis. These results suggest the existence of a tumor promoting role of a B-cell immune response induced by shed tumor associated antigens of solid, nonlymphoid tumors.

  2. Radiation-induced enlargement of granulocytic and macrophage progenitor cells in mouse bone marrow

    Energy Technology Data Exchange (ETDEWEB)

    Metcalf, D; Johnson, G R; Wilson, J [Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia)

    1977-01-01

    The peak sedimentation velocity of C/sub 57/BL mouse bone marrow progenitors of granulocytes and macrophages (GM-colony-forming cells, GM-CFC's) increased from 4.3 mm/h to 7 to 8 mm/h by 2 days after 250 rad whole body irradiation and slowly returned to normal over the next 3 weeks. Preliminary irradiation and/or endotoxin injection did not prevent this radiation-induced change. Some change in sedimentation velocity was seen with as little as 100 rad irradiation. Neither buoyant density nor cell cycle changes could account for the sedimentation velocity data which therefore indicate a major volume increase in the GM-CFC's. This size enlargement affected all subpopulations of GM-CFC's which consequently maintained their size relationship with one another.

  3. Regulation of wound healing by granulocyte-macrophage colony-stimulating factor after vocal fold injury.

    Directory of Open Access Journals (Sweden)

    Jae-Yol Lim

    Full Text Available OBJECTIVES: Vocal fold (VF scarring remains a therapeutic challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF facilitates epithelial wound healing, and recently, growth factor therapy has been applied to promote tissue repair. This study was undertaken to investigate the effect of GM-CSF on VF wound healing in vivo and in vitro. METHODS: VF scarring was induced in New Zealand white rabbits by direct injury. Immediately thereafter, either GM-CSF or PBS was injected into the VFs of rabbits. Endoscopic, histopathological, immunohistochemical, and biomechanical evaluations of VFs were performed at 3 months post-injury. Human vocal fold fibroblasts (hVFFs were cultured with GM-CSF. Production of type I and III collagen was examined immunocytochemically, and the synthesis of elastin and hyaluronic acids was evaluated by ELISA. The mRNA levels of genes related to ECM components and ECM production-related growth factors, such as HGF and TGF-ß1, were examined by real time RT-PCR. RESULTS: The GM-CSF-treated VFs showed reduced collagen deposition in comparison to the PBS-injected controls (P<0.05. Immunohistochemical staining revealed lower amounts of type I collagen and fibronectin in the GM-CSF-treated VFs (P<0.05 and P<0.01, respectively. Viscous and elastic shear moduli of VF samples were significantly lower in the GM-CSF group than in the PBS-injected group (P<0.001 and P<0.01, respectively. Mucosal waves in the GM-CSF group showed significant improvement when compared to the PBS group (P = 0.0446. GM-CSF inhibited TGF-β1-induced collagen synthesis by hVFFs (P<0.05 and the production of hyaluronic acids increased at 72 hours post-treatment (P<0.05. The expressions of HAS-2, tropoelastin, MMP-1, HGF, and c-Met mRNA were significantly increased by GM-CSF, although at different time points (P<0.05. CONCLUSION: The present study shows that GM-CSF offers therapeutic potential for the remodeling of VF wounds and the promotion of VF

  4. A methylcellulose microculture assay for the in vitro assessment of drug toxicity on granulocyte/macrophage progenitors (CFU-GM).

    Science.gov (United States)

    Pessina, Augusto; Croera, Cristina; Bayo, Maria; Malerba, Ilaria; Passardi, Laura; Cavicchini, Loredana; Neri, Maria G; Gribaldo, Laura

    2004-03-01

    In a recent prevalidation study, the use of a methylcellulose colony-forming unit-granulocyte/macrophage (CFU-GM) macroassay for two independent in vitro tests (human and murine cell based) was suggested for quantifying the potential haematotoxicity of xenobiotics. In this paper, we describe the transfer of the macroassay to a 96-well plate microassay, in which the linearity of the response was studied (both in terms of CFU-GM and optical density [OD] versus the number of cells cultured), and the inhibitory concentration (IC) values for doxorubicin, 5-fluorouracil and taxol were determined and compared with those obtained by using the original macroassay. Fresh murine bone marrow and human umbilical cord blood mononuclear cells were used as a source of myeloid progenitors. The cells were cultured in methylcellulose containing granulocyte/macrophage-colony-stimulating factor, and in the presence of increasing drug concentrations. The cloning capacity of the progenitors was measured both as the number of colonies counted manually (CFU-GM), and as OD evaluated with an automated plate reader in an MTT test. Our results show that, in the microassay, up to 20 colonies/well could be easily counted, and that this range (20 to zero) gave a regression line from which IC values were calculated, which were very close to those obtained by using the macroassay (where the range of colony numbers was from 100 to zero). The test did not give good results when the OD (instead of the colony count) was used as the endpoint, because, although a high coefficient of determination was obtained, the OD values ranged from 0.6 to zero and the IC values determined were not comparable to those obtained by manual counts. The use of the microassay dramatically reduces the quantity of methylcellulose needed, and permits hundreds of cultures to be processed in the same experiment, contributing to significant reductions in both the work involved and the cost. A further important benefit is a

  5. Granulocyte macrophage-colony-stimulating factor mouthwashes heal oral ulcers during head and neck radiotherapy

    International Nuclear Information System (INIS)

    Rovirosa, Angeles; Ferre, Jorge; Biete, Albert

    1998-01-01

    Purpose: To evaluate the effectiveness of granulocyte macrophage-colony-stimulating factor GM-CSF mouthwashes in the epithelization of radiation-induced oral mucosal ulceration, control of pain, and weight loss. Methods and Materials: Twelve patients received curative radiotherapy for head and neck carcinoma. All had oropharyngeal and/or oral mucosa irradiation, with a median dose of 72 Gy (range 50-74), with conventional fractionation. A total of 300 μg of GM-CSF in 250 cc of water for 1 h of mouthwashing was prescribed. The procedure started once oral ulceration in the irradiation field was detected. Patients, examined twice a week, were evaluated for oral ulceration, pain, and weight loss. Blood tests were taken weekly during GM-CSF administration. A comparison was carried out with 12 retrospective case-matched controls. Results: In the GM-CSF group, mucosa ulcerations healed in 9 of 12 (75%) of the patients during the course of the radiotherapy. Fifty percent of the patients said they felt less pain during the GM-CSF treatment; 30% needed morphine. The mean and median weight loss as a percentage of baseline weight in addition to the actual weight were 4.2% and 3%, respectively (variation ranged between a gain of 1% and a loss of 13%). No GM-CSF-related side effects were found. In the case control group, in the 12 cases, oral ulcerations increased during radiotherapy and two patients needed intubation intake and hospital admission, as opposed to the GM-CSF group. The mean and median percentage of weight loss were 5.8% and 5%, respectively. Sixty percent of patients needed morphine, as opposed to 30% in the GM-CSF group. Conclusions: Granulocyte macrophage-colony-stimulating factor was effective in curing mucosal ulcerations during the course of radiotherapy. This is the first time we have seen a drug with this capacity. Although the GM-CSF seems to be effective in the control of pain, oral intake, and weight loss, we need further studies with a greater number

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

    Energy Technology Data Exchange (ETDEWEB)

    Gallin, J I

    1974-01-01

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

  7. Granulocyte Macrophage Colony-Stimulating Factor-Activated Eosinophils Promote Interleukin-23 Driven Chronic Colitis

    Science.gov (United States)

    Griseri, Thibault; Arnold, Isabelle C.; Pearson, Claire; Krausgruber, Thomas; Schiering, Chris; Franchini, Fanny; Schulthess, Julie; McKenzie, Brent S.; Crocker, Paul R.; Powrie, Fiona

    2015-01-01

    Summary The role of intestinal eosinophils in immune homeostasis is enigmatic and the molecular signals that drive them from protective to tissue damaging are unknown. Most commonly associated with Th2 cell-mediated diseases, we describe a role for eosinophils as crucial effectors of the interleukin-23 (IL-23)-granulocyte macrophage colony-stimulating factor (GM-CSF) axis in colitis. Chronic intestinal inflammation was characterized by increased bone marrow eosinopoiesis and accumulation of activated intestinal eosinophils. IL-5 blockade or eosinophil depletion ameliorated colitis, implicating eosinophils in disease pathogenesis. GM-CSF was a potent activator of eosinophil effector functions and intestinal accumulation, and GM-CSF blockade inhibited chronic colitis. By contrast neutrophil accumulation was GM-CSF independent and dispensable for colitis. In addition to TNF secretion, release of eosinophil peroxidase promoted colitis identifying direct tissue-toxic mechanisms. Thus, eosinophils are key perpetrators of chronic inflammation and tissue damage in IL-23-mediated immune diseases and it suggests the GM-CSF-eosinophil axis as an attractive therapeutic target. PMID:26200014

  8. Chimeric HIV-1 Envelope Glycoproteins with Potent Intrinsic Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Activity*

    Science.gov (United States)

    Boot, Maikel; Cobos Jiménez, Viviana; Kootstra, Neeltje A.; Sanders, Rogier W.

    2013-01-01

    HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed EnvGM-CSF chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized EnvGM-CSF proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric EnvGM-CSF should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins. PMID:23565193

  9. Chimeric HIV-1 envelope glycoproteins with potent intrinsic granulocyte-macrophage colony-stimulating factor (GM-CSF activity.

    Directory of Open Access Journals (Sweden)

    Gözde Isik

    Full Text Available HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs that target the envelope glycoprotein complex (Env. An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env(GM-CSF chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized Env(GM-CSF proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric Env(GM-CSF should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins.

  10. Granulocyte Macrophage Colony Stimulating Factor Supplementation in Culture Media for Subfertile Women Undergoing Assisted Reproduction Technologies: A Systematic Review

    Science.gov (United States)

    Siristatidis, Charalampos; Vogiatzi, Paraskevi; Salamalekis, George; Creatsa, Maria; Vrachnis, Nikos; Glujovsky, Demián; Iliodromiti, Zoe; Chrelias, Charalampos

    2013-01-01

    Granulocyte macrophage colony stimulating factor (GM-CSF) is a cytokine/growth factor produced by epithelial cells that exerts embryotrophic effects during the early stages of embryo development. We performed a systematic review, and six studies that were performed in humans undergoing assisted reproduction technologies (ART) were located. We wanted to evaluate if embryo culture media supplementation with GM-CSF could improve success rates. As the type of studies and the outcome parameters investigated were heterogeneous, we decided not to perform a meta-analysis. Most of them had a trend favoring the supplementation with GM-CSF, when outcomes were measured in terms of increased percentage of good-quality embryos reaching the blastocyst stage, improved hatching initiation and number of cells in the blastocyst, and reduction of cell death. However, no statistically significant differences were found in implantation and pregnancy rates in all apart from one large multicenter trial, which reported favorable outcomes, in terms of implantation and live birth rates. We propose properly conducted and adequately powered randomized controlled trials (RCTs) to further validate and extrapolate the current findings with the live birth rate to be the primary outcome measure. PMID:23509457

  11. Ontogeny of the granulocyte/macrophage progenitor cell (GM-CFC) pools in the beagle.

    Science.gov (United States)

    Nothdurft, W; Braasch, E; Calvo, W; Prümmer, O; Carbonell, F; Grilli, G; Fliedner, T M

    1984-04-01

    The pattern of development of the granulocyte/macrophage progenitor cell (GM-CFC) pools in the course of canine ontogeny was studied by means of the agar culture technique. Colony formation was stimulated by colony stimulating activity (CSA) in serum from lethally irradiated dogs in combination with erythrocyte-depleted peripheral blood leukocytes from normal adult dogs. The colonies thus obtained in cultures from the different organs were in general large (estimated maximum 50 000 cells) and consisted predominantly of mononucleated macrophages, suggesting that, in these studies, a progenitor cell with high proliferative potential (HPP-CFC) has been monitored. In the yolk sac, a transitory GM-CFC pool became established between day 23 and day 48 of gestation, reaching maximum numbers of approximately 41 X 10(3) per organ on days 36/37. At the same time the GM-CFC concentration in blood collected from the heart also reached a maximum of about 31 X 10(3)/ml, indicating its carrier function for the migration of GM-CFC. In the liver a quasi-exponential increase in the GM-CFC numbers took place between days 36/37 and days 57 to 59 when a total of about 15.2 X 10(6) was found but thereafter and up to day 4 post partum the GM-CFC numbers decreased by almost two orders of magnitude. A continuous increase in the GM-CFC numbers was found in the spleen between day 42 of gestation and day 4 post partum when a maximum of 5.1 X 10(6) to 8.7 X 10(6) was reached. In contrast to the GM-CFC numbers in the liver, the splenic GM-CFC dropped only by 50% of peak values when the dogs reached adulthood. The bone marrow always had the highest incidence of GM-CFC, the concentration per 10(6) cells being 18.7 X 10(3)/10(6) cells on days 45/46, the earliest time point at which cultures could be set up. The absolute GM-CFC numbers in the two femora increased continuously between days 45/46 and day 4 post partum in parallel with the growth of the bones. In the thymus a relatively small

  12. Role of Granulocyte-Macrophage Colony-Stimulating Factor Production by T Cells during Mycobacterium tuberculosis Infection.

    Science.gov (United States)

    Rothchild, Alissa C; Stowell, Britni; Goyal, Girija; Nunes-Alves, Cláudio; Yang, Qianting; Papavinasasundaram, Kadamba; Sassetti, Christopher M; Dranoff, Glenn; Chen, Xinchun; Lee, Jinhee; Behar, Samuel M

    2017-10-24

    Mice deficient for granulocyte-macrophage colony-stimulating factor (GM-CSF -/- ) are highly susceptible to infection with Mycobacterium tuberculosis , and clinical data have shown that anti-GM-CSF neutralizing antibodies can lead to increased susceptibility to tuberculosis in otherwise healthy people. GM-CSF activates human and murine macrophages to inhibit intracellular M. tuberculosis growth. We have previously shown that GM-CSF produced by iNKT cells inhibits growth of M. tuberculosis However, the more general role of T cell-derived GM-CSF during infection has not been defined and how GM-CSF activates macrophages to inhibit bacterial growth is unknown. Here we demonstrate that, in addition to nonconventional T cells, conventional T cells also produce GM-CSF during M. tuberculosis infection. Early during infection, nonconventional iNKT cells and γδ T cells are the main source of GM-CSF, a role subsequently assumed by conventional CD4 + T cells as the infection progresses. M. tuberculosis -specific T cells producing GM-CSF are also detected in the peripheral blood of infected people. Under conditions where nonhematopoietic production of GM-CSF is deficient, T cell production of GM-CSF is protective and required for control of M. tuberculosis infection. However, GM-CSF is not required for T cell-mediated protection in settings where GM-CSF is produced by other cell types. Finally, using an in vitro macrophage infection model, we demonstrate that GM-CSF inhibition of M. tuberculosis growth requires the expression of peroxisome proliferator-activated receptor gamma (PPARγ). Thus, we identified GM-CSF production as a novel T cell effector function. These findings suggest that a strategy augmenting T cell production of GM-CSF could enhance host resistance against M. tuberculosis IMPORTANCE Mycobacterium tuberculosis is the bacterium that causes tuberculosis, the leading cause of death by any infection worldwide. T cells are critical components of the immune

  13. Serologic Evidence of Human Monocytic and Granulocytic Ehrlichiosis in Israel

    Science.gov (United States)

    Keysary, Avi; Amram, Lili; Keren, Gershon; Sthoeger, Zev; Potasman, Israel; Jacob, Amir; Strenger, Carmella; Dawson, Jacqueline E.

    1999-01-01

    We conducted a retrospective serosurvey of 1,000 persons in Israel who had fever of undetermined cause to look for Ehrlichia chaffeensis antibodies. Four of five cases with antibodies reactive to E. chaffeensis were diagnosed in the summer, when ticks are more active. All patients had influenzalike symptoms with high fever. None of the cases was fatal. Three serum samples were also seroreactive for antibodies to E. canis, and one was also reactive to the human granulocytic ehrlichiosis (HGE) agent. The titer to the HGE agent in this patient was higher than the serum titer to E. chaffeensis, and the Western blot analysis also indicated that the HGE agent was the primary cause of infection. We present the first serologic evidence that the agents of human monocytic ehrlichiosis (HME) and HGE are present in Israel. Therefore, human ehrlichiosis should be included in the differential diagnoses for persons in Israel who have been exposed to ticks and have influenzalike symptoms. PMID:10603210

  14. Thermo-radiosensitivity of the granulocyte and macrophage precursor cells of mice. II. - X irradiation effects and influence of hyperthermia on the radiosensitivity

    International Nuclear Information System (INIS)

    Bueren, J.A.; Nieto, M.

    1983-01-01

    The effects of the X-irradiation on the viability of the granulocyte-macrophage precursors, has been determined by means of the agar diffusion chamber culture technique. The results show the high radiosensitivity of these cells, with survival parameter similar to those previously reported in the literature about different granulocyte-macrophage precursors. When a hyperthermic treatment is performed prior to the X-irradiation, a radiosensitization phenomenon is observed due to the synergism existent between hyperthermia and X rays on the lethality of the precursors. (Authors) 37 refs

  15. A karyometric note on nucleoli in human early granulocytic precursors.

    Science.gov (United States)

    Smetana, K; Mikulenková, D; Jirásková, I; Klamová, H

    2006-01-01

    The diameter of nucleoli was measured in human bone marrow early granulocytic precursors after visualization by a simple cytochemical method for demonstration of RNA. Such method facilitated to clearly see nucleolar bodies without perinucleolar chromatin, including those of micronucleoli. The bone marrow of patients suffering from chronic myeloid leukaemia (untreated with cytostatics) provided a satisfactory number of both myeloblasts and promyelocytes for nucleolar measurements because of prevailing granulopoiesis. The direct nucleolar measurement was carried out on digitized and processed images on the screen at magnification 4,300x. It seems to be likely that the nucleolar size is directly related to the number of nucleoli per cell. The largest nucleoli were present in both myeloblasts and promyelocytes that possessed a single nucleolus. In contrast, the nucleolar diameter was significantly smaller in cells with multiple nucleoli. However, in cells with small multiple nucleoli, one of them was always larger and dominant with a large number of AgNORs. Such large nucleoli are possibly visible in specimens stained with panoptic procedures or methods staining nuclear chromatin or DNA. It should also be mentioned that both myeloblasts and promyelocytes mostly possessed two nucleoli with the mean diameter close to 1.5 microm. The incidence of early granulocytic precursors classified according to the nucleolar number and size strongly suggested that the various nucleolar number and nucleolar size in these cells might be related to the different stage of the cell cycle and might also explain their heterogeneity.

  16. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

    Directory of Open Access Journals (Sweden)

    Chang Rae Rho

    Full Text Available Granulocyte-macrophage colony-stimulating factor (GM-CSF is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs. We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF. An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml. MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

  17. Biological properties in vitro of a combination of recombinant murine interleukin-3 and granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Riklis, I; Kletter, Y; Bleiberg, I; Fabian, I

    1989-04-01

    The effect of recombinant murine interleukin-3 (rIL-3) and recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) on in vitro murine myeloid progenitor cell (CFU-C) growth and on the function of murine resident peritoneal macrophages was investigated. Both rIL-3 and rGM-CSF are known to support the growth of CFU-C and, when combined, were found to act synergistically to induce the development of an increased number of CFU-C. The distribution pattern of myeloid colonies in the presence of these two growth factors was in general similar to that in the presence of rGM-CSF alone. Both rGM-CSF and rIL-3 enhanced the phagocytosis of Candida albicans (CA) by mature macrophages producing an increase in the percentage of phagocytosing cells as well as an increase in the number of yeast particles ingested per cell. No additive effect on the phagocytosis was observed when the two growth factors were added concurrently. rGM-CSF, but not rIL-3, enhanced the killing of CA by macrophages. This killing was inhibited by scavengers of oxygen radicals.

  18. Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine

    Science.gov (United States)

    Hellerstein, Michael; Xu, Yongxian; Marino, Tracie; Lu, Shan; Yi, Hong; Wright, Elizabeth R.; Robinson, Harriet L.

    2012-01-01

    Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection. PMID:23111169

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

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

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

  20. MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor, in the treatment of patients with moderate rheumatoid arthritis: results of a phase Ib/IIa randomised, double-blind, placebo-controlled, dose-escalation trial.

    Science.gov (United States)

    Behrens, Frank; Tak, Paul P; Østergaard, Mikkel; Stoilov, Rumen; Wiland, Piotr; Huizinga, Thomas W; Berenfus, Vadym Y; Vladeva, Stoyanka; Rech, Juergen; Rubbert-Roth, Andrea; Korkosz, Mariusz; Rekalov, Dmitriy; Zupanets, Igor A; Ejbjerg, Bo J; Geiseler, Jens; Fresenius, Julia; Korolkiewicz, Roman P; Schottelius, Arndt J; Burkhardt, Harald

    2015-06-01

    To determine the safety, tolerability and signs of efficacy of MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), in patients with rheumatoid arthritis (RA). Patients with active, moderate RA were enrolled in a randomised, multicentre, double-blind, placebo-controlled, dose-escalation trial of intravenous MOR103 (0.3, 1.0 or 1.5 mg/kg) once a week for 4 weeks, with follow-up to 16 weeks. The primary outcome was safety. Of the 96 randomised and treated subjects, 85 completed the trial (n=27, 24, 22 and 23 for pooled placebo and MOR103 0.3, 1.0 and 1.5 mg/kg, respectively). Treatment emergent adverse events (AEs) in the MOR103 groups were mild or moderate in intensity and generally reported at frequencies similar to those in the placebo group. The most common AE was nasopharyngitis. In two cases, AEs were classified as serious because of hospitalisation: paronychia in a placebo subject and pleurisy in a MOR103 0.3 mg/kg subject. Both patients recovered fully. In exploratory efficacy analyses, subjects in the MOR103 1.0 and 1.5 mg/kg groups showed significant improvements in Disease Activity Score-28 scores and joint counts and significantly higher European League Against Rheumatism response rates than subjects receiving placebo. MOR103 1.0 mg/kg was associated with the largest reductions in disease activity parameters. MOR103 was well tolerated and showed preliminary evidence of efficacy in patients with active RA. The data support further investigation of this monoclonal antibody to GM-CSF in RA patients and potentially in those with other immune-mediated inflammatory diseases. NCT01023256. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  1. Nicotine can skew the characterization of the macrophage type-1 (MΦ1) phenotype differentiated with granulocyte-macrophage colony-stimulating factor to the MΦ2 phenotype

    International Nuclear Information System (INIS)

    Yanagita, Manabu; Kobayashi, Ryohei; Murakami, Shinya

    2009-01-01

    Macrophages (MΦs) exhibit functional heterogeneity and plasticity in the local microenvironment. Recently, it was reported that MΦs can be divided into proinflammatory MΦs (MΦ1) and anti-inflammatory MΦs (MΦ2) based on their polarized functional properties. Here, we report that nicotine, the major ingredient of cigarette smoke, can modulate the characteristics of MΦ1. Granulocyte-macrophage colony-stimulating factor-driven MΦ1 with nicotine (Ni-MΦ1) showed the phenotypic characteristics of MΦ2. Like MΦ2, Ni-MΦ1 exhibited antigen-uptake activities. Ni-MΦ1 suppressed IL-12, but maintained IL-10 and produced high amounts of MCP-1 upon lipopolysaccharide stimulation compared with MΦ1. Moreover, we observed strong proliferative responses of T cells to lipopolysaccharide-stimulated MΦ1, whereas Ni-MΦ1 reduced T cell proliferation and inhibited IFN-γ production by T cells. These results suggest that nicotine can change the functional characteristics of MΦ and skew the MΦ1 phenotype to MΦ2. We propose that nicotine is a potent regulator that modulates immune responses in microenvironments.

  2. Thermo-radiosensitivity of the granulocyte and macrophage precursor cells of mice. I.-Development of the in vivo culture and effects induced by the hyperthermia

    International Nuclear Information System (INIS)

    Bueren, J. A.; Nieto, M.

    1983-01-01

    The present report shows the agar diffusion chamber technique for culturing granulocyte- macrophage precursor cells, obtained from mice bone marrow. Diffusion chambers containing the bone marrow suspension are implanted intraperitoneally Into mice and constitute a compartment which avoids the migration of cells, but allows the transit of the mouse biological fluxes, necessary for the cellular proliferation. By means of this technique, we studied the lethal effects of the hyperthermia on the precursors and their capacity to repair sublethal damage. (Author) 129 refs

  3. Substance P enhances tissue factor release from granulocyte-macrophage colony-stimulating factor-dependent macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway.

    Science.gov (United States)

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

    2016-03-01

    Granulocyte-macrophage colony stimulating factor (GM-CSF) induces procoagulant activity of macrophages. Tissue factor (TF) is a membrane-bound glycoprotein and substance P (SP) is a pro-inflammatory neuropeptide involved in the formation of membrane blebs. This study investigated the role of SP in TF release by GM-CSF-dependent macrophages. SP significantly decreased TF levels in whole-cell lysates of GM-CSF-dependent macrophages. TF was detected in the culture supernatant by enzyme-linked immunosorbent assay after stimulation of macrophages by SP. Aprepitant (an SP/neurokinin 1 receptor antagonist) reduced TF release from macrophages stimulated with SP. Pretreatment of macrophages with a radical scavenger(pyrrolidinedithiocarbamate) also limited the decrease of TF in whole-cell lysates after stimulation with SP. A protein kinase C inhibitor (rottlerin) partially blocked this macrophage response to SP, while it was significantly inhibited by a ROCK inhibitor (Y-27632) or a dynamin inhibitor (dinasore). An Akt inhibitor (perifosine) also partially blocked this response. Furthermore, siRNA targeting p22phox, β-arrestin 2, or Rho A, blunted the release of TF from macrophages stimulated with SP. In other experiments, visceral adipocytes derived from cryopreserved preadipocytes were found to produce SP. In conclusion, SP enhances the release of TF from macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. The optimal use of granulocyte macrophage colony stimulating factor in radiation induced mucositis in head and neck squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Patni Nidhi

    2005-01-01

    Full Text Available Objective: Evaluation of response of granulocyte macrophage colony stimulating factor (GM-CSF on acute radiation toxicity profile in head and neck squamous cell carcinoma. Methods and Materials: Thirty three patients with proven stage I or II head & neck carcinoma received conventional external beam radiation therapy. Out of these, six patients received postoperative adjuvant therapy while remaining 27 received definitive RT. Patients were given 100 mcg GM-CSF subcutaneously per day along with radiation after they developed grade 2 mucositis and /or grade 2 dysphagia and / or complained of moderate pain. GM-CSF was administered till there was a subjective relief or objective response. Patients were evaluated for oral ulceration, swallowing status, pain and weight loss. Response to the treatment and patient outcome was assessed. Results: There was a decreased severity of mucositis and dysphagia in the evaluated patients. None of the patients suffered severe pain or required opioids. The mean weight loss was only 1.94%. Minimal side effects were experienced with GM-CSF. Conclusions: GM-CSF reduces the severity of acute side effects of radiation therapy thereby allowing completion of the treatment without interruption. Its remarkable response needs to be evaluated further in large randomized trials. The time of initiation and cessation of GM-CSF during radiation therapy and the required dose needs to be established.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  6. Effects of granulocyte-macrophage colony-stimulating factor and interleukin 6 on the growth of leukemic blasts in suspension culture.

    Science.gov (United States)

    Tsao, C J; Cheng, T Y; Chang, S L; Su, W J; Tseng, J Y

    1992-05-01

    We examined the stimulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL)-6 on the in vitro proliferation of leukemic blast cells from patients with acute leukemia. Bone marrow or peripheral blood leukemic blast cells were obtained from 21 patients, including 14 cases of acute myeloblastic leukemia (AML), four cases of acute lymphoblastic leukemia (ALL), two cases of acute undifferentiated leukemia, and one case of acute mixed-lineage leukemia. The proliferation of leukemic blast cells was evaluated by measuring the incorporation of 3H-thymidine into cells incubated with various concentrations of cytokines for 3 days. GM-CSF stimulated the DNA synthesis (with greater than 2.0 stimulation index) of blast cells in 9 of 14 (64%) AML cases, two cases of acute undifferentiated leukemia and one case of acute mixed-lineage leukemia. Only two cases of AML blasts responded to IL-6 to grow in the short-term suspension cultures. GM-CSF and IL-6 did not display a synergistic effect on the growth of leukemic cells. Moreover, GM-CSF and IL-6 did not stimulate the proliferation of ALL blast cells. Binding study also revealed the specific binding of GM-CSF on the blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia. Our results indicated that leukemic blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia possessed functional GM-CSF receptors.

  7. Soluble human leukocyte antigen G5 polarizes differentiation of macrophages toward a decidual macrophage-like phenotype.

    Science.gov (United States)

    Lee, Cheuk-Lun; Guo, YiFan; So, Kam-Hei; Vijayan, Madhavi; Guo, Yue; Wong, Vera H H; Yao, YuanQing; Lee, Kai-Fai; Chiu, Philip C N; Yeung, William S B

    2015-10-01

    What are the actions of soluble human leukocyte antigen G5 (sHLAG5) on macrophage differentiation? sHLAG5 polarizes the differentiation of macrophages toward a decidual macrophage-like phenotype, which could regulate fetomaternal tolerance and placental development. sHLAG5 is a full-length soluble isoform of human leukocyte antigen implicated in immune tolerance during pregnancy. Low or undetectable circulating level of sHLAG5 in first trimester of pregnancy is associated with pregnancy complications such as pre-eclampsia and spontaneous abortion. Decidual macrophages are located in close proximity to invasive trophoblasts, and are involved in regulating fetomaternal tolerance and placental development. Human peripheral blood monocytes were differentiated into macrophages by treatment with granulocyte macrophage colony-stimulating factor in the presence or absence of recombinant sHLAG5 during the differentiation process. The phenotypes and the biological activities of the resulting macrophages were compared. Recombinant sHLAG5 was produced in Escherichia coli BL21 and the protein identity was verified by tandem mass spectrometry. The expression of macrophage markers were analyzed by flow cytometry and quantitative PCR. Phagocytosis was determined by flow cytometry. Indoleamine 2,3-dioxygenase 1 expression and activity were measured by western blot analysis and kynurenine assay, respectively. Cell proliferation and cell cycling were determined by fluorometric cell proliferation assay and flow cytometry, respectively. Cytokine secretion was determined by cytokine array and ELISA kits. Intracellular cytokine expression was measured by flow cytometry. Cell invasion and migration were determined by trans-well invasion and migration assay, respectively. sHLAG5 drove the differentiation of macrophages with 'immuno-modulatory' characteristics, including reduced expression of M1 macrophage marker CD86 and increased expression of M2 macrophage marker CD163. sHLAG5-polarized

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

    NARCIS (Netherlands)

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

    1997-01-01

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

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

    Science.gov (United States)

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

    2013-04-01

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

  10. Benefits of gene transduction of granulocyte macrophage colony-stimulating factor in cancer vaccine using genetically modified dendritic cells.

    Science.gov (United States)

    Ojima, Toshiyasu; Iwahashi, Makoto; Nakamura, Masaki; Matsuda, Kenji; Nakamori, Mikihito; Ueda, Kentaro; Naka, Teiji; Katsuda, Masahiro; Miyazawa, Motoki; Yamaue, Hiroki

    2007-10-01

    Granulocyte macrophage colony-stimulating factor (GM-CSF) is a key cytokine for the generation and stimulation of dendritic cells (DCs), and it may also play a pivotal role in promoting the survival of DCs. In this study, the feasibility of creating a cancer vaccine using DCs adenovirally transduced with the carcinoembryonic antigen (CEA) gene and the GM-CSF gene was examined. In addition, the effect of the co-transduction of GM-CSF gene on the lifespan of these genetically modified DCs was determined. A cytotoxic assay using peripheral blood mononuclear cell (PBMC)-derived cytotoxic T lymphocytes (CTLs) was performed in a 4-h 51Cr release assay. The apoptosis of DCs was examined by TdT-mediated dUTP-FITC nick end labeling (TUNEL) assay. CEA-specific CTLs were generated from PBMCs stimulated with genetically modified DCs expressing CEA. The cytotoxicity of these CTLs was augmented by co-transduction of DCs with the GM-CSF gene. Co-transduction of the GM-CSF gene into DCs inhibited apoptosis of these DCs themselves via up-regulation of Bcl-x(L) expression, leading to the extension of the lifespan of these DCs. Furthermore, the transduction of the GM-CSF gene into DCs also suppressed the incidence of apoptosis of DCs induced by transforming growth factor-beta1 (TGFbeta-1). Immunotherapy using these genetically modified DCs may therefore be useful with several advantages as follows: i) adenoviral toxicity to DCs can be reduced; ii) the lifespan of vaccinated DCs can be prolonged; and iii) GM-CSF may protect DCs from apoptosis induced by tumor-derived TGFbeta-1 in the regional lymph nodes.

  11. The combined effect of erythropoietin and granulocyte macrophage colony stimulating factor on liver regeneration after major hepatectomy in rats

    Directory of Open Access Journals (Sweden)

    Frangou Matrona

    2010-07-01

    Full Text Available Abstract Background The liver presents a remarkable capacity for regeneration after hepatectomy but the exact mechanisms and mediators involved are not yet fully clarified. Erythropoietin (EPO and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF have been shown to promote liver regeneration after major hepatectomy. Aim of this experimental study is to compare the impact of exogenous administration of EPO, GM-CSF, as well as their combination on the promotion of liver regeneration after major hepatectomy. Methods Wistar rats were submitted to 70% major hepatectomy. The animals were assigned to 4 experimental groups: a control group (n = 21 that received normal saline, an EPO group (n = 21, that received EPO 500 IU/kg, a GM-CSF group (n = 21 that received 20 mcg/kg of GM-CSF and a EPO+GMCSF group (n = 21 which received a combination of the above. Seven animals of each group were killed on the 1st, 3rd and 7th postoperative day and their remnant liver was removed to evaluate liver regeneration by immunochemistry for PCNA and Ki 67. Results Our data suggest that EPO and GM-CSF increases liver regeneration following major hepatectomy when administered perioperatively. EPO has a more significant effect than GM-CSF (p Conclusion EPO, GM-CSF and their combination enhance liver regeneration after hepatectomy in rats when administered perioperatively. However their combination has a weaker effect on liver regeneration compared to EPO alone. Further investigation is needed to assess the exact mechanisms that mediate this finding.

  12. Thermal sensitivity and thermally enhanced radiosensitivity of murine bone marrow granulocyte-macrophage colony-forming units (CFU-GM)

    International Nuclear Information System (INIS)

    Yoshida, Hiroshi

    1994-01-01

    This study was to evaluate thermal response of granulocyte-macrophage colony-forming unit (CFU-GM) in vitro and to investigate the difference of thermally enhanced radiosensitivity on cell survivals of CFU-GM between in vitro and in vivo. In in vitro heating exposure, bone marrow suspensions, obtained from mouse femora or tibiae, were incubated; and in vivo heating exposure, the lower half-body of mice were immersed in a circulating hot water bath. For irradiation schedules, cell suspensions were irradiated in vitro or in vivo (whole-body irradiation). Thermal sensitivity curve, obtained by in vivo heating exposure, showed a shoulder region at short exposures followed by an exponential decline during longer heating exposures. The Arrhenius curve showed a break at 42.3deg C and inactivation enthalpy was 1836 kJ/mol (438 kcal/mole) below the break point and 704 kJ/mole (168 kcal/mole) above the point. When bone marrow suspensions, obtained after either in vitro or in vivo irradiation, were heated in vitro at 42deg C for 60 min, supura-additive effect on cell survivals was observed by in vivo irradiation, but not observed by in vitro irradiation. Thermal enhancement ratio (TER), defined as D 0 of combined in vivo irradiation and in vitro heating divided by D 0 of the sole in vivo irradiation, was 1.12. In vivo heating following in vivo irradiation also showed supra-additive effect, giving TER of 1.66. These findings indicated that murine marrow CFU-GM is sensitive to hyperthermia and that thermal radiosensitization is never negligible when hyperthermia is employed with preceding X-irradiation. Thus, combined use of radiotherapy and hyperthermia may decrease bone marrow function. (N.K.)

  13. Adjuvant therapy for melanoma in dogs: results of randomized clinical trials using surgery, liposome-encapsulated muramyl tripeptide, and granulocyte macrophage colony-stimulating factor.

    Science.gov (United States)

    MacEwen, E G; Kurzman, I D; Vail, D M; Dubielzig, R R; Everlith, K; Madewell, B R; Rodriguez, C O; Phillips, B; Zwahlen, C H; Obradovich, J; Rosenthal, R C; Fox, L E; Rosenberg, M; Henry, C; Fidel, J

    1999-12-01

    Spontaneous canine oral melanoma (COM) is a highly metastatic cancer, resistant to chemotherapy, and can serve as a model for cancer immunotherapy. Liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (L-MTP-PE) can activate the tumoricidal activity of the monocyte-macrophage system following i.v. injection. The objective of these studies was to evaluate the therapeutic effectiveness of L-MTP-PE administered alone and combined with recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) in dogs undergoing surgery for oral melanoma. Ninety-eight dogs with histologically confirmed, clinically staged, oral melanoma were entered into two randomized, double-blind, surgical adjuvant trials. In trial 1, 50 dogs were stratified based on clinical stage and randomized to once a week L-MTP-PE or lipid equivalent (control). When all of the clinical stages were combined, no difference in disease-free survival or in survival time (ST) were detected. However, within stage I, dogs receiving L-MTP-PE had a significant increase in ST compared with control, with 80% of the dogs treated with L-MTP-PE still alive at >2 years. Within each stage II and stage III, there was no difference detected between the treatment groups. In trial 2, 48 dogs were stratified on the basis of clinical stage and extent of surgery (simple resection or radical excision), treated with L-MTP-PE two times a week, and randomized to rcGM-CSF or saline (placebo) given s.c. daily for 9 weeks. Within each stage and when all of the stages were combined, there was no difference between the treatment groups. In both studies, stage I COM is associated with a better prognosis. No effect on survival was observed with regard to tumor location in the oral cavity, sex, type/extent of surgery, or age. In a subset of dogs tested, pulmonary alveolar macrophage cytotoxicity was enhanced with combined rcGM-CSF and L-MTP-PE but not in dogs treated with L-MTP-PE alone. The present study

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

    Science.gov (United States)

    Vian, Alex M; Higgins, Adam Z

    2014-02-01

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

  15. Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

    Directory of Open Access Journals (Sweden)

    Magda Paula Pereira do Nascimento

    2011-09-01

    Full Text Available Multinucleated giant cells (MGC are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF in association with other cytokines such as interferon-gamma (IFN-γ, tumour necrosis factor-alpha, interleukin (IL-10 or transforming growth factor beta (TGF-β1 on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg. The generation of MGC was determined by fusion index (FI and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18. The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

  16. Interleukin-6 and granulocyte-macrophage colony-stimulating factor in apical periodontitis: correlation with clinical and histologic findings of the involved teeth.

    Science.gov (United States)

    Radics, T; Kiss, C; Tar, I; Márton, I J

    2003-02-01

    Apical periodontitis is characterized by the presence of immunocompetent cells producing a wide variety of inflammatory mediators. Releasing cytokines with long-range action, such as interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF), apical periodontitis may induce changes in remote organs of the host. This study quantified the levels of IL-6 and GM-CSF in symptomatic and asymptomatic human periradicular lesions. Lesions were also characterized by size and histologic findings. Tissue samples were homogenized and supernatants were assayed using an enzyme-linked immunosorbent assay (ELISA). Correlations between cytokine levels and characteristic features (as single variables) of the lesions were analysed. There was a trend for higher levels of IL-6 and GM-CSF in symptomatic than in asymptomatic lesions, but the difference was not significant. Levels also tended to be higher in large than in small lesions, in polymorphonuclear (PMN) cell-rich than in PMN cell-poor samples, and in epithelialized than in non-epithelialized lesions. Significantly higher levels of IL-6 (778.1 +/- 220.5 pg/microg) and GM-CSF (363.3 +/- 98.4 pg/microg) were found in samples coincidentally possessing symptomatic and epithelialized features than in asymptomatic, small, PMN cell-poor, non-epithelialized lesions (IL-6: 45.2 +/- 13.1 pg/microg and GM-CSF: 135.1 +/- 26.4 pg/microg). These results suggest that symptomatic lesions containing epithelial cells represent an immunologically active stage of apical periodontitis, whereas asymptomatic, small, PMN cell-poor, non-epithelialized lesions represent healing apical lesions.

  17. Human Granulocyte Colony-Stimulating Factor (hG-CSF) Expression in Plastids of Lactuca sativa

    OpenAIRE

    Sharifi Tabar, Mehdi; Habashi, Ali Akbar; Rajabi Memari, Hamid

    2013-01-01

    Background: Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. Methods: hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA ter...

  18. X-ray-induced production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by mouse spleen cells in culture

    International Nuclear Information System (INIS)

    Onoda, M.; Shinoda, M.; Tsuneoka, K.; Shikita, M.

    1980-01-01

    Spleen cells were collected from normal mice and cultured in a medium containing 20% calf serum. Addition of lipopolysaccharide (LPS) in the culture significantly increased the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), and a maximum induction was attained in 5 days. Irradiation of the spleen cells with 300 to 3000 R x rays also enhanced the production of GM-CSF, but there was a latent period of about 5 days before the factor appeared in the culture medium. The observed difference between LPS and x rays in the timing of inducing GM-CSF production in the spleen cell culture was consistent with the difference observed in animals. These results suggest that different mechanisms of GM-CSF production operate in the spleen in response to either LPS or x rays

  19. Human Induced Pluripotent Stem Cell-Derived Macrophages for Unraveling Human Macrophage Biology.

    Science.gov (United States)

    Zhang, Hanrui; Reilly, Muredach P

    2017-11-01

    Despite a substantial appreciation for the critical role of macrophages in cardiometabolic diseases, understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (iPSC)-derived macrophages (IPSDM), as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of the role of macrophages in human diseases. In this review, we summarize current literature in the differentiation and characterization of IPSDM at phenotypic, functional, and transcriptomic levels. We emphasize the progress in differentiating iPSC to tissue resident macrophages, and in understanding the ontogeny of in vitro differentiated IPSDM that resembles primitive hematopoiesis, rather than adult definitive hematopoiesis. We review the application of IPSDM in modeling both Mendelian genetic disorders and host-pathogen interactions. Finally, we highlighted the potential areas of research using IPSDM in functional validation of coronary artery disease loci in genome-wide association studies, functional genomic analyses, drug testing, and cell therapeutics in cardiovascular diseases. © 2017 American Heart Association, Inc.

  20. Leucine supplementation attenuates macrophage foam-cell formation: Studies in humans, mice, and cultured macrophages.

    Science.gov (United States)

    Grajeda-Iglesias, Claudia; Rom, Oren; Hamoud, Shadi; Volkova, Nina; Hayek, Tony; Abu-Saleh, Niroz; Aviram, Michael

    2018-02-05

    Whereas atherogenicity of dietary lipids has been largely studied, relatively little is known about the possible contribution of dietary amino acids to macrophage foam-cell formation, a hallmark of early atherogenesis. Recently, we showed that leucine has antiatherogenic properties in the macrophage model system. In this study, an in-depth investigation of the role of leucine in macrophage lipid metabolism was conducted by supplementing humans, mice, or cultured macrophages with leucine. Macrophage incubation with serum obtained from healthy adults supplemented with leucine (5 g/d, 3 weeks) significantly decreased cellular cholesterol mass by inhibiting the rate of cholesterol biosynthesis and increasing cholesterol efflux from macrophages. Similarly, leucine supplementation to C57BL/6 mice (8 weeks) resulted in decreased cholesterol content in their harvested peritoneal macrophages (MPM) in relation with reduced cholesterol biosynthesis rate. Studies in J774A.1 murine macrophages revealed that leucine dose-dependently decreased cellular cholesterol and triglyceride mass. Macrophages treated with leucine (0.2 mM) showed attenuated uptake of very low-density lipoproteins and triglyceride biosynthesis rate, with a concurrent down-regulation of diacylglycerol acyltransferase-1, a key enzyme catalyzing triglyceride biosynthesis in macrophages. Similar effects were observed when macrophages were treated with α-ketoisocaproate, a key leucine metabolite. Finally, both in vivo and in vitro leucine supplementation significantly improved macrophage mitochondrial respiration and ATP production. The above studies, conducted in human, mice, and cultured macrophages, highlight a protective role for leucine attenuating macrophage foam-cell formation by mechanisms related to the metabolism of cholesterol, triglycerides, and energy production. © 2018 BioFactors, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

  1. A randomized case-controlled study of recombinant human granulocyte colony stimulating factor for the treatment of sepsis in preterm neutropenic infants.

    Science.gov (United States)

    Aktaş, Doğukan; Demirel, Bilge; Gürsoy, Tuğba; Ovalı, Fahri

    2015-06-01

    To investigate the efficacy and safety of recombinant human granulocyte colony-stimulating factor, recombinant human granulocyte-macrophage colony-stimulating factor (rhG-CSF) to treat sepsis in neutropenic preterm infants. Fifty-six neutropenic preterm infants with suspected or culture-proven sepsis hospitalized in Zeynep Kamil Maternity and Children's Educational and Training Hospital, Kozyatağı/Istanbul, Turkey between January 2008 and January 2010 were enrolled. Patients were randomized either to receive rhG-CSF plus empirical antibiotics (Group I) or empirical antibiotics alone (Group II). Clinical features were recorded. Daily complete blood count was performed until neutropenia subsided. Data were analyzed using SPSS version 11.5. Thirty-three infants received rhG-CSF plus antibiotic treatment and 23 infants received antibiotic treatment. No drug-related adverse event was recorded. Absolute neutrophil count values were significantly higher on the 2(nd) study day and 3(rd) study day in Group I. Short-term mortality did not differ between the groups. Treatment with rhG-CSF resulted in a more rapid recovery of ANC in neutropenic preterm infants. However, no reduction in short-term mortality was documented. Copyright © 2014. Published by Elsevier B.V.

  2. Short-term exposure of umbilical cord blood CD34+ cells to granulocyte-macrophage colony-stimulating factor early in culture improves ex vivo expansion of neutrophils.

    Science.gov (United States)

    Marturana, Flavia; Timmins, Nicholas E; Nielsen, Lars K

    2011-03-01

    Despite the availability of modern antibiotics/antimycotics and cytokine support, neutropenic infection accounts for the majority of chemotherapy-associated deaths. While transfusion support with donor neutrophils is possible, cost and complicated logistics make such an option unrealistic on a routine basis. A manufactured neutrophil product could enable routine prophylactic administration of neutrophils, preventing the onset of neutropenia and substantially reducing the risk of infection. We examined the use of pre-culture strategies and various cytokine/modulator combinations to improve neutrophil expansion from umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HPC). Enriched UCB HPC were cultured using either two-phase pre-culture strategies or a single phase using various cytokine/modulator combinations. Outcome was assessed with respect to numerical expansion, cell morphology, granulation and respiratory burst activity. Pre-culture in the absence of strong differentiation signals (e.g. granulocyte colony-stimulating factor; G-CSF) failed to provide any improvement to final neutrophil yields. Similarly, removal of differentiating cells during pre-culture failed to improve neutrophil yields to an appreciable extent. Of the cytokine/modulator combinations, the addition of granulocyte-macrophage (GM)-colony-stimulating factor (CSF) alone gave the greatest increase. In order to avoid production of monocytes, it was necessary to remove GM-CSF on day 5. Using this strategy, neutrophil expansion improved 2.7-fold. Although all cytokines and culture strategies employed have been reported previously to enhance HPC expansion, we found that the addition of GM-CSF alone was sufficient to improve total cell yields maximally. The need to remove GM-CSF on day 5 to avoid monocyte differentiation highlights the context and time-dependent complexity of exogenous signaling in hematopoietic cell differentiation and growth.

  3. The immunomodulatory effect of inhaled granulocyte-macrophage colony-stimulating factor in cystic fibrosis. A new treatment paradigm

    DEFF Research Database (Denmark)

    Heslet, Lars; Bay, Christiane; Nepper-Christensen, Steen

    2012-01-01

    Patients with cystic fibrosis (CF) experience recurrent infections and develop chronically infected lungs, which initiates an altered immunological alveolar environment. End-stage pulmonary dysfunction is a result of a long sequence of complex events in CF, progressing to alveolar macrophage dysf...

  4. Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage

    Science.gov (United States)

    Ushach, Irina; Zlotnik, Albert

    2016-01-01

    M-CSF and GM-CSF are 2 important cytokines that regulate macrophage numbers and function. Here, we review their known effects on cells of the macrophage-monocyte lineage. Important clues to their function come from their expression patterns. M-CSF exhibits a mostly homeostatic expression pattern, whereas GM-CSF is a product of cells activated during inflammatory or pathologic conditions. Accordingly, M-CSF regulates the numbers of various tissue macrophage and monocyte populations without altering their "activation" status. Conversely, GM-CSF induces activation of monocytes/macrophages and also mediates differentiation to other states that participate in immune responses [i.e., dendritic cells (DCs)]. Further insights into their function have come from analyses of mice deficient in either cytokine. M-CSF signals through its receptor (CSF-1R). Interestingly, mice deficient in CSF-1R expression exhibit a more significant phenotype than mice deficient in M-CSF. This observation was explained by the discovery of a novel cytokine (IL-34) that represents a second ligand of CSF-1R. Information about the function of these ligands/receptor system is still developing, but its complexity is intriguing and strongly suggests that more interesting biology remains to be elucidated. Based on our current knowledge, several therapeutic molecules targeting either the M-CSF or the GM-CSF pathways have been developed and are currently being tested in clinical trials targeting either autoimmune diseases or cancer. It is intriguing to consider how evolution has directed these pathways to develop; their complexity likely mirrors the multiple functions in which cells of the monocyte/macrophage system are involved. PMID:27354413

  5. Synthesis, structural characterization and effect on human granulocyte intracellular cAMP levels of abscisic acid analogs.

    Science.gov (United States)

    Bellotti, Marta; Salis, Annalisa; Grozio, Alessia; Damonte, Gianluca; Vigliarolo, Tiziana; Galatini, Andrea; Zocchi, Elena; Benatti, Umberto; Millo, Enrico

    2015-01-01

    The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. (±)-2-Chloropropionic acid elevates reactive oxygen species formation in human neutrophil granulocytes

    International Nuclear Information System (INIS)

    Aam, B.B.; Fonnum, F.

    2006-01-01

    (±)-2-Chloropropionic acid (2-CPA) is a neurotoxic compound which kills cerebellar granule cells in vivo, and makes cerebellar granule cells in vitro produce reactive oxygen species (ROS). We have studied the effect of 2-CPA on ROS formation in human neutrophil granulocytes in vitro. We found an increased formation of ROS after 2-CPA exposure using three different methods; the fluorescent probe DCFH-DA and the chemiluminescent probes lucigenin and luminol. Four different inhibitors of ROS formation were tested on the cells in combination with 2-CPA to characterize the signalling pathways. The spin-trap s-PBN, the ERK1/2 inhibitor U0126 and the antioxidant Vitamin E inhibited the 2-CPA-induced ROS formation completely, while the mitochondrial transition permeability pore blocker cyclosporine A inhibited the ROS formation partly. We also found that 2-CPA induced an increased nitric oxide production in the cells by using the Griess reagent. The level of reduced glutathione, measured with the DTNB assay, was decreased after exposure to high concentrations of 2-CPA. Western blotting analysis showed that 2-CPA exposure led to an elevated phosphorylation of ERK MAP kinase. This phosphorylation was inhibited by U0126. Based on these experiments it seems like the mechanisms for 2-CPA induced toxicity involves ROS formation and is similar in neutrophil granulocytes as earlier shown in cerebellar granule cells. This also implies that 2-CPA may be immunotoxic

  7. Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Jücker, M; Feldman, R A

    1995-11-17

    Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

  8. Nicotine can skew the characterization of the macrophage type-1 (M{Phi}1) phenotype differentiated with granulocyte-macrophage colony-stimulating factor to the M{Phi}2 phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Yanagita, Manabu; Kobayashi, Ryohei [Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, Osaka 565-0871 (Japan); Murakami, Shinya, E-mail: ipshinya@dent.osaka-u.ac.jp [Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, Osaka 565-0871 (Japan)

    2009-10-09

    Macrophages (M{Phi}s) exhibit functional heterogeneity and plasticity in the local microenvironment. Recently, it was reported that M{Phi}s can be divided into proinflammatory M{Phi}s (M{Phi}1) and anti-inflammatory M{Phi}s (M{Phi}2) based on their polarized functional properties. Here, we report that nicotine, the major ingredient of cigarette smoke, can modulate the characteristics of M{Phi}1. Granulocyte-macrophage colony-stimulating factor-driven M{Phi}1 with nicotine (Ni-M{Phi}1) showed the phenotypic characteristics of M{Phi}2. Like M{Phi}2, Ni-M{Phi}1 exhibited antigen-uptake activities. Ni-M{Phi}1 suppressed IL-12, but maintained IL-10 and produced high amounts of MCP-1 upon lipopolysaccharide stimulation compared with M{Phi}1. Moreover, we observed strong proliferative responses of T cells to lipopolysaccharide-stimulated M{Phi}1, whereas Ni-M{Phi}1 reduced T cell proliferation and inhibited IFN-{gamma} production by T cells. These results suggest that nicotine can change the functional characteristics of M{Phi} and skew the M{Phi}1 phenotype to M{Phi}2. We propose that nicotine is a potent regulator that modulates immune responses in microenvironments.

  9. The β-hemolysin and intracellular survival of Streptococcus agalactiae in human macrophages.

    Science.gov (United States)

    Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara

    2013-01-01

    S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.

  10. The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

    Science.gov (United States)

    Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara

    2013-01-01

    S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches. PMID:23593170

  11. The β-hemolysin and intracellular survival of Streptococcus agalactiae in human macrophages.

    Directory of Open Access Journals (Sweden)

    Anubha Sagar

    Full Text Available S. agalactiae (group B streptococci, GBS is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.

  12. The role of granulocyte macrophage colony stimulating factor (GM-CSF) in radiation-induced tumor cell migration.

    Science.gov (United States)

    Vilalta, Marta; Brune, Jourdan; Rafat, Marjan; Soto, Luis; Graves, Edward E

    2018-03-13

    Recently it has been observed in preclinical models that that radiation enhances the recruitment of circulating tumor cells to primary tumors, and results in tumor regrowth after treatment. This process may have implications for clinical radiotherapy, which improves control of a number of tumor types but which, despite continued dose escalation and aggressive fractionation, is unable to fully prevent local recurrences. By irradiating a single tumor within an animal bearing multiple lesions, we observed an increase in tumor cell migration to irradiated and unirradiated sites, suggesting a systemic component to this process. Previous work has identified the cytokine GM-CSF, produced by tumor cells following irradiation, as a key effector of this process. We evaluated the ability of systemic injections of a PEGylated form of GM-CSF to stimulate tumor cell migration. While increases in invasion and migration were observed for tumor cells in a transwell assay, we found that daily injections of PEG-GM-CSF to tumor-bearing animals did not increase migration of cells to tumors, despite the anticipated changes in circulating levels of granulocytes and monocytes produced by this treatment. Combination of PEG-GM-CSF treatment with radiation also did not increase tumor cell migration. These findings suggest that clinical use of GM-CSF to treat neutropenia in cancer patients will not have negative effects on the aggressiveness of residual cancer cells. However, further work is needed to characterize the mechanism by which GM-CSF facilitates systemic recruitment of trafficking tumor cells to tumors.

  13. Enhancement of the grafting efficiency of transplanted marrow cells by preincubation with interleukin-3 and granulocyte-macrophage colony-stimulating factor

    Energy Technology Data Exchange (ETDEWEB)

    Tavassoli, M.; Konno, M.; Shiota, Y.; Omoto, E.; Minguell, J.J.; Zanjani, E.D.

    1991-04-01

    To improve the grafting efficiency of transplanted murine hematopoietic progenitors, we briefly preincubated mouse bone marrow cells with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) ex vivo before their transplantation into irradiated recipients. This treatment was translated into an increase in the seeding efficiency of colony-forming unit-spleen (CFU-S) and CFU-GM after transplantation. Not only was the concentration of CFU-S in the tibia increased 2 and 24 hours after transplantation, but the total cell number and CFU-S and CFU-GM concentrations were persistently higher in IL-3- and GM-CSF-treated groups 1 to 3 weeks after transplantation. In addition, the survival of animals as a function of transplanted cell number was persistently higher in IL-3- and GM-CSF-treated groups compared with controls. The data indicate that the pretreatment of marrow cells with IL-3 and GM-CSF before transplantation increases the seeding efficiency of hematopoietic stem cells and probably other progenitor cells after transplantation. This increased efficiency may be mediated by upward modulation of homing receptors. Therefore, ex vivo preincubation of donor marrow cells with IL-3 and GM-CSF may be a useful tactic in bone marrow transplantation.

  14. The effect of interleukin-8 and granulocyte macrophage colony stimulating factor on the response of neutrophils to formyl methionyl leucyl phenylalanine.

    Science.gov (United States)

    Mikami, M; Llewellyn-Jones, C G; Stockley, R A

    1998-08-14

    Neutrophils isolated from patients with chronic bronchitis and emphysema have been shown to have enhanced responses to formyl peptides when assessed in vitro compared to age, sex matched controls. It is currently unclear whether the observed differences are due to a 'priming' effect by a second agent in vivo, or whether this is a primary difference in the neutrophils. We have studied the effects of interleukin-8, which is thought to be one of the major pro-inflammatory cytokines in chronic lung disease and granulocyte macrophage colony stimulating factor (GMCSF), in order to assess their effects on neutrophil chemotaxis and connective tissue degradation. In addition, we have assessed the effect of preincubation of these agents with neutrophils for 30 min followed by stimulation with F-Met-Leu-Phe (FMLP) to investigate any possible 'priming' effect that may be relevant to our clinical data. We report suppression of neutrophil chemotaxis to FMLP following incubation of the neutrophils with both IL-8 and GMCSF. However, we have observed an additive effect of IL-8 and FMLP for neutrophil degranulation leading to fibronectin degradation. The results suggest that IL-8 does not 'prime' neutrophils for subsequent FMLP stimulation as observed in vivo. Although the results for GMCSF were similar for the chemotactic response, the agent also had a synergistic effect on connective tissue degradation. However, it is concluded that neither agent could explain the enhanced neutrophil responses seen in our patients.

  15. The frequency of clinical pregnancy and implantation rate after cultivation of embryos in a medium with granulocyte macrophage colony-stimulating factor (GM-CSF) in patients with preceding failed attempts of ART.

    Science.gov (United States)

    Tevkin, S; Lokshin, V; Shishimorova, M; Polumiskov, V

    2014-10-01

    The application in IVF practice of modern techniques can improve positive outcome of each cycle in the assisted reproductive technology (ART) programs and the effectiveness of treatment as a whole. There are embryos in the female reproductive tract in physiological medium which contain various cytokines and growth factors. It plays an important role in the regulation of normal embryonic development, improve implantation and subsequently optimizing the development of the fetus and the placenta. Granulocyte macrophage colony-stimulating factor (GM-CSF is one of the cytokines playing an important role in reproductive function. Addition of recombinant GM-CSF to the culture medium can makes closer human embryos culture to in vivo conditions and improve the efficacy ART cycles. The analysis of culture embryos in EmbryoGen medium has shown that fertilization rate embryo culture and transfer to patients with previous unsuccessful attempts increases clinical pregnancy rate compared to the control group 39.1 versus 27.8%, respectively. It is noted that the implantation rate (on 7 weeks' gestation) and progressive clinical pregnancy rate (on 12 weeks' gestation) were significantly higher in group embryos culture in EmbryoGen medium compared to standard combination of medium (ISM1+VA), and were 20.4 and 17.4% versus 11.6 and 9.1%, respectively.

  16. Mobilization of peripheral blood progenitor cells by chemotherapy and granulocyte-macrophage colony-stimulating factor for hematologic support after high-dose intensification for breast cancer.

    Science.gov (United States)

    Elias, A D; Ayash, L; Anderson, K C; Hunt, M; Wheeler, C; Schwartz, G; Tepler, I; Mazanet, R; Lynch, C; Pap, S

    1992-06-01

    High-dose therapy with autologous marrow support results in durable complete remissions in selected patients with relapsed lymphoma and leukemia who cannot be cured with conventional dose therapy. However, substantial morbidity and mortality result from the 3- to 6-week period of marrow aplasia until the reinfused marrow recovers adequate hematopoietic function. Hematopoietic growth factors, particularly used after chemotherapy, can increase the number of peripheral blood progenitor cells (PBPCs) present in systemic circulation. The reinfusion of PBPCs with marrow has recently been reported to reduce the time to recovery of adequate marrow function. This study was designed to determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF)-mobilized PBPCs alone (without marrow) would result in rapid and reliable hematopoietic reconstitution. Sixteen patients with metastatic breast cancer were treated with four cycles of doxorubicin, 5-fluorouracil, and methotrexate (AFM induction). Patients responding after the first two cycles were administered GM-CSF after the third and fourth cycles to recruit PBPCs for collection by two leukapheresis per cycle. These PBPCs were reinfused as the sole source of hematopoietic support after high doses of cyclophosphamide, thiotepa, and carboplatin. No marrow or hematopoietic cytokines were used after progenitor cell reinfusion. Granulocytes greater than or equal to 500/microL was observed on a median of day 14 (range, 8 to 57). Transfusion independence of platelets greater than or equal to 20,000/microL occurred on a median day of 12 (range, 8 to 134). However, three patients required the use of a reserve marrow for slow platelet engraftment. In retrospect, these patients were characterized by poor baseline bone marrow cellularity and poor platelet recovery after AFM induction therapy. When compared with 29 historical control patients who had received the same high-dose intensification chemotherapy using autologous

  17. Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.

    Science.gov (United States)

    Soldano, Stefano; Pizzorni, Carmen; Paolino, Sabrina; Trombetta, Amelia Chiara; Montagna, Paola; Brizzolara, Renata; Ruaro, Barbara; Sulli, Alberto; Cutolo, Maurizio

    2016-01-01

    Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells. Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography. ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were

  18. [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.

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

    Science.gov (United States)

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

    2012-10-01

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

  20. Human granulocyte colony-stimulating factor (hG-CSF) expression in plastids of Lactuca sativa.

    Science.gov (United States)

    Sharifi Tabar, Mehdi; Habashi, Ali Akbar; Rajabi Memari, Hamid

    2013-01-01

    Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.

  1. Vitamin E--a selective inhibitor of the NADPH oxidoreductase enzyme system in human granulocytes

    International Nuclear Information System (INIS)

    Butterick, C.J.; Baehner, R.L.; Boxer, L.A.; Jersild, R.A. Jr.

    1983-01-01

    The cellular sites of H 2 O 2 formation in phagocytizing granulocytes have been identified with cerium chloride. A precipitate was visible in phagosomes and on plasma membranes from intact normal cells in the presence of either 0.71 mM NADH or NADPH. X-ray microanalysis permitted identification of cerium deposition within the phagosomes even in the absence of reduced pyridine nucleotides. Catalase ablated the formation of the reaction product. Intact granulocytes obtained from subjects receiving 1600 units of vitamin E daily for 2 weeks exhibited reaction product in the presence of NADH but not NADPH. Intact cells from subjects treated with vitamin E demonstrated diminished numbers of phagocytic vesicles containing reaction product. During phagocytosis the granulocytes treated with vitamin E consumed oxygen but exhibited significantly reduced rates of hydrogen-peroxide-dependent glucose-1- 14 C oxidation to 14 CO 2 . Isolated phagocytic vesicles obtained from granulocytes after ingestion of opsonized lipopolysaccharide-paraffin oil droplets contained reaction product when exposed to 0.71 mM NADPH. No reaction product was evident at 0.71 mM NADH but was evident at 2.0 mM NADH. Isolated phagocytic vesicles from the granulocytes of subjects receiving vitamin E exhibited reaction product only in the presence of NADH. These observations suggest that vitamin E interferes with the electron transport chain apparently required for the oxidation of NADPH to form H 2 O 2 in the phagocytizing granulocyte

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associate...... significantly to the antiprotease levels in tissues during inflammation. Impaired binding of neutrophil A1AT to serine proteases in patients with (PI)ZZ mutations may enhance their susceptibility to the development of emphysema....

  3. Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

    Science.gov (United States)

    Pessina, A; Albella, B; Bueren, J; Brantom, P; Casati, S; Gribaldo, L; Croera, C; Gagliardi, G; Foti, P; Parchment, R; Parent-Massin, D; Sibiril, Y; Van Den Heuvel, R

    2001-12-01

    This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional

  4. Rhinovirus infection induces distinct transcriptome profiles in polarized human macrophages.

    Science.gov (United States)

    Rajput, Charu; Walsh, Megan P; Eder, Breanna N; Metitiri, Ediri E; Popova, Antonia P; Hershenson, Marc B

    2018-05-01

    Infections with rhinovirus (RV) cause asthma exacerbations. Recent studies suggest that macrophages play a role in asthmatic airway inflammation and the innate immune response to RV infection. Macrophages exhibit phenotypes based on surface markers and gene expression. We hypothesized that macrophage polarization state alters gene expression in response to RV infection. Cells were derived from human peripheral blood derived monocytes. M1 and M2 polarization was carried out by using IFN-γ and IL-4, respectively, and RNA was extracted for Affymetrix Human Gene ST2.1 exon arrays. Selected genes were validated by quantitative (q)PCR. Treatment of nonactivated (M0) macrophages with IFN-γ and IL-4 induced the expression of 252 and 153 distinct genes, respectively, including previously-identified M1 and M2 markers. RV infection of M0 macrophages induced upregulation of 232 genes; pathway analysis showed significant overrepresentation of genes involved in IFN-α/β signaling and cytokine signaling in the immune system. RV infection induced differential expression of 195 distinct genes in M1-like macrophages but only seven distinct genes in M2-like-polarized cells. In a secondary analysis, comparison between M0-, RV-infected, and M1-like-polarized, RV-infected macrophages revealed differential expression of 227 genes including those associated with asthma and its exacerbation. qPCR demonstrated increased expression of CCL8, CXCL10, TNFSF10, TNFSF18, IL6, NOD2, and GSDMD and reduced expression of VNN1, AGO1, and AGO2. Together, these data show that, in contrast to M2-like-polarized macrophages, gene expression of M1-like macrophages is highly regulated by RV.

  5. Modulation of human macrophage activity by Ascaris antigens is dependent on macrophage polarization state

    DEFF Research Database (Denmark)

    Almeida, Sara; Nejsum, Peter; Williams, Andrew R.

    2018-01-01

    Parasitic worms (helminths) are known to actively modulate host immune responses and inflammation. The aim of this study was to investigate if adult body fluid (ABF) from the helminth Ascaris suum has immunomodulatory effects on different subtypes of human monocyte-derived macrophages (Mɸ) in vitro...

  6. Growth regulation on human acute myeloid leukemia effects of five recombinant hematopoietic factors in a serum-free culture system

    NARCIS (Netherlands)

    Delwel, E.; Salem, M.; Pellens, C.; Dorssers, L.; Wagemaker, G.; Clark, S.; Loewenberg, B

    1988-01-01

    The response of human acute myeloid leukemia (AML) cells to the distinct hematopoietic growth factors (HGFs), ie, recombinant interleukin-3 (IL-3), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), and erythropoietin (Epo) was investigated under well-defined

  7. DMPD: Differential responses of human monocytes and macrophages to IL-4 and IL-13. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10534111 Differential responses of human monocytes and macrophages to IL-4 and IL-1...):575-8. (.png) (.svg) (.html) (.csml) Show Differential responses of human monocytes and macrophages to IL-...4 and IL-13. PubmedID 10534111 Title Differential responses of human monocytes an

  8. Use of a Granulocyte Immunofluorescence Assay Designed for Humans for Detection of Antineutrophil Cytoplasmic Antibodies in Dogs with Chronic Enteropathies.

    Science.gov (United States)

    Florey, J; Viall, A; Streu, S; DiMuro, V; Riddle, A; Kirk, J; Perazzotti, L; Affeldt, K; Wagner, R; Vaden, S; Harris, T; Allenspach, K

    2017-07-01

    Perinuclear antineutrophil cytoplasmic antibodies (pANCA) previously have been shown to be serum markers in dogs with chronic enteropathies, with dogs that have food-responsive disease (FRD) having higher frequencies of seropositivity than dogs with steroid-responsive disease (SRD). The indirect immunofluorescence (IIF) assay used in previous publications is time-consuming to perform, with low interobserver agreement. We hypothesized that a commercially available granulocyte IIF assay designed for humans could be used to detect perinuclear antineutrophil cytoplasmic antibodies in dogs. Forty-four dogs with FRD, 20 dogs with SRD, 20 control dogs, and 38 soft-coated wheaten terrier (SCWT) or SCWT-cross dogs. A granulocyte assay designed for humans was used to detect pANCA, cANCA, and antinuclear antibodies (ANA), as well as antibodies against proteinase-3 protein (PR-3) and myeloperoxidase protein (MPO) in archived serum samples. Sensitivity of the granulocyte assay to predict FRD in dogs was 0.61 (95% confidence interval (CI), 0.45, 0.75), and specificity was 1.00 (95% CI, 0.91, 1.00). A significant association was identified between positive pANCA or cANCA result and diagnosis of FRD (P < 0.0001). Agreement between the two assays to detect ANCA in the same serum samples from SCWT with protein-losing enteropathy/protein-losing nephropathy (PLE/PLN) was substantial (kappa, 0.77; 95% CI, 0.53, 1.00). Eight ANCA-positive cases were positive for MPO or PR-3 antibodies. The granulocyte immunofluorescence assay used in our pilot study was easy and quick to perform. Agreement with the previously published method was good. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  9. Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells.

    Science.gov (United States)

    Na, Yi Rang; Hong, Ji Hye; Lee, Min Yong; Jung, Jae Hun; Jung, Daun; Kim, Young Won; Son, Dain; Choi, Murim; Kim, Kwang Pyo; Seok, Seung Hyeok

    2015-10-01

    Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Thermo-radiosensitivity of the granulocyte and macrophage precursor cells of mice. I I . - X- irradiation effects and influence of hyperthermia on the radiosensitivity; Termo-radiosensibilidad del precursor hematopoyetico que origina las series granulocitica y macrofaga de raton. II. - Efectos producidos por la radiacion X e influencia de la hipertermia sobre la radiosensibilidad celular

    Energy Technology Data Exchange (ETDEWEB)

    Bueren, J A; Nieto, M

    1983-07-01

    The effects of the X-irradiation on the viability of the granulocyte-macrophage precursors, has been determined by means of the agar diffusion chamber culture technique. The results show the high radiosensitivity of these cells, with survival parameter similar to those previously reported in the literature about different granulocyte-macrophage precursors. When a hyperthermic treatment is performed prior to the X-irradiation, a radiosensitization phenomenon is observed due to the synergism existent between hyperthermia and X rays on the lethality of the precursors. (Authors) 37 refs.

  11. Immune-enhancing effect of nano-DNA vaccine encoding a gene of the prME protein of Japanese encephalitis virus and BALB/c mouse granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Zhai, Yongzhen; Zhou, Yan; Li, Ximei; Feng, Guohe

    2015-07-01

    Plasmid-encoded granulocyte-macrophage colony-stimulating factor (GM‑CSF) is an adjuvant for genetic vaccines; however, how GM-CSF enhances immunogenicity remains to be elucidated. In the present study, it was demonstrated that injection of a plasmid encoding the premembrane (prM) and envelope (E) protein of Japanese encephalitis virus and mouse GM-CSF (pJME/GM-CSF) into mouse muscle recruited large and multifocal conglomerates of macrophages and granulocytes, predominantly neutrophils. During the peak of the infiltration, an appreciable number of immature dendritic cells (DCs) appeared, although no T and B-cells was detected. pJME/GM-CSF increased the number of splenic DCs and the expression of major histocompatibility complex class II (MHCII) on splenic DC, and enhanced the antigenic capture, processing and presentation functions of splenic DCs, and the cell-mediated immunity induced by the vaccine. These findings suggested that the immune-enhancing effect by pJME/GM-CSF was associated with infiltrate size and the appearance of integrin αx (CD11c)+cells. Chitosan-pJME/GM-CSF nanoparticles, prepared by coacervation via intramuscular injection, outperformed standard pJME/GM-CSF administrations in DC recruitment, antigen processing and presentation, and vaccine enhancement. This revealed that muscular injection of chitosan‑pJME/GM-CSF nanoparticles may enhance the immunoadjuvant properties of GM-CSF.

  12. Granulocyte kinetics

    International Nuclear Information System (INIS)

    Peters, A.M.; Lavender, J.P.; Saverymuttu, S.H.

    1985-01-01

    By using density gradient materials enriched with autologous plasma, the authors have been able to isolate granulocutes from other cellular elements and label them with In-111 without separation from a plasma environment. The kinetic behavior of these cells suggests that phenomena attributed to granulocyte activation are greatly reduced by this labeling. Here, they review their study of granulocyte kinetics in health and disease in hope of quantifying sites of margination and identifying principal sites of destruction. The three principle headings of the paper are distribution, life-span, and destruction

  13. Curcumin enhances human macrophage control of Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Bai, Xiyuan; Oberley-Deegan, Rebecca E; Bai, An; Ovrutsky, Alida R; Kinney, William H; Weaver, Michael; Zhang, Gong; Honda, Jennifer R; Chan, Edward D

    2016-07-01

    With the worldwide emergence of highly drug-resistant tuberculosis (TB), novel agents that have direct antimycobacterial effects or that enhance host immunity are urgently needed. Curcumin is a polyphenol responsible for the bright yellow-orange colour of turmeric, a spice derived from the root of the perennial herb Curcuma longa. Curcumin is a potent inducer of apoptosis-an effector mechanism used by macrophages to kill intracellular Mycobacterium tuberculosis (MTB). An in vitro human macrophage infection model was used to determine the effects of curcumin on MTB survival. We found that curcumin enhanced the clearance of MTB in differentiated THP-1 human monocytes and in primary human alveolar macrophages. We also found that curcumin was an inducer of caspase-3-dependent apoptosis and autophagy. Curcumin mediated these anti-MTB cellular functions, in part, via inhibition of nuclear factor-kappa B (NFκB) activation. Curcumin protects against MTB infection in human macrophages. The host-protective role of curcumin against MTB in macrophages needs confirmation in an animal model; if validated, the immunomodulatory anti-TB effects of curcumin would be less prone to drug resistance development. © 2016 Asian Pacific Society of Respirology.

  14. Effects of human granulocyte-colony stimulating factor on fracture healing in rats

    International Nuclear Information System (INIS)

    Bozlar, M.; Aslan, B.; Kalaci, A.; Yanat, Ahmet N.; Baktiroglu, L.; Tasci, A.

    2005-01-01

    Granulocyte colony stimulation factor (G-CSF) is generally used to prevent and cure the neutropenia associated with chemotherapy and bone marrow transplantation. In addition to its effects on neutrophil function, G-CSF was found to have the characteristic of modulating the cytokines in the inflammatory response. Then, the question to answer is whether it has any effect on fracture healing and to what extent? In this study, we test the effects of G-CSF on the healing of tibia fracture in a rat model. This study was performed at Harran University, Sanliurfa, Turkey between July 2003 and August 2004. Twenty female, healthy Sprague-Dawley rats, weighing between 250 and 300 gm were divided into 2 groups, and their tibiae broken. The rats in the G-CSF group were injected subcutaneous with 25ug/kg/day of recombinant human G-CSF for 7 days, and the ones in the control group with 0.9% sodium chloride. Rats were sacrificed 3 weeks after surgery and then radiological, histological and biomechanical evaluations were performed. Biomechanical tests were performed at the Middle East Technical University, Ankara, Turkey.The median radiographic scores for the control group were calculated as 4.1, and 6.1 for the G-CSF group (p = 0.016). Cortex remodeling, callus formation, bone union and marrow changes values did not differ significantly (p > 0.05). Mechanical parameter (mean max-Load) values for the control group were found to be 24.0 +/- 3.0 N, and 241.5 +/-75.7 N for the G-CSF group (p 0.001). We found that G-CSF has an important effect on fracture healing. However, this effect requires further study. (author)

  15. Human macrophage hemoglobin-iron metabolism in vitro

    International Nuclear Information System (INIS)

    Custer, G.; Balcerzak, S.; Rinehart, J.

    1982-01-01

    An entirely in vitro technique was employed to characterize hemoglobin-iron metabolism by human macrophages obtained by culture of blood monocytes and pulmonary alveolar macrophages. Macrophages phagocytized about three times as many erythrocytes as monocytes and six times as many erythrocytes as pulmonary alveolar macrophages. The rate of subsequent release of 59 Fe to the extracellular transferrin pool was two- to fourfold greater for macrophages as compared to the other two cell types. The kinetics of 59 Fe-transferrin release were characterized by a relatively rapid early phase (hours 1-4) followed by a slow phase (hours 4-72) for all three cell types. Intracellular movement of iron was characterized by a rapid shift from hemoglobin to ferritin that was complete with the onset of the slow phase of extracellular release. A transient increase in 59 Fe associated with an intracellular protein eluting with transferrin was also observed within 1 hour after phagocytosis. The process of hemoglobin-iron release to extracellular transferrin was inhibited at 4 degrees C but was unaffected by inhibitory of protein synthesis, glycolysis, microtubule function, and microfilament function. These data emphasize the rapidity of macrophage hemoglobin iron metabolism, provide a model for characterization of this process in vitro, and in general confirm data obtained utilizing in vivo animal models

  16. Granulocytic sarcoma.

    Science.gov (United States)

    Hutchison, R E; Kurec, A S; Davey, F R

    1990-12-01

    Granulocytic sarcoma is a variant presentation of acute myeloblastic leukemia, occurring in extramedullary locations. It is uncommon, but it may occur at any site and at any age, which necessitates its inclusion in the differential diagnosis of all undifferentiated tumors. Histology, touch-imprint cytology, cytochemistry, immunocytochemistry, electron microscopy, and molecular studies all contribute to the diagnosis.

  17. Peptide secreted by human alveolar macrophages releases neutrophil granule contents

    International Nuclear Information System (INIS)

    MacArthur, C.K.; Miller, E.J.; Cohen, A.B.

    1987-01-01

    A monoclonal antibody was developed against an 8000-kDa enzyme-releasing peptide (ERP) released from human alveolar macrophages. ERP was isolated on an immunoaffinity column containing the antibody bound to staphylococcal protein A-Sepharose, and by autoradiography. Release of ERP from the macrophages is not changed by plastic adherence, phagocytosis, calcium ionophore, or phorbol esters. The peptide was not antigenically similar to interferon-γ, tumor necrosis factor, or interleukin lα or 1β. The release of constituents from azurophilic and specific granules was the main identified biologic function of ERP. ERP was a more effective secretagogue in the untreated neutrophils and f-met-leu-phe was more effective in the cytochalasin B-treated neutrophils. Absorption of ERP from macrophage-conditioned medium removed a small amount of the chemotactic activity; however, the immunopurified peptide was not chemotactic or chemokinetic for neutrophils, and at high concentrations, it suppressed base line chemokinesis. Treatment of washed macrophages with trypsin released active ERP of approximately the same m.w. of spontaneously secreted ERP. These studies showed that human alveolar macrophages release a peptide which is a secretagogue for human neutrophils under conditions which may be encountered in the lungs during certain disease states. Proteolytic enzymes which are free in the lungs may release the peptide and lead to the secretion of neutrophil enzymes

  18. Moderate restriction of macrophage-tropic human immunodeficiency virus type 1 by SAMHD1 in monocyte-derived macrophages.

    Science.gov (United States)

    Taya, Kahoru; Nakayama, Emi E; Shioda, Tatsuo

    2014-01-01

    Macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains are able to grow to high titers in human monocyte-derived macrophages. However, it was recently reported that cellular protein SAMHD1 restricts HIV-1 replication in human cells of the myeloid lineage, including monocyte-derived macrophages. Here we show that degradation of SAMHD1 in monocyte-derived macrophages was associated with moderately enhanced growth of the macrophage-tropic HIV-1 strain. SAMHD1 degradation was induced by treating target macrophages with vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 2 (HIV-2) particles containing viral protein X. For undifferentiated monocytes, HIV-2 particle treatment allowed undifferentiated monocytes to be fully permissive for productive infection by the macrophage-tropic HIV-1 strain. In contrast, untreated monocytes were totally resistant to HIV-1 replication. These results indicated that SAMHD1 moderately restricts even a macrophage-tropic HIV-1 strain in monocyte-derived macrophages, whereas the protein potently restricts HIV-1 replication in undifferentiated monocytes.

  19. Recombinant Granulocyte-Macrophage Colony-Stimulating Factor (rGM-CSF) : A Review of its Pharmacological Properties and Prospective Role in the Management of Myelosuppression.

    Science.gov (United States)

    Grant, Susan M; Heel, Rennie C

    1992-04-01

    Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) is a polypeptide hormone produced through recombinant DNA technologies in glycosylated (yeast or mammalian expression systems) or nonglycosylated (Escherichia coli expression system) form. It is a multilineage haematopoietin which stimulates proliferation and differentiation of bone marrow myeloid progenitors and increases peripheral white blood cell counts when administered systemically. Treatment is generally well tolerated, although mild to moderate flu-like symptoms are common and rGM-CSF-induced fever and fluid retention may be problematic in occasional patients. rGM-CSF accelerates recovery of peripheral neutrophil counts after bone marrow transplantation, and results of a placebo-controlled randomised trial correlate this with reduced infectious episodes and shortened length of hospitalisation in patients with lymphoid malignancies. A substantial number of patients with graft failure after bone marrow transplantation also respond to rGM-CSF. The duration of myelosuppression secondary to cancer chemotherapy can be significantly reduced by rGM-CSF which has permitted investigation of antineoplastic dose-intensity escalation. In some haematopoietic disorders (e.g. aplastic anaemia, myelodysplasia and neutropenia secondary to HIV infection and antiviral therapy), rGM-CSF produces clinically useful increases in peripheral blood granulocyte counts, although the effect is generally not sustained after drug withdrawal. The potential for rGM-CSF to stimulate proliferation of the abnormal clone in myelodysplasia and in acute myelogenous leukaemia following induction therapy is of concern. Available data suggest, however, that with appropriate monitoring and exclusion of high-risk patients this serious potential risk can be avoided, and that myelopoiesis is enhanced in such patients by rGM-CSF treatment. Recombinant colony-stimulating factors are a new therapeutic modality; hence many aspects of

  20. Effect of Granulocyte-Macrophage Colony-Stimulating Factor With or Without Supervised Exercise on Walking Performance in Patients With Peripheral Artery Disease: The PROPEL Randomized Clinical Trial.

    Science.gov (United States)

    McDermott, Mary M; Ferrucci, Luigi; Tian, Lu; Guralnik, Jack M; Lloyd-Jones, Donald; Kibbe, Melina R; Polonsky, Tamar S; Domanchuk, Kathryn; Stein, James H; Zhao, Lihui; Taylor, Doris; Skelly, Christopher; Pearce, William; Perlman, Harris; McCarthy, Walter; Li, Lingyu; Gao, Ying; Sufit, Robert; Bloomfield, Christina L; Criqui, Michael H

    2017-12-05

    Benefits of granulocyte-macrophage colony-stimulating factor (GM-CSF) for improving walking ability in people with lower extremity peripheral artery disease (PAD) are unclear. Walking exercise may augment the effects of GM-CSF in PAD, since exercise-induced ischemia enhances progenitor cell release and may promote progenitor cell homing to ischemic calf muscle. To determine whether GM-CSF combined with supervised treadmill exercise improves 6-minute walk distance, compared with exercise alone and compared with GM-CSF alone; to determine whether GM-CSF alone improves 6-minute walk more than placebo and whether exercise improves 6-minute walk more than an attention control intervention. Randomized clinical trial with 2 × 2 factorial design. Participants were identified from the Chicago metropolitan area and randomized between January 6, 2012, and December 22, 2016, to 1 of 4 groups: supervised exercise + GM-CSF (exercise + GM-CSF) (n = 53), supervised exercise + placebo (exercise alone) (n = 53), attention control  + GM-CSF (GM-CSF alone) (n = 53), attention control + placebo (n = 51). The final follow-up visit was on August 15, 2017. Supervised exercise consisted of treadmill exercise 3 times weekly for 6 months. The attention control consisted of weekly educational lectures by clinicians for 6 months. GM-CSF (250 μg/m2/d) or placebo were administered subcutaneously (double-blinded) 3 times/wk for the first 2 weeks of the intervention. The primary outcome was change in 6-minute walk distance at 12-week follow-up (minimum clinically important difference, 20 m). P values were adjusted based on the Hochberg step-up method. Of 827 persons evaluated, 210 participants with PAD were randomized (mean age, 67.0 [SD, 8.6] years; 141 [67%] black, 82 [39%] women). One hundred ninety-five (93%) completed 12-week follow-up. At 12-week follow-up, exercise + GM-CSF did not significantly improve 6-minute walk distance more than

  1. Ionic channels and membrane hyperpolarization in human macrophages

    NARCIS (Netherlands)

    Ince, C.; van Duijn, B.; Ypey, D. L.; van Bavel, E.; Weidema, F.; Leijh, P. C.

    1987-01-01

    Microelectrode impalement of human macrophages evokes a transient hyperpolarizing response (HR) of the membrane potential. This HR was found to be dependent on the extracellular concentration of K+ but not on that of Na+ or Cl-. It was not influenced by low temperature (12 degrees C) or by 0.2 mM

  2. Diagnosis, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: A Review.

    Science.gov (United States)

    Sanchez, Edgar; Vannier, Edouard; Wormser, Gary P; Hu, Linden T

    2016-04-26

    Lyme disease, human granulocytic anaplasmosis (HGA), and babesiosis are emerging tick-borne infections. To provide an update on diagnosis, treatment, and prevention of tick-borne infections. Search of PubMed and Scopus for articles on diagnosis, treatment, and prevention of tick-borne infections published in English from January 2005 through December 2015. The search yielded 3550 articles for diagnosis and treatment and 752 articles for prevention. Of these articles, 361 were reviewed in depth. Evidence supports the use of US Food and Drug Administration-approved serologic tests, such as an enzyme immunoassay (EIA), followed by Western blot testing, to diagnose extracutaneous manifestations of Lyme disease. Microscopy and polymerase chain reaction assay of blood specimens are used to diagnose active HGA and babesiosis. The efficacy of oral doxycycline, amoxicillin, and cefuroxime axetil for treating Lyme disease has been established in multiple trials. Ceftriaxone is recommended when parenteral antibiotic therapy is recommended. Multiple trials have shown efficacy for a 10-day course of oral doxycycline for treatment of erythema migrans and for a 14-day course for treatment of early neurologic Lyme disease in ambulatory patients. Evidence indicates that a 10-day course of oral doxycycline is effective for HGA and that a 7- to 10-day course of azithromycin plus atovaquone is effective for mild babesiosis. Based on multiple case reports, a 7- to 10-day course of clindamycin plus quinine is often used to treat severe babesiosis. A recent study supports a minimum of 6 weeks of antibiotics for highly immunocompromised patients with babesiosis, with no parasites detected on blood smear for at least the final 2 weeks of treatment. Evidence is evolving regarding the diagnosis, treatment, and prevention of Lyme disease, HGA, and babesiosis. Recent evidence supports treating patients with erythema migrans for no longer than 10 days when doxycycline is used and prescription

  3. Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

    Science.gov (United States)

    Odai, H; Sasaki, K; Iwamatsu, A; Nakamoto, T; Ueno, H; Yamagata, T; Mitani, K; Yazaki, Y; Hirai, H

    1997-04-15

    Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine

  4. Angiogenic potential of human macrophages on electrospun bioresorbable vascular grafts

    Energy Technology Data Exchange (ETDEWEB)

    Garg, K; Sell, S A; Madurantakam, P; Bowlin, G L, E-mail: glbowlin@vcu.ed [Virginia Commonwealth University, Richmond, VA 23284 (United States)

    2009-06-15

    The aim of this study was to investigate macrophage interactions with electrospun scaffolds and quantify the expression of key angiogenic growth factors in vitro. This study will further help in evaluating the potential of these electrospun constructs as vascular grafts for tissue repair and regeneration in situ. Human peripheral blood macrophages were seeded in serum free media on electrospun (10 mm) discs of polydioxanone (PDO), elastin and PDO:elastin blends (50:50, 70:30 and 90:10). The growth factor secretion was analyzed by ELISA. Macrophages produced high levels of vascular endothelial growth factor and acidic fibroblast growth factor. Transforming growth factor beta-1 (TGF-beta1) secretion was relatively low and there was negligible production of basic fibroblast growth factor. Therefore, it can be anticipated that these scaffolds will support tissue regeneration and angiogenesis. (communication)

  5. Dopamine receptor activation increases HIV entry into primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Peter J Gaskill

    Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

  6. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    Science.gov (United States)

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  7. G-CSF regulates macrophage phenotype and associates with poor overall survival in human triple-negative breast cancer

    Science.gov (United States)

    Hollmén, Maija; Karaman, Sinem; Schwager, Simon; Lisibach, Angela; Christiansen, Ailsa J.; Maksimow, Mikael; Varga, Zsuzsanna; Jalkanen, Sirpa; Detmar, Michael

    2016-01-01

    ABSTRACT Tumor-associated macrophages (TAMs) have been implicated in the promotion of breast cancer growth and metastasis, and a strong infiltration by TAMs has been associated with estrogen receptor (ER)-negative tumors and poor prognosis. However, the molecular mechanisms behind these observations are unclear. We investigated macrophage activation in response to co-culture with several breast cancer cell lines (T47D, MCF-7, BT-474, SKBR-3, Cal-51 and MDA-MB-231) and found that high granulocyte colony-stimulating factor (G-CSF) secretion by the triple-negative breast cancer (TNBC) cell line MDA-MB-231 gave rise to immunosuppressive HLA-DRlo macrophages that promoted migration of breast cancer cells via secretion of TGF-α. In human breast cancer samples (n = 548), G-CSF was highly expressed in TNBC (p CSF blockade in the 4T1 mammary tumor model promoted maturation of MHCIIhi blood monocytes and TAMs and significantly reduced lung metastasis, anti-CSF-1R treatment promoted MHCIIloF4/80hiMRhi anti-inflammatory TAMs and enhanced lung metastasis in the presence of high G-CSF levels. Combined anti-G-CSF and anti-CSF-1R therapy significantly increased lymph node metastases, possibly via depletion of the so-called “gate-keeper” subcapsular sinus macrophages. These results indicate that G-CSF promotes the anti-inflammatory phenotype of tumor-induced macrophages when CSF-1R is inhibited and therefore caution against the use of M-CSF/CSF-1R targeting agents in tumors with high G-CSF expression. PMID:27141367

  8. Adipose tissue macrophages impair preadipocyte differentiation in humans.

    Directory of Open Access Journals (Sweden)

    Li Fen Liu

    Full Text Available The physiologic mechanisms underlying the relationship between obesity and insulin resistance are not fully understood. Impaired adipocyte differentiation and localized inflammation characterize adipose tissue from obese, insulin-resistant humans. The directionality of this relationship is not known, however. The aim of the current study was to investigate whether adipose tissue inflammation is causally-related to impaired adipocyte differentiation.Abdominal subcutaneous(SAT and visceral(VAT adipose tissue was obtained from 20 human participants undergoing bariatric surgery. Preadipocytes were isolated, and cultured in the presence or absence of CD14+ macrophages obtained from the same adipose tissue sample. Adipocyte differentiation was quantified after 14 days via immunofluorescence, Oil-Red O, and adipogenic gene expression. Cytokine secretion by mature adipocytes cultured with or without CD14+macrophages was quantified.Adipocyte differentiation was significantly lower in VAT than SAT by all measures (p<0.001. With macrophage removal, SAT preadipocyte differentiation increased significantly as measured by immunofluorescence and gene expression, whereas VAT preadipocyte differentiation was unchanged. Adipocyte-secreted proinflammatory cytokines were higher and adiponectin lower in media from VAT vs SAT: macrophage removal reduced inflammatory cytokine and increased adiponectin secretion from both SAT and VAT adipocytes. Differentiation of preadipocytes from SAT but not VAT correlated inversely with systemic insulin resistance.The current results reveal that proinflammatory immune cells in human SAT are causally-related to impaired preadipocyte differentiation, which in turn is associated with systemic insulin resistance. In VAT, preadipocyte differentiation is poor even in the absence of tissue macrophages, pointing to inherent differences in fat storage potential between the two depots.

  9. Human Induced Pluripotent Stem Cell-Derived Macrophages Share Ontogeny with MYB-Independent Tissue-Resident Macrophages

    Directory of Open Access Journals (Sweden)

    Julian Buchrieser

    2017-02-01

    Full Text Available Tissue-resident macrophages, such as microglia, Kupffer cells, and Langerhans cells, derive from Myb-independent yolk sac (YS progenitors generated before the emergence of hematopoietic stem cells (HSCs. Myb-independent YS-derived resident macrophages self-renew locally, independently of circulating monocytes and HSCs. In contrast, adult blood monocytes, as well as infiltrating, gut, and dermal macrophages, derive from Myb-dependent HSCs. These findings are derived from the mouse, using gene knockouts and lineage tracing, but their applicability to human development has not been formally demonstrated. Here, we use human induced pluripotent stem cells (iPSCs as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy, we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent, RUNX1-, and SPI1 (PU.1-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages, such as alveolar and kidney macrophages, microglia, Kupffer cells, and Langerhans cells.

  10. Identification of the Common Origins of Osteoclasts, Macrophages, and Dendritic Cells in Human Hematopoiesis

    Directory of Open Access Journals (Sweden)

    Yanling Xiao

    2015-06-01

    Full Text Available Osteoclasts (OCs originate from the myeloid cell lineage, but the successive steps in their lineage commitment are ill-defined, especially in humans. To clarify OC origin, we sorted cell populations from pediatric bone marrow (BM by flow cytometry and assessed their differentiation potential in vitro. Within the CD11b−CD34+c-KIT+ BM cell population, OC-differentiation potential was restricted to FLT3+ cells and enriched in an IL3 receptor (Rαhigh subset that constituted less than 0.5% of total BM. These IL3Rαhigh cells also generated macrophages (MΦs and dendritic cells (DCs but lacked granulocyte (GR-differentiation potential, as demonstrated at the clonal level. The IL3Rαlow subset was re-defined as common progenitor of GR, MΦ, OC, and DC (GMODP and gave rise to the IL3Rαhigh subset that was identified as common progenitor of MΦ, OC, and DC (MODP. Unbiased transcriptome analysis of CD11b−CD34+c-KIT+FLT3+ IL3Rαlow and IL3Rαhigh subsets corroborated our definitions of the GMODP and MODP and their developmental relationship.

  11. Human immunodeficiency virus impairs reverse cholesterol transport from macrophages.

    Directory of Open Access Journals (Sweden)

    Zahedi Mujawar

    2006-10-01

    Full Text Available Several steps of HIV-1 replication critically depend on cholesterol. HIV infection is associated with profound changes in lipid and lipoprotein metabolism and an increased risk of coronary artery disease. Whereas numerous studies have investigated the role of anti-HIV drugs in lipodystrophy and dyslipidemia, the effects of HIV infection on cellular cholesterol metabolism remain uncharacterized. Here, we demonstrate that HIV-1 impairs ATP-binding cassette transporter A1 (ABCA1-dependent cholesterol efflux from human macrophages, a condition previously shown to be highly atherogenic. In HIV-1-infected cells, this effect was mediated by Nef. Transfection of murine macrophages with Nef impaired cholesterol efflux from these cells. At least two mechanisms were found to be responsible for this phenomenon: first, HIV infection and transfection with Nef induced post-transcriptional down-regulation of ABCA1; and second, Nef caused redistribution of ABCA1 to the plasma membrane and inhibited internalization of apolipoprotein A-I. Binding of Nef to ABCA1 was required for down-regulation and redistribution of ABCA1. HIV-infected and Nef-transfected macrophages accumulated substantial amounts of lipids, thus resembling foam cells. The contribution of HIV-infected macrophages to the pathogenesis of atherosclerosis was supported by the presence of HIV-positive foam cells in atherosclerotic plaques of HIV-infected patients. Stimulation of cholesterol efflux from macrophages significantly reduced infectivity of the virions produced by these cells, and this effect correlated with a decreased amount of virion-associated cholesterol, suggesting that impairment of cholesterol efflux is essential to ensure proper cholesterol content in nascent HIV particles. These results reveal a previously unrecognized dysregulation of intracellular lipid metabolism in HIV-infected macrophages and identify Nef and ABCA1 as the key players responsible for this effect. Our findings

  12. Neuroprotective effects of recombinant human granulocyte colony-stimulating factor (G-CSF) in a rat model of anterior ischemic optic neuropathy (rAION).

    Science.gov (United States)

    Chang, Chung-Hsing; Huang, Tzu-Lun; Huang, Shun-Ping; Tsai, Rong-Kung

    2014-01-01

    The purpose of this study was to investigate the neuroprotective effects of recombinant human granulocyte colony stimulating factor (G-CSF), as administered in a rat model of anterior ischemic optic neuropathy (rAION). Using laser-induced photoactivation of intravenously administered Rose Bengal in the optic nerve head of 60 adult male Wistar rats, an anterior ischemic optic neuropathy (rAION) was inducted. Rats either immediately received G-CSF (subcutaneous injections) or phosphate buffered saline (PBS) for 5 consecutive days. Rats were euthanized at 4 weeks post infarct. Density of retinal ganglion cells (RGCs) was counted using retrograde labeling of Fluoro-gold. Visual function was assessed by flash visual-evoked potentials (FVEP) at 4 weeks. TUNEL assay in the retinal sections and immunohistochemical staining of ED1 (marker of macrophage/microglia) were investigated in the optic nerve (ON) specimens. The RGC densities in the central and mid-peripheral retinas in the G-CSF treated rats were significantly higher than those of the PBS-treated rats (survival rate was 71.4% vs. 33.2% in the central retina; 61.8% vs. 22.7% in the mid-peripheral retina, respectively; both p optic nerve sections of G-CSF-treated rats (16 ± 6/HPF vs. 35 ± 10/HPF; p = 0.016). In conclusion, administration of G-CSF is neuroprotective in the rat model of anterior ischemic optic neuropathy, as demonstrated both structurally by RGC density and functionally by FVEP. G-CSF may work via the dual actions of anti-apoptosis for RGC surviving as well as anti-inflammation in the optic nerves as evidenced by less infiltration of ED1-povitive cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Effects of recombinant human granulocyte colony-stimulating factor on leucopenia in zidovudine-treated patients with AIDS and AIDS related complex, a phase I/II study

    NARCIS (Netherlands)

    van der Wouw, P. A.; van Leeuwen, R.; van Oers, R. H.; Lange, J. M.; Danner, S. A.

    1991-01-01

    Twelve male patients, eight with the acquired immunodeficiency syndrome (AIDS) and four with AIDS related complex (ARC), who had zidovudine associated neutropenia (less than 1 x 10(9) neutrophils/l) were treated with recombinant human granulocyte colony-stimulating factor (G-CSF) in a phase I/II

  14. Macrophage Activation Mechanisms in Human Monocytic Cell Line-derived Macrophages.

    Science.gov (United States)

    Sumiya, Yu; Ishikawa, Mami; Inoue, Takahiro; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

    2015-08-01

    Although the mechanisms of macrophage activation are important for cancer immunotherapy, they are poorly understood. Recently, easy and robust assay systems for assessing the macrophage-activating factor (MAF) using monocytic cell line-derived macrophages were established. Gene-expression profiles of U937- and THP-1-derived macrophages were compared using gene expression microarray analysis and their responses against several MAFs were examined by in vitro experiments. Activated states of these macrophages could not be assigned to a specific sub-type but showed, however, different unique characteristics. The unique of monocytic cell line-derived macrophages could provide clues to understand the activation mechanism of macrophages and, therefore, help to develop effective cancer immunotherapy with MAFs. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  15. Granulocyte-Colony Stimulating Factor Receptor, Tissue Factor, and VEGF-R Bound VEGF in Human Breast Cancer In Loco.

    Science.gov (United States)

    Wojtukiewicz, Marek Z; Sierko, Ewa; Skalij, Piotr; Kamińska, Magda; Zimnoch, Lech; Brekken, Ralf A; Thorpe, Philip E

    2016-01-01

    Doxorubicin and docetaxel-based chemotherapy regimens used in breast cancer patients are associated with high risk of febrile neutropenia (FN). Granulocyte colony-stimulating factors (G-CSF) are recommended for both treating and preventing chemotherapy-induced neutropenia. Increased thrombosis incidence in G-CSF treated patients was reported; however, the underlying mechanisms remain unclear. The principal activator of blood coagulation in cancer is tissue factor (TF). It additionally contributes to cancer progression and stimulates angiogenesis. The main proangiogenic factor is vascular endothelial growth factor (VEGF). The aim of the study was to evaluate granulocyte-colony stimulating factor receptor (G-CSFR), tissue factor (TF) expression and vascular endothelial growth factor receptor (VEGF-R) bound VEGF in human breast cancer in loco. G-CSFR, TF and VEGFR bound VEGF (VEGF: VEGFR) were assessed in 28 breast cancer tissue samples. Immunohistochemical (IHC) methodologies according to ABC technique and double staining IHC procedure were employed utilizing antibodies against G-CSFR, TF and VEGF associated with VEGFR (VEGF: VEGFR). Expression of G-CSFR was demonstrated in 20 breast cancer tissue specimens (71%). In 6 cases (21%) the expression was strong (IRS 9-12). Strong expression of TF was observed in all investigated cases (100%). Moreover, expression of VEGF: VEGFR was visualized in cancer cells (IRS 5-8). No presence of G-CSFR, TF or VEGF: VEGFR was detected on healthy breast cells. Double staining IHC studies revealed co-localization of G-CSFR and TF, G-CSFR and VEGF: VEGFR, as well as TF and VEGF: VEGFR on breast cancer cells and ECs. The results of the study indicate that GCSFR, TF and VEGF: VEGFR expression as well as their co-expression might influence breast cancer biology, and may increase thromboembolic adverse events incidence.

  16. Legionella pneumophila transcriptome during intracellular multiplication in human macrophages

    Directory of Open Access Journals (Sweden)

    Sebastien P Faucher

    2011-04-01

    Full Text Available Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot Type IVB secretion system (TFBSS, which is used to translocate more than 150 effector proteins to host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called SCOTS to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to L-arginine, L-histidine and L-proline as well as many transport systems involved in amino acid and iron uptake. Gene involved in catabolism of glycerol is also induced during intracellular growth and could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.

  17. Thermo-radiosensitivity of the granulocyte and macrophage precursor cells of mice. I.-Development of the in vivo culture and effects induced by the hyperthermia; Termo-radiosensibilidad del precursor hematopoyetico que origina las series granulocitica y macrofaga de raton. I.- Desarrollo del cultivo in vivo y efectos producidos por la hipertermia

    Energy Technology Data Exchange (ETDEWEB)

    Bueren, J A; Nieto, M

    1983-07-01

    The present report shows the agar diffusion chamber technique for culturing granulocyte- macrophage precursor cells, obtained from mice bone marrow. Diffusion chambers containing the bone marrow suspension are implanted intraperitoneally Into mice and constitute a compartment which avoids the migration of cells, but allows the transit of the mouse biological fluxes, necessary for the cellular proliferation. By means of this technique, we studied the lethal effects of the hyperthermia on the precursors and their capacity to repair sublethal damage. (Author) 129 refs.

  18. Supernatants from oral epithelial cells and gingival fibroblasts modulate human immunodeficiency virus type 1 promoter activation induced by periodontopathogens in monocytes/macrophages.

    Science.gov (United States)

    González, O A; Ebersole, J L; Huang, C B

    2010-04-01

    Bacterial and host cell products during coinfections of Human Immunodeficiency Virus type 1-positive (HIV-1(+)) patients regulate HIV-1 recrudescence in latently infected cells (e.g. T cells, monocytes/macrophages), impacting highly active antiretroviral therapy (HAART) failure and progression of acquired immunodeficiency syndrome. A high frequency of oral opportunistic infections (e.g. periodontitis) in HIV-1(+) patients has been demonstrated; however, their potential to impact HIV-1 exacerbation is unclear. We sought to determine the ability of supernatants derived from oral epithelial cells (OKF4) and human gingival fibroblasts (Gin-4) challenged with periodontal pathogens, to modulate the HIV-1 promoter activation in monocytes/macrophages. BF24 monocytes/macrophages transfected with the HIV-1 promoter driving the expression of chloramphenicol acetyltransferase (CAT) were stimulated with Porphyromonas gingivalis, Fusobacterium nucleatum, or Treponema denticola in the presence of supernatants from OKF4 or Gin4 cells either unstimulated or previously pulsed with bacteria. CAT levels were determined by enzyme-linked immunosorbent assay and cytokine production was evaluated by Luminex beadlyte assays. OKF4 and Gin4 supernatants enhanced HIV-1 promoter activation particularly related to F. nucleatum challenge. An additive effect was observed in HIV-1 promoter activation when monocytes/macrophages were simultaneously stimulated with gingival cell supernatants and bacterial extracts. OKF4 cells produced higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins -6 and -8 in response to F. nucleatum and P. gingivalis. Preincubation of OKF4 supernatants with anti-GM-CSF reduced the additive effect in periodontopathogen-induced HIV-1 promoter activation. These results suggest that soluble mediators produced by gingival resident cells in response to periodontopathogens could contribute to HIV-1 promoter activation in monocytes/macrophages

  19. Time-dependent labelling course of human eosinophilic granulocytes after 3H thymidine application

    International Nuclear Information System (INIS)

    Walle, A.J.

    1975-01-01

    After intravenous injection of 0.1 μCi/g body weight 3 H-Thymidine and taking of blood samples in intervals of 6-12 hrs. on three test persons with healthy blood, the labelling course of the eosinophilic granulocytes was studied. The cells were classified in four groups, according to the relative frequency of the different degrees of labelling. The time-dependent labelling index curves showed a nawe-sheped course. Elimination of the eosinophilics from the blood is carried out according to the 'At-random'-principle. 12 hrs. p.i. already 10% of the eosinophilics in the blood were labelled with maximally 5 grains. The cell flow-in phase of 13 hrs. was succeeded by a flow-out phase of nearly the same duration, afthr the first labelling maximum of 17%. 80 hrs. p.i. the first massive in-flow of high-labelled cells containing more than 30 grains. After reaching the labelling maximum of 58%, the labelling index values decreased continuously. Until the 11th day p.i., appr. 50% of the eosinophilics were still labelled, after 17 days appr. 25%, more than 65% of which consisted of cells with only 2-4 grains. Comparison of the labelling index curves of the grain groups with each other shows at first a synchronous, then an increasingly asynchronous course, according to the desynchronization of the several eosinophilic generation cycles in the bone marrow which gets more significant in the course of time. (orig.) [de

  20. Promotive effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on recovery from neutropenia induced by fractionated irradiation in mice

    Energy Technology Data Exchange (ETDEWEB)

    Kabaya, Koji; Watanabe, Masahiko; Kusaka, Masaru; Seki, Masatoshi (Kirin Brewery Co., Ltd., Gunma (Japan). Pharmaceutical Research Laboratory); Fushiki, Masato

    1994-08-01

    The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the recovery from neutropenia induced by fractionated whole-body irradiation was investigated in mice. Male 7-week old C3H/HeN mice received a total of ten exposures of 0.25 Gy/day from day 1 to 5 and from day 8 to 12. Peripheral neutropenia with a nadir on day 17 was caused by the fractionated irradiation. Daily subcutaneous injections of rhG-CSF at 0.25 and 2.5 [mu]g/body/day from day from day 1 to 21 promoted the recovery of neutrophils in a dose-dependent manner. The kinetics of morphologically identifiable bone marrow cells were studied to clarify the mechanism behind the promotive effect of this factor. A slight decrease in mitotic immature granulocytes, such as myeloblasts, promyelocytes and myelocytes on day 5, and a drastic decrease in metamyelocytes and marrow neutrophils on days 5, 9, and 17 were seen in the femur of irradiated mice. Treatment using rhG-CSF caused an increase in immature granulocytes of all differential stages in the femur. Microscopic findings of the femurs and spleens also reveals an increase in immature granulocytes in these organs in mice injected with rhG-CSF. These results indicate that rhG-CSF accelerates granulopoiesis in the femur and spleen, thereby promoting recovery from neutropenia induced by fractionated irradiation. (author).

  1. Promotive effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on recovery from neutropenia induced by fractionated irradiation in mice

    International Nuclear Information System (INIS)

    Kabaya, Koji; Watanabe, Masahiko; Kusaka, Masaru; Seki, Masatoshi; Fushiki, Masato.

    1994-01-01

    The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the recovery from neutropenia induced by fractionated whole-body irradiation was investigated in mice. Male 7-week old C3H/HeN mice received a total of ten exposures of 0.25 Gy/day from day 1 to 5 and from day 8 to 12. Peripheral neutropenia with a nadir on day 17 was caused by the fractionated irradiation. Daily subcutaneous injections of rhG-CSF at 0.25 and 2.5 μg/body/day from day from day 1 to 21 promoted the recovery of neutrophils in a dose-dependent manner. The kinetics of morphologically identifiable bone marrow cells were studied to clarify the mechanism behind the promotive effect of this factor. A slight decrease in mitotic immature granulocytes, such as myeloblasts, promyelocytes and myelocytes on day 5, and a drastic decrease in metamyelocytes and marrow neutrophils on days 5, 9, and 17 were seen in the femur of irradiated mice. Treatment using rhG-CSF caused an increase in immature granulocytes of all differential stages in the femur. Microscopic findings of the femurs and spleens also reveals an increase in immature granulocytes in these organs in mice injected with rhG-CSF. These results indicate that rhG-CSF accelerates granulopoiesis in the femur and spleen, thereby promoting recovery from neutropenia induced by fractionated irradiation. (author)

  2. Inductive potential of recombinant human granulocyte colony-stimulating factor to mature neutrophils from X-irradiated human peripheral blood hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Katsumori, Takeo; Yoshino, Hironori; Hayashi, Masako; Takahashi, Kenji; Kashiwakura, Ikuo

    2009-01-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been used for treatment of neutropenia. Filgrastim, Nartograstim, and Lenograstim are clinically available in Japan. However, the differences in potential benefit for radiation-induced disorder between these types of rhG-CSFs remain unknown. Therefore, the effects of three different types of rhG-CSFs on granulocyte progenitor cells and expansion of neutrophils from nonirradiated or 2 Gy X-irradiated human CD34 + hematopoietic progenitor cells were examined. For analysis of granulocyte colony-forming units (CFU-G) and a surviving fraction of CFU-G, nonirradiated or X-irradiated CD34 + cells were cultured in methylcellulose containing rhG-CSF. These cells were cultured in serum-free medium supplemented with rhG-CSF, and the expansion and characteristics of neutrophils were analyzed. All three types of rhG-CSFs increased the number of CFU-G in a dose-dependent manner; however, Lenograstim is superior to others because of CFU-G-derived colony formation at relatively low doses. The surviving fraction of CFU-G was independent of the types of rhG-CSFs. Expansion of neutrophils by rhG-CSF was largely attenuated by X-irradiation, though no significant difference in neutrophil number was observed between the three types of rhG-CSFs under both nonirradiation and X-irradiation conditions. In terms of functional characteristics of neutrophils, Lenograstim-induced neutrophils produced high levels of reactive oxygen species compared to Filgrastim, when rhG-CSF was applied to nonirradiated CD34 + cells. In conclusion, different types of rhG-CSFs lead to different effects when rhG-CSF is applied to nonirradiated CD34 + cells, though Filgrastim, Nartograstim, and Lenograstim show equal effects on X-irradiated CD34 + cells. (author)

  3. Highly Expressed Granulocyte Colony-Stimulating Factor (G-CSF) and Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) in Human Gastric Cancer Leads to Poor Survival.

    Science.gov (United States)

    Fan, Zhisong; Li, Yong; Zhao, Qun; Fan, Liqiao; Tan, Bibo; Zuo, Jing; Hua, Kelei; Ji, Qiang

    2018-03-23

    BACKGROUND Chemotherapy for advanced gastric cancer (GC) patients has been the mainstay of therapy for many years. Although adding anti-angiogenic drugs to chemotherapy improves patient survival slightly, identifying anti-angiogenic therapy-sensitive patients remains challenging for oncologists. Granulocyte colony-stimulating factor (G-CSF) promotes tumor growth and angiogenesis, which can be minimized with the anti-G-CSF antibody. Thus, G-CSF might be a potential tumor marker. However, the effects of G-CSF and G-CSFR expression on GC patient survival remain unclear. MATERIAL AND METHODS Seventy GC tissue samples were collected for G-CSF and G-CSFR detection by immunohistochemistry. A total of 40 paired GC tissues and matched adjacent mucosa were used to measure the G-CSF and G-CSFR levels by ELISA. Correlations between G-CSF/G-CSFR and clinical characteristics, VEGF-A levels and overall survival were analyzed. Biological function and underlying mechanistic investigations were carried out using SGC7901 cell lines, and the effects of G-CSF on tumor proliferation, migration, and tube formation were examined. RESULTS The levels of G-CSFR were upregulated in GC tissues compared to normal mucosa tissues. Higher G-CSF expression was associated with later tumor stages and higher tumor VEGF-A and serum CA724 levels, whereas higher G-CSFR expression was associated with lymph node metastasis. Patients with higher G-CSF expression had shorter overall survival times. In vitro, G-CSF stimulated SGC7901 proliferation and migration through the JAK2/STAT3 pathway and accelerated HUVEC tube formation. CONCLUSIONS These data suggest that increased G-CSF and G-CSFR in tumors leads to unfavorable outcomes for GC patients by stimulating tumor proliferation, migration, and angiogenesis, indicating that these factors are potential tumor targets for cancer treatment.

  4. CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

    Science.gov (United States)

    Zhang, Hanrui; Shi, Jianting; Hachet, Melanie A; Xue, Chenyi; Bauer, Robert C; Jiang, Hongfeng; Li, Wenjun; Tohyama, Junichiro; Millar, John; Billheimer, Jeffrey; Phillips, Michael C; Razani, Babak; Rader, Daniel J; Reilly, Muredach P

    2017-11-01

    To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA ( LIPA -/- ) had barely detectable LAL enzymatic activity. Control and LIPA -/- IPSDM were loaded with [ 3 H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [ 3 H]-cholesterol to apolipoprotein A-I was abolished in LIPA -/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [ 3 H]-cholesterol-labeled AcLDL, [ 3 H]-cholesterol efflux was, however, not different between control and LIPA -/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA -/- IPSDM. In nonlipid loaded state, LIPA -/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA -/- IPSDM. LIPA -/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B , IL6 , and CCL5. CONCLUSIONS: LIPA -/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human

  5. Comparative analysis of the internalization of the macrophage receptor sialoadhesin in human and mouse primary macrophages and cell lines.

    Science.gov (United States)

    De Schryver, Marjorie; Leemans, Annelies; Pintelon, Isabel; Cappoen, Davie; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

    2017-06-01

    Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab') 2 fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can

  6. The safety and clinical efficacy of recombinant human granulocyte colony stimulating factor injection for colon cancer patients undergoing chemotherapy

    Directory of Open Access Journals (Sweden)

    Jie Chen

    Full Text Available Summary Objective: The present study was designed to evaluate safety and efficacy of recombinant human granulocyte colony stimulating factor (G-CSF injection and whether this regimen could reduce the incidence of adverse events caused by chemotherapy. Method: A total of 100 patients with colon cancer who were treated with chemotherapy in our hospital from January 2011 to December 2014 were randomly divided into two groups, with 50 patients in each group. The patients in the treatment group received G-CSF 24 hours after chemotherapy for consecutive three days; the patients in the control group received the same dose of normal saline. Routine blood tests were performed 7 days and 14 days after chemotherapy. Results: Compared with the control group, the incidences of febrile neutropenia and leukocytopenia in the treatment group were significantly lower (p<0.05. In addition, the incidence of liver dysfunction in the treatment group was lower than that of the control group, without statistical significance. The incidence of myalgia in the treatment was higher than that of the control group without statistical significance. Conclusion: The present study indicated that G-CSF injection after chemotherapy is safe and effective for preventing adverse events in colon cancer patients with chemotherapy.

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

    Science.gov (United States)

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

    2015-01-30

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

  8. CCR8 signaling influences Toll-like receptor 4 responses in human macrophages in inflammatory diseases.

    Science.gov (United States)

    Reimer, Martina Kvist; Brange, Charlotte; Rosendahl, Alexander

    2011-12-01

    CCR8 immunity is generally associated with Th2 responses in allergic diseases. In this study, we demonstrate for the first time a pronounced attenuated influx of macrophages in ovalbumin (OVA)-challenged CCR8 knockout mice. To explore whether macrophages in human inflamed lung tissue also were CCR8 positive, human lung tissue from patients with chronic obstructive pulmonary disease (COPD) was evaluated. Indeed, CCR8 expression was pronounced in invading monocytes/macrophages from lungs of patients with Global Initiative for Obstructive Lung Disease (GOLD) stage IV COPD. Given this expression pattern, the functional role of CCR8 on human macrophages was evaluated in vitro. Human peripheral blood monocytes expressed low levels of CCR8, while macrophage colony-stimulating factor (M-CSF)-derived human macrophages expressed significantly elevated surface levels of CCR8. Importantly, CCL1 directly regulated the expression of CD18 and CD49b and hence influenced the adhesion capacity of human macrophages. CCL1 drives chemotaxis in M-CSF-derived macrophages, and this could be completely inhibited by lipopolysaccharide (LPS). Whereas both CCL1 and LPS monotreatment inhibited spontaneous superoxide release in macrophages, CCL1 significantly induced superoxide release in the presence of LPS in a dose-dependent manner. Finally, CCL1 induced production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and could inhibit LPS-induced cytokine production in a dose-dependent manner. Our data demonstrate, for the first time, the presence of CCR8 on inflammatory macrophages in human COPD lung tissue. Importantly, the functional data from human macrophages suggest a potential cross talk between the CCR8 and the Toll-like receptor 4 (TLR4) pathways, both of which are present in COPD patients.

  9. Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.

    Science.gov (United States)

    Anzinger, Joshua J; Chang, Janet; Xu, Qing; Buono, Chiara; Li, Yifu; Leyva, Francisco J; Park, Bum-Chan; Greene, Lois E; Kruth, Howard S

    2010-10-01

    To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

  10. Use of recombinant granulocyte-macrophage colony-stimulating factor during and after remission induction chemotherapy in patients aged 61 years and older with acute myeloid leukemia (AML) : Final report of AML-11, a phase III randomized study of the Leukemia Cooperative Group of European Organisation for the Research and Treatment of Cancer (EORTC-LCG) and the Dutch Belgian Hemato-Oncology Cooperative Group (HOVON)

    NARCIS (Netherlands)

    Lowenberg, B; Suciu, S; Archimbaud, E; Ossenkoppele, G; Verhoef, GEG; Vellenga, E; Wijermans, P; Berneman, Z; Dekker, AW; Stryckmans, P; Jehn, U; Muus, P; Sonneveld, P; Dardenne, M; Zittoun, R

    1997-01-01

    We conducted a prospective randomized multicenter clinical trial comparing the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjunct to intensive chemotherapy in patients of 61 years and older with untreated newly diagnosed acute myeloid leukemia (AML). Patients were

  11. Human Adipose Tissue Macrophages Are Enhanced but Changed to an Anti-Inflammatory Profile in Obesity

    Directory of Open Access Journals (Sweden)

    Karen Fjeldborg

    2014-01-01

    Full Text Available Objective. Adipose tissue (AT macrophages are increased in obesity and associated with low grade inflammation. We aimed to characterize the phenotype of AT macrophages in humans in relation to obesity and insulin resistance. Design. Gene-expression levels of general macrophage markers (CD68 and CD14, proinflammatory markers/M1 (TNF-α, MCP-1, and IL-6, and anti-inflammatory markers/M2 (CD163, CD206, and IL-10 were determined by RT-PCR in subcutaneous AT samples from lean and obese subjects. Insulin resistance was determined by HOMA-IR. Results. All the macrophage markers were elevated in the AT from obese compared to lean subjects (P<0.001. To determine the phenotype of the macrophages the level of CD14 was used to adjust the total number of macrophages. The relative expression of CD163 and IL-10 was elevated, and TNF-α and IL-6 were reduced in AT from obese subjects (all P<0.05. In a multivariate regression analysis CD163 was the only macrophage marker significantly associated with HOMA-IR (β: 0.57; P<0.05. Conclusion. Obesity is associated with elevated numbers of macrophages in the AT. Unexpectedly, the macrophages change phenotype by obesity, with a preponderance of M2 and a decrement of M1 markers in AT from obese subjects. Moreover, CD163 was the only macrophage marker associated with HOMA-IR after multiple adjustments.

  12. Adaptive T cell responses induced by oncolytic Herpes Simplex Virus-granulocyte macrophage-colony-stimulating factor therapy expanded by dendritic cell and cytokine-induced killer cell adoptive therapy.

    Science.gov (United States)

    Ren, Jun; Gwin, William R; Zhou, Xinna; Wang, Xiaoli; Huang, Hongyan; Jiang, Ni; Zhou, Lei; Agarwal, Pankaj; Hobeika, Amy; Crosby, Erika; Hartman, Zachary C; Morse, Michael A; H Eng, Kevin; Lyerly, H Kim

    2017-01-01

    Purpose : Although local oncolytic viral therapy (OVT) may enhance tumor lysis, antigen release, and adaptive immune responses, systemic antitumor responses post-therapy are limited. Adoptive immunotherapy with autologous dendritic cells (DC) and cytokine-induced killer cells (DC-CIK) synergizes with systemic therapies. We hypothesized that OVT with Herpes Simplex Virus-granulocyte macrophage-colony-stimulating factor (HSV-GM-CSF) would induce adaptive T cell responses that could be expanded systemically with sequential DC-CIK therapy. Patients and Methods : We performed a pilot study of intratumoral HSV-GM-CSF OVT followed by autologous DC-CIK cell therapy. In addition to safety and clinical endpoints, we monitored adaptive T cell responses by quantifying T cell receptor (TCR) populations in pre-oncolytic therapy, post-oncolytic therapy, and after DC-CIK therapy. Results : Nine patients with advanced malignancy were treated with OVT (OrienX010), of whom seven experienced stable disease (SD). Five of the OVT treated patients underwent leukapheresis, generation, and delivery of DC-CIKs, and two had SD, whereas three progressed. T cell receptor sequencing of TCR β sequences one month after OVT therapy demonstrates a dynamic TCR repertoire in response to OVT therapy in the majority of patients with the systematic expansion of multiple T cell clone populations following DC-CIK therapy. This treatment was well tolerated and long-term event free and overall survival was observed in six of the nine patients. Conclusions : Strategies inducing the local activation of tumor-specific immune responses can be combined with adoptive cellular therapies to expand the adaptive T cell responses systemically and further studies are warranted.

  13. A pilot study of the effect of granulocyte-macrophage colony-stimulating factor on oral mucositis in head and neck cancer patients during x-radiation therapy: a preliminary report

    International Nuclear Information System (INIS)

    Nicolatou, Ourania; Sotiropoulou-Lontou, Anastasia; Skarlatos, John; Kyprianou, Konstantinos; Kolitsi, Georgia; Dardoufas, Konstantinos

    1998-01-01

    Purpose: To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in reduction of radiotherapy-induced oral mucositis. Methods and Materials: Seventeen patients who were going to be irradiated with a total dose of 50-70 Gy for head and neck malignancies were included in the study. After the second week of radiotherapy, with the experience of oral pain, GM-CSF 400 μg was administered locally, once a day, until completion of radiotherapy. Patients were evaluated weekly for mucosal reaction and functional impairment. Results: Three patients with gross and functional mucositis grade I after the second week, completed the planned radiotherapy showing mucositis grade I. Eleven patients who experienced, after 2 weeks of radiotherapy, mucositis grade II and III, presented after the third week with gross mucositis grade I and II and functional impairment grade I. One of these 11 patients was then lost to follow-up and the remaining 10 completed their planned radiotherapy having an almost asymptomatic mucositis grade I. The 15th patient with gross mucositis grade III after the 2 weeks of radiotherapy, had a 2-day interruption because of painful mucositis and then continued and completed radiotherapy with gross and functional mucositis grade I. The 16th patient with mucositis grade III after the second week, did not show any improvement, and completed her planned radiotherapy with mucositis grade III which finally healed after the administration of acyclovir. The last, 17th patient discontinued radiotherapy at the third week because of mucositis grade IV and severe ulceration in apposition to an extensive gold prosthesis. Conclusion: The local administration of GM-CSF significantly reduced and almost healed radiation-induced oral mucositis in 14 of 17 patients during the radiotherapy, which was completed within the preplanned time and without any significant patient weight loss or functional impairment

  14. Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-κB translocation

    International Nuclear Information System (INIS)

    Jawan, Bruno; Kao, Y.-H.; Goto, Shigeru; Pan, M.-C.; Lin, Y.-C.; Hsu, L.-W.; Nakano, Toshiaki; Lai, C.-Y.; Sun, C.-K.; Cheng, Y.-F.; Tai, M.-H.

    2008-01-01

    Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 μM after 48 h incubation. Pretreatment with 100 μM PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstrated that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and IκBα, as well as the nuclear translocation of NF-κB primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-κB nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers

  15. The impact of concurrent granulocyte macrophage-colony stimulating factor on radiation-induced mucositis in head and neck cancer patients: A double-blind placebo-controlled prospective Phase III study by Radiation Therapy Oncology Group 9901

    International Nuclear Information System (INIS)

    Ryu, Janice K.; Swann, Suzanne; LeVeque, Francis; Scarantino, Charles W.; Johnson, Darlene J.; Chen, Allan; Fortin, Andre; Pollock, JonDavid; Kim, Harold; Ang, Kian K.

    2007-01-01

    Purpose: Based on early clinical evidence of potential mucosal protection by granulocyte-macrophage colony stimulating factor (GM-CSF), the Radiation Therapy Oncology Group conducted a double-blind, placebo-controlled, randomized study to test the efficacy and safety of GM-CSF in reducing the severity and duration of mucosal injury and pain (mucositis) associated with curative radiotherapy (RT) in head-and-neck cancer patients. Methods and Materials: Eligible patients included those with head-and-neck cancer with radiation ports encompassing >50% of oral cavity and/or oropharynx. Standard RT ports were used to cover the primary tumor and regional lymphatics at risk in standard fractionation to 60-70 Gy. Concurrent cisplatin chemotherapy was allowed. Patients were randomized to receive subcutaneous injection of GM-CSF 250 μg/m 2 or placebo 3 times a week. Mucosal reaction was assessed during the course of RT using the National Cancer Institute Common Toxicity Criteria and the protocol-specific scoring system. Results: Between October 2000 and September 2002, 130 patients from 36 institutions were accrued. Nine patients (7%) were excluded from the analysis, 3 as a result of drug unavailability. More than 80% of the patients participated in the quality-of-life endpoint of this study. The GM-CSF did not cause any increase in toxicity compared with placebo. There was no statistically significant difference in the average mean mucositis score in the GM-CSF and placebo arms by a t test (p = 0.4006). Conclusion: This placebo-controlled, randomized study demonstrated no significant effect of GM-CSF given concurrently compared with placebo in reducing the severity or duration of RT-induced mucositis in patients undergoing definitive RT for head-and-neck cancer

  16. MOR103, a human monoclonal antibody to granulocyte–macrophage colony-stimulating factor, in the treatment of patients with moderate rheumatoid arthritis

    DEFF Research Database (Denmark)

    Behrens, Frank; Tak, Paul P; Ostergaard, Mikkel

    2015-01-01

    OBJECTIVES: To determine the safety, tolerability and signs of efficacy of MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), in patients with rheumatoid arthritis (RA). METHODS: Patients with active, moderate RA were enrolled in a randomised...... placebo and MOR103 0.3, 1.0 and 1.5 mg/kg, respectively). Treatment emergent adverse events (AEs) in the MOR103 groups were mild or moderate in intensity and generally reported at frequencies similar to those in the placebo group. The most common AE was nasopharyngitis. In two cases, AEs were classified...... with active RA. The data support further investigation of this monoclonal antibody to GM-CSF in RA patients and potentially in those with other immune-mediated inflammatory diseases. TRIAL REGISTRATION NUMBER: NCT01023256....

  17. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis.

    Science.gov (United States)

    Shaw, Catherine A; Webb, David J; Rossi, Adriano G; Megson, Ian L

    2009-05-07

    Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMvarphi), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type. Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMvarphi. Resultant MDMvarphi were treated for 24 h with DETA/NO (100 - 1000 muM) or GEA-3162 (10 - 300 muM) in the presence or absence of BAY 41-2272 (1 muM), isobutylmethylxanthine (IBMX; 1 muM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 muM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMvarphi was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDMvarphi. Preconditioning of MDMvarphi with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41-2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner. These results

  18. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Rossi Adriano G

    2009-05-01

    Full Text Available Abstract Background Nitric oxide (NO can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-. In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMϕ, and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP is able to limit apoptosis in this cell type. Methods Characterisation of the NO-related species generated by (Z-1- [2-(2-aminoethyl-N-(2-ammonioethylamino]diazen-1-ium-1,2-diolate (DETA/NO and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl-, chloride (GEA-3162 was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM or GEA-3162 (10 – 300 μM in the presence or absence of BAY 41–2272 (1 μM, isobutylmethylxanthine (IBMX; 1 μM, 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM or 8-bromo-cGMP (1 mM. Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Results Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO had no effect on cell viability, but ONOO- (GEA-3162 caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX, or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner

  19. Purity assessment of recombinant human granulocyte colony-stimulating factor in finished drug product by capillary zone electrophoresis.

    Science.gov (United States)

    Benković, Goran; Skrlin, Ana; Madić, Tomislav; Debeljak, Zeljko; Medić-Šarić, Marica

    2014-09-01

    Current methods for determination of impurities with different charge-to-volume ratio are limited especially in terms of sensitivity and precision. The main goal of this research was to establish a quantitative method for determination of impurities with charges differing from that of recombinant human granulocyte colony-stimulating factor (rhG-CSF, filgrastim) with superior precision and sensitivity compared to existing methods. A CZE method has been developed, optimized, and validated for a purity assessment of filgrastim in liquid pharmaceutical formulations. Optimal separation of filgrastim from the related impurities with different charges was achieved on a 50 μm id fused-silica capillary of a total length of 80.5 cm. A BGE that contains 100 mM phosphoric acid adjusted to pH 7.0 with triethanolamine was used. The applied voltage was 20 kV while the temperature was maintained at 25°C. UV detection was set to 200 nm. Method was validated in terms of selectivity/specificity, linearity, precision, LOD, LOQ, stability, and robustness. Linearity was observed in the concentration range of 6-600 μg/mL and the LOQ was determined to be 0.3% relative to the concentration of filgrastim of 0.6 mg/mL. Other validation parameters were also found to be acceptable; thus the method was successfully applied for a quantitative purity assessment of filgrastim in a finished drug product. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    DEFF Research Database (Denmark)

    Nielsen, H

    1993-01-01

    After 10 weeks of treatment with clozapine, severe agranulocytosis was diagnosed in a 33-year-old female. The patient was treated with filgrastim (granulocyte colony-stimulating factor [G-CSF]) 5 micrograms kg-1 day-1. The neutrophil count was 0.234 x 10(9) l-1 on admission, with a further decrease...

  1. Expression of the stem cell factor in fibroblasts, endothelial cells, and macrophages in periapical tissues in human chronic periapical diseases.

    Science.gov (United States)

    Shen, S Q; Wang, R; Huang, S G

    2017-03-08

    Stem cell factor (SCF), an important stem cell cytokine, has multiple functions. Fibroblasts (FBs), mature mast cells, endothelial cells (ECs), and eosinophil granulocytes can produce SCF in the inflammatory process. Therefore, we aimed to observe SCF expression in FBs, ECs, and macrophages (MPs) in periapical tissues in human chronic periapical disease and investigate the effects of cells expressing SCF in pathogenesis of the disease. Healthy (N = 20), periapical cyst (N = 15), and periapical granuloma (N = 15) tissues were fixed in 10% formalin for 48 h, embedded in paraffin, and stained with hematoxylin and eosin to observe histological changes. SCF expression was observed in FBs, ECs, and MPs in periapical tissues by double immunofluorescence. CD334, CD31, and CD14 are specific markers of FBs, ECs, and MPs, respectively. Results showed that densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs were significantly increased in periapical tissue groups (P periapical tissue groups (P > 0.05). CD14-SCF double-positive MP density was considerably higher in periapical granulomas than in cysts (P periapical tissues, suggesting that the cells might be related to occurrence, development, and pathogenesis of chronic periapical disease.

  2. T3 Regulates a Human Macrophage-Derived TSH-β Splice Variant: Implications for Human Bone Biology.

    Science.gov (United States)

    Baliram, R; Latif, R; Morshed, S A; Zaidi, M; Davies, T F

    2016-09-01

    TSH and thyroid hormones (T3 and T4) are intimately involved in bone biology. We have previously reported the presence of a murine TSH-β splice variant (TSH-βv) expressed specifically in bone marrow-derived macrophages and that exerted an osteoprotective effect by inducing osteoblastogenesis. To extend this observation and its relevance to human bone biology, we set out to identify and characterize a TSH-β variant in human macrophages. Real-time PCR analyses using human TSH-β-specific primers identified a 364-bp product in macrophages, bone marrow, and peripheral blood mononuclear cells that was sequence verified and was homologous to a human TSH-βv previously reported. We then examined TSH-βv regulation using the THP-1 human monocyte cell line matured into macrophages. After 4 days, 46.1% of the THP-1 cells expressed the macrophage markers CD-14 and macrophage colony-stimulating factor and exhibited typical morphological characteristics of macrophages. Real-time PCR analyses of these cells treated in a dose-dependent manner with T3 showed a 14-fold induction of human TSH-βv mRNA and variant protein. Furthermore, these human TSH-βv-positive cells, induced by T3 exposure, had categorized into both M1 and M2 macrophage phenotypes as evidenced by the expression of macrophage colony-stimulating factor for M1 and CCL-22 for M2. These data indicate that in hyperthyroidism, bone marrow resident macrophages have the potential to exert enhanced osteoprotective effects by oversecreting human TSH-βv, which may exert its local osteoprotective role via osteoblast and osteoclast TSH receptors.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    We have previously shown that gadolinium oxide (Gd 2 O 3 ) nanoparticles are promising candidates to be used as contrast agents in magnetic resonance (MR) imaging applications. In this study, these nanoparticles were investigated in a cellular system, as possible probes for visualization and targeting intended for bioimaging applications. We evaluated the impact of the presence of Gd 2 O 3 nanoparticles on the production of reactive oxygen species (ROS) from human neutrophils, by means of luminol-dependent chemiluminescence. Three sets of Gd 2 O 3 nanoparticles were studied, i.e. as synthesized, dialyzed and both PEG-functionalized and dialyzed Gd 2 O 3 nanoparticles. In addition, neutrophil morphology was evaluated by fluorescent staining of the actin cytoskeleton and fluorescence microscopy. We show that surface modification of these nanoparticles with polyethylene glycol (PEG) is essential in order to increase their biocompatibility. We observed that the as synthesized nanoparticles markedly decreased the ROS production from neutrophils challenged with prey (opsonized yeast particles) compared to controls without nanoparticles. After functionalization and dialysis, more moderate inhibitory effects were observed at a corresponding concentration of gadolinium. At lower gadolinium concentration the response was similar to that of the control cells. We suggest that the diethylene glycol (DEG) present in the as synthesized nanoparticle preparation is responsible for the inhibitory effects on the neutrophil oxidative burst. Indeed, in the present study we also show that even a low concentration of DEG, 0.3%, severely inhibits neutrophil function. In summary, the low cellular response upon PEG-functionalized Gd 2 O 3 nanoparticle exposure indicates that these nanoparticles are promising candidates for MR-imaging purposes. (paper)

  4. Cysteamine-mediated clearance of antibiotic-resistant pathogens in human cystic fibrosis macrophages.

    Directory of Open Access Journals (Sweden)

    Chandra L Shrestha

    Full Text Available Members of the Burkholderia cepacia complex are virulent, multi-drug resistant pathogens that survive and replicate intracellularly in patients with cystic fibrosis (CF. We have discovered that B. cenocepacia cannot be cleared from CF macrophages due to defective autophagy, causing continued systemic inflammation and infection. Defective autophagy in CF is mediated through constitutive reactive oxygen species (ROS activation of transglutaminase-2 (TG2, which causes the sequestration (accumulation of essential autophagy initiating proteins. Cysteamine is a TG2 inhibitor and proteostasis regulator with the potential to restore autophagy. Therefore, we sought to examine the impact of cysteamine on CF macrophage autophagy and bacterial killing. Human peripheral blood monocyte-derived macrophages (MDMs and alveolar macrophages were isolated from CF and non-CF donors. Macrophages were infected with clinical isolates of relevant CF pathogens. Cysteamine caused direct bacterial growth killing of live B. cenocepacia, B. multivorans, P. aeruginosa and MRSA in the absence of cells. Additionally, B. cenocepacia, B. multivorans, and P. aeruginosa invasion were significantly decreased in CF MDMs treated with cysteamine. Finally, cysteamine decreased TG2, p62, and beclin-1 accumulation in CF, leading to increased Burkholderia uptake into autophagosomes, increased macrophage CFTR expression, and decreased ROS and IL-1β production. Cysteamine has direct anti-bacterial growth killing and improves human CF macrophage autophagy resulting in increased macrophage-mediated bacterial clearance, decreased inflammation, and reduced constitutive ROS production. Thus, cysteamine may be an effective adjunct to antibiotic regimens in CF.

  5. Hyperglycemia induces mixed M1/M2 cytokine profile in primary human monocyte-derived macrophages.

    Science.gov (United States)

    Moganti, Kondaiah; Li, Feng; Schmuttermaier, Christina; Riemann, Sarah; Klüter, Harald; Gratchev, Alexei; Harmsen, Martin C; Kzhyshkowska, Julia

    2017-10-01

    Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytokine profile that can support of diabetes-associated inflammation and development of vascular complications. Copyright © 2016 Elsevier GmbH. All rights reserved.

  6. Kinetic data of in-vivo labeled granulocytes in humans with a murine Tc-99m-labelled monoclonal antibody

    International Nuclear Information System (INIS)

    Becker, W.; Boerner, W.; Borst, U.; Schaefer, R.; Fischbach, W.; Pasurka, B.

    1989-01-01

    Twenty-five patients were examined in vivo with 99m Tc labelled monoclonal antibodies; 15 with suspected infections with an antigranulocyte antibody (BW 250/183), 10 with suspected recurrence of a colorectal carcinoma with an anti CEA antibody (BW 431/26). Both antibodies were IgG1 isotypes. In the patients with suspected infections no change of the peripheral leukocyte count could be observed after the antibody injection (1 mg, n=9; 0.05 mg, n=1; 0.25 mg, n=6). In 2 patients examined with the anti CEA antibody (2 mg), a significant decrease of the peripheral leukocyte count could be observed. The recovery rate of the 99m Tc antibody labelled granulocytes was calculated to be about 10%. The increase of the antibody-antigen binding was calculated to be 0.2%/min. In vivo the organ distribution curves demonstrated an increase of 99m Tc activity over spleen and bone marrow of 1.1%/min, which was interpreted as antigen-antibody reactivity. The organ distribution curves of the anti granulocyte antibody over spleen and bone marrow showed typical binding characteristics to the local granulocyte epitopes. The curves over other organs showed a simple perfusion pattern. The curves of the anti CEA antibody showed a perfusion pattern over all the examined organs. A sham dialysis model in one patient with renal insufficiency undergoing regular dialysis treatment demonstrated the viability of 99m Tc antibody labelled granulocytes in vivo. The kinetic patterns of the 99m Tc antibody in patients with Crohn's disease were interpreted as CEA binding of the antibody in the bowel wall. (orig.)

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

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    Yoshitaka Sunami

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

  8. Comparison of granulocyte-macrophage colony-stimulating factor and sucralfate mouthwashes in the prevention of radiation-induced mucositis: a double-blind prospective randomized phase III study

    International Nuclear Information System (INIS)

    Saarilahti, Kauko; Kajanti, Mikael; Joensuu, Timo; Kouri, Mauri; Joensuu, Heikki

    2002-01-01

    Purpose: To compare granulocyte-macrophage colony-stimulating factor (GM-CSF) mouthwashes with sucralfate mouthwashes in the prevention of radiation-induced mucositis. Methods and Materials: Forty patients with radically operated head-and-neck cancer were randomly allocated to use either GM-CSF (n=21) or sucralfate (n=19) mouthwashes during postoperative radiotherapy (RT). All patients received conventionally fractionated RT to a total dose of 50-60 Gy in 2-Gy daily fractions during 5-6 weeks to the primary site and regional lymphatics. A minimum of 50% of the oral cavity and oropharyngeal mucosa was included in the clinical target volume. GM-CSF mouthwashes consisted of 37.5 μg GM-CSF and sucralfate mouthwashes of 1.0 g of sucralfate distilled in water. Both washes were used 4 times daily, beginning after the first week of RT and continued to the end of the RT course. Symptoms related to radiation mucositis and body weight, serum prealbumin level, and blood cell counts were monitored weekly. Results: Oral mucositis tended to be less severe in the GM-CSF group (p=0.072). Complete (n=1) or partial (n=4) healing of mucositis occurred during the RT course in 5 patients (24%) in the GM-CSF group and in none of the patients in the sucralfate group (p=0.049). Patients who received GM-CSF had less mucosal pain (p=0.058) and were less often prescribed opioids for pain (p=0.042). Three patients in the sucralfate group needed hospitalization for mucositis during RT compared with none in the GM-CSF group. Four patients (21%) in the sucralfate group and none in the GM-CSF group required an interruption in the RT course (p=0.042). No significant differences in weight, prealbumin level, or blood cell count were found between the groups, and both mouthwashes were well tolerated. Conclusion: GM-CSF mouthwashes may be moderately more effective than sucralfate mouthwashes in preventing radiation-induced mucositis and mucositis-related pain, and their use may lead to less frequent

  9. Efficacy and safety of talimogene laherparepvec versus granulocyte-macrophage colony-stimulating factor in patients with stage IIIB/C and IVM1a melanoma: subanalysis of the Phase III OPTiM trial

    Directory of Open Access Journals (Sweden)

    Harrington KJ

    2016-11-01

    Full Text Available Kevin J Harrington,1 Robert HI Andtbacka,2 Frances Collichio,3 Gerald Downey,4 Lisa Chen,5 Zsolt Szabo,6 Howard L Kaufman7 1The Institute of Cancer Research/The Royal Marsden Hospital NIHR Biomedical Research Centre, London, UK; 2Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA; 3Division of Hematology and Oncology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 4Amgen Ltd, Cambridge, UK; 5Amgen Inc, Thousand Oaks, CA, USA; 6Amgen GmbH, Zug, Switzerland; 7Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA Objectives: Talimogene laherparepvec is the first oncolytic immunotherapy to receive approval in Europe, the USA and Australia. In the randomized, open-label Phase III OPTiM trial (NCT00769704, talimogene laherparepvec significantly improved durable response rate (DRR versus granulocyte-macrophage colony-stimulating factor (GM-CSF in 436 patients with unresectable stage IIIB–IVM1c melanoma. The median overall survival (OS was longer versus GM-CSF in patients with earlier-stage melanoma (IIIB–IVM1a. Here, we report a detailed subgroup analysis of the OPTiM study in patients with IIIB–IVM1a disease. Patients and methods: The patients were randomized (2:1 ratio to intralesional talimogene laherparepvec or subcutaneous GM-CSF and were evaluated for DRR, overall response rate (ORR, OS, safety, benefit–risk and numbers needed to treat. Descriptive statistics were used for subgroup comparisons. Results: Among 249 evaluated patients with stage IIIB–IVM1a melanoma, DRR was higher with talimogene laherparepvec compared with GM-CSF (25.2% versus 1.2%; P<0.0001. ORR was also higher in the talimogene laherparepvec arm (40.5% versus 2.3%; P<0.0001, and 27 patients in the talimogene laherparepvec arm had a complete response, compared with none in GM-CSF-treated patients. The incidence rates of exposure-adjusted adverse events (AE and serious AEs were similar with both treatments. Conclusion

  10. 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

    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...... 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...

  11. Viral infection of human lung macrophages increases PDL1 expression via IFNβ.

    Directory of Open Access Journals (Sweden)

    Karl J Staples

    Full Text Available Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.

  12. Tie2 signaling cooperates with TNF to promote the pro-inflammatory activation of human macrophages independently of macrophage functional phenotype.

    Science.gov (United States)

    García, Samuel; Krausz, Sarah; Ambarus, Carmen A; Fernández, Beatriz Malvar; Hartkamp, Linda M; van Es, Inge E; Hamann, Jörg; Baeten, Dominique L; Tak, Paul P; Reedquist, Kris A

    2014-01-01

    Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 -differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 -differentiated macrophages, expression of multiple chemokines and cytokines, including CXCL3, CXCL5, CXCL8, IL6, and IL12B was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.

  13. Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

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    Xiyuan Bai

    Full Text Available Nuclear factor-kappa B (NFκB is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB. However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

  14. Anti-human neutrophil antigen-1a, -1b, and -2 antibodies in neonates and children with immune neutropenias analyzed by extracted granulocyte antigen immunofluorescence assay.

    Science.gov (United States)

    Onodera, Rie; Kurita, Emi; Taniguchi, Kikuyo; Karakawa, Shuhei; Okada, Satoshi; Kihara, Hirotaka; Fujii, Teruhisa; Kobayashi, Masao

    2017-11-01

    Anti-human neutrophil antigen (HNA) antibodies have been implicated in the development of neonatal alloimmune neutropenia (NAN) and autoimmune neutropenia (AIN). There are many conventional assay methods that detect anti-HNA antibodies. However, a method to measure multiple samples and detect several anti-HNA antibodies simultaneously is needed. We developed a new method, the extracted granulocyte antigen immunofluorescence assay (EGIFA), to analyze anti-HNA-1a, -1b, and -2 antibodies in sera. The results obtained by EGIFA were evaluated in comparison with those from several standard assay methods. Anti-HNA antibodies in serum samples from nine familial cases with suspected NAN (n = 19) and children with suspected AIN (n = 88) were also measured by EGIFA. The evaluation of nine serum samples with anti-HNA antibodies suggested that EGIFA demonstrated equivalent specificity and superior sensitivity to monoclonal antibody-specific immobilization of granulocyte antigens and had comparable sensitivity to the granulocyte indirect immunofluorescence test. EGIFA successfully detected anti-HNA-1a or -1b antibodies in seven of nine familial cases with suspected NAN. EGIFA detected anti-HNA antibodies in 40.9% of children with suspected AIN. Among them, isolated anti-HNA-1a or -1b antibody was detected in 4.5 or 12.5% of children, respectively, and anti-HNA-2 antibody was identified in 3.4% of children. The 30.8% (16 of 52) of children negative for anti-HNA antibody by EGIFA were positive for anti-HLA antibody. EGIFA facilitated the measurement of anti-HNA-1a, -1b, and/or -2 antibodies in sera. The prompt measurement of anti-HNA antibodies will improve the diagnosis and clinical management of patients with suspected NAN or AIN. © 2017 AABB.

  15. Chlamydia pneumoniae hides inside apoptotic neutrophils to silently infect and propagate in macrophages.

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    Jan Rupp

    Full Text Available BACKGROUND: Intracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae. METHODOLOGY/PRINCIPAL FINDINGS: We could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ss production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-alpha response. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.

  16. Cigarette smoking decreases global microRNA expression in human alveolar macrophages.

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    Joel W Graff

    Full Text Available Human alveolar macrophages are critical components of the innate immune system. Cigarette smoking-induced changes in alveolar macrophage gene expression are linked to reduced resistance to pulmonary infections and to the development of emphysema/COPD. We hypothesized that microRNAs (miRNAs could control, in part, the unique messenger RNA (mRNA expression profiles found in alveolar macrophages of cigarette smokers. Activation of macrophages with different stimuli in vitro leads to a diverse range of M1 (inflammatory and M2 (anti-inflammatory polarized phenotypes that are thought to mimic activated macrophages in distinct tissue environments. Microarray mRNA data indicated that smoking promoted an "inverse" M1 mRNA expression program, defined by decreased expression of M1-induced transcripts and increased expression of M1-repressed transcripts with few changes in M2-regulated transcripts. RT-PCR arrays identified altered expression of many miRNAs in alveolar macrophages of smokers and a decrease in global miRNA abundance. Stratification of human subjects suggested that the magnitude of the global decrease in miRNA abundance was associated with smoking history. We found that many of the miRNAs with reduced expression in alveolar macrophages of smokers were predicted to target mRNAs upregulated in alveolar macrophages of smokers. For example, miR-452 is predicted to target the transcript encoding MMP12, an important effector of smoking-related diseases. Experimental antagonism of miR-452 in differentiated monocytic cells resulted in increased expression of MMP12. The comprehensive mRNA and miRNA expression profiles described here provide insight into gene expression regulation that may underlie the adverse effects cigarette smoking has on alveolar macrophages.

  17. CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes

    OpenAIRE

    Arthos, James; Rubbert, Andrea; Rabin, Ronald L.; Cicala, Claudia; Machado, Elizabeth; Wildt, Kathryne; Hanbach, Meredith; Steenbeke, Tavis D.; Swofford, Ruth; Farber, Joshua M.; Fauci, Anthony S.

    2000-01-01

    The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity o...

  18. Transcriptomic analysis of human polarized macrophages: more than one role of alternative activation?

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    Eleonora Derlindati

    Full Text Available Macrophages are a heterogeneous cell population which in response to the cytokine milieu polarize in either classically activated macrophages (M1 or alternatively activated macrophages (M2. This plasticity makes macrophages essential in regulating inflammation, immune response and tissue remodeling and a novel therapeutic target in inflammatory diseases such as atherosclerosis. The aim of the study was to describe the transcriptomic profiles of differently polarized human macrophages to generate new hypotheses on the biological function of the different macrophage subtypes.Polarization of circulating monocytes/macrophages of blood donors was induced in vitro by IFN-γ and LPS (M1, by IL-4 (M2a, and by IL-10 (M2c. Unstimulated cells (RM served as time controls. Gene expression profile of M1, M2a, M2c and RM was assessed at 6, 12 and 24h after polarization with Whole Human Genome Agilent Microarray technique. When compared to RM, M1 significantly upregulated pathways involved in immunity and inflammation, whereas M2a did the opposite. Conversely, decreased and increased expression of mitochondrial metabolism, consistent with insulin resistant and insulin sensitive patterns, was seen in M1 and M2a, respectively. The time sequence in the expression of some pathways appeared to have some specific bearing on M1 function. Finally, canonical and non-canonical Wnt genes and gene groups, promoting inflammation and tissue remodeling, were upregulated in M2a compared to RM.Our data in in vitro polarized human macrophages: 1. confirm and extend known inflammatory and anti-inflammatory gene expression patterns; 2. demonstrate changes in mitochondrial metabolism associated to insulin resistance and insulin sensitivity in M1 and M2a, respectively; 3. highlight the potential relevance of gene expression timing in M1 function; 4. unveil enhanced expression of Wnt pathways in M2a suggesting a potential dual (pro-inflammatory and anti-inflammatory role of M2a in

  19. The impact of splenectomy on human coronary artery atherosclerosis and vascular macrophage distribution.

    Science.gov (United States)

    Li, Yu; Stone, James R

    Splenectomy can potentially impact atherosclerosis through multiple mechanisms including altered lipid homeostasis, increased coagulation, and altered macrophage recruitment to the plaque. In patients, splenectomy has been associated with increased rates of coronary artery events, while in experimental mice, splenectomy causes increased atherosclerosis but reduces systemic monocyte supply. In this study, the direct impact of splenectomy on human coronary artery atherosclerotic plaque severity and macrophage content was investigated. Coronary artery atherosclerotic plaque severity was determined at autopsy in 18 long-term (≥10 years) splenectomy patients and 90 matched control patients. Coronary artery macrophage content was evaluated in mild atherosclerotic plaques of 11 mid- to long-term (≥1 year) splenectomy patients and 11 matched control patients. Splenectomy was associated with reduced coronary artery atherosclerosis (P=.03). The association was most pronounced for the subgroup of patients who had undergone splenectomy 20 years or more prior to death (P=.02). There was no difference in the density of macrophages in the plaque, media, or adventitia upon comparing splenectomy and control patients. In the control group, there was no correlation between the macrophage densities in the three arterial layers. However, in the splenectomy patients, there was a strong correlation in the macrophage densities across the plaque, media, and adventitia (P≤.0002), with resulting slopes that were significantly greater than seen in the control patients (P=.0007-.011). These findings indicate that, in humans, splenectomy is associated with lower coronary artery atherosclerotic plaque severity and altered coronary artery macrophage distribution. These results suggest that the spleen can modulate the recruitment of macrophages into human coronary arteries and the progression of atherosclerosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Dual Transcriptome Profiling of Leishmania-Infected Human Macrophages Reveals Distinct Reprogramming Signatures.

    Science.gov (United States)

    Fernandes, Maria Cecilia; Dillon, Laura A L; Belew, Ashton Trey; Bravo, Hector Corrada; Mosser, David M; El-Sayed, Najib M

    2016-05-10

    Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome. Little is known about the transcriptional changes that occur within mammalian cells harboring intracellular pathogens. This study characterizes the gene expression signatures of Leishmania spp. parasites and the coordinated response of infected human macrophages as the pathogen enters and persists within them. After accounting for the generic effects of large-particle phagocytosis, we observed a parasite-specific response of the human macrophages early in

  1. Human macrophage foam cells degrade atherosclerotic plaques through cathepsin K mediated processes

    DEFF Research Database (Denmark)

    Barascuk, Natasha; Skjøt-Arkil, Helene; Register, Thomas C

    2010-01-01

    BACKGROUND: Proteolytic degradation of Type I Collagen by proteases may play an important role in remodeling of atherosclerotic plaques, contributing to increased risk of plaque rupture.The aim of the current study was to investigate whether human macrophage foam cells degrade the extracellular...... matrix (ECM) of atherosclerotic plaques by cathepsin K mediated processes. METHODS: We 1) cultured human macrophages on ECM and measured cathepsin K generated fragments of type I collagen (C-terminal fragments of Type I collagen (CTX-I) 2) investigated the presence of CTX-I in human coronary arteries......-I in areas of intimal hyperplasia and in shoulder regions of advanced plaques. Treatment of human monocytes with M-CSF or M-CSF+LDL generated macrophages and foam cells producing CTX-I when cultured on type I collagen enriched matrix. Circulating levels of CTX-I were not significantly different in women...

  2. Electronegative L5-LDL induces the production of G-CSF and GM-CSF in human macrophages through LOX-1 involving NF-κB and ERK2 activation.

    Science.gov (United States)

    Yang, Tzu-Ching; Chang, Po-Yuan; Kuo, Tzu-Ling; Lu, Shao-Chun

    2017-12-01

    Circulating levels of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) are associated with the severity of acute myocardial infarction (AMI). However, what causes increases in G-CSF and GM-CSF is unclear. In this study, we investigated whether L5-low-density lipoprotein (LDL), a mildly oxidized LDL from AMI, can induce G-CSF and GM-CSF production in human macrophages. L1-LDL and L5-LDL were isolated through anion-exchange chromatography from AMI plasma. Human macrophages derived from THP-1 and peripheral blood mononuclear cells were treated with L1-LDL, L5-LDL, or copper-oxidized LDL (Cu-oxLDL) and G-CSF and GM-CSF protein levels in the medium were determined. In addition, the effects of L5-LDL on G-CSF and GM-CSF production were tested in lectin-type oxidized LDL receptor-1 (LOX-1), CD36, extracellular signal-regulated kinase (ERK) 1, and ERK2 knockdown THP-1 macrophages. L5-LDL but not L1-LDL or Cu-oxLDL significantly induced production of G-CSF and GM-CSF in macrophages. In vitro oxidation of L1-LDL and L5-LDL altered their ability to induce G-CSF and GM-CSF, suggesting that the degree of oxidation is critical for the effects. Knockdown and antibody neutralization experiments suggested that the effects were caused by LOX-1. In addition, nuclear factor (NF)-κB and ERK1/2 inhibition resulted in marked reductions of L5-LDL-induced G-CSF and GM-CSF production. Moreover, knockdown of ERK2, but not ERK1, hindered L5-LDL-induced G-CSF and GM-CSF production. The results indicate that L5-LDL, a naturally occurring mild oxidized LDL, induced G-CSF and GM-CSF production in human macrophages through LOX-1, ERK2, and NF-κB dependent pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. The effects of exogenous fatty acids and niacin on human monocyte-macrophage plasticity.

    Science.gov (United States)

    Montserrat-de la Paz, Sergio; Rodriguez, Dolores; Cardelo, Magdalena P; Naranjo, Maria C; Bermudez, Beatriz; Abia, Rocio; Muriana, Francisco J G; Lopez, Sergio

    2017-08-01

    Macrophage plasticity allows adapting to different environments, having a dual activity in inflammatory-related diseases. Our hypothesis is that the type of dietary fatty acids into human postprandial triglyceride-rich lipoproteins (TRLs), alone or in combination with niacin (vitamin B3), could modulate the plasticity of monocytes-macrophages. We isolated TRLs at the postprandial peak from blood samples of healthy volunteers after the ingestion of a meal rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) or MUFAs plus omega-3 long-chain polyunsaturated fatty acids (LCPUFAs). Autologous monocytes isolated at fasting were first induced to differentiate into naïve macrophages. We observed that postprandial TRL-MUFAs, particularly in combination with niacin, enhance competence to monocytes to differentiate and polarise into M2 macrophages. Postprandial TRL-SFAs made polarised macrophages prone to an M1 phenotype. In contrast to dietary SFAs, dietary MUFAs in the meals plus immediate-release niacin primed circulating monocytes for a reduced postprandial pro-inflammatory profile. Our study underlines a role of postprandial TRLs as a metabolic entity in regulating the plasticity of the monocyte-macrophage lineage and also brings an understanding of the mechanisms by which dietary fatty acids are environmental factors fostering the innate immune responsiveness in humans. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Progastrin represses the alternative activation of human macrophages and modulates their influence on colon cancer epithelial cells.

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    Carlos Hernández

    Full Text Available Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive to M2 (tolerogenic, pro-tumoral. Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10 with normal amounts of M1-markers (CD86, IL12. Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization.

  5. Human macrophages support persistent transcription from unintegrated HIV-1 DNA

    International Nuclear Information System (INIS)

    Kelly, Jeremy; Beddall, Margaret H.; Yu Dongyang; Iyer, Subashini R.; Marsh, Jon W.; Wu Yuntao

    2008-01-01

    Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells, unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally, there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines

  6. Modulation of cytokine expression in human macrophages by endocrine-disrupting chemical Bisphenol-A

    International Nuclear Information System (INIS)

    Liu, Yanzhen; Mei, Chenfang; Liu, Hao; Wang, Hongsheng; Zeng, Guoqu; Lin, Jianhui; Xu, Meiying

    2014-01-01

    Highlights: • Effects of BPA on the cytokines expression of human macrophages were investigated. • BPA increased pro-inflammation cytokines TNF-α and IL-6 production. • BPA decreased anti-inflammation IL-10 and TGF-β production. • ERα/β/ERK/NF-κB signaling involved in BPA-mediated cytokines expression. - Abstract: Exposure to environmental endocrine-disrupting chemical Bisphenol-A (BPA) is often associated with dysregulated immune homeostasis, but the mechanisms remain unclear. In the present study, the effects of BPA on the cytokines responses of human macrophages were investigated. Treatment with BPA increased pro-inflammation cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production, but decreased anti-inflammation cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production in THP1 macrophages, as well as in primary human macrophages. BPA effected cytokines expression through estrogen receptor α/β (ERα/β)-dependent mechanism with the evidence of ERα/β antagonist reversed the expression of cytokines. We also identified that activation of extracellular regulated protein kinases (ERK)/nuclear factor κB (NF-κB) signal cascade marked the effects of BPA on cytokines expression. Our results indicated that BPA effected inflammatory responses of macrophages via modulating of cytokines expression, and provided a new insight into the link between exposure to BPA and human health

  7. Modulation of cytokine expression in human macrophages by endocrine-disrupting chemical Bisphenol-A

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yanzhen; Mei, Chenfang [State Key Laboratory of Applied Microbiology Southern China, Guangzhou 510070 (China); Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070 (China); Liu, Hao [Affiliated Cancer Hospital and Cancer Research Institute, Guangzhou Medical University, Guangzhou 510095 (China); Wang, Hongsheng [Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Zeng, Guoqu; Lin, Jianhui [State Key Laboratory of Applied Microbiology Southern China, Guangzhou 510070 (China); Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070 (China); Xu, Meiying, E-mail: xumy@gdim.cn [State Key Laboratory of Applied Microbiology Southern China, Guangzhou 510070 (China); Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070 (China)

    2014-09-05

    Highlights: • Effects of BPA on the cytokines expression of human macrophages were investigated. • BPA increased pro-inflammation cytokines TNF-α and IL-6 production. • BPA decreased anti-inflammation IL-10 and TGF-β production. • ERα/β/ERK/NF-κB signaling involved in BPA-mediated cytokines expression. - Abstract: Exposure to environmental endocrine-disrupting chemical Bisphenol-A (BPA) is often associated with dysregulated immune homeostasis, but the mechanisms remain unclear. In the present study, the effects of BPA on the cytokines responses of human macrophages were investigated. Treatment with BPA increased pro-inflammation cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production, but decreased anti-inflammation cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production in THP1 macrophages, as well as in primary human macrophages. BPA effected cytokines expression through estrogen receptor α/β (ERα/β)-dependent mechanism with the evidence of ERα/β antagonist reversed the expression of cytokines. We also identified that activation of extracellular regulated protein kinases (ERK)/nuclear factor κB (NF-κB) signal cascade marked the effects of BPA on cytokines expression. Our results indicated that BPA effected inflammatory responses of macrophages via modulating of cytokines expression, and provided a new insight into the link between exposure to BPA and human health.

  8. Entrance and Survival of Brucella pinnipedialis Hooded Seal Strain in Human Macrophages and Epithelial Cells

    Science.gov (United States)

    Briquemont, Benjamin; Sørensen, Karen K.; Godfroid, Jacques

    2013-01-01

    Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary. PMID:24376851

  9. Influence of UV-radiation on granulocytic phagocytosis in vitro

    International Nuclear Information System (INIS)

    Walther, T.; Rytter, M.; Gast, W.; Haustein, U.F.

    1987-01-01

    The influence of UV radiation on the vitality, the performance of phagocytosis and the ability to reduce nitro-blue tetrazolium test (NBT) by human granulocytes was investigated in vitro. Already by low doses of UVA (8% UVB) the percentage of phagocytizing granulocytes was decreased more distinctly than their cell vitality. The number of ingested Candida albicans particles was 4.5 particles per granulocyte in the controls. It was reduced to about 1.4 particles per cell by UV radiation independent of the dosis applied. On the other hand the ability of granulocytes to reduce NBT intracellularly remained completely unchanged. (author)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-15

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  13. Application of microchip CGE for the analysis of PEG-modified recombinant human granulocyte-colony stimulating factors.

    Science.gov (United States)

    Park, Eun Ji; Lee, Kyung Soo; Lee, Kang Choon; Na, Dong Hee

    2010-11-01

    The purpose of this study was to evaluate the microchip CGE (MCGE) for the analysis of PEG-modified granulocyte-colony stimulating factor (PEG-G-CSF) prepared with PEG-aldehydes. The unmodified and PEG-modified G-CSFs were analyzed by Protein 80 and 230 Labchips on the Agilent 2100 Bioanalyzer. The MCGE allowed size-based separation and quantitation of PEG-G-CSF. The Protein 80 Labchip was useful for PEG-5K-G-CSF, while the Protein 230 Labchip was more suitable for PEG-20K-G-CSF. The MCGE was also used to monitor a search for optimal PEG-modification (PEGylation) conditions to produce mono-PEG-G-CSF. This study demonstrates the usefulness of MCGE for monitoring and optimizing the PEGylation of G-CSF with the advantages of speed, minimal sample consumption, and automatic quantitation.

  14. Anti-inflammatory effects of octadecylamine-functionalized nanodiamond on primary human macrophages.

    Science.gov (United States)

    Pentecost, A E; Witherel, C E; Gogotsi, Y; Spiller, K L

    2017-09-26

    Chronic inflammatory disorders such as rheumatoid arthritis are characterized by excessive pro-inflammatory or "M1" activation of macrophages, the primary cells of the innate immune system. Current treatments include delivery of glucocorticoids (e.g. dexamethasone - Dex), which reduce pro-inflammatory M1 behaviour in macrophages. However, these treatments have many off-target effects on cells other than macrophages, resulting in broad immunosuppression. To limit such side effects, drug-incorporated nano- and microparticles may be used to selectively target macrophages via phagocytosis, because of their roles as highly effective phagocytes in the body. In this study, surface-modified nanodiamond (ND) was explored as a platform for the delivery of dexamethasone to macrophages because of ND's rich surface chemistry, which contributes to ND's high potential as a versatile drug delivery platform. After finding that octadecylamine-functionalized nanodiamond (ND-ODA) enhanced adsorption of Dex compared to carboxylated ND, the effects of Dex, ND-ODA, and Dex-adsorbed ND-ODA on primary human macrophage gene expression were characterized. Surprisingly, even in the absence of Dex, ND-ODA had strong anti-inflammatory effects, as determined by multiplex gene expression via NanoString and by protein secretion analysis via ELISA. ND-ODA also inhibited expression of M2a markers yet increased the expression of M2c markers and phagocytic receptors. Interestingly, the adsorption of Dex to ND-ODA further increased some anti-inflammatory effects, but abrogated the effect on phagocytic receptors, compared to its individual components. Overall, the ability of ND-ODA to promote anti-inflammatory and pro-phagocytic behaviour in macrophages, even in the absence of loaded drugs, suggests its potential for use as an anti-inflammatory therapeutic to directly target macrophages through phagocytosis.

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

    Directory of Open Access Journals (Sweden)

    E.I. Alexeeva

    2010-01-01

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

  16. Granulocyte-platelet interactions and platelet fibrinogen receptor exposure

    International Nuclear Information System (INIS)

    Kornecki, E.; Ehrlich, Y.H.; Egbring, R.; Gramse, M.; Seitz, R.; Eckardt, A.; Lukasiewicz, H.; Niewiarowski, S.

    1988-01-01

    The authors have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125 I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase. Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen-binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125 I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. They conclude that (1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and (2) human granulocyte elastase exposes active fibrongen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen

  17. Effect of filgrastim (recombinant human granulocyte colony stimulating factor) on IgE responses in human asthma: a case study.

    Science.gov (United States)

    Smith-Norowitz, Tamar A; Joks, Rauno; Norowitz, Kevin B; Chice, Seto; Durkin, Helen G; Bluth, Martin H

    2013-10-01

    The role of peripheral blood progenitor cell mobilization on Immunoglobulin E (IgE) responses has not been studied. Distributions of blood lymphocytes (CD4+, CD8+, CD8+CD60+, CD19+, CD23+, CD16/56+, CD25, CD45RA+, CD45RO+, CD34+), and levels of serum immunoglobulins (IgM, IgG, IgA, IgE) were studied in an allergic asthmatic serum IgE+ (181IU/mL) adult (m/45 y/o) donor undergoing routine stem cell mobilization protocol (American Society of Hematology) before (day-30), during (day 4), and after (1 wk post last dose) filgrastim (subcutaneous, 480 mcg, 2qd) treatment (flow cytometry, nephelometry, UniCAP Total IgE Fluoro enzyme immunoassay). On day 4 of filgrastim treatment, numbers of CD8+CD60+T cells and CD23+ blood cells dramatically increased (98% and 240% respectively) compared with pre treatment. In contrast on day 4 of treatment, serum IgE levels decreased (>50%) compared with pre treatment. CD8+CD60+T cells and CD23+ blood cells and serum IgE levels approached pre-treatment levels at 1 week post treatment. Filgrastim treatment transiently increases numbers of CD8+CD60+T and CD23+ expressing cells, which are known to regulate human IgE responses, while also transiently suppressing ongoing IgE responses. These results suggest that filgrastim affects IgE related responses, and may be useful in modulating allergic responses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.

    Science.gov (United States)

    Iqbal, Asif J; McNeill, Eileen; Kapellos, Theodore S; Regan-Komito, Daniel; Norman, Sophie; Burd, Sarah; Smart, Nicola; Machemer, Daniel E W; Stylianou, Elena; McShane, Helen; Channon, Keith M; Chawla, Ajay; Greaves, David R

    2014-10-09

    The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. © 2014 by The American Society of Hematology.

  19. NOD2 enhances the innate response of alveolar macrophages to Mycobacterium tuberculosis in humans.

    Science.gov (United States)

    Juárez, Esmeralda; Carranza, Claudia; Hernández-Sánchez, Fernando; León-Contreras, Juan C; Hernández-Pando, Rogelio; Escobedo, Dante; Torres, Martha; Sada, Eduardo

    2012-04-01

    A role for the nucleotide-binding oligomerization domain 2 (NOD2) receptor in pulmonary innate immune responses has recently been explored. In the present study, we investigated the role that NOD2 plays in human alveolar macrophage innate responses and determined its involvement in the response to infection with virulent Mycobacterium tuberculosis. Our results showed that NOD2 was expressed in human alveolar macrophages, and significant amounts of IL-1β, IL-6, and TNF-α were produced upon ligand recognition with muramyldipeptide (MDP). NOD2 ligation induced the transcription and protein expression of the antimicrobial peptide LL37 and the autophagy enzyme IRGM in alveolar macrophages, demonstrating a novel function for this receptor in these cells. MDP treatment of alveolar macrophages improved the intracellular growth control of virulent M. tuberculosis; this was associated with a significant release of TNF-α and IL-6 and overexpression of bactericidal LL37. In addition, the autophagy proteins IRGM, LC3 and ATG16L1 were recruited to the bacteria-containing autophagosome after treatment with MDP. In conclusion, our results suggest that NOD2 can modulate the innate immune response of alveolar macrophages and play a role in the initial control of respiratory M. tuberculosis infections. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. CCR8 Signaling Influences Toll-Like Receptor 4 Responses in Human Macrophages in Inflammatory Diseases ▿

    Science.gov (United States)

    Kvist Reimer, Martina; Brange, Charlotte; Rosendahl, Alexander

    2011-01-01

    CCR8 immunity is generally associated with Th2 responses in allergic diseases. In this study, we demonstrate for the first time a pronounced attenuated influx of macrophages in ovalbumin (OVA)-challenged CCR8 knockout mice. To explore whether macrophages in human inflamed lung tissue also were CCR8 positive, human lung tissue from patients with chronic obstructive pulmonary disease (COPD) was evaluated. Indeed, CCR8 expression was pronounced in invading monocytes/macrophages from lungs of patients with Global Initiative for Obstructive Lung Disease (GOLD) stage IV COPD. Given this expression pattern, the functional role of CCR8 on human macrophages was evaluated in vitro. Human peripheral blood monocytes expressed low levels of CCR8, while macrophage colony-stimulating factor (M-CSF)-derived human macrophages expressed significantly elevated surface levels of CCR8. Importantly, CCL1 directly regulated the expression of CD18 and CD49b and hence influenced the adhesion capacity of human macrophages. CCL1 drives chemotaxis in M-CSF-derived macrophages, and this could be completely inhibited by lipopolysaccharide (LPS). Whereas both CCL1 and LPS monotreatment inhibited spontaneous superoxide release in macrophages, CCL1 significantly induced superoxide release in the presence of LPS in a dose-dependent manner. Finally, CCL1 induced production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and could inhibit LPS-induced cytokine production in a dose-dependent manner. Our data demonstrate, for the first time, the presence of CCR8 on inflammatory macrophages in human COPD lung tissue. Importantly, the functional data from human macrophages suggest a potential cross talk between the CCR8 and the Toll-like receptor 4 (TLR4) pathways, both of which are present in COPD patients. PMID:21976223

  1. Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

    Science.gov (United States)

    Kuchiike, Daisuke; Uto, Yoshihiro; Mukai, Hirotaka; Ishiyama, Noriko; Abe, Chiaki; Tanaka, Daichi; Kawai, Tomohito; Kubo, Kentaro; Mette, Martin; Inui, Toshio; Endo, Yoshio; Hori, Hitoshi

    2013-07-01

    The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

  2. Foamy macrophages and the progression of the human TB granuloma

    Science.gov (United States)

    Russell, David G.; Cardona, Pere-Joan; Kim, Mi-Jeong; Allain, Sophie; Altare, Frédéric

    2009-01-01

    The progression of tuberculosis from a latent, sub-clinical infection to active disease that culminates in transmission of infectious bacilli is determined locally at the level of the granuloma. This progression takes place even in the face of a robust immune response that, while it contains infection, is unable to eliminate the bacterium. The factors or environmental conditions that influence this progression remain to be determined. Recent advances have indicated that pathogen-induced dysregulation of host lipid synthesis and sequestration plays a critical role in this transition. The foamy macrophage appears to be a key player in both sustaining persistent bacteria and contributing to the tissue pathology that leads to cavitation and release of infectious bacilli. PMID:19692995

  3. Cloning of a cDNA encoding chitotriosidase, a human chitinase produced by macrophages

    NARCIS (Netherlands)

    Boot, R. G.; Renkema, G. H.; Strijland, A.; van Zonneveld, A. J.; Aerts, J. M.

    1995-01-01

    We have recently observed that chitotriosidase, a chitinolytic enzyme, is secreted by activated human macrophages and is markedly elevated in plasma of Gaucher disease patients (Hollak, C. E. M., van Weely, S., van Oers, M. H. J., and Aerts, J. M. F. G. (1994) J. Clin. Invest. 93, 1288-1292). Here,

  4. 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.

  5. Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

    Science.gov (United States)

    Alvarado-Vazquez, Perla Abigail; Bernal, Laura; Paige, Candler A; Grosick, Rachel L; Moracho Vilrriales, Carolina; Ferreira, David Wilson; Ulecia-Morón, Cristina; Romero-Sandoval, E Alfonso

    2017-08-01

    M1 macrophages release proinflammatory factors during inflammation. They transit to an M2 phenotype and release anti-inflammatory factors to resolve inflammation. An imbalance in the transition from M1 to M2 phenotype in macrophages contributes to the development of persistent inflammation. CD163, a member of the scavenger receptor cysteine-rich family, is an M2 macrophage marker. The functional role of CD163 during the resolution of inflammation is not completely known. We postulate that CD163 contributes to the transition from M1 to M2 phenotype in macrophages. We induced CD163 gene in THP-1 and primary human macrophages using polyethylenimine nanoparticles grafted with a mannose ligand (Man-PEI). This nanoparticle specifically targets cells of monocytic origin via mannose receptors. Cells were challenged with a single or a double stimulation of lipopolysaccharide (LPS). A CD163 or empty plasmid was complexed with Man-PEI nanoparticles for cell transfections. Quantitative RT-PCR, immunocytochemistry, and ELISAs were used for molecular assessments. CD163-overexpressing macrophages displayed reduced levels of tumor necrosis factor-alpha (TNF)-α and monocytes chemoattractant protein (MCP)-1 after a single stimulation with LPS. Following a double stimulation paradigm, CD163-overexpressing macrophages showed an increase of interleukin (IL)-10 and IL-1ra and a reduction of MCP-1. This anti-inflammatory phenotype was partially blocked by an anti-CD163 antibody (effects on IL-10 and IL-1ra). A decrease in the release of TNF-α, IL-1β, and IL-6 was observed in CD163-overexpressing human primary macrophages. The release of IL-6 was blocked by an anti-CD163 antibody in the CD163-overexpressing group. Our data show that the induction of the CD163 gene in human macrophages under inflammatory conditions produces changes in cytokine secretion in favor of an anti-inflammatory phenotype. Targeting macrophages to induce CD163 using cell-directed nanotechnology is an attractive

  6. The Reactive Oxygen Species in Macrophage Polarization: Reflecting Its Dual Role in Progression and Treatment of Human Diseases

    Science.gov (United States)

    Tan, Hor-Yue; Li, Sha; Hong, Ming; Wang, Xuanbin

    2016-01-01

    High heterogeneity of macrophage is associated with its functions in polarization to different functional phenotypes depending on environmental cues. Macrophages remain in balanced state in healthy subject and thus macrophage polarization may be crucial in determining the tissue fate. The two distinct populations, classically M1 and alternatively M2 activated, representing the opposing ends of the full activation spectrum, have been extensively studied for their associations with several disease progressions. Accumulating evidences have postulated that the redox signalling has implication in macrophage polarization and the key roles of M1 and M2 macrophages in tissue environment have provided the clue for the reasons of ROS abundance in certain phenotype. M1 macrophages majorly clearing the pathogens and ROS may be crucial for the regulation of M1 phenotype, whereas M2 macrophages resolve inflammation which favours oxidative metabolism. Therefore how ROS play its role in maintaining the homeostatic functions of macrophage and in particular macrophage polarization will be reviewed here. We also review the biology of macrophage polarization and the disturbance of M1/M2 balance in human diseases. The potential therapeutic opportunities targeting ROS will also be discussed, hoping to provide insights for development of target-specific delivery system or immunomodulatory antioxidant for the treatment of ROS-related diseases. PMID:27143992

  7. Conservation of myeloid surface antigens on primate granulocytes.

    Science.gov (United States)

    Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

    1983-02-01

    Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

  8. Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages.

    Directory of Open Access Journals (Sweden)

    Punsiri M Colonne

    2016-10-01

    Full Text Available Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV. C. burnetii manipulates host cAMP-dependent protein kinase (PKA signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E mutant protein prevented optimal PV formation, whereas VASP (S157E mutant expression had no effect. VASP (S239E expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA

  9. Benzo(a)pyrene activation and detoxification by human pulmonary alveolar macrophages and lymphocytes

    International Nuclear Information System (INIS)

    Marshall, M.V.; McLemore, T.L.; Martin, R.R.; Marshall, M.H.; Wray, N.P.; Busbee, D.L.; Cantrell, E.T.; Arnott, M.S.; Griffin, A.C.

    1980-01-01

    Comparisons of pulmonary alveolar macrophages and circulating lymphocytes from five smokers and five nonsmokers for their ability to metabolize benzo(a)pyrene as determined by high pressure liquid chromatography were carried out. Utilizing this approach, further investigation of activation and detoxification by several human cell types could provide the basis for more precise and comprehensive studies of carcinogen and drug metabolism in the human lung, and for a better assessment of cancer risk in selected populations

  10. Synthesis of pro-inflammatory cytokines and adhesion molecules expression by the irradiated human monocyte/macrophage

    International Nuclear Information System (INIS)

    Pons, I.

    1997-09-01

    As lesions induced by ionizing radiations are essentially noticed in organs the functional and structural organisation of which depend on the highly proliferative stem cell pool, the author reports an in-vivo investigation of the effect of a gamma irradiation on the expression and secretion of pro-inflammatory cytokines par human monocytes/macrophages. In order to study the role of the cell environment in the radiation-induced inflammation, the author studied whether a co-stimulation of monocytes/macrophages by gamma irradiation, or the exposure of co-cultures of monocytes/macrophages and lymphocytes, could modulate the regulation of inflammatory cytokines. The author also studied the modulation of the expression of adhesion molecules mainly expressed by the monocyte/macrophage, and the membrane density of the CD14 receptor after irradiation of monocytes/macrophages during 24 hours, and of totally differentiated macrophages after seven days of culture

  11. Enhanced M1 macrophage polarization in human helicobacter pylori-associated atrophic gastritis and in vaccinated mice.

    Directory of Open Access Journals (Sweden)

    Marianne Quiding-Järbrink

    Full Text Available BACKGROUND: Infection with Helicobacter pylori triggers a chronic gastric inflammation that can progress to atrophy and gastric adenocarcinoma. Polarization of macrophages is a characteristic of both cancer and infection, and may promote progression or resolution of disease. However, the role of macrophages and their polarization during H. pylori infection has not been well defined. METHODOLOGY/PRINCIPAL FINDINGS: By using a mouse model of infection and gastric biopsies from 29 individuals, we have analyzed macrophage recruitment and polarization during H. pylori infection by flow cytometry and real-time PCR. We found a sequential recruitment of neutrophils, eosinophils and macrophages to the gastric mucosa of infected mice. Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination. Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages. However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion. CONCLUSIONS/SIGNIFICANCE: These results show that vaccination of mice against H. pylori amplifies M1 polarization of gastric macrophages, and that a similar enhanced M1 polarization is present in human H. pylori-induced atrophic gastritis.

  12. Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages : similarities and differences

    NARCIS (Netherlands)

    Martinez, Fernando O.; Helming, Laura; Milde, Ronny; Varin, Audrey; Melgert, Barbro N.; Draijer, Christina; Thomas, Benjamin; Fabbri, Marco; Crawshaw, Anjali; Ho, Ling Pei; Ten Hacken, Nick H.; Jimenez, Viviana Cobos; Kootstra, Neeltje A.; Hamann, Jorg; Greaves, David R.; Locati, Massimo; Mantovani, Alberto; Gordon, Siamon

    2013-01-01

    The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4-activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues.

  13. Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences

    NARCIS (Netherlands)

    Martinez, Fernando O.; Helming, Laura; Milde, Ronny; Varin, Audrey; Melgert, Barbro N.; Draijer, Christina; Thomas, Benjamin; Fabbri, Marco; Crawshaw, Anjali; Ho, Ling Pei; ten Hacken, Nick H.; Cobos Jiménez, Viviana; Kootstra, Neeltje A.; Hamann, Jörg; Greaves, David R.; Locati, Massimo; Mantovani, Alberto; Gordon, Siamon

    2013-01-01

    The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4-activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues.

  14. Vitamin D Is Required for IFN-γ–Mediated Antimicrobial Activity of Human Macrophages

    Science.gov (United States)

    Fabri, Mario; Stenger, Steffen; Shin, Dong-Min; Yuk, Jae-Min; Liu, Philip T.; Realegeno, Susan; Lee, Hye-Mi; Krutzik, Stephan R.; Schenk, Mirjam; Sieling, Peter A.; Teles, Rosane; Montoya, Dennis; Iyer, Shankar S.; Bruns, Heiko; Lewinsohn, David M.; Hollis, Bruce W.; Hewison, Martin; Adams, John S.; Steinmeyer, Andreas; Zügel, Ulrich; Cheng, Genhong; Jo, Eun-Kyeong; Bloom, Barry R.; Modlin, Robert L.

    2012-01-01

    Control of tuberculosis worldwide depends on our understanding of human immune mechanisms, which combat the infection. Acquired T cell responses are critical for host defense against microbial pathogens, yet the mechanisms by which they act in humans remain unclear. We report that T cells, by the release of interferon-γ (IFN-γ), induce autophagy, phagosomal maturation, the production of antimicrobial peptides such as cathelicidin, and antimicrobial activity against Mycobacterium tuberculosis in human macrophages via a vitamin D–dependent pathway. IFN-γ induced the antimicrobial pathway in human macrophages cultured in vitamin D–sufficient sera, but not in sera from African-Americans that have lower amounts of vitamin D and who are more susceptible to tuberculosis. In vitro supplementation of vitamin D–deficient serum with 25-hydroxyvitamin D3 restored IFN-γ–induced antimicrobial peptide expression, autophagy, phagosome-lysosome fusion, and antimicrobial activity. These results suggest a mechanism in which vitamin D is required for acquired immunity to overcome the ability of intracellular pathogens to evade macrophage-mediated antimicrobial responses. The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection. PMID:21998409

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

    Science.gov (United States)

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

    2012-03-01

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

  16. Efficient internalization of mesoporous silica particles of different sizes by primary human macrophages without impairment of macrophage clearance of apoptotic or antibody-opsonized target cells

    International Nuclear Information System (INIS)

    Witasp, Erika; Kupferschmidt, Natalia; Bengtsson, Linnea; Hultenby, Kjell; Smedman, Christian; Paulie, Staffan; Garcia-Bennett, Alfonso E.; Fadeel, Bengt

    2009-01-01

    Macrophage recognition and ingestion of apoptotic cell corpses, a process referred to as programmed cell clearance, is of considerable importance for the maintenance of tissue homeostasis and in the resolution of inflammation. Moreover, macrophages are the first line of defense against microorganisms and other foreign materials including particles. However, there is sparse information on the mode of uptake of engineered nanomaterials by primary macrophages. In this study, mesoporous silica particles with cubic pore geometries and covalently fluorescein-grafted particles were synthesized through a novel route, and their interactions with primary human monocyte-derived macrophages were assessed. Efficient and active internalization of mesoporous silica particles of different sizes was observed by transmission electron microscopic and flow cytometric analysis and studies using pharmacological inhibitors suggested that uptake occurred through a process of endocytosis. Moreover, uptake of silica particles was independent of serum factors. The silica particles with very high surface areas due to their porous structure did not impair cell viability or function of macrophages, including the ingestion of different classes of apoptotic or opsonized target cells. The current findings are relevant to the development of mesoporous materials for drug delivery and other biomedical applications.

  17. 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.

  18. Ocimum basilicum ethanolic extract decreases cholesterol synthesis and lipid accumulation in human macrophages.

    Science.gov (United States)

    Bravo, Elena; Amrani, Souliman; Aziz, Mohammed; Harnafi, Hicham; Napolitano, Mariarosaria

    2008-12-01

    Macrophage lipid accumulation induced by low density lipoproteins (LDL) plays a pivotal role in atherosclerotic plaque development. Previous work showed that Ocimum basilicum extract, used as hypocholesterolemic agent by traditional medicine in Morocco, has hypolipidemic activity in rat acute hyperlipimidemia. This study investigated the effects of ethanolic extract of O. basilicum on lipid accumulation in human macrophages. As modification of LDL increase atherogenicity of the particles we evaluated the effects of the extract on LDL oxidation. The extract caused a dose-related increase of LDL-resistance to Cu(2+)-induced oxidation. Furthermore, at the dose of 60 microg/ml, significantly decreases the accumulation of macrophage lipid droplets induced by modified LDL evaluated as by red-oil staining. Cholesterol esterification and triacylglycerol synthesis in the cells were not affected. Macrophage treatment with 60 microg/ml, but not 20 microg/ml, of the extract reduced newly synthesized unesterified cholesterol by about 60% and decreased scavenger receptors activity by about 20-30%, evaluated by the internalization of cholesterol carried by [(3)H]CE-aggregated-LDL. The results suggest that O. basilicum ethanolic extract has the capability to reduce foam cell formation through the reduction of cholesterol synthesis and the modulation of the activity of surface scavenger receptors.

  19. Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

    Science.gov (United States)

    Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

    2014-04-01

    Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Rats and mice immunised with chimeric human/mouse proteinase 3 produce autoantibodies to mouse Pr3 and rat granulocytes

    NARCIS (Netherlands)

    van der Geld, Ymke M.; Hellmark, Thomas; Selga, Daina; Heeringa, Peter; Huitema, Minke G.; Limburg, Pieter C.; Kallenberg, Cees G. M.

    2007-01-01

    Aim: In this study, we employed chimeric human/ mouse Proteinase 3 ( PR3) proteins as tools to induce an autoantibody response to PR3 in rats and mice. Method: Rats and mice were immunised with recombinant human PR3 ( HPR3), recombinant murine PR3 ( mPR3), single chimeric human/ mouse PR3 ( HHm,

  1. Influence of ER leak on resting cytoplasmic Ca2+ and receptor-mediated Ca2+ signalling in human macrophage.

    Science.gov (United States)

    Layhadi, Janice A; Fountain, Samuel J

    2017-06-03

    Mechanisms controlling endoplasmic reticulum (ER) Ca 2+ homeostasis are important regulators of resting cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyto ) and receptor-mediated Ca 2+ signalling. Here we investigate channels responsible for ER Ca 2+ leak in THP-1 macrophage and human primary macrophage. In the absence of extracellular Ca 2+ we employ ionomycin action at the plasma membrane to stimulate ER Ca 2+ leak. Under these conditions ionomycin elevates [Ca 2+ ] cyto revealing a Ca 2+ leak response which is abolished by thapsigargin. IP 3 receptors (Xestospongin C, 2-APB), ryanodine receptors (dantrolene), and translocon (anisomycin) inhibition facilitated ER Ca 2+ leak in model macrophage, with translocon inhibition also reducing resting [Ca 2+ ] cyto . In primary macrophage, translocon inhibition blocks Ca 2+ leak but does not influence resting [Ca 2+ ] cyto . We identify a role for translocon-mediated ER Ca 2+ leak in receptor-mediated Ca 2+ signalling in both model and primary human macrophage, whereby the Ca 2+ response to ADP (P2Y receptor agonist) is augmented following anisomycin treatment. In conclusion, we demonstrate a role of ER Ca 2+ leak via the translocon in controlling resting cytoplasmic Ca 2+ in model macrophage and receptor-mediated Ca 2+ signalling in model macrophage and primary macrophage. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

    Science.gov (United States)

    Nair, Nisha R; Chidambareswaren, M; Manjula, S

    2014-09-01

    Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

  3. Unlike PPARγ, PPARα or PPARβ/δ activation does not promote human monocyte differentiation toward alternative macrophages

    International Nuclear Information System (INIS)

    Bouhlel, Mohamed Amine; Brozek, John; Derudas, Bruno; Zawadzki, Christophe; Jude, Brigitte; Staels, Bart; Chinetti-Gbaguidi, Giulia

    2009-01-01

    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 PPARγ promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPARβ/δ in this process has been reported only in mice and no data are available for PPARα. Here, we show that in contrast to PPARγ, expression of PPARα and PPARβ/δ 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 PPARγ, PPARα or PPARβ/δ activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPARα and PPARβ/δ do not appear to modulate the alternative differentiation of human macrophages.

  4. Direct anti-inflammatory effects of granulocyte colony-stimulating factor (G-CSF) on activation and functional properties of human T cell subpopulations in vitro.

    Science.gov (United States)

    Malashchenko, Vladimir Vladimirovich; Meniailo, Maxsim Evgenievich; Shmarov, Viacheslav Anatolievich; Gazatova, Natalia Dinislamovna; Melashchenko, Olga Borisovna; Goncharov, Andrei Gennadievich; Seledtsova, Galina Victorovna; Seledtsov, Victor Ivanovich

    2018-03-01

    We investigated the direct effects of human granulocyte colony-stimulating factor (G-CSF) on functionality of human T-cell subsets. CD3 + T-lymphocytes were isolated from blood of healthy donors by positive magnetic separation. T cell activation with particles conjugated with antibodies (Abs) to human CD3, CD28 and CD2 molecules increased the proportion of cells expressing G-CSF receptor (G-CSFR, CD114) in all T cell subpopulations studied (CD45RA + /CD197 + naive T cells, CD45RA - /CD197 + central memory T cells, CD45RA - /CD197 - effector memory T cells and CD45RA + /CD197 - terminally differentiated effector T cells). Upon T-cell activation in vitro, G-CSF (10.0 ng/ml) significantly and specifically enhanced the proportion of CD114 + T cells in central memory CD4 + T cell compartment. A dilution series of G-CSF (range, 0.1-10.0 ng/ml) was tested, with no effect on the expression of CD25 (interleukin-2 receptor α-chain) on activated T cells. Meanwhile, G-CSF treatment enhanced the proportion of CD38 + T cells in CD4 + naïve T cell, effector memory T cell and terminally differentiated effector T cell subsets, as well as in CD4 - central memory T cells and terminally differentiated effector T cells. G-CSF did not affect IL-2 production by T cells; relatively low concentrations of G-CSF down-regulated INF-γ production, while high concentrations of this cytokine up-regulated IL-4 production in activated T cells. The data obtained suggests that G-CSF could play a significant role both in preventing the development of excessive and potentially damaging inflammatory reactivity, and in constraining the expansion of potentially cytodestructive T cells. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Anti-Inflammatory Effects of Lactobacillus Rahmnosus and Bifidobacterium Breve on Cigarette Smoke Activated Human Macrophages.

    Science.gov (United States)

    Mortaz, Esmaeil; Adcock, Ian M; Ricciardolo, Fabio L M; Varahram, Mohammad; Jamaati, Hamidreza; Velayati, Ali Akbar; Folkerts, Gert; Garssen, Johan

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is a major global health problem with cigarette smoke (CS) as the main risk factor for its development. Airway inflammation in COPD involves the increased expression of inflammatory mediators such as CXCL-8 and IL-1β which are important mediators for neutrophil recruitment. Macrophages are an important source of these mediators in COPD. Lactobacillus rhamnosus (L. rhamnosus) and Befidobacterium breve (B. breve) attenuate the development of 'allergic asthma' in animals but their effects in COPD are unknown. To determine the anti-inflammatory effects of L. rhamnosus and B. breve on CS and Toll-like receptor (TLR) activation. We stimulated the human macrophage cell line THP-1 with CS extract in the presence and absence of L. rhamnosus and B. breve and measured the expression and release of inflammatory mediators by RT-qPCR and ELISA respectively. An activity assay and Western blotting were used to examine NF-κB activation. Both L. rhamnosus and B. breve were efficiently phagocytized by human macrophages. L. rhamnosus and B. breve significantly suppressed the ability of CS to induce the expression of IL-1β, IL-6, IL-10, IL-23, TNFα, CXCL-8 and HMGB1 release (all p<0.05) in human THP-1 macrophages. Similar suppression of TLR4- and TLR9-induced CXCL8 expression was also observed (p<0.05). The effect of L. rhamnosus and B. breve on inflammatory mediator release was associated with the suppression of CS-induced NF-κB activation (p<0.05). This data indicate that these probiotics may be useful anti-inflammatory agents in CS-associated disease such as COPD.

  6. Anti-Inflammatory Effects of Lactobacillus Rahmnosus and Bifidobacterium Breve on Cigarette Smoke Activated Human Macrophages.

    Directory of Open Access Journals (Sweden)

    Esmaeil Mortaz

    Full Text Available Chronic obstructive pulmonary disease (COPD is a major global health problem with cigarette smoke (CS as the main risk factor for its development. Airway inflammation in COPD involves the increased expression of inflammatory mediators such as CXCL-8 and IL-1β which are important mediators for neutrophil recruitment. Macrophages are an important source of these mediators in COPD. Lactobacillus rhamnosus (L. rhamnosus and Befidobacterium breve (B. breve attenuate the development of 'allergic asthma' in animals but their effects in COPD are unknown.To determine the anti-inflammatory effects of L. rhamnosus and B. breve on CS and Toll-like receptor (TLR activation.We stimulated the human macrophage cell line THP-1 with CS extract in the presence and absence of L. rhamnosus and B. breve and measured the expression and release of inflammatory mediators by RT-qPCR and ELISA respectively. An activity assay and Western blotting were used to examine NF-κB activation.Both L. rhamnosus and B. breve were efficiently phagocytized by human macrophages. L. rhamnosus and B. breve significantly suppressed the ability of CS to induce the expression of IL-1β, IL-6, IL-10, IL-23, TNFα, CXCL-8 and HMGB1 release (all p<0.05 in human THP-1 macrophages. Similar suppression of TLR4- and TLR9-induced CXCL8 expression was also observed (p<0.05. The effect of L. rhamnosus and B. breve on inflammatory mediator release was associated with the suppression of CS-induced NF-κB activation (p<0.05.This data indicate that these probiotics may be useful anti-inflammatory agents in CS-associated disease such as COPD.

  7. Human macrophage scavenger receptors: Primary structure, expression, and localization in atherosclerotic lesions

    International Nuclear Information System (INIS)

    Matsumoto, Akiyo; Itakura, Hiroshige; Kodama, Tatsuhiko; Naito, Makoto; Takahashi, Kiyoshi; Ikemoto, Shinji; Asaoka, Hitoshi; Hayakawa, Ikuho; Kanamori, Hiroshi; Takaku, Fumimaro; Aburatani, Hiroyuki; Suzuki, Hiroshi; Kobari, Yukage; Miyai, Tatsuya; Cohen, E.H.; Wydro, R.; Housman, D.E.

    1990-01-01

    Two types of cDNAs for human macrophage scavenger receptors were cloned from a cDNA library derived from the phorbol ester-treated human monocytic cell line THP-1. The type I and type II human scavenger receptors encoded by these cDNAs are homologous (73% and 71% amino acid identity) to their previously characterized bovine counterparts and consist of six domains: cytoplasmic (I), membrane-spanning (II), spacer (III), α-helical coiled-coil (IV), collagen-like (V), and a type-specific C-terminal (VI). The receptor gene is located on human chromosome 8. The human receptors expressed in CHO-K1 cells mediated endocytosis of modified low density lipoproteins. Two mRNAs, 4.0 and 3.2 kilobases, have been detected in human liver, placenta, and brain. Immunohistochemical studies using an anti-peptide antibody which recognizes human scavenger receptors indicated the presence of the scavenger receptors in the macrophages of lipid-rich atherosclerotic lesions, suggesting the involvement of scavenger receptors in atherogenesis

  8. In vitro cytotoxicity of Manville Code 100 glass fibers: Effect of fiber length on human alveolar macrophages

    Directory of Open Access Journals (Sweden)

    Jones William

    2006-03-01

    Full Text Available Abstract Background Synthetic vitreous fibers (SVFs are inorganic noncrystalline materials widely used in residential and industrial settings for insulation, filtration, and reinforcement purposes. SVFs conventionally include three major categories: fibrous glass, rock/slag/stone (mineral wool, and ceramic fibers. Previous in vitro studies from our laboratory demonstrated length-dependent cytotoxic effects of glass fibers on rat alveolar macrophages which were possibly associated with incomplete phagocytosis of fibers ≥ 17 μm in length. The purpose of this study was to examine the influence of fiber length on primary human alveolar macrophages, which are larger in diameter than rat macrophages, using length-classified Manville Code 100 glass fibers (8, 10, 16, and 20 μm. It was hypothesized that complete engulfment of fibers by human alveolar macrophages could decrease fiber cytotoxicity; i.e. shorter fibers that can be completely engulfed might not be as cytotoxic as longer fibers. Human alveolar macrophages, obtained by segmental bronchoalveolar lavage of healthy, non-smoking volunteers, were treated with three different concentrations (determined by fiber number of the sized fibers in vitro. Cytotoxicity was assessed by monitoring cytosolic lactate dehydrogenase release and loss of function as indicated by a decrease in zymosan-stimulated chemiluminescence. Results Microscopic analysis indicated that human alveolar macrophages completely engulfed glass fibers of the 20 μm length. All fiber length fractions tested exhibited equal cytotoxicity on a per fiber basis, i.e. increasing lactate dehydrogenase and decreasing chemiluminescence in the same concentration-dependent fashion. Conclusion The data suggest that due to the larger diameter of human alveolar macrophages, compared to rat alveolar macrophages, complete phagocytosis of longer fibers can occur with the human cells. Neither incomplete phagocytosis nor length-dependent toxicity was

  9. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages.

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    Weibin Zha

    Full Text Available HIV protease inhibitor (PI-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR, a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp in HIV PI-mediated accumulation of BBR in macrophages.Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT and human P-gp transfected (MDCK/P-gp cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123 efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

  10. Matrix metalloproteinase-12 gene regulation by a PPAR alpha agonist in human monocyte-derived macrophages

    International Nuclear Information System (INIS)

    Souissi, Imen Jguirim; Billiet, Ludivine; Cuaz-Perolin, Clarisse; Slimane, Mohamed-Naceur; Rouis, Mustapha

    2008-01-01

    MMP-12, a macrophage-specific matrix metalloproteinase with large substrate specificity, has been reported to be highly expressed in mice, rabbits and human atherosclerotic lesions. Increased MMP-12 from inflammatory macrophages is associated with several degenerative diseases such as atherosclerosis. In this manuscript, we show that IL-1β, a proinflammatory cytokine found in atherosclerotic plaques, increases both mRNA and protein levels of MMP-12 in human monocyte-derived macrophages (HMDM). Since peroxisome proliferator-activated receptors (PPARs), such as PPARα and PPARγ, are expressed in macrophages and because PPAR activation exerts an anti-inflammatory effect on vascular cells, we have investigated the effect of PPARα and γ isoforms on MMP-12 regulation in HMDM. Our results show that MMP-12 expression (mRNA and protein) is down regulated in IL-1β-treated macrophages only in the presence of a specific PPARα agonist, GW647, in a dose-dependent manner. In contrast, this inhibitory effect was abolished in IL-1β-stimulated peritoneal macrophages isolated from PPARα -/- mice and treated with the PPARα agonist, GW647. Moreover, reporter gene transfection experiments using different MMP-12 promoter constructs showed a reduction of the promoter activities by ∼ 50% in IL-1β-stimulated PPARα-pre-treated cells. However, MMP-12 promoter analysis did not reveal the presence of a PPRE response element. The IL-1β effect is known to be mediated through the AP-1 binding site. Mutation of the AP-1 site, located at - 81 in the MMP-12 promoter region relative to the transcription start site, followed by transfection analysis, gel shift and ChIP experiments revealed that the inhibitory effect was the consequence of the protein-protein interaction between GW 647-activated PPARα and c-Fos or c-Jun transcription factors, leading to inhibition of their binding to the AP-1 motif. These studies suggest that PPARα agonists may be used therapeutically, not only for lipid

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

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    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.

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

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    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.

  13. Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity.

    Directory of Open Access Journals (Sweden)

    Svantje Tauber

    Full Text Available The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC-TOF-MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface-bound fucose. The reduced ICAM-1 expression and the loss of cell surface-bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small

  14. Evaluation of a nanotechnology-based approach to induce gene-expression in human THP-1 macrophages under inflammatory conditions.

    Science.gov (United States)

    Bernal, Laura; Alvarado-Vázquez, Abigail; Ferreira, David Wilson; Paige, Candler A; Ulecia-Morón, Cristina; Hill, Bailey; Caesar, Marina; Romero-Sandoval, E Alfonso

    2017-02-01

    Macrophages orchestrate the initiation and resolution of inflammation by producing pro- and anti-inflammatory products. An imbalance in these mediators may originate from a deficient or excessive immune response. Therefore, macrophages are valid therapeutic targets to restore homeostasis under inflammatory conditions. We hypothesize that a specific mannosylated nanoparticle effectively induces gene expression in human macrophages under inflammatory conditions without undesirable immunogenic responses. THP-1 macrophages were challenged with lipopolysaccharide (LPS, 5μg/mL). Polyethylenimine (PEI) nanoparticles grafted with a mannose receptor ligand (Man-PEI) were used as a gene delivery method. Nanoparticle toxicity, Man-PEI cellular uptake rate and gene induction efficiency (GFP, CD14 or CD68) were studied. Potential immunogenic responses were evaluated by measuring the production of tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-10. Man-PEI did not produce cytotoxicity, and it was effectively up-taken by THP-1 macrophages (69%). This approach produced a significant expression of GFP (mRNA and protein), CD14 and CD68 (mRNA), and transiently and mildly reduced IL-6 and IL-10 levels in LPS-challenged macrophages. Our results indicate that Man-PEI is suitable for inducing an efficient gene overexpression in human macrophages under inflammatory conditions with limited immunogenic responses. Our promising results set the foundation to test this technology to induce functional anti-inflammatory genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  15. Enhanced uptake of multiple sclerosis-derived myelin by THP-1 macrophages and primary human microglia.

    Science.gov (United States)

    Hendrickx, Debbie A E; Schuurman, Karianne G; van Draanen, Michael; Hamann, Jörg; Huitinga, Inge

    2014-03-31

    The pathological hallmark of multiple sclerosis (MS) is myelin phagocytosis. It remains unclear why microglia and macrophages demyelinate axons in MS, but previously found or yet-unknown changes in the myelin of MS patients could contribute to this process. We therefore studied whether myelin from normal-appearing white matter (NAWM) of MS donors is phagocytosed more efficiently than myelin from control donors. Myelin was isolated from 11 MS and 12 control brain donors and labeled with the pH-sensitive fluorescent dye pHrodo to quantify uptake in lysosomes. Phagocytosis by differentiated THP-1 macrophages and by primary human microglia was quantified with flow cytometry. Whereas myelin uptake by THP-1 macrophages reached a plateau after approximately 24 hours, uptake by primary human microglia showed an almost linear increase over a 72-hour period. Data were statistically analyzed with the Mann-Whitney U test. MS-derived myelin was phagocytosed more efficiently by THP-1 macrophages after 6-hour incubation (P = 0.001 for the percentage of myelin-phagocytosing cells and P = 0.0005 for total myelin uptake) and after 24-hour incubation (P = 0.0006 and P = 0.0001, respectively), and by microglia after 24-hour incubation (P = 0.0106 for total myelin uptake). This enhanced uptake was not due to differences in the oxidation status of the myelin. Interestingly, myelin phagocytosis correlated negatively with the age of myelin donors, whereas the age of microglia donors showed a positive trend with myelin phagocytosis. Myelin isolated from normal-appearing white matter of MS donors was phagocytosed more efficiently than was myelin isolated from control brain donors by both THP-1 macrophages and primary human microglia. These data indicate that changes in MS myelin might precede phagocyte activation and subsequent demyelination in MS. Identifying these myelin changes responsible for enhancing phagocytic ability could be an interesting therapeutic target to

  16. Apoptotic death of Listeria monocytogenes-infected human macrophages induced by lactoferricin B, a bovine lactoferrin-derived peptide.

    Science.gov (United States)

    Longhi, C; Conte, M P; Ranaldi, S; Penta, M; Valenti, P; Tinari, A; Superti, F; Seganti, L

    2005-01-01

    Listeria monocytogenes, an intracellular facultative food-borne pathogen, was reported to induce apoptosis in vitro and in vivo in a variety of cell types with the exception of murine macrophages. These cells represent the predominant compartment of bacterial multiplication and die as a result of necrosis. In this study we showed that human non-activated and IFN-gamma-activated macrophagic-like (THP-1) cells infected with L. monocytogenes, mainly die by necrosis rather than by an apoptotic process. Two natural products derived from bovine milk, lactoferrin and its derivative peptide lactoferricin B, are capable of regulating the fate of infected human macrophages. Bovine lactoferrin treatment of macrophages protects them from L. monocytogenes-induced death whereas lactoferricin B, its derivative peptide, determines a shifting of the equilibrium from necrosis to apoptosis.

  17. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    Science.gov (United States)

    Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Renée; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle; Beauséjour, Christian; Stroncek, David; Le Deist, Françoise; Haddad, Elie

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation. PMID:27070086

  18. Interaction of differentiated human adipocytes with macrophages leads to trogocytosis and selective IL-6 secretion.

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    Sárvári, A K; Doan-Xuan, Q-M; Bacsó, Z; Csomós, I; Balajthy, Z; Fésüs, L

    2015-01-22

    Obesity leads to adipose tissue inflammation that is characterized by increased release of proinflammatory molecules and the recruitment of activated immune cells. Although macrophages are present in the highest number among the immune cells in obese adipose tissue, not much is known about their direct interaction with adipocytes. We have introduced an ex vivo experimental system to characterize the cellular interactions and the profile of secreted cytokines in cocultures of macrophages and human adipocytes differentiated from either mesenchymal stem cells or a preadipocyte cell line. As observed by time-lapse microscopy, flow, and laser-scanning cytometry, macrophages phagocytosed bites of adipocytes (trogocytosis), which led to their de novo, phagocytosis and NF-κB-dependent synthesis, then release of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1. IL-6 secretion was not accompanied by secretion of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-8, except MCP-1. LPS-induced release of TNF-α, IL-8 and MCP-1 was decreased in the presence of the differentiated adipocytes but the IL-6 level did not subside suggesting that phagocytosis-dependent IL-6 secretion may have significant regulatory function in the inflamed adipose tissue.

  19. Quantification of sterol-specific response in human macrophages using automated imaged-based analysis.

    Science.gov (United States)

    Gater, Deborah L; Widatalla, Namareq; Islam, Kinza; AlRaeesi, Maryam; Teo, Jeremy C M; Pearson, Yanthe E

    2017-12-13

    The transformation of normal macrophage cells into lipid-laden foam cells is an important step in the progression of atherosclerosis. One major contributor to foam cell formation in vivo is the intracellular accumulation of cholesterol. Here, we report the effects of various combinations of low-density lipoprotein, sterols, lipids and other factors on human macrophages, using an automated image analysis program to quantitatively compare single cell properties, such as cell size and lipid content, in different conditions. We observed that the addition of cholesterol caused an increase in average cell lipid content across a range of conditions. All of the sterol-lipid mixtures examined were capable of inducing increases in average cell lipid content, with variations in the distribution of the response, in cytotoxicity and in how the sterol-lipid combination interacted with other activating factors. For example, cholesterol and lipopolysaccharide acted synergistically to increase cell lipid content while also increasing cell survival compared with the addition of lipopolysaccharide alone. Additionally, ergosterol and cholesteryl hemisuccinate caused similar increases in lipid content but also exhibited considerably greater cytotoxicity than cholesterol. The use of automated image analysis enables us to assess not only changes in average cell size and content, but also to rapidly and automatically compare population distributions based on simple fluorescence images. Our observations add to increasing understanding of the complex and multifactorial nature of foam-cell formation and provide a novel approach to assessing the heterogeneity of macrophage response to a variety of factors.

  20. Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors

    International Nuclear Information System (INIS)

    Zanocco-Marani, Tommaso; Vignudelli, Tatiana; Gemelli, Claudia; Pirondi, Sara; Testa, Anna; Montanari, Monica; Parenti, Sandra; Tenedini, Elena; Grande, Alexis; Ferrari, Sergio

    2006-01-01

    The MItf-Tfe family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage

  1. Mobilization of primitive and committed hematopoietic progenitors in nonhuman primates treated with defibrotide and recombinant human granulocyte colony-stimulating factor.

    Science.gov (United States)

    Carlo-Stella, Carmelo; Di Nicola, Massimo; Longoni, Paolo; Milani, Raffaella; Milanesi, Marco; Guidetti, Anna; Haanstra, Krista; Jonker, Margaret; Cleris, Loredana; Magni, Michele; Formelli, Franca; Gianni, Alesssandro M

    2004-01-01

    The aim of this study was to evaluate the capacity of defibrotide in enhancing cytokine-induced hematopoietic mobilization in rhesus monkeys. Animals received recombinant human granulocyte colony-stimulating factor (rhG-CSF, 100 microg/kg/day SC for 5 days) and, after a 4- to 6-week washout period, were remobilized with defibrotide (15 mg/kg/hour continuous intravenous for 5 days) plus rhG-CSF. Hematopoietic mobilization was evaluated by complete blood counts, differential counts, as well as frequency and absolute numbers of colony-forming cells (CFCs), high-proliferative potential CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). Compared to baseline values, rhG-CSF increased circulating CFCs, HPP-CFCs, and LTC-ICs by 158-, 125-, and 67-fold, respectively; the same figures for defibrotide/rhG-CSF were 299-, 1452-, and 295-fold, respectively. Defibrotide/rhG-CSF treatment compared to rhG-CSF alone increased CFCs, HPP-CFCs, and LTC-ICs by 1.4- (35,089 vs 25,825, pdefibrotide treatment associated with a 5-day rhG-CSF treatment. Compared to rhG-CSF, defibrotide/rhG-CSF increased the mobilization of CFCs, HPP-CFCs, and LTC-ICs by 2- (31,128 vs 15,527, pdefibrotide enhances rhG-CSF-elicited mobilization of primitive and committed progenitors; and 2) a 2-day defibrotide injection is as effective as a 5-day injection.

  2. Imatinib and Nilotinib Off-Target Effects on Human NK Cells, Monocytes, and M2 Macrophages.

    Science.gov (United States)

    Bellora, Francesca; Dondero, Alessandra; Corrias, Maria Valeria; Casu, Beatrice; Regis, Stefano; Caliendo, Fabio; Moretta, Alessandro; Cazzola, Mario; Elena, Chiara; Vinti, Luciana; Locatelli, Franco; Bottino, Cristina; Castriconi, Roberta

    2017-08-15

    Tyrosine kinase inhibitors (TKIs) are used in the clinical management of hematological neoplasms. Moreover, in solid tumors such as stage 4 neuroblastomas (NB), imatinib showed benefits that might depend on both on-target and immunological off-target effects. We investigated the effects of imatinib and nilotinib on human NK cells, monocytes, and macrophages. High numbers of monocytes died upon exposure to TKI concentrations similar to those achieved in patients. Conversely, NK cells were highly resistant to the TKI cytotoxic effect, were properly activated by immunostimulatory cytokines, and degranulated in the presence of NB cells. In NB, neither drug reduced the expression of ligands for activating NK receptors or upregulated that of HLA class I, B7-H3, PD-L1, and PD-L2, molecules that might limit NK cell function. Interestingly, TKIs modulated the chemokine receptor repertoire of immune cells. Acting at the transcriptional level, they increased the surface expression of CXCR4, an effect observed also in NK cells and monocytes of patients receiving imatinib for chronic myeloid leukemia. Moreover, TKIs reduced the expression of CXCR3 (in NK cells) and CCR1 (in monocytes). Monocytes also decreased the expression of M-CSFR, and low numbers of cells underwent differentiation toward macrophages. M0 and M2 macrophages were highly resistant to TKIs and maintained their phenotypic and functional characteristics. Importantly, also in the presence of TKIs, the M2 immunosuppressive polarization was reverted by TLR engagement, and M1-oriented macrophages fully activated autologous NK cells. Our results contribute to better interpreting the off-target efficacy of TKIs in tumors and to envisaging strategies aimed at facilitating antitumor immune responses. Copyright © 2017 by The American Association of Immunologists, Inc.

  3. Viral Inhibition of Bacterial Phagocytosis by Human Macrophages: Redundant Role of CD36.

    Directory of Open Access Journals (Sweden)

    Grace E Cooper

    Full Text Available Macrophages are essential to maintaining lung homoeostasis and recent work has demonstrated that influenza-infected lung macrophages downregulate their expression of the scavenger receptor CD36. This receptor has also been shown to be involved in phagocytosis of Streptococcus pneumoniae, a primary agent associated with pneumonia secondary to viral infection. The aim of this study was to investigate the role of CD36 in the effects of viral infection on macrophage phagocytic function. Human monocyte-derived macrophages (MDM were exposed to H3N2 X31 influenza virus, M37 respiratory syncytial virus (RSV or UV-irradiated virus. No infection of MDM was seen upon exposure to UV-irradiated virus but incubation with live X31 or M37 resulted in significant levels of viral detection by flow cytometry or RT-PCR respectively. Infection resulted in significantly diminished uptake of S. pneumoniae by MDM and significantly decreased expression of CD36 at both the cell surface and mRNA level. Concurrently, there was a significant increase in IFNβ gene expression in response to infection and we observed a significant decrease in bacterial phagocytosis (p = 0.031 and CD36 gene expression (p = 0.031 by MDM cultured for 24 h in 50IU/ml IFNβ. Knockdown of CD36 by siRNA resulted in decreased phagocytosis, but this was mimicked by transfection reagent alone. When MDM were incubated with CD36 blocking antibodies no effect on phagocytic ability was observed. These data indicate that autologous IFNβ production by virally-infected cells can inhibit bacterial phagocytosis, but that decreased CD36 expression by these cells does not play a major role in this functional deficiency.

  4. Mycobacterium tuberculosis decreases human macrophage IFN-γ responsiveness through miR-132 and miR-26a.

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    Ni, Bin; Rajaram, Murugesan V S; Lafuse, William P; Landes, Michelle B; Schlesinger, Larry S

    2014-11-01

    IFN-γ-activated macrophages play an essential role in controlling intracellular pathogens; however, macrophages also serve as the cellular home for the intracellular pathogen Mycobacterium tuberculosis. Based on previous evidence that M. tuberculosis can modulate host microRNA (miRNA) expression, we examined the miRNA expression profile of M. tuberculosis-infected primary human macrophages. We identified 31 differentially expressed miRNAs in primary human macrophages during M. tuberculosis infection by NanoString and confirmed our findings by quantitative real-time RT-PCR. In addition, we determined a role for two miRNAs upregulated upon M. tuberculosis infection, miR-132 and miR-26a, as negative regulators of transcriptional coactivator p300, a component of the IFN-γ signaling cascade. Knockdown expression of miR-132 and miR-26a increased p300 protein levels and improved transcriptional, translational, and functional responses to IFN-γ in human macrophages. Collectively, these data validate p300 as a target of miR-132 and miR-26a, and demonstrate a mechanism by which M. tuberculosis can limit macrophage responses to IFN-γ by altering host miRNA expression. Copyright © 2014 by The American Association of Immunologists, Inc.

  5. Acquired agranulocytosis with granulocyte specific cytotoxic autoantibody.

    Science.gov (United States)

    Blaschke, J; Goeken, N E; Thompson, J S; Dick, F R; Gingrich, R D

    1979-05-01

    Multiple infections and severe neutropenia were found in a previously healthy 29 year old man with no history of similar syndromes in the family, drug ingestion or exposure to environmental toxins. There was no evidence at the time of presentation of diseases previously associated with agranulocytosis (e.g., neoplasia, thyrotoxicosis, chronic infection, collagen-vascular disease or leukoagglutinating antibody). His serum contained a nonagglutinating, complement-dependent, cytotoxic antibody, however, reactive with peripheral blood granulocytes from 35 per cent of normal donors. The neutropenia was not affected by steroids but resolved promptly after splenectomy. Microscopic examination of the spleen revealed ingestion of polymorphonuclear leukocytes by splenic macrophages. Family studies indicated that the target antigen was non-HLA and that the antibody was not absorbed by lymphocytes or platelets. We conclude that the agranulocytosis was autoimmune in origin and suggest that similar myeloid-specific immune responses could influence granulocyte tranfusion and bone marrow transplantation by alloimmune "rejection" that would not be avoided by matching only for HLA specificities.

  6. Osteogenesis differentiation of human periodontal ligament cells by CO2 laser-treatment stimulating macrophages via BMP2 signalling pathway

    International Nuclear Information System (INIS)

    Hsieh, Wen-Hui; Chen, Yi-Jyun; Hung, Chi-Jr; Huang, Tsui-Hsien; Kao, Chia-Tze; Shie, Ming-You

    2014-01-01

    Immune reactions play an important role in determining the biostimulation of bone formation, either in new bone formation or inflammatory fibrous tissue encapsulation. Macrophage cell, the important effector cells in the immune reaction, which are indispensable for osteogenesis and their heterogeneity and plasticity, render macrophages a primer target for immune system modulation. However, there are very few studies about the effects of macrophage cells on laser treatment-regulated osteogenesis. In this study, we used CO 2 laser as a model biostimulation to investigate the role of macrophage cells on the CO 2 laser stimulated osteogenesis. Bone morphogenetic protein 2 (BMP2) was also significantly up regulated by the CO 2 laser stimulation, indicating that macrophage may participate in the CO 2 laser stimulated osteogenesis. Interestingly, when laser treatment macrophage-conditioned medium were applied to human periodontal ligament cells (hPDLs), the osteogenesis differentiation of hPDLs was significantly enhanced, indicating the important role of macrophages in CO 2 laser-induced osteogenesis. These findings provided valuable insights into the mechanism of CO 2 laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment. (paper)

  7. Combination Immunotherapy of B16 Melanoma Using Anti–Cytotoxic T Lymphocyte–Associated Antigen 4 (Ctla-4) and Granulocyte/Macrophage Colony-Stimulating Factor (Gm-Csf)-Producing Vaccines Induces Rejection of Subcutaneous and Metastatic Tumors Accompanied by Autoimmune Depigmentation

    Science.gov (United States)

    van Elsas, Andrea; Hurwitz, Arthur A.; Allison, James P.

    1999-01-01

    We examined the effectiveness of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) blockade, alone or in combination with a granulocyte/macrophage colony-stimulating factor (GM-CSF)–expressing tumor cell vaccine, on rejection of the highly tumorigenic, poorly immunogenic murine melanoma B16-BL6. Recently established tumors could be eradicated in 80% (68/85) of the cases using combination treatment, whereas each treatment by itself showed little or no effect. Tumor rejection was dependent on CD8+ and NK1.1+ cells but occurred irrespective of the presence of CD4+ T cells. Mice surviving a primary challenge rejected a secondary challenge with B16-BL6 or the parental B16-F0 line. The same treatment regimen was found to be therapeutically effective against outgrowth of preestablished B16-F10 lung metastases, inducing long-term survival. Of all mice surviving B16-BL6 or B16-F10 tumors after combination treatment, 56% (38/68) developed depigmentation, starting at the site of vaccination or challenge and in most cases progressing to distant locations. Depigmentation was found to occur in CD4-depleted mice, strongly suggesting that the effect was mediated by CTLs. This study shows that CTLA-4 blockade provides a powerful tool to enhance T cell activation and memory against a poorly immunogenic spontaneous murine tumor and that this may involve recruitment of autoreactive T cells. PMID:10430624

  8. Soluble immune complexes shift the TLR-induced cytokine production of distinct polarized human macrophage subsets towards IL-10.

    Directory of Open Access Journals (Sweden)

    Carmen A Ambarus

    Full Text Available BACKGROUND: Costimulation of murine macrophages with immune complexes (ICs and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. MATERIALS AND METHODS: Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs. Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR. RESULTS: HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦ(IL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2. The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. CONCLUSION: HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.

  9. Nicotinamide: a vitamin able to shift macrophage differentiation toward macrophages with restricted inflammatory features.

    Science.gov (United States)

    Weiss, Ronald; Schilling, Erik; Grahnert, Anja; Kölling, Valeen; Dorow, Juliane; Ceglarek, Uta; Sack, Ulrich; Hauschildt, Sunna

    2015-11-01

    The differentiation of human monocytes into macrophages is influenced by environmental signals. Here we asked in how far nicotinamide (NAM), a vitamin B3 derivative known to play a major role in nicotinamide adenine dinucleotide (NAD)-mediated signaling events, is able to modulate monocyte differentiation into macrophages developed in the presence of granulocyte macrophage colony-stimulating factor (GM-MØ) or macrophage colony-stimulating factor (M-MØ). We found that GM-MØ undergo biochemical, morphological and functional modifications in response to NAM, whereas M-MØ were hardly affected. GM-MØ exposed to NAM acquired an M-MØ-like structure while the LPS-induced production of pro-inflammatory cytokines and COX-derived eicosanoids were down-regulated. In contrast, NAM had no effect on the production of IL-10 or the cytochrome P450-derived eicosanoids. Administration of NAM enhanced intracellular NAD concentrations; however, it did not prevent the LPS-mediated drain on NAD pools. In search of intracellular molecular targets of NAM known to be involved in LPS-induced cytokine and eicosanoid synthesis, we found NF-κB activity to be diminished. In conclusion, our data show that vitamin B3, when present during the differentiation of monocytes into GM-MØ, interferes with biochemical pathways resulting in strongly reduced pro-inflammatory features. © The Author(s) 2015.

  10. Culture of normal human blood cells in diffusion chamber systems. I. Granulocyte survival and proliferation. [X radiation, mice

    Energy Technology Data Exchange (ETDEWEB)

    Chikkappa, G.; Carsten, A.L.; Chanana, A.D.; Cronkite, E.P.

    1978-01-01

    Blood cells from four normal volunteers were cultured in diffusion chambers (DC), made of Millipore (MDC) or Nuclepore (NDC) filters, in the peritoneal cavities of whole body X-irradiated (700 rad) mice. The total nucleated cell recovery from the two types of DC over 18 days indicates that the cells in DC persist and proliferate. The mature neutrophilic cells, metamyelocytes (M/sub 5/) + band forms (M/sub 6/) + segmented forms (M/sub 7/), survived with T/sup 1///sub 2/ of 29 and 34 h in MDC and NDC, respectively. The reduction of the cells in the DC was surmised to be due to degeneration and death of the M/sub 7/. The /sup 3/H-diisopropylfluorophosphate (/sup 3/HDFP) labeled M/sub /sub 6/+/sub 7// survival in MDC was slightly shorter than that of unlabeled cells, which may be explained on the basis of the loss of /sup 3/HDFP (5.1%/day) from the cells. The eosinophils survived with an average T/sup 1///sub 2/ of 7.2 days (range 4.8 to 9.6), and the results were comparable in both types of DC. Formation of myeloblasts, promyelocytes, and neutrophilic, eosinophilic and basophilic myelocytes, occasional megakaryocytes and rare normoblasts in DC indicated that the normal human blood contains progenitors (pluripotent and/or committed stem cells) of hemopoietic cells. The neutrophilic cell recovery pattern was similar from both types of DC, but the total number recovered was always greater from NDC than from MDC.

  11. Ebola virion attachment and entry into human macrophages profoundly effects early cellular gene expression.

    Directory of Open Access Journals (Sweden)

    Victoria Wahl-Jensen

    2011-10-01

    Full Text Available Zaire ebolavirus (ZEBOV infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP(1,2 is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP(1,2 (VLP(VP40-GP triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLP(VP40 (particles lacking GP(1,2 caused an aberrant response. This suggests that GP(1,2 binding to macrophages plays an important role in the immediate cellular response.

  12. Triglyceride-rich lipoprotein regulates APOB48 receptor gene expression in human THP-1 monocytes and macrophages.

    Science.gov (United States)

    Bermudez, Beatriz; Lopez, Sergio; Varela, Lourdes M; Ortega, Almudena; Pacheco, Yolanda M; Moreda, Wenceslao; Moreno-Luna, Rafael; Abia, Rocio; Muriana, Francisco J G

    2012-02-01

    The postprandial metabolism of dietary fats implies that the production of TG-rich lipoproteins (TRL) contributes to the progression of plaque development. TRL and their remnants cause rapid receptor-mediated monocyte/macrophage lipid engorgement via the cell surface apoB48 receptor (apoB48R). However, the mechanistic basis for apoB48 receptor (APOB48R) regulation by postprandial TRL in monocytes and macrophages is not well established. In this study, we investigated the effects of postprandial TRL from healthy volunteers on the expression of APOB48R mRNA and lipid uptake in human THP-1 monocytes and THP-1-derived macrophages. The expression of APOB48R mRNA was upregulated in THP-1 monocytes, but downregulated in THP-1-derived macrophages when treated with postprandial TRL (P < 0.05), in a dose- and time-dependent manner. TG and free cholesterol were dramatically increased in THP-1-derived macrophages (140 and 50%, respectively; P < 0.05) and in THP-1 monocytes (160 and 95%, respectively; P < 0.05). This lipid accumulation was severely decreased (~50%; P < 0.05) in THP-1-derived macrophages by small interfering RNA (siRNA) targeting of APOB48R. Using PPAR and retinoid X receptor (RXR) agonists, antagonists, and siRNA, our data indicate that PPARα, PPARγ, and RXRα are involved in postprandial TRL-induced APOB48R transcriptional regulation. Co-incubation with acyl-CoA synthetase or acyl-CoA:cholesterol acyltransferase inhibitors potentiated the effects of postprandial TRL on the expression of APOB48R mRNA in THP-1 monocytes and THP-1-derived macrophages. Our findings collectively suggest that APOB48R represents a molecular target of postprandial TRL via PPAR-dependent pathways in human THP-1 monocytes and macrophages and advance a potentially important link between postprandial metabolism of dietary fats and atherogenesis.

  13. Toxicity of silver nanoparticles in human macrophages: uptake, intracellular distribution and cellular responses

    Science.gov (United States)

    Haase, A.; Tentschert, J.; Jungnickel, H.; Graf, P.; Mantion, A.; Draude, F.; Plendl, J.; Goetz, M. E.; Galla, S.; Mašić, A.; Thuenemann, A. F.; Taubert, A.; Arlinghaus, H. F.; Luch, A.

    2011-07-01

    Silver nanoparticles (SNP) are among the most commercialized nanoparticles worldwide. They can be found in many diverse products, mostly because of their antibacterial properties. Despite its widespread use only little data on possible adverse health effects exist. It is difficult to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles. Here, we applied a novel synthesis approach to obtain SNP, which are covalently stabilized by a small peptide. This enables a tight control of both size and shape. We applied these SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages. Similar gold nanoparticles with the same coating (Au20Pep) were used for comparison and found to be non-toxic. We assessed the cytotoxicity of particles and confirmed their cellular uptake via transmission electron microscopy and confocal Raman microscopy. Importantly a majority of the SNP could be detected as individual particles spread throughout the cells. Furthermore we studied several types of oxidative stress related responses such as induction of heme oxygenase I or formation of protein carbonyls. In summary, our data demonstrate that even low doses of SNP exerted adverse effects in human macrophages.

  14. Toxicity of silver nanoparticles in human macrophages: uptake, intracellular distribution and cellular responses

    International Nuclear Information System (INIS)

    Haase, A; Tentschert, J; Jungnickel, H; Goetz, M E; Luch, A; Graf, P; Mantion, A; Thuenemann, A F; Draude, F; Galla, S; Arlinghaus, H F; Plendl, J; Masic, A; Taubert, A

    2011-01-01

    Silver nanoparticles (SNP) are among the most commercialized nanoparticles worldwide. They can be found in many diverse products, mostly because of their antibacterial properties. Despite its widespread use only little data on possible adverse health effects exist. It is difficult to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles. Here, we applied a novel synthesis approach to obtain SNP, which are covalently stabilized by a small peptide. This enables a tight control of both size and shape. We applied these SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages. Similar gold nanoparticles with the same coating (Au20Pep) were used for comparison and found to be non-toxic. We assessed the cytotoxicity of particles and confirmed their cellular uptake via transmission electron microscopy and confocal Raman microscopy. Importantly a majority of the SNP could be detected as individual particles spread throughout the cells. Furthermore we studied several types of oxidative stress related responses such as induction of heme oxygenase I or formation of protein carbonyls. In summary, our data demonstrate that even low doses of SNP exerted adverse effects in human macrophages.

  15. CD14-dependent monocyte isolation enhances phagocytosis of listeria monocytogenes by proinflammatory, GM-CSF-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Caroline Neu

    Full Text Available Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF or macrophage colony-stimulating factor (M-CSF into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ stained positive for CD206 and M-CSF-derived macrophages (M-Mφ for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most

  16. Inhibition of transglutaminase 2 reduces efferocytosis in human macrophages: Role of CD14 and SR-AI receptors.

    Science.gov (United States)

    Eligini, S; Fiorelli, S; Tremoli, E; Colli, S

    2016-10-01

    Transglutaminase 2 (TGM2), a member of the transglutaminase family of enzymes, is a multifunctional protein involved in numerous events spanning from cell differentiation, to signal transduction, apoptosis, and wound healing. It is expressed in a variety of cells, macrophages included. Macrophage TGM2 promotes the clearance of apoptotic cells (efferocytosis) and emerging evidence suggests that defective efferocytosis contributes to the consequences of inflammation-associated diseases, including atherosclerotic lesion progression and its sequelae. Of interest, active TGM2 identified in human atherosclerotic lesions plays critical roles in plaque stability through effects on matrix cross-linking and TGFβ activity. This study explores the mechanisms by which TGM2 controls efferocytosis in human macrophages. Herein we show that TGM2 increases progressively during monocyte differentiation towards macrophages and controls their efferocytic potential as well as morphology and viability. Two experimental approaches that took advantage of the inhibition of TGM2 activity and protein silencing give proof that TGM2 reduction significantly impairs macrophage efferocytosis. Among the mechanisms involved we highlighted a role of the receptors CD14 and SR-AI whose levels were markedly reduced by TGM2 inhibition. Conversely, CD36 receptor and αvβ3 integrin levels were not influenced. Of note, lipid accumulation and IL-10 secretion were reduced in macrophages displaying defective efferocytosis. Overall, our data define a crucial role of TGM2 activity during macrophage differentiation via mechanisms involving CD14 and SR-AI receptors and show that TGM2 inhibition triggers a pro-inflammatory phenotype. Copyright © 2016 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    James A Cotton

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

  18. Human Subcutaneous Tissue Response to Glucose Sensors: Macrophages Accumulation Impact on Sensor Accuracy.

    Science.gov (United States)

    Rigla, Mercedes; Pons, Belén; Rebasa, Pere; Luna, Alexis; Pozo, Francisco Javier; Caixàs, Assumpta; Villaplana, Maria; Subías, David; Bella, Maria Rosa; Combalia, Neus

    2018-04-01

    Subcutaneous (s.c.) glucose sensors have become a key component in type 1 diabetes management. However, their usability is limited by the impact of foreign body response (FBR) on their duration, reliability, and accuracy. Our study gives the first description of human acute and subacute s.c. response to glucose sensors, showing the changes observed in the sensor surface, the inflammatory cells involved in the FBR and their relationship with sensor performance. Twelve obese patients (seven type 2 diabetes) underwent two abdominal biopsies comprising the surrounding area where they had worn two glucose sensors: the first one inserted 7 days before and the second one 24 h before biopsy procedure. Samples were processed and studied to describe tissue changes by two independent pathologists (blind regarding sensor duration). Macrophages quantification was studied by immunohistochemistry methods in the area surrounding the sensor (CD68, CD163). Sensor surface changes were studied by scanning electron microscopy. Seven-day continuous glucose monitoring records were considered inaccurate when mean absolute relative difference was higher than 10%. Pathologists were able to correctly classify all the biopsies regarding sensor duration. Acute response (24 h) was characterized by the presence of neutrophils while macrophages were the main cell involved in subacute inflammation. The number of macrophages around the insertion hole was higher for less accurate sensors compared with those performing more accurately (32.6 ± 14 vs. 10.6 ± 1 cells/0.01 mm 2 ; P sensor-tissue interface is related with decrease in accuracy of the glucose measure.

  19. Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Maaike Kockx

    Full Text Available Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs or directly target the GTPase domain (Dyngo or Dynole series, dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

  20. Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243.

    Directory of Open Access Journals (Sweden)

    Andrea J Dowling

    2010-12-01

    Full Text Available Burkholderia pseudomallei is an important human pathogen whose infection biology is still poorly understood. The bacterium is endemic to tropical regions, including South East Asia and Northern Australia, where it causes melioidosis, a serious disease associated with both high mortality and antibiotic resistance. B. pseudomallei is a Gram-negative facultative intracellular pathogen that is able to replicate in macrophages. However despite the critical nature of its interaction with macrophages, few anti-macrophage factors have been characterized to date. Here we perform a genome-wide gain of function screen of B. pseudomallei strain K96243 to identify loci encoding factors with anti-macrophage activity. We identify a total of 113 such loci scattered across both chromosomes, with positive gene clusters encoding transporters and secretion systems, enzymes/toxins, secondary metabolite, biofilm, adhesion and signal response related factors. Further phenotypic analysis of four of these regions shows that the encoded factors cause striking cellular phenotypes relevant to infection biology, including apoptosis, formation of actin 'tails' and multi-nucleation within treated macrophages. The detailed analysis of the remaining host of loci will facilitate genetic dissection of the interaction of this important pathogen with host macrophages and thus further elucidate this critical part of its infection cycle.

  1. Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration

    DEFF Research Database (Denmark)

    Saclier, Marielle; Yacoub-Youssef, Houda; Mackey, Abigail

    2013-01-01

    , we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory...... anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates......Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here...

  2. 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...

  3. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    DEFF Research Database (Denmark)

    Bokil, Nilesh J.; Totsika, Makrina; Carey, Alison J.

    2011-01-01

    assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50...... or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1+ vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data...

  4. Extracts of human atherosclerotic lesions modify LDL inducing enhanced macrophage uptake

    International Nuclear Information System (INIS)

    Hoff, H.F.; O'Neill, J.

    1986-01-01

    Both an LDL-like fraction isolated from human aortic plaques and LDL incubated with cultured aortic endothelial or smooth muscle cells have been shown to be internalized by macrophages in vitro in an unregulated fashion leading to foam cell formation. Lipid peroxidation induced by free radicals released from cells was shown to be responsible for cell-modified LDL. The authors incubated LDL with a supernatant fraction of leached, i.e. non-homogenized, extracts of aortic plaques for one hour at 37 0 C, to determine whether extracellular components present in arteries were also capable of modifying LDL. Extract-treated LDL showed the following changes relative to untreated LDL: 1) increased electrophretic mobility, 2) altered pattern of B-100 on SDS-PAGE, i.e. presence of a doublet with higher M/sub r/ than B-100, and 3) enhanced uptake by cultured mouse peritoneal macrophages as measured by increased degradation of 125 I-LDL, and increased stimulation of cholesterol esterification using 14 C-oleate. Extracts from homogenized plaques and grossly normal intima induced similar changes. The modification was tissue specific in that extracts of arteries but not of liver, muscle or skin modified LDL. Protease degradation of LDL during incubation was probably not responsible since inhibitors did not prevent modification. It is possible that products of lipid peroxidation present in extracellular lipid of arteries may propagate free radicals or be incorporated into LDL, leading to modifications similar to those found in cell-modified LDL

  5. Cigarette smoke regulates the expression of TLR4 and IL-8 production by human macrophages

    Directory of Open Access Journals (Sweden)

    Rahman Irfan

    2009-05-01

    Full Text Available Abstract Background Toll-like receptors (TLRs are present on monocytes and alveolar macrophages that form the first line of defense against inhaled particles. The importance of those cells in the pathophysiology of chronic obstructive pulmonary disease (COPD has well been documented. Cigarette smoke contains high concentration of oxidants which can stimulate immune cells to produce reactive oxygen species, cytokines and chemokines. Methods In this study, we evaluated the effects of cigarette smoke medium (CSM on TLR4 expression and interleukin (IL-8 production by human macrophages investigating the involvement of ROS. Results and Discussion TLR4 surface expression was downregulated on short term exposure (1 h of CSM. The downregulation could be explained by internalization of the TLR4 and the upregulation by an increase in TLR4 mRNA. IL-8 mRNA and protein were also increased by CSM. CSM stimulation increased intracellular ROS-production and decreased glutathione (GSH levels. The modulation of TLR4 mRNA and surface receptors expression, IRAK activation, IκB-α degradation, IL-8 mRNA and protein, GSH depletion and ROS production were all prevented by antioxidants such as N-acetyl-L-cysteine (NAC. Conclusion TLR4 may be involved in the pathogenesis of lung emphysema and oxidative stress and seems to be a crucial contributor in lung inflammation.

  6. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    KAUST Repository

    Bokil, Nilesh J.

    2011-11-01

    Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1 + vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival. © 2011 Elsevier GmbH.

  7. Th1-like human T-cell clones recognizing Leishmania gp63 inhibit Leishmania major in human macrophages

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Bendtzen, K

    1994-01-01

    The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T-cell clones recognizing this protein were generated from individuals who had previously had...... resembling Th1 cells. Autologous mononuclear cells and Epstein-Barr virus-transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells...... recognizing gp63 can take part in the host defence against L. major infections....

  8. Type 3 Deiodinase Is Highly Expressed in Infiltrating Neutrophilic Granulocytes in Response to Acute Bacterial Infection

    NARCIS (Netherlands)

    Boelen, Anita; Boorsma, Jeffrey; Kwakkel, Joan; Wieland, Catharina W.; Renckens, Rosemarijn; Visser, Theo J.; Fliers, Eric; Wiersinga, Wilmar M.

    2008-01-01

    Background: Macrophages and polymorphonuclear cells (PMNs) play an important role in the first line of defense against bacteria by infiltrating the infected organ in order to clear the harmful pathogen. Our earlier studies showed that granulocytes express type 3 deiodinase (D3) when activated during

  9. Decreased expression of liver X receptor-α in macrophages infected with Chlamydia pneumoniae in human atherosclerotic arteries in situ.

    Science.gov (United States)

    Bobryshev, Yuri V; Orekhov, Alexander N; Killingsworth, Murray C; Lu, Jinhua

    2011-01-01

    In in vitro experiments, Chlamydia pneumoniae has been shown to infect macrophages and to accelerate foam cell formation. It has been hypothesized that the C. pneumoniae infection affects foam cell formation by suppressing the expression of liver X receptors (LXR), but whether such an event occurs in human atherosclerosis is not known. In this study we examined carotid artery segments, obtained by endarterectomy, in which the presence of C. pneumoniae was confirmed by both polymerase chain reaction and immunohistochemistry. The expression of LXR-α in macrophages infected with C. pneumoniae and macrophages that were not infected was compared using a quantitative immunohistochemical analysis. The analysis revealed a 2.2-fold reduction in the expression of LXR-α in C. pneumoniae-infected cells around the lipid cores in atherosclerotic plaques. In the cytoplasm of laser-capture microdissected cells that were immunopositive for C. pneumoniae, electron microscopy demonstrated the presence of structures with the appearance of elementary, reticulate and aberrant bodies of C. pneumoniae. We conclude that LXR-α expression is reduced in C. pneumoniae-infected macrophages in human atherosclerotic lesions which supports the hypothesis that C. pneumoniae infection might suppress LXR expression in macrophages transforming into foam cells. Copyright © 2011 S. Karger AG, Basel.

  10. Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis.

    Science.gov (United States)

    Hoffman, Ewelina; Patel, Aateka; Ball, Doug; Klapwijk, Jan; Millar, Val; Kumar, Abhinav; Martin, Abigail; Mahendran, Rhamiya; Dailey, Lea Ann; Forbes, Ben; Hutter, Victoria

    2017-12-01

    Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. Cell health, morphology and lipid content were comparable (p content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

  11. Improved gene expression in resting macrophages using an oligopeptide derived from Vpr of human immunodeficiency virus type-1

    International Nuclear Information System (INIS)

    Mizoguchi, Izuru; Ooe, Yoshihiro; Hoshino, Shigeki; Shimura, Mari; Kasahara, Tadashi; Kano, Shigeyuki; Ohta, Toshiko; Takaku, Fumimaro; Nakayama, Yasuhide; Ishizaka, Yukihito

    2005-01-01

    Vpr, an accessory gene product of human immunodeficiency virus type-1, is thought to transport a viral DNA from the cytoplasm to the nucleus in resting macrophages. Previously, we reported that a peptide encompassing amino acids 52-78 of Vpr (C45D18) promotes the nuclear trafficking of recombinant proteins that are conjugated with C45D18. Here, we present evidence that C45D18, when conjugated with a six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide)-block-oligo(4-aminostyrene) (SV: star vector), facilitates gene expression in resting macrophages. Although there was no difference between SV alone and C45D18-SV with respect to gene transduction into growing cells, C45D18-SV resulted in more than 40-fold greater expression of the exogenous gene upon transduction into chemically differentiated macrophages and human quiescent monocyte-derived macrophages. The data suggest that C45D18 contributes to improving the ability of a non-viral vector to transduce macrophages with exogenous genes and we discuss its further application

  12. Preservation Analysis of Macrophage Gene Coexpression Between Human and Mouse Identifies PARK2 as a Genetically Controlled Master Regulator of Oxidative Phosphorylation in Humans

    Directory of Open Access Journals (Sweden)

    Veronica Codoni

    2016-10-01

    Full Text Available Macrophages are key players involved in numerous pathophysiological pathways and an in-depth characterization of their gene regulatory networks can help in better understanding how their dysfunction may impact on human diseases. We here conducted a cross-species network analysis of macrophage gene expression data between human and mouse to identify conserved networks across both species, and assessed whether such networks could reveal new disease-associated regulatory mechanisms. From a sample of 684 individuals processed for genome-wide macrophage gene expression profiling, we identified 27 groups of coexpressed genes (modules. Six modules were found preserved (P < 10−4 in macrophages from 86 mice of the Hybrid Mouse Diversity Panel. One of these modules was significantly [false discovery rate (FDR = 8.9 × 10−11] enriched for genes belonging to the oxidative phosphorylation (OXPHOS pathway. This pathway was also found significantly (FDR < 10−4 enriched in susceptibility genes for Alzheimer, Parkinson, and Huntington diseases. We further conducted an expression quantitative trait loci analysis to identify SNP that could regulate macrophage OXPHOS gene expression in humans. This analysis identified the PARK2 rs192804963 as a trans-acting variant influencing (minimal P-value = 4.3 × 10−8 the expression of most OXPHOS genes in humans. Further experimental work demonstrated that PARK2 knockdown expression was associated with increased OXPHOS gene expression in THP1 human macrophages. This work provided strong new evidence that PARK2 participates to the regulatory networks associated with oxidative phosphorylation and suggested that PARK2 genetic variations could act as a trans regulator of OXPHOS gene macrophage expression in humans.

  13. Volcanic ash activates the NLRP3 inflammasome in murine and human macrophages

    Science.gov (United States)

    Damby, David; Horwell, Claire J.; Baxter, Peter J.; Kueppers, Ulrich; Schnurr, Max; Dingwell, Donald B.; Duewell, Peter

    2018-01-01

    Volcanic ash is a heterogeneous mineral dust that is typically composed of a mixture of amorphous (glass) and crystalline (mineral) fragments. It commonly contains an abundance of the crystalline silica (SiO2) polymorph cristobalite. Inhalation of crystalline silica can induce inflammation by stimulating the NLRP3 inflammasome, a cytosolic receptor complex that plays a critical role in driving inflammatory immune responses. Ingested material results in the assembly of NLRP3, ASC, and caspase-1 with subsequent secretion of the interleukin-1 family cytokine IL-1β. Previous toxicology work suggests that cristobalite-bearing volcanic ash is minimally reactive, calling into question the reactivity of volcanically derived crystalline silica, in general. In this study, we target the NLRP3 inflammasome as a crystalline silica responsive element to clarify volcanic cristobalite reactivity. We expose immortalized bone marrow-derived macrophages of genetically engineered mice and primary human peripheral blood mononuclear cells (PBMCs) to ash from the Soufrière Hills volcano as well as representative, pure-phase samples of its primary componentry (volcanic glass, feldspar, cristobalite) and measure NLRP3 inflammasome activation. We demonstrate that respirable Soufrière Hills volcanic ash induces the activation of caspase-1 with subsequent release of mature IL-1β in a NLRP3 inflammasome-dependent manner. Macrophages deficient in NLRP3 inflammasome components are incapable of secreting IL-1β in response to volcanic ash ingestion. Cellular uptake induces lysosomal destabilization involving cysteine proteases. Furthermore, the response involves activation of mitochondrial stress pathways leading to the generation of reactive oxygen species. Considering ash componentry, cristobalite is the most reactive pure-phase with other components inducing only low-level IL-1β secretion. Inflammasome activation mediated by inhaled ash and its potential relevance in chronic pulmonary

  14. Human mesenchymal stem cells alter macrophage phenotype and promote regeneration via homing to the kidney following ischemia-reperfusion injury

    NARCIS (Netherlands)

    Wise, Andrea F; Williams, Timothy M; Kiewiet, Mensiena B G; Payne, Natalie L; Siatskas, Christopher; Samuel, Chrishan S; Ricardo, Sharon D

    2014-01-01

    Mesenchymal stem cells (MSCs) ameliorate injury and accelerate repair in many organs, including the kidney, although the reparative mechanisms and interaction with macrophages have not been elucidated. This study investigated the reparative potential of human bone marrow-derived MSCs and traced

  15. Organic UV filters exposure induces the production of inflammatory cytokines in human macrophages.

    Science.gov (United States)

    Ao, Junjie; Yuan, Tao; Gao, Li; Yu, Xiaodan; Zhao, Xiaodong; Tian, Ying; Ding, Wenjin; Ma, Yuning; Shen, Zhemin

    2018-09-01

    Organic ultraviolet (UV) filters, found in many personal care products, are considered emerging contaminants due to growing concerns about potential long-term deleterious effects. We investigated the immunomodulatory effects of four commonly used organic UV filters (2-hydroxy-4-methoxybenzophenone, BP-3; 4-methylbenzylidene camphor, 4-MBC; 2-ethylhexyl 4-methoxycinnamate, EHMC; and butyl-methoxydibenzoylmethane, BDM) on human macrophages. Our results indicated that exposure to these four UV filters significantly increased the production of various inflammatory cytokines in macrophages, particular tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). After exposure to the UV filters, a significant 1.1-1.5 fold increase were found in TNF-α and IL-6 mRNA expression. In addition, both the p38 MAPK and the NF-κB signaling pathways were enhanced 2 to 10 times in terms of phosphorylation after exposure to the UV filters, suggesting that these pathways are involved in the release of TNF-α and IL-6. Molecular docking analysis predicted that all four UV filter molecules would efficiently bind transforming growth factor beta-activated kinase 1 (TAK1), which is responsible for the activation of the p38 MAPK and NF-κB pathways. Our results therefore demonstrate that exposure to the four organic UV filters investigated may alter human immune system function. It provides new clue for the development of asthma or allergic diseases in terms of the environmental pollutants. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Involvement of macrophage migration inhibitory factor and its receptor (CD74) in human breast cancer.

    Science.gov (United States)

    Richard, Vincent; Kindt, Nadège; Decaestecker, Christine; Gabius, Hans-Joachim; Laurent, Guy; Noël, Jean-Christophe; Saussez, Sven

    2014-08-01

    Macrophage migration inhibitory factor (MIF) and its receptor CD74 appear to be involved in tumorigenesis. We evaluated, by immunohistochemical staining, the tissue expression and distribution of MIF and CD74 in serial sections of human invasive breast cancer tumor specimens. The serum MIF level was also determined in breast cancer patients. We showed a significant increase in serum MIF average levels in breast cancer patients compared to healthy individuals. MIF tissue expression, quantified by a modified Allred score, was strongly increased in carcinoma compared to tumor-free specimens, in the cancer cells and in the peritumoral stroma, with fibroblasts the most intensely stained. We did not find any significant correlation with histoprognostic factors, except for a significant inverse correlation between tumor size and MIF stromal positivity. CD74 staining was heterogeneous and significantly decreased in cancer cells but increased in the surrounding stroma, namely in lymphocytes, macrophages and vessel endothelium. There was no significant variation according to classical histoprognostic factors, except that CD74 stromal expression was significantly correlated with triple-negative receptor (TRN) status and the absence of estrogen receptors. In conclusion, our data support the concept of a functional role of MIF in human breast cancer. In addition to auto- and paracrine effects on cancer cells, MIF could contribute to shape the tumor microenvironment leading to immunomodulation and angiogenesis. Interfering with MIF effects in breast tumors in a therapeutic perspective remains an attractive but complex challenge. Level of co-expression of MIF and CD74 could be a surrogate marker for efficacy of anti-angiogenic drugs, particularly in TRN breast cancer tumor.

  17. Responses of murine and human macrophages to leptospiral infection: a study using comparative array analysis.

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    Feng Xue

    Full Text Available Leptospirosis is a re-emerging tropical infectious disease caused by pathogenic Leptospira spp. The different host innate immune responses are partially related to the different severities of leptospirosis. In this study, we employed transcriptomics and cytokine arrays to comparatively calculate the responses of murine peritoneal macrophages (MPMs and human peripheral blood monocytes (HBMs to leptospiral infection. We uncovered a series of different expression profiles of these two immune cells. The percentages of regulated genes in several biological processes of MPMs, such as antigen processing and presentation, membrane potential regulation, and the innate immune response, etc., were much greater than those of HBMs (>2-fold. In MPMs and HBMs, the caspase-8 and Fas-associated protein with death domain (FADD-like apoptosis regulator genes were significantly up-regulated, which supported previous results that the caspase-8 and caspase-3 pathways play an important role in macrophage apoptosis during leptospiral infection. In addition, the key component of the complement pathway, C3, was only up-regulated in MPMs. Furthermore, several cytokines, e.g. interleukin 10 (IL-10 and tumor necrosis factor alpha (TNF-alpha, were differentially expressed at both mRNA and protein levels in MPMs and HBMs. Some of the differential expressions were proved to be pathogenic Leptospira-specific regulations at mRNA level or protein level. Though it is still unclear why some animals are resistant and others are susceptible to leptospiral infection, this comparative study based on transcriptomics and cytokine arrays partially uncovered the differences of murine resistance and human susceptibility to leptospirosis. Taken together, these findings will facilitate further molecular studies on the innate immune response to leptospiral infection.

  18. Responses of Murine and Human Macrophages to Leptospiral Infection: A Study Using Comparative Array Analysis

    Science.gov (United States)

    Yang, Yingchao; Zhao, Jinping; Yang, Yutao; Cao, Yongguo; Hong, Cailing; Liu, Yuan; Sun, Lan; Huang, Minjun; Gu, Junchao

    2013-01-01

    Leptospirosis is a re-emerging tropical infectious disease caused by pathogenic Leptospira spp. The different host innate immune responses are partially related to the different severities of leptospirosis. In this study, we employed transcriptomics and cytokine arrays to comparatively calculate the responses of murine peritoneal macrophages (MPMs) and human peripheral blood monocytes (HBMs) to leptospiral infection. We uncovered a series of different expression profiles of these two immune cells. The percentages of regulated genes in several biological processes of MPMs, such as antigen processing and presentation, membrane potential regulation, and the innate immune response, etc., were much greater than those of HBMs (>2-fold). In MPMs and HBMs, the caspase-8 and Fas-associated protein with death domain (FADD)-like apoptosis regulator genes were significantly up-regulated, which supported previous results that the caspase-8 and caspase-3 pathways play an important role in macrophage apoptosis during leptospiral infection. In addition, the key component of the complement pathway, C3, was only up-regulated in MPMs. Furthermore, several cytokines, e.g. interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-alpha), were differentially expressed at both mRNA and protein levels in MPMs and HBMs. Some of the differential expressions were proved to be pathogenic Leptospira-specific regulations at mRNA level or protein level. Though it is still unclear why some animals are resistant and others are susceptible to leptospiral infection, this comparative study based on transcriptomics and cytokine arrays partially uncovered the differences of murine resistance and human susceptibility to leptospirosis. Taken together, these findings will facilitate further molecular studies on the innate immune response to leptospiral infection. PMID:24130911

  19. 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.

  20. Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease

    Science.gov (United States)

    Bustamante, Jacinta; Arias, Andres A; Vogt, Guillaume; Picard, Capucine; Galicia, Lizbeth Blancas; Prando, Carolina; Grant, Audrey V; Marchal, Christophe C; Hubeau, Marjorie; Chapgier, Ariane; de Beaucoudrey, Ludovic; Puel, Anne; Feinberg, Jacqueline; Valinetz, Ethan; Jannière, Lucile; Besse, Céline; Boland, Anne; Brisseau, Jean-Marie; Blanche, Stéphane; Lortholary, Olivier; Fieschi, Claire; Emile, Jean-François; Boisson-Dupuis, Stéphanie; Al-Muhsen, Saleh; Woda, Bruce; Newburger, Peter E; Condino-Neto, Antonio; Dinauer, Mary C; Abel, Laurent; Casanova, Jean-Laurent

    2011-01-01

    Germline mutations in CYBB, the human gene encoding the gp91phox subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This ‘experiment of nature’ indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria. PMID:21278736

  1. Adsorption of recombinant human granulocyte colony stimulating factor (rhG-CSF) to polyvinyl chloride, polypropylene, and glass: effect of solvent additives.

    Science.gov (United States)

    Johnston, T P

    1996-01-01

    The adsorption of recombinant-derived proteins to glass and polymeric materials used in their packaging and delivery remains a problem. Loss of these very expensive proteins to surface adsorption not only results in reduced yields during purification and scale-up, but also to decreased therapeutic efficacy. The purpose of the present investigation was to inhibit/minimize adsorption of a model protein, namely, recombinant human granulocyte colony stimulating factor (rhG-CSF) to glass, polyvinyl chloride (PVC), and polypropylene by inclusion of select solvent additives. Solvent additives used to inhibit/minimize surface adsorption included glycerin, U.S.P. (0.5%, 1%, 5%, and 25% v/v), Pluronic F-127 (0.005%, 0.05%, and 0.5% w/w), Pluronic F-68 (0.005%, 0.05%, and 0.5% w/w), Tween 80 (0.005% and 0.05% w/w) and Tween 20 (0.005%, 0.05%, and 0.5% w/w). Over the rhG-CSF concentration range of 0.0 ng/ml to 300 ng/ml, the amount of rhG-CSF bound per cm2 of PVC increased with an increase in the rhG-CSF concentration tested. At rhG-CSF equilibrium concentrations of 262 +/- 3.7 ng/ml and 136 +/- 1.9 ng/ml, the rhG-CSF bound/cm2 of PVC at 22 degrees C and 45 degrees C reached a maximum of 37.6 +/- 9.8 ng/cm2 and 165.2 +/- 11.7 ng/cm2, respectively. The adsorption isotherms determined at each temperature were described by the classic Freundlich equation. Moreover, the rate of adsorption of rhG-CSF to PVC was extremely rapid. The mean values of the percent of rhG-CSF bound to PVC after only 10 minutes of equilibration at 22 degrees C and 45 degrees C were 92.8 +/- 9.2 percent and 97.3 +/- 17.9 percent, respectively. The mean values of the percent of rhG-CSF bound to PVC at 22 degrees C and 45 degrees C after 24 hours were 52.4 +/- 10.9% and 70.0 +/- 9.7%, respectively, indicating that some desorption of rhG-CSF does occur during 24 hr. However, surface adsorption of rhG-CSF to PVC was shown to be irreversible over a 1 hr time period. Using viscometry, an estimate of the thickness

  2. Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage.

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    Valentina Guerrini

    Full Text Available Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10-9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth.

  3. Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model

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    Laura Rindi

    2018-01-01

    Full Text Available Background: Mycobacterium avium subsp. hominissuis (MAH is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro. Methods: Cultures of human peripheral blood mononuclear cells and phagocytic human leukemic cell line THP-1 cells were infected with each MAH isolate and intracellular colony-forming units (CFU were determined. Results: At 2 h after infection, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types. At 5 days, Mav-A organisms, sharing highly related VNTR-MIRU genotypes, yielded numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types. MIRU-VNTR-based minimum spanning tree analysis of the MAH isolates showed a divergent phylogenetic pathway of Mav-A and Mav-B organisms. Conclusion: Mav-A and Mav-B sequevars might have evolved different pathogenetic properties that might account for their association with different human infections.

  4. The differential role of human macrophage in triggering secondary bystander effects after either gamma-ray or carbon beam irradiation.

    Science.gov (United States)

    Dong, Chen; He, Mingyuan; Tu, Wenzhi; Konishi, Teruaki; Liu, Weili; Xie, Yuexia; Dang, Bingrong; Li, Wenjian; Uchihori, Yukio; Hei, Tom K; Shao, Chunlin

    2015-07-10

    The abscopal effect could be an underlying factor in evaluating prognosis of radiotherapy. This study established an in vitro system to examine whether tumor-generated bystander signals could be transmitted by macrophages to further trigger secondary cellular responses after different irradiations, where human lung cancer NCI-H446 cells were irradiated with either γ-rays or carbon ions and co-cultured with human macrophage U937 cells, then these U937 cells were used as a bystander signal transmitter and co-cultured with human bronchial epithelial cells BEAS-2B. Results showed that U937 cells were only activated by γ-irradiated NCI-H446 cells so that the secondary injuries in BEAS-2B cells under carbon ion irradiation were weaker than γ-rays. Both TNF-α and IL-1α were involved in the γ-irradiation induced secondary bystander effect but only TNF-α contributed to the carbon ion induced response. Further assay disclosed that IL-1α but not TNF-α was largely responsible for the activation of macrophages and the formation of micronucleus in BEAS-2B cells. These data suggest that macrophages could transfer secondary bystander signals and play a key role in the secondary bystander effect of photon irradiation, while carbon ion irradiation has conspicuous advantage due to its reduced secondary injury. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Specific Kv1.3 blockade modulates key cholesterol-metabolism-associated molecules in human macrophages exposed to ox-LDL.

    Science.gov (United States)

    Yang, Yong; Wang, Yan-Fu; Yang, Xiao-Fang; Wang, Zhao-Hui; Lian, Yi-Tian; Yang, Ying; Li, Xiao-Wei; Gao, Xiang; Chen, Jian; Shu, Yan-Wen; Cheng, Long-Xian; Liao, Yu-Hua; Liu, Kun

    2013-01-01

    Cholesterol-metabolism-associated molecules, including scavenger receptor class A (SR-A), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), CD36, ACAT1, ABCA1, ABCG1, and scavenger receptor class B type I, can modulate cholesterol metabolism in the transformation from macrophages to foam cells. Voltage-gated potassium channel Kv1.3 has increasingly been demonstrated to play an important role in the modulation of macrophage function. Here, we investigate the role of Kv1.3 in modulating cholesterol-metabolism-associated molecules in human acute monocytic leukemia cell-derived macrophages (THP-1 macrophages) and human monocyte-derived macrophages exposed to oxidized LDL (ox-LDL). Human Kv1.3 and Kv1.5 channels (hKv1.3 and hKv1.5) are expressed in macrophages and form a heteromultimeric channel. The hKv1.3-E314 antibody that we had generated as a specific hKv1.3 blocker inhibited outward delayed rectifier potassium currents, whereas the hKv1.5-E313 antibody that we had generated as a specific hKv1.5 blocker failed. Accordingly, the hKv1.3-E314 antibody reduced percentage of cholesterol ester and enhanced apoA-I-mediated cholesterol efflux in THP-1 macrophages and human monocyte-derived macrophages exposed to ox-LDL. The hKv1.3-E314 antibody downregulated SR-A, LOX-1, and ACAT1 expression and upregulated ABCA1 expression in THP-1 macrophages and human monocyte-derived macrophages. Our results reveal that specific Kv1.3 blockade represents a novel strategy modulating cholesterol metabolism in macrophages, which benefits the treatment of atherosclerotic lesions.

  6. Specific Kv1.3 blockade modulates key cholesterol-metabolism-associated molecules in human macrophages exposed to ox-LDL[S

    Science.gov (United States)

    Yang, Yong; Wang, Yan-Fu; Yang, Xiao-Fang; Wang, Zhao-Hui; Lian, Yi-Tian; Yang, Ying; Li, Xiao-Wei; Gao, Xiang; Chen, Jian; Shu, Yan-Wen; Cheng, Long-Xian; Liao, Yu-Hua; Liu, Kun

    2013-01-01

    Cholesterol-metabolism-associated molecules, including scavenger receptor class A (SR-A), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), CD36, ACAT1, ABCA1, ABCG1, and scavenger receptor class B type I, can modulate cholesterol metabolism in the transformation from macrophages to foam cells. Voltage-gated potassium channel Kv1.3 has increasingly been demonstrated to play an important role in the modulation of macrophage function. Here, we investigate the role of Kv1.3 in modulating cholesterol-metabolism-associated molecules in human acute monocytic leukemia cell-derived macrophages (THP-1 macrophages) and human monocyte-derived macrophages exposed to oxidized LDL (ox-LDL). Human Kv1.3 and Kv1.5 channels (hKv1.3 and hKv1.5) are expressed in macrophages and form a heteromultimeric channel. The hKv1.3-E314 antibody that we had generated as a specific hKv1.3 blocker inhibited outward delayed rectifier potassium currents, whereas the hKv1.5-E313 antibody that we had generated as a specific hKv1.5 blocker failed. Accordingly, the hKv1.3-E314 antibody reduced percentage of cholesterol ester and enhanced apoA-I-mediated cholesterol efflux in THP-1 macrophages and human monocyte-derived macrophages exposed to ox-LDL. The hKv1.3-E314 antibody downregulated SR-A, LOX-1, and ACAT1 expression and upregulated ABCA1 expression in THP-1 macrophages and human monocyte-derived macrophages. Our results reveal that specific Kv1.3 blockade represents a novel strategy modulating cholesterol metabolism in macrophages, which benefits the treatment of atherosclerotic lesions. PMID:23099443

  7. Rapid transient expression of human granulocyte-macrophage colony-stimulating factor in two industrial cultivars of tobacco (Nicotiana tabacum L. by agroinfiltration

    Directory of Open Access Journals (Sweden)

    Lea Vojta

    2015-09-01

    Full Text Available We report the production of hGM-CSF cytokine in leaves of industrial tobacco cultivars DH-17 and DH-27 by using Agrobacterium-mediated transient expression. We prove the concept that very high biomass industrial tobacco plants are suitable platforms for rapid, low cost production of foreign proteins. Successful transient expression of the GM-CSF was achieved in less than three months, opening the possibility for future applications of this approach in rapid response production of various proteins of non-plant origin in industrial tobacco.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    to the antibacterial effects of fosfomycin and metronidazole alone. CONCLUSION: The drug combination had neutral and stable pH, was iso-osmotic, and had stable concentrations during 24 h of storage. The antibacterial effect of fosfomycin and metronidazole were not altered when the drugs were mixed....

  9. Retinoic acid-induced granulocytic differentiation of HL60 human promyelocytic leukemia cells is preceded by downregulation of autonomous generation of inositol lipid-derived second messengers

    International Nuclear Information System (INIS)

    Porfiri, E.; Hoffbrand, A.V.; Wickremasinghe, R.G.

    1991-01-01

    Inositol phosphates (InsPs) and diacyglycerol (DAG) are second messengers derived via the breakdown of inositol phospholipids, and which play important signalling roles in the regulation of proliferation of some cell types. The authors have studied the operation of this pathway during the early stages of retionic acid (RA)-induced granulocytic differentiation of HL60 myeloid leukemia cells. The autonomous breakdown of inositol lipids that occurred in HL60 cells labeled with [3H] inositol was completely abolished following 48 hours of RA treatment. The rate of influx of 45Ca2+ was also significantly decreased at 48 hours, consistent with the role of inositol lipid-derived second messengers in regulating Ca2+ entry into cells. The downregulation of inositol lipid metabolism clearly preceded the onset of reduced proliferation induced by RA treatment, and was therefore not a consequence of decreased cell growth. The generation of InsPs in RA-treated cells was reactivated by the fluoroaluminate ion, a direct activator of guanine nucleotide-binding protein(s) (G proteins) that regulate the inositol lipid signalling pathway. Subtle alterations to a regulatory mechanism may therefore mediate the RA-induced downregulation of this pathway. The data are consistent with the hypothesis that the autonomous generation of inositol lipid-derived second messengers may contribute to the continuous proliferation of HL60 cells, and that the RA-induced downregulation of this pathway may, in turn, play a role in signalling the cessation of proliferation that preceedes granulocytic differentiation

  10. C–C Chemokines Released by Lipopolysaccharide (LPS)-stimulated Human Macrophages Suppress HIV-1 Infection in Both Macrophages and T Cells

    Science.gov (United States)

    Verani, Alessia; Scarlatti, Gabriella; Comar, Manola; Tresoldi, Eleonora; Polo, Simona; Giacca, Mauro; Lusso, Paolo; Siccardi, Antonio G.; Vercelli, Donata

    1997-01-01

    Human immunodeficiency virus-1 (HIV-1) expression in monocyte-derived macrophages (MDM) infected in vitro is known to be inhibited by lipopolysaccharide (LPS). However, the mechanisms are incompletely understood. We show here that HIV-1 suppression is mediated by soluble factors released by MDM stimulated with physiologically significant concentrations of LPS. LPS-conditioned supernatants from MDM inhibited HIV-1 replication in both MDM and T cells. Depletion of C–C chemokines (RANTES, MIP-1α, and MIP-1β) neutralized the ability of LPS-conditioned supernatants to inhibit HIV-1 replication in MDM. A combination of recombinant C–C chemokines blocked HIV-1 infection as effectively as LPS. Here, we report an inhibitory effect of C–C chemokines on HIV replication in primary macrophages. Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes. PMID:9120386

  11. Changes in the topology of gene expression networks by human immunodeficiency virus type 1 (HIV-1) integration in macrophages.

    Science.gov (United States)

    Soto-Girón, María Juliana; García-Vallejo, Felipe

    2012-01-01

    One key step of human immunodeficiency virus type 1 (HIV-1) infection is the integration of its viral cDNA. This process is mediated through complex networks of host-virus interactions that alter several normal cell functions of the host. To study the complexity of disturbances in cell gene expression networks by HIV-1 integration, we constructed a network of human macrophage genes located close to chromatin regions rich in proviruses. To perform the network analysis, we selected 28 genes previously identified as the target of cDNA integration and their transcriptional profiles were obtained from GEO Profiles (NCBI). A total of 2770 interactions among the 28 genes located around the HIV-1 proviruses in human macrophages formed a highly dense main network connected to five sub-networks. The overall network was significantly enriched by genes associated with signal transduction, cellular communication and regulatory processes. To simulate the effects of HIV-1 integration in infected macrophages, five genes with the most number of interaction in the normal network were turned off by putting in zero the correspondent expression values. The HIV-1 infected network showed changes in its topology and alteration in the macrophage functions reflected in a re-programming of biosynthetic and general metabolic process. Understanding the complex virus-host interactions that occur during HIV-1 integration, may provided valuable genomic information to develop new antiviral treatments focusing on the management of some specific gene expression networks associated with viral integration. This is the first gene network which describes the human macrophages genes interactions related with HIV-1 integration. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Granulocyte-mobilized bone marrow.

    Science.gov (United States)

    Arcese, William; De Angelis, Gottardo; Cerretti, Raffaella

    2012-11-01

    In the last few years, mobilized peripheral blood has overcome bone marrow as a graft source, but, despite the evidence of a more rapid engraftment, the incidence of chronic graft-versus-host disease is significantly higher with, consequently, more transplant-related mortality on the long follow-up. Overall, the posttransplant outcome of mobilized peripheral blood recipients is similar to that of patients who are bone marrow grafted. More recently, the use of bone marrow after granulocyte colony-stimulating factor (G-CSF) donor priming has been introduced in the transplant practice. Herein, we review biological acquisitions and clinical results on the use of G-CSF-primed bone marrow as a source of hematopoietic stem cells (HSC) for allogeneic stem cell transplantation. G-CSF the increases the HSC compartment and exerts an intense immunoregulatory effect on marrow T-cells resulting in the shift from Th1 to Th2 phenotype with higher production of anti-inflammatory cytokines. The potential advantages of these biological effects have been translated in the clinical practice by using G-CSF primed unmanipulated bone marrow in the setting of transplant from human leukocyte antigen (HLA)-haploidentical donor with highly encouraging results. For patients lacking an HLA-identical sibling, the transplant of G-CSF primed unmanipulated bone marrow from a haploidentical donor combined with an intense in-vivo immunosuppression is a valid alternative achieving results that are well comparable with those reported for umbilical cord blood, HLA-matched unrelated peripheral blood/bone marrow or T-cell-depleted haploidentical transplant.

  13. alpha-MSH and its receptors in regulation of tumor necrosis factor-alpha production by human monocyte/macrophages.

    Science.gov (United States)

    Taherzadeh, S; Sharma, S; Chhajlani, V; Gantz, I; Rajora, N; Demitri, M T; Kelly, L; Zhao, H; Ichiyama, T; Catania, A; Lipton, J M

    1999-05-01

    The hypothesis that macrophages contain an autocrine circuit based on melanocortin [ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH)] peptides has major implications for neuroimmunomodulation research and inflammation therapy. To test this hypothesis, cells of the THP-1 human monocyte/macrophage line were stimulated with lipopolysaccharide (LPS) in the presence and absence of alpha-MSH. The inflammatory cytokine tumor necrosis factor (TNF)-alpha was inhibited in relation to alpha-MSH concentration. Similar inhibitory effects on TNF-alpha were observed with ACTH peptides that contain the alpha-MSH amino acid sequence and act on melanocortin receptors. Nuclease protection assays indicated that expression of the human melanocortin-1 receptor subtype (hMC-1R) occurs in THP-1 cells; Southern blots of RT-PCR product revealed that additional subtypes, hMC-3R and hMC-5R, also occur. Incubation of resting macrophages with antibody to hMC-1R increased TNF-alpha concentration; the antibody also markedly reduced the inhibitory influence of alpha-MSH on TNF-alpha in macrophages treated with LPS. These results in cells known to produce alpha-MSH at rest and to increase secretion of the peptide when challenged are consistent with an endogenous regulatory circuit based on melanocortin peptides and their receptors. Targeting of this neuroimmunomodulatory circuit in inflammatory diseases in which myelomonocytic cells are prominent should be beneficial.

  14. Increased platelet reactivity is associated with circulating platelet-monocyte complexes and macrophages in human atherosclerotic plaques.

    Directory of Open Access Journals (Sweden)

    Bert Rutten

    Full Text Available Platelet reactivity, platelet binding to monocytes and monocyte infiltration play a detrimental role in atherosclerotic plaque progression. We investigated whether platelet reactivity was associated with levels of circulating platelet-monocyte complexes (PMCs and macrophages in human atherosclerotic carotid plaques.Platelet reactivity was determined by measuring platelet P-selectin expression after platelet stimulation with increasing concentrations of adenosine diphosphate (ADP, in two independent cohorts: the Circulating Cells cohort (n = 244 and the Athero-Express cohort (n = 91. Levels of PMCs were assessed by flow cytometry in blood samples of patients who were scheduled for percutaneous coronary intervention (Circulating Cells cohort. Monocyte infiltration was semi-quantitatively determined by histological examination of atherosclerotic carotid plaques collected during carotid endarterectomy (Athero-Express cohort.We found increased platelet reactivity in patients with high PMCs as compared to patients with low PMCs (median (interquartile range: 4153 (1585-11267 area under the curve (AUC vs. 9633 (3580-21565 AUC, P<0.001. Also, we observed increased platelet reactivity in patients with high macrophage levels in atherosclerotic plaques as compared to patients with low macrophage levels in atherosclerotic plaques (mean ± SD; 8969 ± 3485 AUC vs. 7020 ± 3442 AUC, P = 0.02. All associations remained significant after adjustment for age, sex and use of drugs against platelet activation.Platelet reactivity towards ADP is associated with levels of PMCs and macrophages in human atherosclerotic carotid plaques.

  15. Peroxisome Proliferator-Activated Receptor γ Induces the Expression of Tissue Factor Pathway Inhibitor-1 (TFPI-1 in Human Macrophages

    Directory of Open Access Journals (Sweden)

    G. Chinetti-Gbaguidi

    2016-01-01

    Full Text Available Tissue factor (TF is the initiator of the blood coagulation cascade after interaction with the activated factor VII (FVIIa. Moreover, the TF/FVIIa complex also activates intracellular signalling pathways leading to the production of inflammatory cytokines. The TF/FVIIa complex is inhibited by the tissue factor pathway inhibitor-1 (TFPI-1. Peroxisome proliferator-activated receptor gamma (PPARγ is a transcription factor that, together with PPARα and PPARβ/δ, controls macrophage functions. However, whether PPARγ activation modulates the expression of TFP1-1 in human macrophages is not known. Here we report that PPARγ activation increases the expression of TFPI-1 in human macrophages in vitro as well as in vivo in circulating peripheral blood mononuclear cells. The induction of TFPI-1 expression by PPARγ ligands, an effect shared by the activation of PPARα and PPARβ/δ, occurs also in proinflammatory M1 and in anti-inflammatory M2 polarized macrophages. As a functional consequence, treatment with PPARγ ligands significantly reduces the inflammatory response induced by FVIIa, as measured by variations in the IL-8, MMP-2, and MCP-1 expression. These data identify a novel role for PPARγ in the control of TF the pathway.

  16. GABA and Topiramate Inhibit the Formation of Human Macrophage-Derived Foam Cells by Modulating Cholesterol-Metabolism-Associated Molecules

    Directory of Open Access Journals (Sweden)

    Ying Yang

    2014-04-01

    Full Text Available Aims: γ-aminobutyric acid (GABA, the principal inhibitory neurotransmitter, acts on GABA receptors to play an important role in the modulation of macrophage functions. The present study examined the effects of GABA and a GABA receptor agonist on modulating cholesterol-metabolism-associated molecules in human monocyte-derived macrophages (HMDMs. Methods: ORO stain, HPLC, qRT-PCR, Western blot and EMSA were carried out using HMDMs exposed to ox-LDL with or without GABAergic agents as the experimental model. Results: GABA and topiramate reduced the percentage of cholesterol ester in lipid-laden HMDMs by down-regulating SR-A, CD36 and LOX-1 expression and up-regulating ABCA1, ABCG1 and SR-BI expression in lipid-laden HMDMs. The production of TNF-a was decreased in GABA-and topiramate-treated lipid-laden HMDMs, and levels of interleukin (IL-6 did not change. The activation of two signaling pathways, p38MAPK and NF-γB, was repressed by GABA and topiramate in lipid-laden HMDMs. Conclusion: GABA and topiramate inhibit the formation of human macrophage-derived foam cells and may be a possibility for macrophage targeted therapy of atherosclerotic lesions.

  17. Cholesterol Oxidase Binds TLR2 and Modulates Functional Responses of Human Macrophages

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    Katarzyna Bednarska

    2014-01-01

    Full Text Available Cholesterol oxidase (ChoD is considered to be an important virulence factor for Mycobacterium tuberculosis (Mtb, but its influence on macrophage activity is unknown. Here we used Nocardia erythropolis ChoD, which is very similar to the Mtb enzyme (70% identity at the amino-acid level, to evaluate the impact of bacterial ChoD on the activity of THP-1-derived macrophages in vitro. We found that ChoD decreased the surface expression of Toll-like receptor type 2 (TLR2 and complement receptor 3 (CR3 on these macrophages. Flow cytometry and confocal microscopy showed that ChoD competed with lipoteichoic acid for ligand binding sites on TLR2 but not on CR3, suggesting that ChoD signaling is mediated via TLR2. Binding of ChoD to the membrane of macrophages had diverse effects on the activity of macrophages, activating p38 mitogen activated kinase and stimulating production of a large amount of interleukin-10. Moreover, ChoD primed macrophages to enhance the production of reactive oxygen species in response to the phorbol myristate acetate, which was reduced by “switching off” TLR-derived signaling through interleukin-1 receptor-associated kinases 1 and 4 inhibition. Our study revealed that ChoD interacts directly with macrophages via TLR2 and influences the biological activity of macrophages during the development of the initial response to infection.

  18. Biocompatibility and Inflammatory Potential of Titanium Alloys Cultivated with Human Osteoblasts, Fibroblasts and Macrophages

    Science.gov (United States)

    Markhoff, Jana; Krogull, Martin; Schulze, Christian; Rotsch, Christian; Hunger, Sandra; Bader, Rainer

    2017-01-01

    The biomaterials used to maintain or replace functions in the human body consist mainly of metals, ceramics or polymers. In orthopedic surgery, metallic materials, especially titanium and its alloys, are the most common, due to their excellent mechanical properties, corrosion resistance, and biocompatibility. Aside from the established Ti6Al4V alloy, shape memory materials such as nickel-titanium (NiTi) have risen in importance, but are also discussed because of the adverse effects of nickel ions. These might be reduced by specific surface modifications. In the present in vitro study, the osteoblastic cell line MG-63 as well as primary human osteoblasts, fibroblasts, and macrophages were cultured on titanium alloys (forged Ti6Al4V, additive manufactured Ti6Al4V, NiTi, and Diamond-Like-Carbon (DLC)-coated NiTi) to verify their specific biocompatibility and inflammatory potential. Additive manufactured Ti6Al4V and NiTi revealed the highest levels of metabolic cell activity. DLC-coated NiTi appeared as a suitable surface for cell growth, showing the highest collagen production. None of the implant materials caused a strong inflammatory response. In general, no distinct cell-specific response could be observed for the materials and surface coating used. In summary, all tested titanium alloys seem to be biologically appropriate for application in orthopedic surgery. PMID:28772412

  19. Biocompatibility and Inflammatory Potential of Titanium Alloys Cultivated with Human Osteoblasts, Fibroblasts and Macrophages

    Directory of Open Access Journals (Sweden)

    Jana Markhoff

    2017-01-01

    Full Text Available The biomaterials used to maintain or replace functions in the human body consist mainly of metals, ceramics or polymers. In orthopedic surgery, metallic materials, especially titanium and its alloys, are the most common, due to their excellent mechanical properties, corrosion resistance, and biocompatibility. Aside from the established Ti6Al4V alloy, shape memory materials such as nickel-titanium (NiTi have risen in importance, but are also discussed because of the adverse effects of nickel ions. These might be reduced by specific surface modifications. In the present in vitro study, the osteoblastic cell line MG-63 as well as primary human osteoblasts, fibroblasts, and macrophages were cultured on titanium alloys (forged Ti6Al4V, additive manufactured Ti6Al4V, NiTi, and Diamond-Like-Carbon (DLC-coated NiTi to verify their specific biocompatibility and inflammatory potential. Additive manufactured Ti6Al4V and NiTi revealed the highest levels of metabolic cell activity. DLC-coated NiTi appeared as a suitable surface for cell growth, showing the highest collagen production. None of the implant materials caused a strong inflammatory response. In general, no distinct cell-specific response could be observed for the materials and surface coating used. In summary, all tested titanium alloys seem to be biologically appropriate for application in orthopedic surgery.

  20. Transforming growth factor beta-1 expression in macrophages of human chronic periapical diseases.

    Science.gov (United States)

    Liang, Z-Z; Li, J; Huang, S-G

    2017-03-30

    The objective of this study was to observe the distribution of macrophages (MPs) expressing transforming growth factor beta-1 (TGF-β1) in tissue samples from patients with different human chronic periapical diseases. In this study, samples were collected from 75 volunteers, who were divided into three groups according to classified standards, namely, healthy control (N = 25), periapical granuloma (N = 25), and periapical cyst (N = 25). The samples were fixed in 10% buffered formalin for more than 48 h, dehydrated, embedded, and stained with hematoxylin and eosin for histopathology. Double immunofluorescence was conducted to analyze the expression of TGF-β-CD14 double-positive MPs in periapical tissues. The number of double-positive cells (cells/mm 2 ) were significantly higher in the chronic periapical disease tissues (P periapical cyst group than in the periapical granuloma group (P periapical diseases. The TGF-β1-CD14 double-positive cells might play an important role in the pathology of human chronic periapical diseases.

  1. Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ.

    Science.gov (United States)

    Pahl, Jens H W; Kwappenberg, Kitty M C; Varypataki, Eleni M; Santos, Susy J; Kuijjer, Marieke L; Mohamed, Susan; Wijnen, Juul T; van Tol, Maarten J D; Cleton-Jansen, Anne-Marie; Egeler, R Maarten; Jiskoot, Wim; Lankester, Arjan C; Schilham, Marco W

    2014-03-10

    In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages. Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/- IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab. M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1β. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ-activated M2-like macrophages had low anti-tumor activity, IL-10-polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis. This study demonstrates that human macrophages can be induced to exert direct anti-tumor activity against osteosarcoma cells. Our

  2. Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions.

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    Bonnie van Wilgenburg

    Full Text Available Human macrophages are specialised hosts for HIV-1, dengue virus, Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC and multiple human induced Pluripotent Stem Cell (hiPSC lines over time periods of up to one year. Cumulatively, up to ∼3×10(7 monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+, CD16(low, CD163(+. Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology.

  3. Studies on mechanism of treatment of granulocyte colony-stimulating factor, recombinant human interleukin-11 and recombinant human interleukin-2 on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

    International Nuclear Information System (INIS)

    Li Ming; Ou Hongling; Xing Shuang; Huang Haixiao; Xiong Guolin; Xie Ling; Zhao Yanfang; Zhao Zhenhu; Wang Ning; Wang Jinxiang; Miao Jingcheng; Zhu Nankang; Luo Qingliang; Cong Yuwen; Zhang Xueguang

    2010-01-01

    Objective: To investigate the mechanism of treatment of granulocyte colony-stimulating factor (rhG-CSF), recombinant human interleukin-11 (rhIL-11) and recombinant human interleukin-2 (rhIL-2) on hematopoietic injuries induced by 4.5 Gy 60 Co γ-ray irradiation in beagles, and to provide experimental evidence for the clinical treatment of extremely severe myeloid acute radiation sickness (ARS). Methods: Sixteen beagle dogs were given 4.5 Gy 60 Co γ-ray total body irradiation (TBI), then randomly assigned into irradiation control group, supportive care group or cytokines + supportive care (abbreviated as cytokines) group. In addition to supportive care, rhG-CSF, rhIL-11 and rhIL-2 were administered subcutaneously to treat dogs in cytokines group. The percentage of CD34 + cells, cell cycle and apoptosis of nucleated cells in peripheral blood were examined by Flow cytometry. Results: After 4.5 Gy 60 Co γ-ray irradiation, the CD34 + cells in peripheral blood declined obviously (61.3% and 52.1% of baseline for irradiation control and supportive care group separately). The cell proportion of nucleated cells in G 0 /G 1 phase was increased notably notably (99.27% and 99.49% respectively). The rate of apoptosis (26.93% and 21.29% separately) and necrosis (3.27% and 4.14%, respectively) of nucleated cells were elevated significantly when compared with values before irradiation (P 0 /G 1 phase blockage of nucleated cells became more serious (99.71%). The rate of apoptosis (5.66%) and necrosis (1.60%) of nucleated cells were significantly lower than that of irradiation control and supportive care groups 1 d after exposure. Conclusions: Cytokines maybe mobilize CD34 + cells in bone marrow to peripheral blood, indce cell block at G 0 /G 1 phase and reduce apoptosis, and eventually cure hematopoietic injuries induced by irradiation. (authors)

  4. Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

    Science.gov (United States)

    Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

    2003-09-01

    Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the

  5. Tumor-cytolytic human macrophages cultured as nonadherent cells: potential for the adoptive immunotherapy of cancer.

    Science.gov (United States)

    Helinski, E H; Hurley, E L; Streck, R J; Bielat, K L; Pauly, J L

    1990-01-01

    Tumor-cytolytic lymphokine (e.g., interleukin-2; IL-2)-activated killer cells are currently being evaluated in IL-2/LAK cell adoptive immunotherapy regimens for the treatment of cancer. Monocyte-derived macrophages (M phi) are also known to be efficient tumor killer cells; accordingly, M phi that have been activated in vitro may also be of therapeutic merit. However, attempts to cultivate M phi for morphological and functional studies have often been compromised because M phi adhere rapidly and tenaciously to cultureware. Studies that we have conducted to address this problem have proven successful in developing procedures for the long-term cultivation of non-adherent immunocompetent M phi in serum-free medium using petri dishes containing a thin Teflon liner. The utility of this technology is documented by the results of studies presented herein in which light and scanning electron microscopy was used to analyze tumor-cytolytic human M phi. In these experiments, we demonstrated that nonadherent immunocompetent human M phi can be prepared for detailed examinations of their pleomorphic membrane architecture. Moreover, nonadherent human M phi could readily be collected for preparing conjugates of M phi and tumor cells. It is anticipated that this technology should prove useful for future structure-function studies defining the topographical location and spatial distribution of antigens and receptors on M phi membrane ultrastructures, particularly the microvilli-like projections that bridge together an immunocompetent effector M phi and target cell (e.g., tumor cells and microbial pathogens) and which provide the physical interaction required for the initial phases of a cellular immune response that includes antigen recognition and cell-to-cell adhesion.

  6. Classification of M1/M2-polarized human macrophages by label-free hyperspectral reflectance confocal microscopy and multivariate analysis.

    Science.gov (United States)

    Bertani, Francesca R; Mozetic, Pamela; Fioramonti, Marco; Iuliani, Michele; Ribelli, Giulia; Pantano, Francesco; Santini, Daniele; Tonini, Giuseppe; Trombetta, Marcella; Businaro, Luca; Selci, Stefano; Rainer, Alberto

    2017-08-21

    The possibility of detecting and classifying living cells in a label-free and non-invasive manner holds significant theranostic potential. In this work, Hyperspectral Imaging (HSI) has been successfully applied to the analysis of macrophagic polarization, given its central role in several pathological settings, including the regulation of tumour microenvironment. Human monocyte derived macrophages have been investigated using hyperspectral reflectance confocal microscopy, and hyperspectral datasets have been analysed in terms of M1 vs. M2 polarization by Principal Components Analysis (PCA). Following PCA, Linear Discriminant Analysis has been implemented for semi-automatic classification of macrophagic polarization from HSI data. Our results confirm the possibility to perform single-cell-level in vitro classification of M1 vs. M2 macrophages in a non-invasive and label-free manner with a high accuracy (above 98% for cells deriving from the same donor), supporting the idea of applying the technique to the study of complex interacting cellular systems, such in the case of tumour-immunity in vitro models.

  7. Human β-defensin 3 inhibits periodontitis development by suppressing inflammatory responses in macrophages.

    Science.gov (United States)

    Cui, Di; Lyu, Jinglu; Li, Houxuan; Lei, Lang; Bian, Tianying; Li, Lili; Yan, Fuhua

    2017-11-01

    Human β-defensin 3 (hBD3) is a cationic peptide with immunomodulatory effects on both innate and acquired immune responses. Periodontitis, an inflammatory disease that extends deep into periodontal tissues, causes the loss of supporting structures around the tooth. The present study assessed the effects of hBD3 as a monotherapy for periodontitis in mice and explored its potential mechanism. In vivo, hBD3 inhibited the levels of tumour necrosis factor (TNF)-α, interleukin-6, and matrix metalloprotease-9 in periodontium exposed to Porphyromonas gingivalis (P.g) in a mouse periodontitis model; reduced osteoclast formation and lower alveolar bone loss were also observed. In addition, hBD3 was related to the expression of polarization signature molecules in circulating monocytes. In vitro, hBD3 notably suppressed the production of TNF-α and interleukin-6 in RAW 264.7 cells stimulated by the lipopolysaccharide of P.g. Moreover, hBD3 attenuated polarization of RAW 264.7 cells into the M1 phenotype, with reduced activation of nuclear factor-κB signal transduction. In conclusion, hBD3 exhibits potent anti-periodontitis properties both in vitro and in vivo, and this effect may be correlated to inhibition of the nuclear factor-κB pathway and macrophage polarization. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  8. Human umbilical cord blood-derived f-macrophages retain pluripotentiality after thrombopoietin expansion

    International Nuclear Information System (INIS)

    Zhao Yong; Mazzone, Theodore

    2005-01-01

    We have previously characterized a new type of stem cell from human peripheral blood, termed fibroblast-like macrophage (f-MΦ). Here, using umbilical cord blood as a source, we identified cells with similar characteristics including expression of surface markers (CD14, CD34, CD45, CD117, and CD163), phagocytosis, and proliferative capacity. Further, thrombopoietin (TPO) significantly stimulated the proliferation of cord blood-derived f-MΦ (CB f-MΦ) at low dosage without inducing a megakaryocytic phenotype. Additional experiments demonstrated that TPO-expanded cord blood-derived f-MΦ (TCB f-MΦ) retained their surface markers and differentiation ability. Treatment with vascular endothelial cell growth factor (VEGF) gave rise to endothelial-like cells, expressing Flt-1, Flk-1, von Willebrand Factor (vWF), CD31, acetylated low density lipoprotein internalization, and the ability to form endothelial-like cell chains. In the presence of lipopolyssacharide (LPS) and 25 mM glucose, the TCB f-MΦ differentiated to express insulin mRNA, C-peptide, and insulin. In vitro functional analysis demonstrated that these insulin-positive cells could release insulin in response to glucose and other secretagogues. These findings demonstrate a potential use of CB f-MΦ and may lead to develop new therapeutic strategy for treating dominant disease

  9. Alteration of human macrophages microRNA expression profile upon infection with Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Lucinda Furci

    2013-01-01

    Conclusions: This study signifies the miRNA host response upon intracellular mycobacterial infection in macrophages, providing new aspects of regulation in host-pathogen interactions, at post-transcriptional levels.

  10. PLASMA AND LUNG MACROPHAGE CAROTENOID RESPONSIVENESS TO SUPPLEMENTATION AND OZONE EXPOSURE IN HUMANS

    Science.gov (United States)

    OBJECTIVE:: To examine the effect of ozone exposure and vegetable juice supplementation on plasma and lung macrophage concentrations of carotenoids. DESIGN:: A randomized trial. SETTING:: Subjects were exposed to ambient air prior to antioxidant supplementation and to ozone after...

  11. Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis.

    Science.gov (United States)

    Bonne-Année, Sandra; Kerepesi, Laura A; Hess, Jessica A; Wesolowski, Jordan; Paumet, Fabienne; Lok, James B; Nolan, Thomas J; Abraham, David

    2014-06-01

    Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  12. MIR144* inhibits antimicrobial responses against Mycobacterium tuberculosis in human monocytes and macrophages by targeting the autophagy protein DRAM2.

    Science.gov (United States)

    Kim, Jin Kyung; Lee, Hye-Mi; Park, Ki-Sun; Shin, Dong-Min; Kim, Tae Sung; Kim, Yi Sak; Suh, Hyun-Woo; Kim, Soo Yeon; Kim, In Soo; Kim, Jin-Man; Son, Ji-Woong; Sohn, Kyung Mok; Jung, Sung Soo; Chung, Chaeuk; Han, Sang-Bae; Yang, Chul-Su; Jo, Eun-Kyeong

    2017-02-01

    Autophagy is an important antimicrobial effector process that defends against Mycobacterium tuberculosis (Mtb), the human pathogen causing tuberculosis (TB). MicroRNAs (miRNAs), endogenous noncoding RNAs, are involved in various biological functions and act as post-transcriptional regulators to target mRNAs. The process by which miRNAs affect antibacterial autophagy and host defense mechanisms against Mtb infections in human monocytes and macrophages is largely uncharacterized. In this study, we show that Mtb significantly induces the expression of MIR144*/hsa-miR-144-5p, which targets the 3'-untranslated region of DRAM2 (DNA damage regulated autophagy modulator 2) in human monocytes and macrophages. Mtb infection downregulated, whereas the autophagy activators upregulated, DRAM2 expression in human monocytes and macrophages by activating AMP-activated protein kinase. In addition, overexpression of MIR144* decreased DRAM2 expression and formation of autophagosomes in human monocytes, whereas inhibition of MIR144* had the opposite effect. Moreover, the levels of MIR144* were elevated, whereas DRAM2 levels were reduced, in human peripheral blood cells and tissues in TB patients, indicating the clinical significance of MIR144* and DRAM2 in human TB. Notably, DRAM2 interacted with BECN1 and UVRAG, essential components of the autophagic machinery, leading to displacement of RUBCN from the BECN1 complex and enhancement of Ptdlns3K activity. Furthermore, MIR144* and DRAM2 were critically involved in phagosomal maturation and enhanced antimicrobial effects against Mtb. Our findings identify a previously unrecognized role of human MIR144* in the inhibition of antibacterial autophagy and the innate host immune response to Mtb. Additionally, these data reveal that DRAM2 is a key coordinator of autophagy activation that enhances antimicrobial activity against Mtb.

  13. Interaction of differentiated human adipocytes with macrophages leads to trogocytosis and selective IL-6 secretion

    OpenAIRE

    Sárvári, Anitta Kinga; Doan-Xuan, Quang-Minh; Bacsó, Zsolt; Csomós, István; Balajthy, Zoltán; Fésüs, László

    2015-01-01

    Obesity leads to adipose tissue inflammation that is characterized by increased release of proinflammatory molecules and the recruitment of activated immune cells. Although macrophages are present in the highest number among the immune cells in obese adipose tissue, not much is known about their direct interaction with adipocytes. We have introduced an ex vivo experimental system to characterize the cellular interactions and the profile of secreted cytokines in cocultures of macrophages and h...

  14. The humanized anti-human AMHRII mAb 3C23K exerts an anti-tumor activity against human ovarian cancer through tumor-associated macrophages.

    Science.gov (United States)

    Bougherara, Houcine; Némati, Fariba; Nicolas, André; Massonnet, Gérald; Pugnière, Martine; Ngô, Charlotte; Le Frère-Belda, Marie-Aude; Leary, Alexandra; Alexandre, Jérôme; Meseure, Didier; Barret, Jean-Marc; Navarro-Teulon, Isabelle; Pèlegrin, André; Roman-Roman, Sergio; Prost, Jean-François; Donnadieu, Emmanuel; Decaudin, Didier

    2017-11-21

    Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, such as human ovarian cancers (OCs). On this basis, a humanized glyco-engineered monoclonal antibody (3C23K) has been developed. The aim of this study was therefore to experimentally confirm the therapeutic potential of 3C23K in human OCs. We first determined by immunofluorescence, immunohistochemistry and cytofluorometry analyses the expression of AMHRII in patient's tumors and found that a majority (60 to 80% depending on the detection technique) of OCs were positive for this marker. We then provided evidence that the tumor stroma of OC is enriched in tumor-associated macrophages and that these cells are responsible for 3C23K-induced killing of tumor cells through ADCP and ADCC mechanisms. In addition, we showed that 3C23K reduced macrophages induced-T cells immunosuppression. Finally, we evaluated the therapeutic efficacy of 3C23K alone and in combination with a carboplatin-paclitaxel chemotherapy in a panel of OC Patient-Derived Xenografts. In those experiments, we showed that 3C23K significantly increased the proportion and the quality of chemotherapy-based in vivo responses. Altogether, our data support the potential interest of AMHRII targeting in human ovarian cancers and the evaluation of 3C23K in further clinical trials.

  15. Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile.

    Directory of Open Access Journals (Sweden)

    María Carolina Ortiz

    Full Text Available Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ or alternative-M2 (M2-MФ activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1, widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies.

  16. Uptake of [18F]fluorodeoxyglucose in human monocyte-macrophages in vitro

    International Nuclear Information System (INIS)

    Deichen, Jan Thiess; Prante, Olaf; Gack, Michaela; Schmiedehausen, Kristin; Kuwert, Torsten

    2003-01-01

    The fact that fluorine-18 fluorodeoxyglucose ([ 18 F]FDG) accumulates in inflammatory lesions as well as in tumours reduces the diagnostic specificity of positron emission tomography (PET) in oncology. The aim of this study was to characterise the uptake of [ 18 F]FDG in isolated human monocyte-macrophages (HMMs) in vitro in comparison with that in human glioblastoma (GLI) and pancreatic carcinoma cells (PAN). The purity of HMM preparations was determined by immunohistochemical staining and their functional integrity was assessed by long-term incubation with iodine-131 acetylated bovine serum albumin. [ 18 F]FDG uptake in HMMs was quantified as percent of whole [ 18 F]FDG activity per well (% ID) or as % ID in relation to total protein mass. [ 18 F]FDG uptake in HMMs significantly increased with culture duration, yielding 7.5%±0.9% (% ID/100 μg) at day 14. Stimulation by lipopolysaccharide further enhanced [ 18 F]FDG uptake in HMMs by a factor of 2. [ 18 F]FDG uptake significantly decreased with increasing glucose concentration in the medium. Radio-thin layer chromatography of intracellular metabolites revealed that [ 18 F]FDG was trapped by HMMs mainly as [ 18 F]FDG-6-phosphate and [ 18 F]FDG-1,6-diphosphate. [ 18 F]FDG uptake was in the range of uptake values measured in GLI and PAN. By accumulating [ 18 F]FDG in a manner analogous to uptake by tumour cells, activated HMMs may contribute to the [ 18 F]FDG uptake values measured by PET in neoplasms. (orig.)

  17. Irradiation for conjunctival granulocytic sarcoma

    International Nuclear Information System (INIS)

    Fleckenstein, K.; Geinitz, H.; Grosu, A.; Molls, M.; Goetze, K.; Werner, M.

    2003-01-01

    Case History and Findings: A 73-year-old woman with a history of myeloproliferative syndrome (MPS) presented with bilateral chemosis, redness and burning of the eyes. The ocular motility was severely impaired. Ophthalmological examination revealed markedly distended conjunctivas on both sides. Biopsy disclosed conjunctival granulocytic sarcoma as an initial symptom of acute myelogenous leukemia (AML). Diagnosis was confirmed by peripheral blood smear and bone marrow aspiration. Treatment and Outcome: The orbital tumor disappeared completely after local external beam irradiation with a total dose of 30 Gy and no further orbital recurrence occurred. With chemotherapy following irradiation transient hematological remission was achieved. 5 months after diagnosis the patient died of respiratory failure following atypical pneumonia as a consequence of her underlying disorder. Conclusion: Detection of orbital granulocytic sarcoma, even in the absence of typical leukemic symptoms is of practical importance, because treatment with irradiation can lead to stabilization or improvement in the patient's vision. (orig.)

  18. The Probiotic Mixture VSL#3 Alters the Morphology and Secretion Profile of Both Polarized and Unpolarized Human Macrophages in a Polarization-Dependent Manner

    Science.gov (United States)

    Isidro, Raymond A.; Bonilla, Fernando J.; Pagan, Hendrick; Cruz, Myrella L.; Lopez, Pablo; Godoy, Lenin; Hernandez, Siomara; Loucil-Alicea, Raisa Y.; Rivera-Amill, Vanessa; Yamamura, Yasuhiro; Isidro, Angel A.; Appleyard, Caroline B.

    2014-01-01

    Background Patients with Inflammatory Bowel Disease (IBD), most commonly Crohn’s disease (CD) or ulcerative colitis (UC), suffer from chronic intestinal inflammation of unknown etiology. Increased proinflammatory macrophages (M1) have been documented in tissue from patients with CD. Anti-inflammatory macrophages (M2) may play a role in UC given the preponderance of Th2 cytokines in this variant of IBD. Animal and clinical studies have shown that the probiotic VSL#3 can ameliorate signs and symptoms of IBD. Although animal data suggests a modulatory effect on macrophage phenotype, the effect of VSL#3 on human macrophages remains unknown. Objective To determine the effect of the probiotic VSL#3 on the phenotype of polarized (M1/M2) and unpolarized (MΦ) human macrophages. Methods Human monocyte-derived macrophages, generated by culturing monocytes with M-CSF, were left unpolarized or were polarized towards an M1 or an M2 phenotype by culture with LPS and IFN-γ or IL-4, respectively, and were then cultured in the presence or absence of VSL#3 for 3 days. Changes in macrophage morphology were assessed. Cytokine and chemokine levels in supernatants were determined by multiplex assay. Results VSL#3 decreased the granuloma-like aggregates of M1 macrophages, increased fibroblast-like M2 macrophages, and decreased fibroblast-like MΦ macrophages. VSL#3 increased the secretion of IL-1β, IL-6, IL-10, and G-CSF by M1, M2, and MΦ macrophages. VSL#3 exposure maintained the proinflammatory phenotype of M1 macrophages, sustaining IL-12 secretion, increasing IL-23 secretion, and decreasing MDC secretion. Both VSL#3-treated M2 and MΦ macrophages secreted higher levels of anti-inflammatory and pro-healing factors such as IL-1Ra, IL-13, EGF, FGF-2, TGF-α, and VEGF, as well as proinflammatory cytokines, including IL-12 and TNF-α. Conclusion Under our experimental conditions VSL#3 induced a mixed proinflammatory and anti-inflammatory phenotype in polarized and unpolarized

  19. Distribution of In-111 in granulocyte and other cellular elements of blood (CEB) in human In-111-labeled mixed white cell (MWC) and platelet preparations

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Chowdhury, S.; Brown, M.L.; Wahner, H.W.

    1984-01-01

    A large number of platelets (PLT), red blood cells (RBC) are present along with granulocyte (GC) in In-111 in CEB was determined by Ficoll-Hypaque gradient (FHG) centrifugation of In-111-MWC and PLT preparation as a quality control procedure. MWC were separated by sedimentation with hydroxyethyl starch; PLT by differential centrifugation. MWC and PLT were labeled with In-111-oxine in saline, ACD-saline or with In-111-tropolone in 0.5 ml of ACD-plasma. 0.3-0.5 ml of labeled cell suspended in plasma was layered on 3 ml FHG of two densities (1.119 and 1.077 gm/ml) and spun in a clear polystyrene tube at 1800 G for 30 min. Four layers (plasma, PLT, GC, and RBC) were separated, and In-111 radioactivity in each fraction was determined with a gamma counter. Simultaneously cell types in MWC and PLT preparations were determined by Coulter counter and differential counting. Most of In-111 in In-MWC is associated with the PLT and RBC, GC/lymphocyte ratio is 6/4. GC has higher extraction efficiency than RBC and PLT. PLT preparation is pure and (96 +- 3)% of In-111 is bound to PLT, (4 +- 3)% to RBC and (0.2 +- 0.1)% to GC; PLT preparation contains PLT (97 +- 3)%, RBC (4 +- 3)% and GC (0.2 +- 0.1)%

  20. Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

    Science.gov (United States)

    Bakherad, Hamid; Gargari, Seyed Latif Mousavi; Sepehrizadeh, Zargham; Aghamollaei, Hossein; Taheri, Ramezan Ali; Torshabi, Maryam; Yazdi, Mojtaba Tabatabaei; Ebrahimizadeh, Walead; Setayesh, Neda

    2017-09-01

    It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models. Copyright © 2017. Published by Elsevier Masson SAS.

  1. Hematopoietic growth factors and human acute leukemia.

    Science.gov (United States)

    Löwenberg, B; Touw, I

    1988-10-22

    The study of myelopoietic maturation arrest in acute myeloblastic leukemia (AML) has been eased by availability of the human recombinant hemopoietic growth factors, macrophage colony stimulating factor (M-CSF), granulocyte-(G-CSF), granulocyte-macrophage-(GM-CSF) and multilineage stimulating factor (IL-3). Nonphysiological expansion of the leukemic population is not due to escape from control by these factors. Proliferation in vitro of AML cells is dependent on the presence of one or several factors in most cases. The pattern of factor-dependency does not correlate with morphological criteria in individual cases, and may thus offer a new tool for classification of AML. Overproduction of undifferentiated cells is not due to abnormal expression of receptors for the stimulating factors acting at an immature level. Rather, autocrine secretion of early acting lymphokines maintains proliferation of the leukemic clone. When looking at causes of leukemic dysregulation, yet undefined inhibitors of differentiation probably are of equal importance as dysequilibrated stimulation by lymphokines.

  2. Environmental Legionella spp. collected in urban test sites of South East Queensland, Australia, are virulent to human macrophages in vitro.

    Science.gov (United States)

    Lawrence, Amba; Eglezos, Sofroni; Huston, Wilhelmina

    2016-01-01

    Legionellae are frequent contaminants of potable water supplies, resulting in sporadic infections and occasional outbreaks. Isolates of Legionella were collected from urban test sites within South East Queensland and evaluated for their virulence potential in vitro. Two strains (from the species Legionella londiniensis and Legionella quinlivanii) were demonstrated to have the ability to infect human macrophages, while a strain from the species Legionella anisa did not maintain an infection over the same time course. This suggests that the spectrum of urban environmentally associated Legionella with potential to cause human disease might be greater than currently considered. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  3. Nanoparticles as Antituberculosis Drugs Carriers: Effect on Activity Against Mycobacterium tuberculosis in Human Monocyte-Derived Macrophages

    International Nuclear Information System (INIS)

    Anisimova, Y.V.; Gelperina, S.I.; Peloquin, C.A.; Heifets, L.B.

    2000-01-01

    This is the first report evaluating the nanoparticle delivery system for three antituberculosis drugs: isoniazid, rifampin, and streptomycin. The typical particle size is 250 nm. We studied accumulation of these drugs in human monocytes as well as their antimicrobial activity against Mycobacterium tuberculosis residing in human monocyte-derived macrophages. Nanoparticle encapsulation increased the intracellular accumulation (cell-association) of all three tested drugs, but it enhanced the antimicrobial activity of isoniazid and streptomycin only. On the other hand, the activity of encapsulated rifampin against intracellular bacteria was not higher than that of the free drug

  4. 15-Lipoxygenases regulate the production of chemokines in human lung macrophages.

    Science.gov (United States)

    Abrial, C; Grassin-Delyle, S; Salvator, H; Brollo, M; Naline, E; Devillier, P

    2015-09-01

    15-Lipoxygenase (15-LOX) activity is associated with inflammation and immune regulation. The objectives of the present study were to investigate the expression of 15-LOX-1 and 15-LOX-2 and evaluate the enzymes' roles in the polarization of human lung macrophages (LMs) in response to LPS and Th2 cytokines (IL-4/-13). LMs were isolated from patients undergoing surgery for carcinoma. The cells were cultured with a 15-LOX inhibitor (PD146176 or ML351), a COX inhibitor (indomethacin), a 5-LOX inhibitor (MK886) or vehicle and then stimulated with LPS (10 ng · mL(-1)), IL-4 (10 ng · mL(-1)) or IL-13 (50 ng · mL(-1)) for 24 h. Levels of ALOX15 (15-LOX-1) and ALOX15B (15-LOX-2) transcripts were determined by real-time quantitative PCR. Immunoassays were used to measure levels of LPS-induced cytokines (TNF-α, CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL10) and Th2 cytokine-induced chemokines (CCL13, CCL18 and CCL22) in the culture supernatant. Stimulation of LMs with LPS was associated with increased expression of ALOX15B, whereas stimulation with IL-4/IL-13 induced the expression of ALOX15. PD146176 and ML351 (10 μM) reduced the release of the chemokines induced by LPS and Th2 cytokines. The effects of these 15-LOX inhibitors were maintained in the presence of indomethacin and MK886. Furthermore, indomethacin revealed the inhibitory effect of PD146176 on TNF-α release. Inhibition of the 15-LOX pathways is involved in the down-regulation of the in vitro production of chemokines in LMs. Our results suggest that the 15-LOX pathways have a role in the pathogenesis of inflammatory lung disorders and may thus constitute a potential drug target. © 2015 The British Pharmacological Society.

  5. A mature macrophage is a principal HIV-1 cellular reservoir in humanized mice after treatment with long acting antiretroviral therapy.

    Science.gov (United States)

    Araínga, Mariluz; Edagwa, Benson; Mosley, R Lee; Poluektova, Larisa Y; Gorantla, Santhi; Gendelman, Howard E

    2017-03-09

    Despite improved clinical outcomes seen following antiretroviral therapy (ART), resting CD4+ T cells continue to harbor latent human immunodeficiency virus type one (HIV-1). However, such cells are not likely the solitary viral reservoir and as such defining where and how others harbor virus is imperative for eradication measures. To such ends, we used HIV-1 ADA -infected NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice reconstituted with a human immune system to explore two long-acting ART regimens investigating their abilities to affect viral cell infection and latency. At 6 weeks of infection animals were divided into four groups. One received long-acting (LA) cabotegravir (CAB) and rilpivirine (RVP) (2ART), a second received LA CAB, lamivudine, abacavir and RVP (4ART), a third were left untreated and a fourth served as an uninfected control. After 4 weeks of LA ART treatment, blood, spleen and bone marrow (BM) cells were collected then phenotypically characterized. CD4+ T cell subsets, macrophages and hematopoietic progenitor cells were analyzed for HIV-1 nucleic acids by droplet digital PCR. Plasma viral loads were reduced by two log 10 or to undetectable levels in the 2 and 4ART regimens, respectively. Numbers and distributions of CD4+ memory and regulatory T cells, macrophages and hematopoietic progenitor cells were significantly altered by HIV-1 infection and by both ART regimens. ART reduced viral DNA and RNA in all cell and tissue compartments. While memory cells were the dominant T cell reservoir, integrated HIV-1 DNA was also detected in the BM and spleen macrophages in both regimen-treated mice. Despite vigorous ART regimens, HIV-1 DNA and RNA were easily detected in mature macrophages supporting their potential role as an infectious viral reservoir.

  6. Human Cord Blood and Bone Marrow CD34+ Cells Generate Macrophages That Support Erythroid Islands.

    Directory of Open Access Journals (Sweden)

    Eyayu Belay

    Full Text Available Recently, we developed a small molecule responsive hyperactive Mpl-based Cell Growth Switch (CGS that drives erythropoiesis associated with macrophages in the absence of exogenous cytokines. Here, we compare the physical, cellular and molecular interaction between the macrophages and erythroid cells in CGS expanded CD34+ cells harvested from cord blood, marrow or G-CSF-mobilized peripheral blood. Results indicated that macrophage based erythroid islands could be generated from cord blood and marrow CD34+ cells but not from G-CSF-mobilized CD34+ cells. Additional studies suggest that the deficiency resides with the G-CSF-mobilized CD34+ derived monocytes. Gene expression and proteomics studies of the in vitro generated erythroid islands detected the expression of erythroblast macrophage protein (EMP, intercellular adhesion molecule 4 (ICAM-4, CD163 and DNASE2. 78% of the erythroblasts in contact with macrophages reached the pre reticulocyte orthochromatic stage of differentiation within 14 days of culture. The addition of conditioned medium from cultures of CD146+ marrow fibroblasts resulted in a 700-fold increase in total cell number and a 90-fold increase in erythroid cell number. This novel CD34+ cell derived erythroid island may serve as a platform to explore the molecular basis of red cell maturation and production under normal, stress and pathological conditions.

  7. Downregulation of host tryptophan-aspartate containing coat (TACO gene restricts the entry and survival of Leishmania donovani in human macrophage model

    Directory of Open Access Journals (Sweden)

    Venkateswara Reddy Gogulamudi

    2015-10-01

    Full Text Available Leishmania are obligate intracellular protozoan parasites of mammalian hosts. Promastigotes of Leishmania are internalized by macrophages and transformed into amastigotes in phagosomes, and replicate in phagolysosomes. Phagosomal maturation arrest is known to play a central role in the survival of pathogenic Leishmania within activated macrophages. Recently, tryptophan-aspartate containing coat (TACO gene has been recognized as playing a crucial role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process. We postulated that a similar association of TACO gene with phagosomes would prevent the vacuole from maturation in the case of Leishmania. In this study we attempted to define the effect of TACO gene downregulation on the uptake/survival of Leishmania donovani intracellularly, by treatment with Vitamin D3/Retinoic acid (RA & Chenodeoxycholic acid (CDCA/Retinoic acid (RA combinations in human THP-1 macrophages (in vitro. Treatment with these molecules downregulated the TACO gene in macrophages, resulting in reduced parasite load and marked reduction of disease progression in L. donovani infected macrophages. Taken together, these results suggest that TACO gene downregulation may play a role in subverting macrophage machinery in establishing the L.donovani replicative niche inside the host. Our study is the first to highlight the importantrole of the TACO gene in Leishmania entry, and to identify TACO gene downregulation as potential drug target against leishmaniasis.

  8. Targeted Delivery of Glucan Particle Encapsulated Gallium Nanoparticles Inhibits HIV Growth in Human Macrophages

    Directory of Open Access Journals (Sweden)

    Ernesto R. Soto

    2016-01-01

    Full Text Available Glucan particles (GPs are hollow, porous 3–5 μm microspheres derived from the cell walls of Baker’s yeast (Saccharomyces cerevisiae. The 1,3-β-glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing β-glucan receptors. GPs have been used for macrophage-targeted delivery of a wide range of payloads (DNA, siRNA, protein, small molecules, and nanoparticles encapsulated inside the hollow GPs or bound to the surface of chemically derivatized GPs. Gallium nanoparticles have been proposed as an inhibitory agent against HIV infection. Here, macrophage targeting of gallium using GPs provides for more efficient delivery of gallium and inhibition of HIV infection in macrophages compared to free gallium nanoparticles.

  9. Effects of recombinant human granulocyte colony-stimulating factor on central and peripheral T lymphocyte reconstitution after sublethal irradiation in mice

    International Nuclear Information System (INIS)

    Zhao Hongxia; Guo Mei; Sun Xuedong; Ai Huisheng; Sun Wanjun; Hu Hailan; Wei Li

    2013-01-01

    Granulocyte colony-stimulating factor (G-CSF) is one of the most critical cytokines used for the treatment of acute radiation syndrome (ARS). In addition to the hematopoietic effects of G-CSF on the differentiation and proliferation of myeloid progenitor cells, G-CSF is also known to have immunomodulatory effects. The aim of the present study was to investigate whether G-CSF could accelerate central and peripheral T lymphocyte recovery after a sublethal dose of irradiation. Female BALB/c mice were subjected to 6 Gy of total body irradiation and then were treated with either 100 μg/kg G-CSF or an equal volume of PBS once daily for 14 days. Percentages of thymocyte subpopulations including CD4- CD8-, CD4+ CD8+, CD4+ CD8- and CD4- CD8+ T cells, peripheral CD3+, CD4+ and CD8+ cells were analyzed by flow cytometry. Recent thymic emigrants (RTEs) were assessed by real-time polymerase chain reaction (PCR) using primers specific to the 257-bp T cell receptor rearrangement excision circles (sjTRECs). The proliferative capacity of splenic mononuclear cells upon exposure to ConA was measured by using the Cell Count Kit-8 (CCK-8). G-CSF treatment promoted thymocyte regeneration, accelerated the recovery of CD4+ CD8+ cells and increased the frequency of thymocyte sjTRECs. These effects were more prominent at early time points (Day 28) after irradiation. G-CSF also increased the rate of recovery of peripheral CD3+, CD4+ and CD8+ cells and shortened the period of severe lymphopenia following irradiation. G-CSF also increased the splenic mononuclear cell mitotic responsiveness to ConA more than control-treated cells. Our results show that G-CSF accelerates T cell recovery through both thymic-dependent and thymic-independent pathways, which could be used to increase the rate of immune reconstitution after sublethal irradiation. (author)

  10. Macrophage specific overexpression of the human macrophage scavenger receptor in transgenic mice, using a 180-kb yeast artificial chromosome, leads to enhanced foam cell formation of isolated peritoneal macrophages

    NARCIS (Netherlands)

    de Winther, M. P.; van Dijk, K. W.; van Vlijmen, B. J.; Gijbels, M. J.; Heus, J. J.; Wijers, E. R.; van den Bos, A. C.; Breuer, M.; Frants, R. R.; Havekes, L. M.; Hofker, M. H.

    1999-01-01

    Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation. To investigate the in vivo role of the MSR in this process, a transgenic

  11. The toxicity of rifampicin polylactic acid nanoparticles against Mycobacterium bovis BCG and human macrophage THP-1 cell line

    International Nuclear Information System (INIS)

    Erokhina, M; Rybalkina, E; Lepekha, L; Barsegyan, G; Onishchenko, G

    2015-01-01

    Tuberculosis is rapidly becoming a major health problem. The rise in tuberculosis incidence stimulates efforts to develop more effective delivery systems for the existing antituberculous drugs while decreasing the side effects. The nanotechnology may provide novel drug delivery tools allowing controlled drug release. Rifampicin is one of the main antituberculous drugs, characterized by high toxicity, and Poly (L-lactic acid) (PLLA) is a biodegradable polymer used for the preparation of encapsulated drugs. The aim of our work was to evaluate the toxicity of rifampicin-PLLA nanoparticles against Mycobacterium bovis BCG using human macrophage THP-1 cell line. Our data demonstrate that rifampicin-PLLA is effective against M. bovis BCG in the infected macrophages. The drug is inducing the dysfunction of mitochondria and apoptosis in the macrophages and is acting as a potential substrate of Pgp thereby modulating cell chemosensitivity. The severity of the toxic effects of the rifampicin-PLLA nanoparticles is increasing in a dose-dependent manner. We suggest that free rifampicin induces death of M. bovis BCG after PLLA degradation and diffusion from phago-lysosomes to cytoplasm causing mitochondria dysfunction and affecting the Pgp activity. (paper)

  12. Effects of Acrolein on Leukotriene Biosynthesis in Human Neutrophils

    OpenAIRE

    Zemski Berry, Karin A.; Henson, Peter M.; Murphy, Robert C.

    2008-01-01

    Acrolein is a toxic, highly reactive α,β-unsaturated aldehyde that is present in high concentrations in cigarette smoke. In the current study, the effect of acrolein on eicosanoid synthesis in stimulated human neutrophils was examined. Eicosanoid synthesis in neutrophils was initiated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) and 5-LO products in addition to small amounts of COX produc...

  13. Haemophilus ducreyi infection induces activation of the NLRP3 inflammasome in nonpolarized but not in polarized human macrophages.

    Science.gov (United States)

    Li, Wei; Katz, Barry P; Bauer, Margaret E; Spinola, Stanley M

    2013-08-01

    Recognition of microbial infection by certain intracellular pattern recognition receptors leads to the formation of a multiprotein complex termed the inflammasome. Inflammasome assembly activates caspase-1 and leads to cleavage and secretion of the proinflammatory cytokines interleukin-1 beta (IL-1β) and IL-18, which help control many bacterial pathogens. However, excessive inflammation mediated by inflammasome activation can also contribute to immunopathology. Here, we investigated whether Haemophilus ducreyi, a Gram-negative bacterium that causes the genital ulcer disease chancroid, activates inflammasomes in experimentally infected human skin and in monocyte-derived macrophages (MDM). Although H. ducreyi is predominantly extracellular during human infection, several inflammasome-related components were transcriptionally upregulated in H. ducreyi-infected skin. Infection of MDM with live, but not heat-killed, H. ducreyi induced caspase-1- and caspase-5-dependent processing and secretion of IL-1β. Blockage of H. ducreyi uptake by cytochalasin D significantly reduced the amount of secreted IL-1β. Knocking down the expression of the inflammasome components NLRP3 and ASC abolished IL-1β production. Consistent with NLRP3-dependent inflammasome activation, blocking ATP signaling, K(+) efflux, cathepsin B activity, and lysosomal acidification all inhibited IL-1β secretion. However, inhibition of the production and function of reactive oxygen species did not decrease IL-1β production. Polarization of macrophages to classically activated M1 or alternatively activated M2 cells abrogated IL-1β secretion elicited by H. ducreyi. Our study data indicate that H. ducreyi induces NLRP3 inflammasome activation via multiple mechanisms and suggest that the heterogeneity of macrophages within human lesions may modulate inflammasome activation during human infection.

  14. Xylitol, an anticaries agent, exhibits potent inhibition of inflammatory responses in human THP-1-derived macrophages infected with Porphyromonas gingivalis.

    Science.gov (United States)

    Park, Eunjoo; Na, Hee Sam; Kim, Sheon Min; Wallet, Shannon; Cha, Seunghee; Chung, Jin

    2014-06-01

    Xylitol is a well-known anticaries agent and has been used for the prevention and treatment of dental caries. In this study, the anti-inflammatory effects of xylitol are evaluated for possible use in the prevention and treatment of periodontal infections. Cytokine expression was stimulated in THP-1 (human monocyte cell line)-derived macrophages by live Porphyromonas gingivalis, and enzyme-linked immunosorbent assay and a commercial multiplex assay kit were used to determine the effects of xylitol on live P. gingivalis-induced production of cytokine. The effects of xylitol on phagocytosis and the production of nitric oxide were determined using phagocytosis assay, viable cell count, and Griess reagent. The effects of xylitol on P. gingivalis adhesion were determined by immunostaining, and costimulatory molecule expression was examined by flow cytometry. Live P. gingivalis infection increased the production of representative proinflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-1β, in a multiplicity of infection- and time-dependent manner. Live P. gingivalis also enhanced the release of cytokines and chemokines, such as IL-12 p40, eotaxin, interferon γ-induced protein 10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1. The pretreatment of xylitol significantly inhibited the P. gingivalis-induced cytokines production and nitric oxide production. In addition, xylitol inhibited the attachment of live P. gingivalis on THP-1-derived macrophages. Furthermore, xylitol exerted antiphagocytic activity against both Escherichia coli and P. gingivalis. These findings suggest that xylitol acts as an anti-inflammatory agent in THP-1-derived macrophages infected with live P. gingivalis, which supports its use in periodontitis.

  15. Cathepsin D Specifically Cleaves the Chemokines Macrophage Inflammatory Protein-1α, Macrophage Inflammatory Protein-1β, and SLC That Are Expressed in Human Breast Cancer

    Science.gov (United States)

    Wolf, Marlene; Clark-Lewis, Ian; Buri, Caroline; Langen, Hanno; Lis, Maddalena; Mazzucchelli, Luca

    2003-01-01

    Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1α and MIP-1β degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu58 to Trp59 bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1β hybrids indicated that processing of MIP-1β might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1α, MIP-1β, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes. PMID:12651610

  16. The role of substrate morphology for the cytokine release profile of immature human primary macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Bartneck, Matthias [Department of Medicine III, Medical Faculty, RWTH Aachen, Pauwelsstr. 30, 52074 Aachen (Germany); Heffels, Karl-Heinz [Department and Chair of Functional Materials in Medicine and Dentistry, University of Würzburg, Pleicherwall 2, 97070 Würzburg (Germany); Bovi, Manfred [Electron Microscopic Facility, Medical Faculty, RWTH Aachen (Germany); Groll, Jürgen [Department and Chair of Functional Materials in Medicine and Dentistry, University of Würzburg, Pleicherwall 2, 97070 Würzburg (Germany); Zwadlo-Klarwasser, Gabriele [Interdisciplinary Center for Clinical Research and Dept. of Dermatology, Medical Faculty, RWTH Aachen, Pauwelsstr. 30, 52056 Aachen (Germany)

    2013-12-01

    There is increasing evidence that the physicochemical nature of any given material is a dominant factor for the release of cytokines by innate immune cells, specifically of macrophages, and thus majorly influences their interaction with other cell types. Recently, we could show that the 3D structure of star shaped polytheylene oxide–polypropylene oxide co-polymers (sP(EO-stat-PO))-hydrogel coated substrates has a stronger influence on the release pattern of cytokines after 7 days of culture than surface chemistry. Here, we focused on the analysis of cytokine release over time and a more detailed analysis of cell morphology by scanning electron microscopy (SEM). Therefore, we compared different strategies for SEM sample preparation and found that using osmium tetroxide combined with aqua bidest led to best preparation results. For cytokine release we show significant changes from day 3 to day 7 of cell culture. After 3 days, the sP(EO-stat-PO)-coated substrates led to an induction of pro-angiogenic CCL3 and CCL4, and of low amounts of the anti-inflammatory IL10, which declined at day 7. In contrast, pleiotropic IL6 and the pro-inflammatory TNFα and IL1β were expressed stronger at day 7 than at day 3. - Highlights: • Strategies for the preparation of macrophages on hydrogel materials (Fig. 1) • Cytokine release of immature macrophages on the substrates (Fig. 2 and Table 1) • Changes in cytokine release during macrophage maturation (Table 2)

  17. Human macrophages primed with angiogenic factors show dynamic plasticity, irrespective of extracellular matrix components

    NARCIS (Netherlands)

    Ploeger, Diana T. A.; van Putten, Sander M.; Koerts, Jasper A.; van Luyn, Marja J. A.; Harmsen, Martin C.

    Macrophages are important in inflammation as well as in tissue repair processes. They can be activated by various stimuli and classified into two major groups: M1 (classically activated) or M2 (alternatively activated). Inflammation, angiogenesis and matrix remodeling play a major role in tissue

  18. Modeling receptor-mediated endocytosis of polymer-functionalized iron oxide nanoparticles by human macrophages

    Czech Academy of Sciences Publication Activity Database

    Lunov, O.; Zablotskyy, Vitaliy A.; Syrovets, T.; Röcker, C.; Tron, K.; Nienhaus, G.U.; Simmet, T.

    2011-01-01

    Roč. 32, č. 2 (2011), s. 547-555 ISSN 0142-9612 Institutional research plan: CEZ:AV0Z10100522 Keywords : macrophage * nanoparticle * bioabsorption * modeling * antisense * MRI (magnetic resonance imaging) Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 7.404, year: 2011 http://www.sciencedirect.com/science/article/pii/S014296121001149X

  19. The role of substrate morphology for the cytokine release profile of immature human primary macrophages

    International Nuclear Information System (INIS)

    Bartneck, Matthias; Heffels, Karl-Heinz; Bovi, Manfred; Groll, Jürgen; Zwadlo-Klarwasser, Gabriele

    2013-01-01

    There is increasing evidence that the physicochemical nature of any given material is a dominant factor for the release of cytokines by innate immune cells, specifically of macrophages, and thus majorly influences their interaction with other cell types. Recently, we could show that the 3D structure of star shaped polytheylene oxide–polypropylene oxide co-polymers (sP(EO-stat-PO))-hydrogel coated substrates has a stronger influence on the release pattern of cytokines after 7 days of culture than surface chemistry. Here, we focused on the analysis of cytokine release over time and a more detailed analysis of cell morphology by scanning electron microscopy (SEM). Therefore, we compared different strategies for SEM sample preparation and found that using osmium tetroxide combined with aqua bidest led to best preparation results. For cytokine release we show significant changes from day 3 to day 7 of cell culture. After 3 days, the sP(EO-stat-PO)-coated substrates led to an induction of pro-angiogenic CCL3 and CCL4, and of low amounts of the anti-inflammatory IL10, which declined at day 7. In contrast, pleiotropic IL6 and the pro-inflammatory TNFα and IL1β were expressed stronger at day 7 than at day 3. - Highlights: • Strategies for the preparation of macrophages on hydrogel materials (Fig. 1) • Cytokine release of immature macrophages on the substrates (Fig. 2 and Table 1) • Changes in cytokine release during macrophage maturation (Table 2)

  20. Placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin-8 and transforming growth factor-beta1.

    Science.gov (United States)

    Bácsi, A; Aranyosi, J; Beck, Z; Ebbesen, P; Andirkó, I; Szabó, J; Lampé, L; Kiss, J; Gergely, L; Tóth, F D

    1999-10-01

    Although syncytiotrophoblast (ST) cells can be infected by human cytomegalovirus (HCMV), in vitro studies have indicated that ST cells do not support the complete viral reproductive cycle, or HCMV replication may occur in less than 3% of ST cells. The present study tested the possibility that placental macrophages might enhance activation of HCMV carried in ST cells and, further, that infected ST cells would be capable of transmitting virus to neighboring macrophages. For this purpose, we studied HCMV replication in ST cells grown alone or cocultured with uninfected placental macrophages. Our results demonstrated that HCMV gene expression in ST cells was markedly upregulated by coculture with macrophages, resulting in release of substantial amounts of infectious virus from HCMV-infected ST cells. After having become permissive for viral replication, ST cells delivered HCMV to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HCMV gene expression in ST cells was mediated by marked interleukin-8 (IL-8) and transforming growth factor-beta1 (TGF-beta1) release from macrophages, an effect caused by contact between the different placental cells. Our findings indicate an interactive role for the ST layer and placental macrophages in the dissemination of HCMV among placental tissue. Eventually, these interactions may contribute to the transmission of HCMV from mother to the fetus.

  1. Functional characterization and phenotypic monitoring of human hematopoietic stem cell expansion and differentiation of monocytes and macrophages by whole-cell mass spectrometry

    Directory of Open Access Journals (Sweden)

    Guido Vogel

    2018-01-01

    Full Text Available The different facets of macrophages allow them to play distinct roles in tissue homeostasis, tissue repair and in response to infections. Individuals displaying dysregulated macrophage functions are proposed to be prone to inflammatory disorders or infections. However, this being a cause or a consequence of the pathology remains often unclear. In this context, we isolated and expanded CD34+ HSCs from healthy blood donors and derived them into CD14+ myeloid progenitors which were further enriched and differentiated into macrophages. Aiming for a comprehensive phenotypic profiling, we generated whole-cell mass spectrometry (WCMS fingerprints of cell samples collected along the different stages of the differentiation process to build a predictive model using a linear discriminant analysis based on principal components. Through the capacity of the model to accurately predict sample's identity of a validation set, we demonstrate that WCMS profiles obtained from bona fide blood monocytes and respectively derived macrophages mirror profiles obtained from equivalent HSC derivatives. Finally, HSC-derived macrophage functionalities were assessed by quantifying cytokine and chemokine responses to a TLR agonist in a 34-plex luminex assay and by measuring their capacity to phagocytise mycobacteria. These functional read-outs could not discriminate blood monocytes-derived from HSC-derived macrophages. To conclude, we propose that this method opens new avenues to distinguish the impact of human genetics on the dysregulated biological properties of macrophages in pathological conditions.

  2. Dual prognostic significance of tumour-associated macrophages in human pancreatic adenocarcinoma treated or untreated with chemotherapy.

    Science.gov (United States)

    Di Caro, Giuseppe; Cortese, Nina; Castino, Giovanni Francesco; Grizzi, Fabio; Gavazzi, Francesca; Ridolfi, Cristina; Capretti, Giovanni; Mineri, Rossana; Todoric, Jelena; Zerbi, Alessandro; Allavena, Paola; Mantovani, Alberto; Marchesi, Federica

    2016-10-01

    Tumour-associated macrophages (TAMs) play key roles in tumour progression. Recent evidence suggests that TAMs critically modulate the efficacy of anticancer therapies, raising the prospect of their targeting in human cancer. In a large retrospective cohort study involving 110 patients with pancreatic ductal adenocarcinoma (PDAC), we assessed the density of CD68-TAM immune reactive area (%IRA) at the tumour-stroma interface and addressed their prognostic relevance in relation to postsurgical adjuvant chemotherapy (CTX). In vitro, we dissected the synergism of CTX and TAMs. In human PDAC, TAMs predominantly exhibited an immunoregulatory profile, characterised by expression of scavenger receptors (CD206, CD163) and production of interleukin 10 (IL-10). Surprisingly, while the density of TAMs associated to worse prognosis and distant metastasis, CTX restrained their protumour prognostic significance. High density of TAMs at the tumour-stroma interface positively dictated prognostic responsiveness to CTX independently of T-cell density. Accordingly, in vitro, gemcitabine-treated macrophages became tumoricidal, activating a cytotoxic gene expression programme, inhibiting their protumoural effect and switching to an antitumour phenotype. In patients with human PDAC, neoadjuvant CTX was associated to a decreased density of CD206(+) and IL-10(+) TAMs at the tumour-stroma interface. Overall, our data highlight TAMs as critical determinants of prognostic responsiveness to CTX and provide clinical and in vitro evidence that CTX overall directly re-educates TAMs to restrain tumour progression. These results suggest that the quantification of TAMs could be exploited to select patients more likely to respond to CTX and provide the basis for novel strategies aimed at re-educating macrophages in the context of CTX. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  3. Testicular granulocytic sarcoma without systemic leukemia

    NARCIS (Netherlands)

    Lagerveld, B. W.; Wauters, C. A. P.; Karthaus, H. F. M.

    2005-01-01

    This case report describes a unilateral testicular granulocytic sarcoma or chloroma. Because of the relatively immature nature of the tumor cells, the histological diagnosis can be difficult. Granulocytic sarcomas are well known in patients with systemic leukemia and can sometimes precede a systemic

  4. Human-induced pluripotent stem cell-derived macrophages and their immunological function in response to tuberculosis infection.

    Science.gov (United States)

    Hong, Danping; Ding, Jiongyan; Li, Ouyang; He, Quan; Ke, Minxia; Zhu, Mengyi; Liu, Lili; Ou, Wen-Bin; He, Yulong; Wu, Yuehong

    2018-02-26

    Induced pluripotent stem cells (iPS) represent an innovative source for the standardized in vitro generation of macrophages (Mφ). Mφ show great promise in disease pathogenesis, particularly tuberculosis. However, there is no information about human iPS-derived (hiPS) macrophages (hiPS-Mφ) in response to tuberculosis infection. In the present study, macrophages derived from hiPS were established via embryoid body (EB) formation by using feeder-free culture conditions, and the human monocyte cell line THP-1 (THP-1-Mφ) was used as control. iPS-Mφ were characterized by using morphology, Giemsa staining, nonspecific esterase staining (α-NAE), phagocytosis, and surface phenotype. Additionally, after treatment with Bacillus Calmette-Guérin (BCG) for 24 h, cell apoptosis was detected by using an Annexin V-FITC Apoptosis Detection assay. The production of nitric oxide (NO), expression of tumor necrosis factor alpha (TNF-α), activity of apoptosis-related protein cysteine-3 (Caspase-3) and expression of B-cell lymphoma-2 (Bcl-2) were analyzed. With respect to morphology, surface phenotype, and function, the iPS-Mφ closely resembled their counterparts generated in vitro from a human monocyte cell line. iPS-Mφ exhibited the typically morphological characteristics of macrophages, such as round, oval, fusiform and irregular characteristics. The cells were Giemsa-stained-positive, α-NAE-positive, and possessed phagocytic ability. iPS-Mφ express high levels of CD14, CD11b, CD40, CD68, and major histocompatibility complex II (MHC-II). Moreover, with regard to the apoptotic rate, the production of NO, expression of TNF-α, and activity of Caspase-3 and Bcl-2, iPS-Mφ closely resemble that of their counterparts generated in vitro from human monocyte cell line in response to BCG infection. The rate of apoptosis of BCG-treated iPS-Mφ was 37.77 ± 7.94% compared to that of the untreated group at 4.97 ± 1.60% (P immunological function in response to Bacillus Calmette

  5. Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages.

    Science.gov (United States)

    Sorrentino, Flavia; Gonzalez del Rio, Ruben; Zheng, Xingji; Presa Matilla, Jesus; Torres Gomez, Pedro; Martinez Hoyos, Maria; Perez Herran, Maria Esther; Mendoza Losana, Alfonso; Av-Gay, Yossef

    2016-01-01

    Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

    Science.gov (United States)

    Sung, Ho Joong; Son, Sin Jee; Yang, Seung-ju; Rhee, Ki-Jong; Kim, Yoon Suk

    2012-07-01

    Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.

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

    Directory of Open Access Journals (Sweden)

    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

  8. Macrophage Functions in Early Dissemination and Dormancy of Breast Cancer

    Science.gov (United States)

    2016-09-01

    mammary gland development 17,18, 69   arguing that normal mammary epithelial cells cooperate with these innate immune cells 70   for invasive... cells lacking 218     11   lymphoid and granulocytic markers (Supplementary Fig.3B). viSNE plots 30 of myelo-219   monocytic cells (Fig.5A...macrophages are actively recruited by pre-malignant ErbB2 overexpressing cancer cells and that these intra-epithelial macrophages then produce factors

  9. Effect of granulocyte colony-stimulating factor (G-CSF) in human immunodeficiency virus-infected patients: increase in numbers of naive CD4 cells and CD34 cells makes G-CSF a candidate for use in gene therapy or to support antiretroviral therapy

    DEFF Research Database (Denmark)

    Nielsen, S D; Afzelius, P; Dam-Larsen, S

    1998-01-01

    The potential of granulocyte colony-stimulating factor (G-CSF) to mobilize CD4 cells and/or CD34 cells for use in gene therapy or to support antiretroviral therapy was examined. Ten human immunodeficiency virus-infected patients were treated with G-CSF (300 microg/day) for 5 days. Numbers of CD4.......01/microL (P CSF induced increases in numbers of CD34 cells and CD4 cells in HIV-infected patients...

  10. The Effects of First-Line Anti-Tuberculosis Drugs on the Actions of Vitamin D in Human Macrophages.

    Science.gov (United States)

    Chesdachai, Supavit; Zughaier, Susu M; Hao, Li; Kempker, Russell R; Blumberg, Henry M; Ziegler, Thomas R; Tangpricha, Vin

    2016-12-01

    Tuberculosis (TB) is a major global health problem. Patients with TB have a high rate of vitamin D deficiency, both at diagnosis and during the course of treatment with anti-tuberculosis drugs. Although data on the efficacy of vitamin D supplementation on Mycobacterium tuberculosis (Mtb) clearance is uncertain from randomized controlled trials (RCTs), vitamin D enhances the expression of the anti-microbial peptide human cathelicidin (hCAP18) in cultured macrophages in vitro. One possible explanation for the mixed (primarily negative) results of RCTs examining vitamin D treatment in TB infection is that anti-TB drugs given to enrolled subjects may impact actions of vitamin D to enhance cathelicidin in macrophages. To address this hypothesis, human macrophage-like monocytic (THP-1) cells were treated with varying doses of first-line anti-tuberculosis drugs in the presence of the active form of vitamin D, 1N1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). The expression of hCAP18 was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). 1,25(OH) 2 D 3 strongly induced expression of hCAP18 mRNA in THP-1 cells (fold-change from control). The combination of the standard 4-drug TB therapy (isoniazid, rifampicin, pyrazinamide and ethambutol) in the cultured THP-1 cells demonstrated a significant decrease of hCAP18 mRNA at the dosage of 10 ug/mL. In 31 subjects with newly diagnosed drug-sensitive TB randomized to either high-dose vitamin D 3 (1.2 million IU over 8 weeks, n=13) versus placebo (n=18), there was no change from baseline to week 8 in hCAP18 mRNA levels in peripheral blood mononuclear cells or in plasma concentrations of LL-37, the protein product of hCAP18.These data suggest that first-line anti-TB drugs may alter the vitamin D-dependent increase in hCAP18 and LL-37 human macrophages.

  11. Human native lipoprotein-induced de novo DNA methylation is associated with repression of inflammatory genes in THP-1 macrophages.

    Science.gov (United States)

    Rangel-Salazar, Rubén; Wickström-Lindholm, Marie; Aguilar-Salinas, Carlos A; Alvarado-Caudillo, Yolanda; Døssing, Kristina B V; Esteller, Manel; Labourier, Emmanuel; Lund, Gertrud; Nielsen, Finn C; Rodríguez-Ríos, Dalia; Solís-Martínez, Martha O; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio

    2011-11-25

    We previously showed that a VLDL- and LDL-rich mix of human native lipoproteins induces a set of repressive epigenetic marks, i.e. de novo DNA methylation, histone 4 hypoacetylation and histone 4 lysine 20 (H4K20) hypermethylation in THP-1 macrophages. Here, we: 1) ask what gene expression changes accompany these epigenetic responses; 2) test the involvement of candidate factors mediating the latter. We exploited genome expression arrays to identify target genes for lipoprotein-induced silencing, in addition to RNAi and expression studies to test the involvement of candidate mediating factors. The study was conducted in human THP-1 macrophages. Native lipoprotein-induced de novo DNA methylation was associated with a general repression of various critical genes for macrophage function, including pro-inflammatory genes. Lipoproteins showed differential effects on epigenetic marks, as de novo DNA methylation was induced by VLDL and to a lesser extent by LDL, but not by HDL, and VLDL induced H4K20 hypermethylation, while HDL caused H4 deacetylation. The analysis of candidate factors mediating VLDL-induced DNA hypermethylation revealed that this response was: 1) surprisingly, mediated exclusively by the canonical maintenance DNA methyltransferase DNMT1, and 2) independent of the Dicer/micro-RNA pathway. Our work provides novel insights into epigenetic gene regulation by native lipoproteins. Furthermore, we provide an example of DNMT1 acting as a de novo DNA methyltransferase independently of canonical de novo enzymes, and show proof of principle that de novo DNA methylation can occur independently of a functional Dicer/micro-RNA pathway in mammals.

  12. Human native lipoprotein-induced de novo DNA methylation is associated with repression of inflammatory genes in THP-1 macrophages

    Directory of Open Access Journals (Sweden)

    Rangel-Salazar Rubén

    2011-11-01

    Full Text Available Abstract Background We previously showed that a VLDL- and LDL-rich mix of human native lipoproteins induces a set of repressive epigenetic marks, i.e. de novo DNA methylation, histone 4 hypoacetylation and histone 4 lysine 20 (H4K20 hypermethylation in THP-1 macrophages. Here, we: 1 ask what gene expression changes accompany these epigenetic responses; 2 test the involvement of candidate factors mediating the latter. We exploited genome expression arrays to identify target genes for lipoprotein-induced silencing, in addition to RNAi and expression studies to test the involvement of candidate mediating factors. The study was conducted in human THP-1 macrophages. Results Native lipoprotein-induced de novo DNA methylation was associated with a general repression of various critical genes for macrophage function, including pro-inflammatory genes. Lipoproteins showed differential effects on epigenetic marks, as de novo DNA methylation was induced by VLDL and to a lesser extent by LDL, but not by HDL, and VLDL induced H4K20 hypermethylation, while HDL caused H4 deacetylation. The analysis of candidate factors mediating VLDL-induced DNA hypermethylation revealed that this response was: 1 surprisingly, mediated exclusively by the canonical maintenance DNA methyltransferase DNMT1, and 2 independent of the Dicer/micro-RNA pathway. Conclusions Our work provides novel insights into epigenetic gene regulation by native lipoproteins. Furthermore, we provide an example of DNMT1 acting as a de novo DNA methyltransferase independently of canonical de novo enzymes, and show proof of principle that de novo DNA methylation can occur independently of a functional Dicer/micro-RNA pathway in mammals.

  13. Insulin resistance is associated with MCP1-mediated macrophage accumulation in skeletal muscle in mice and humans.

    Directory of Open Access Journals (Sweden)

    David Patsouris

    Full Text Available Inflammation is now recognized as a major factor contributing to type 2 diabetes (T2D. However, while the mechanisms and consequences associated with white adipose tissue inflammation are well described, very little is known concerning the situation in skeletal muscle. The aim of this study was to investigate, in vitro and in vivo, how skeletal muscle inflammation develops and how in turn it modulates local and systemic insulin sensitivity in different mice models of T2D and in humans, focusing on the role of the chemokine MCP1. Here, we found that skeletal muscle inflammation and macrophage markers are increased and associated with insulin resistance in mice models and humans. In addition, we demonstrated that intra-muscular TNFα expression is exclusively restricted to the population of intramuscular leukocytes and that the chemokine MCP1 was associated with skeletal muscle inflammatory markers in these models. Furthermore, we demonstrated that exposure of C2C12 myotubes to palmitate elevated the production of the chemokine MCP1 and that the muscle-specific overexpression of MCP1 in transgenic mice induced the local recruitment of macrophages and altered local insulin sensitivity. Overall our study demonstrates that skeletal muscle inflammation is clearly increased in the context of T2D in each one of the models we investigated, which is likely consecutive to the lipotoxic environment generated by peripheral insulin resistance, further increasing MCP1 expression in muscle. Consequently, our results suggest that MCP1-mediated skeletal muscle macrophages recruitment plays a role in the etiology of T2D.

  14. Nucleotide-oligomerizing domain-1 (NOD1) receptor activation induces pro-inflammatory responses and autophagy in human alveolar macrophages.

    Science.gov (United States)

    Juárez, Esmeralda; Carranza, Claudia; Hernández-Sánchez, Fernando; Loyola, Elva; Escobedo, Dante; León-Contreras, Juan Carlos; Hernández-Pando, Rogelio; Torres, Martha; Sada, Eduardo

    2014-09-25

    Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan fragments that localize to the cytosol. NOD1 activation triggers inflammation, antimicrobial mechanisms and autophagy in both epithelial cells and murine macrophages. NOD1 mediates intracellular pathogen clearance in the lungs of mice; however, little is known about NOD1's role in human alveolar macrophages (AMs) or its involvement in Mycobacterium tuberculosis (Mtb) infection. AMs, monocytes (MNs), and monocyte-derived macrophages (MDMs) from healthy subjects were assayed for NOD1 expression. Cells were stimulated with the NOD1 ligand Tri-DAP and cytokine production and autophagy were assessed. Cells were infected with Mtb and treated with Tri-DAP post-infection. CFUs counting determined growth control, and autophagy protein recruitment to pathogen localization sites was analyzed by immunoelectron microscopy. NOD1 was expressed in AMs, MDMs and to a lesser extent MNs. Tri-DAP stimulation induced NOD1 up-regulation and a significant production of IL1β, IL6, IL8, and TNFα in AMs and MDMs; however, the level of NOD1-dependent response in MNs was limited. Autophagy activity determined by expression of proteins Atg9, LC3, IRGM and p62 degradation was induced in a NOD1-dependent manner in AMs and MDMs but not in MNs. Infected AMs could be activated by stimulation with Tri-DAP to control the intracellular growth of Mtb. In addition, recruitment of NOD1 and the autophagy proteins IRGM and LC3 to the Mtb localization site was observed in infected AMs after treatment with Tri-DAP. NOD1 is involved in AM and MDM innate responses, which include proinflammatory cytokines and autophagy, with potential implications in the killing of Mtb in humans.

  15. Comparison of autologous cell therapy and granulocyte-colony stimulating factor (G-CSF) injection vs. G-CSF injection alone for the treatment of acute radiation syndrome in a non-human primate model

    International Nuclear Information System (INIS)

    Bertho, Jean-Marc; Frick, Johanna; Prat, Marie; Demarquay, Christelle; Dudoignon, Nicolas; Trompier, Francois; Gorin, Norbert-Claude; Thierry, Dominique; Gourmelon, Patrick

    2005-01-01

    Purpose: To compare the efficacy of autologous cell therapy after irradiation combined with granulocyte-colony stimulating factor (G-CSF) injections with G-CSF treatment alone in a heterogeneous model of irradiation representative of an accidental situation. Material and Methods: Non-human primates were irradiated at 8.7 Gy whole-body dose with the right arm shielded to receive 4.8 Gy. The first group of animals received G-CSF (lenograstim) injections starting 6 h after irradiation, and a second group received a combination of G-CSF (lenograstim) injections and autologous expanded hematopoietic cells. Animals were followed up for blood cell counts, circulating progenitors, and bone marrow cellularity. Results: No significant differences were seen between the two treatment groups, whatever the parameter observed: time to leukocyte or platelet recovery and duration and severity of aplasia. Conclusion: Our results indicated that identical recovery kinetic was observed when irradiated animals are treated with G-CSF independently of the reinjection of ex vivo expanded autologous hematopoietic cells. Thus G-CSF injections might be chosen as a first-line therapeutic strategy in the treatment of accidental acute radiation victims

  16. Anti-Inflammatory Effects of Lactobacillus Rahmnosus and Bifidobacterium Breve on Cigarette Smoke Activated Human Macrophages

    OpenAIRE

    Mortaz, E; Adcock, IM; Ricciardolo, FLM; Varahram, M; Jamaati, H; Velayati, AA; Folkerts, G; Garssen, J

    2015-01-01

    BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a major global health problem with cigarette smoke (CS) as the main risk factor for its development. Airway inflammation in COPD involves the increased expression of inflammatory mediators such as CXCL-8 and IL-1β which are important mediators for neutrophil recruitment. Macrophages are an important source of these mediators in COPD. Lactobacillus rhamnosus (L. rhamnosus) and Befidobacterium breve (B. breve) attenuate the development...

  17. Colony-stimulating factor (CSF) radioimmunoassay: detection of a CSF subclass stimulating macrophage production

    International Nuclear Information System (INIS)

    Stanley, E.R.

    1979-01-01

    Colony-stimulating factors (CSFs) stimulate the differentiation of immature precursor cells to mature granulocytes and macrophages. Purified 125 I-labeled murine L cell CSF has been used to develop a radioimmunoassay (RIA) that detects a subclass of CSFs that stimulates macrophage production. Murine CSF preparations that contain this subclass of CSF compete for all of the CSF binding sites on anti-L cell CSF antibody. With the exception of mouse serum, which can contain inhibitors of the bioassay, there is complete correspondence between activities determined by RIA and those determined by bioassay. The RIA is slightly more sensitive than the bioassay, detecting approximately 0.3 fmol of purified L cell CSF. It can also detect this subclass of CSF in chickens, rats, and humans. In the mouse, the subclass is distinguished from other CSFs by a murine cell bioassay dose-response curve in which 90% of the response occurs over a 10-fold (rather than a 100-fold) increase in concentration, by stimulating the formations of colonies contaning a high proportion of mononuclear (rather than granulocytic) cells, and by certain physical characteristics

  18. TAM receptor-dependent regulation of SOCS3 and MAPKs contributes to proinflammatory cytokine downregulation following chronic NOD2 stimulation of human macrophages.

    Science.gov (United States)

    Zheng, Shasha; Hedl, Matija; Abraham, Clara

    2015-02-15

    Microbial-induced cytokine regulation is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, cytokines are attenuated after chronic NOD2 and pattern recognition receptor stimulation of macrophages; similar attenuation is observed in intestinal macrophages. The role of Tyro3, Axl, and Mer (TAM) receptors in regulating chronic pattern recognition receptor stimulation and NOD2-induced outcomes has not been examined. Moreover, TAM receptors have been relatively less investigated in human macrophages. Whereas TAM receptors did not downregulate acute NOD2-induced cytokines in primary human macrophages, they were essential for downregulating signaling and proinflammatory cytokine secretion after chronic NOD2 and TLR4 stimulation. Axl and Mer were similarly required in mice for cytokine downregulation after chronic NOD2 stimulation in vivo and in intestinal tissues. Consistently, TAM expression was increased in human intestinal myeloid-derived cells. Chronic NOD2 stimulation led to IL-10- and TGF-β-dependent TAM upregulation in human macrophages, which, in turn, upregulated suppressor of cytokine signaling 3 expression. Restoring suppressor of cytokine signaling 3 expression under TAM knockdown conditions restored chronic NOD2-mediated proinflammatory cytokine downregulation. In contrast to the upregulated proinflammatory cytokines, attenuated IL-10 secretion was maintained in TAM-deficient macrophages upon chronic NOD2 stimulation. The level of MAPK activation in TAM-deficient macrophages after chronic NOD2 stimulation was insufficient to upregulate IL-10 secretion; however, full restoration of MAPK activation under these conditions restored c-Fos, c-Jun, musculoaponeurotic fibrosarcoma oncogene homolog K, and PU.1 binding to the IL-10 promoter and IL-10 secretion. Therefore, TAM receptors are critical for

  19. Comparative effects of metal oxide nanoparticles on human airway epithelial cells and macrophages

    Science.gov (United States)

    Rotoli, Bianca Maria; Bussolati, Ovidio; Costa, Anna Luisa; Blosi, Magda; Di Cristo, Luisana; Zanello, Pier Paolo; Bianchi, Massimiliano G.; Visigalli, Rossana; Bergamaschi, Enrico

    2012-09-01

    Among nanomaterials of industrial relevance, metal-based nanoparticles (NPs) are widely used, but their effects on airway cells are relatively poorly characterized. To compare the effects of metal NPs on cells representative of the lung-blood barrier, Calu-3 epithelial cells and Raw264.7 macrophages were incubated with three industrially relevant preparations of TiO2 NPs (size range 4-33 nm), two preparations of CeO2 NPs (9-36 nm) and CuO NPs (25 nm). While Raw264.7 were grown on standard plasticware, Calu-3 cells were seeded on permeable filters, where they form a high-resistance monolayer, providing an in vitro model of the airway barrier. Metal NPs, obtained from industrial sources, were characterized under the conditions adopted for the biological tests. Cytotoxicity was assessed with resazurin method in both epithelial and macrophage cells, while epithelial barrier permeability was monitored measuring the trans-epithelial electrical resistance (TEER). In macrophages, titania and ceria had no significant effect on viability in the whole range of nominal doses tested (15-240 μg/cm2 of monolayer), while CuO NPs produced a marked viability loss. Moreover, only CuO NPs, but not the other NPs, lowered TEER of Calu-3 monolayers, pointing to the impairment of the epithelial barrier. TEER decreased by 30 % at the dose of 10 μg/cm2 of CuO NPs, compared to untreated control, and was abolished at doses ≥80 μg/cm2, in strict correlation with changes in cell viability. These results indicate that (1) CuO NPs increase airway epithelium permeability even at relatively low doses and are significantly toxic for macrophages and airway epithelial cells, likely through the release of Cu ions in the medium; (2) TiO2 and CeO2 NPs do not affect TEER and exhibit little acute toxicity for airway epithelial cells and macrophages; and (3) TEER measurement can provide a simple method to assess the impairment of in vitro airway epithelial barrier model by manufactured nanomaterials.

  20. Comparative effects of metal oxide nanoparticles on human airway epithelial cells and macrophages

    International Nuclear Information System (INIS)

    Rotoli, Bianca Maria; Bussolati, Ovidio; Costa, Anna Luisa; Blosi, Magda; Di Cristo, Luisana; Zanello, Pier Paolo; Bianchi, Massimiliano G.; Visigalli, Rossana; Bergamaschi, Enrico

    2012-01-01

    Among nanomaterials of industrial relevance, metal-based nanoparticles (NPs) are widely used, but their effects on airway cells are relatively poorly characterized. To compare the effects of metal NPs on cells representative of the lung-blood barrier, Calu-3 epithelial cells and Raw264.7 macrophages were incubated with three industrially relevant preparations of TiO 2 NPs (size range 4–33 nm), two preparations of CeO 2 NPs (9–36 nm) and CuO NPs (25 nm). While Raw264.7 were grown on standard plasticware, Calu-3 cells were seeded on permeable filters, where they form a high-resistance monolayer, providing an in vitro model of the airway barrier. Metal NPs, obtained from industrial sources, were characterized under the conditions adopted for the biological tests. Cytotoxicity was assessed with resazurin method in both epithelial and macrophage cells, while epithelial barrier permeability was monitored measuring the trans-epithelial electrical resistance (TEER). In macrophages, titania and ceria had no significant effect on viability in the whole range of nominal doses tested (15–240 μg/cm 2 of monolayer), while CuO NPs produced a marked viability loss. Moreover, only CuO NPs, but not the other NPs, lowered TEER of Calu-3 monolayers, pointing to the impairment of the epithelial barrier. TEER decreased by 30 % at the dose of 10 μg/cm 2 of CuO NPs, compared to untreated control, and was abolished at doses ≥80 μg/cm 2 , in strict correlation with changes in cell viability. These results indicate that (1) CuO NPs increase airway epithelium permeability even at relatively low doses and are significantly toxic for macrophages and airway epithelial cells, likely through the release of Cu ions in the medium; (2) TiO 2 and CeO 2 NPs do not affect TEER and exhibit little acute toxicity for airway epithelial cells and macrophages; and (3) TEER measurement can provide a simple method to assess the impairment of in vitro airway epithelial barrier model by manufactured

  1. The Anti-Inflammatory Effect of Algae-Derived Lipid Extracts on Lipopolysaccharide (LPS)-Stimulated Human THP-1 Macrophages.

    Science.gov (United States)

    Robertson, Ruairi C; Guihéneuf, Freddy; Bahar, Bojlul; Schmid, Matthias; Stengel, Dagmar B; Fitzgerald, Gerald F; Ross, R Paul; Stanton, Catherine

    2015-08-20

    Algae contain a number of anti-inflammatory bioactive compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) and chlorophyll a, hence as dietary ingredients, their extracts may be effective in chronic inflammation-linked metabolic diseases such as cardiovascular disease. In this study, anti-inflammatory potential of lipid extracts from three red seaweeds (Porphyra dioica, Palmaria palmata and Chondrus crispus) and one microalga (Pavlova lutheri) were assessed in lipopolysaccharide (LPS)-stimulated human THP-1 macrophages. Extracts contained 34%-42% total fatty acids as n-3 PUFA and 5%-7% crude extract as pigments, including chlorophyll a, β-carotene and fucoxanthin. Pretreatment of the THP-1 cells with lipid extract from P. palmata inhibited production of the pro-inflammatory cytokines interleukin (IL)-6 (p lipid extracts. The lipid extracts effectively inhibited the LPS-induced pro-inflammatory signaling pathways mediated via toll-like receptors, chemokines and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling molecules. These results suggest that lipid extracts from P. lutheri, P. palmata, P. dioica and C. crispus can inhibit LPS-induced inflammatory pathways in human macrophages. Therefore, algal lipid extracts should be further explored as anti-inflammatory ingredients for chronic inflammation-linked metabolic diseases.

  2. The Local Inflammatory Responses to Infection of the Peritoneal Cavity in Humans: Their Regulation by Cytokines, Macrophages, and Other Leukocytes

    Directory of Open Access Journals (Sweden)

    Marien Willem Johan Adriaan Fieren

    2012-01-01

    Full Text Available Studies on infection-induced inflammatory reactions in humans rely largely on findings in the blood compartment. Peritoneal leukocytes from patients treated with peritoneal dialysis offer a unique opportunity to study in humans the inflammatory responses taking place at the site of infection. Compared with peritoneal macrophages (pM from uninfected patients, pM from infected patients display ex vivo an upregulation and downregulation of proinflammatory and anti-inflammatory mediators, respectively. Pro-IL-1 processing and secretion rather than synthesis proves to be increased in pM from infectious peritonitis suggesting up-regulation of caspase-1 in vivo. A crosstalk between pM, γ T cells, and neutrophils has been found to be involved in augmented TNF expression and production during infection. The recent finding in experimental studies that alternatively activated macrophages (M2 increase by proliferation rather than recruitment may have significant implications for the understanding and treatment of chronic inflammatory conditions such as encapsulating peritoneal sclerosis (EPS.

  3. Caprine arthritis encephalitis virus dysregulates the expression of cytokines in macrophages.

    Science.gov (United States)

    Lechner, F; Machado, J; Bertoni, G; Seow, H F; Dobbelaere, D A; Peterhans, E

    1997-01-01

    Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues, in particular, the radiocarpal joints. Cells of the monocyte/macrophage lineage are the major host cells of CAEV in vivo. We have shown that infection of cultured goat macrophages with CAEV results in an alteration of cytokine expression in vitro. Constitutive expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) was increased in infected macrophages, whereas transforming growth factor beta1 (TGF-beta1) mRNA was down-regulated. When macrophages were infected with a CAEV clone lacking the trans-acting nuclear regulatory gene tat, IL-8 and MCP-1 were also increased. No significant differences from cells infected with the wild-type clone were observed, suggesting that Tat is not required for the increased expression of IL-8 and MCP-1 in infected macrophages. Furthermore, infection with CAEV led to an altered pattern of cytokine expression in response to lipopolysaccharide (LPS), heat-killed Listeria monocytogenes plus gamma interferon, or fixed cells of Staphylococcus aureus Cowan I. In infected macrophages, tumor necrosis factor alpha, IL-1beta, IL-6, and IL-12 p40 mRNA expression was reduced in response to all stimuli tested whereas changes in expression of granulocyte-macrophage colony-stimulating factor depended on the stimulating agent. Electrophoretic mobility shift assays demonstrated that, in contrast to effects of human immunodeficiency virus infection of macrophages, CAEV infection had no effect on the level of constitutive nuclear factor-kappaB (NF-kappaB) activity or on the level of LPS-stimulated NF-kappaB activity, suggesting that NF-kappaB is not involved in altered regulation of cytokine expression in CAEV-infected cells. In contrast, activator protein 1 (AP-1) binding activity was decreased in infected macrophages. These data show that CAEV infection may result in a dysregulation of

  4. Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages

    KAUST Repository

    Bokil, Nilesh J.; Totsika, Makrina; Carey, Alison J.; Stacey, Katryn J.; Hancock, Viktoria; Saunders, Bernadette M.; Ravasi, Timothy; Ulett, Glen C.; Schembri, Mark A.; Sweet, Matthew J.

    2011-01-01

    within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes

  5. Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Bianca Maria Rotoli

    2018-03-01

    Full Text Available Lysinuric protein intolerance (LPI is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y+L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA, a significant induction of the expression and release of the inflammatory mediators IL1β and TNFα was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1β has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation.

  6. Evaluating the evidence for macrophage presence in skeletal muscle and its relation to insulin resistance in obese mice and humans: a systematic review protocol.

    Science.gov (United States)

    Bhatt, Meha; Rudrapatna, Srikesh; Banfield, Laura; Bierbrier, Rachel; Wang, Pei-Wen; Wang, Kuan-Wen; Thabane, Lehana; Samaan, M Constantine

    2017-08-08

    The current global rates of obesity and type 2 diabetes are staggering. In order to implement effective management strategies, it is imperative to understand the mechanisms of obesity-induced insulin resistance and diabetes. Macrophage infiltration and inflammation of the adipose tissue in obesity is a well-established paradigm, yet the role of macrophages in muscle inflammation, insulin resistance and diabetes is not adequately studied. In this systematic review, we will examine the evidence for the presence of macrophages in skeletal muscle of obese humans and mice, and will assess the association between muscle macrophages and insulin resistance. We will identify published studies that address muscle macrophage content and phenotype, and its association with insulin resistance. We will search MEDLINE/PubMed, EMBASE, and Web of Science for eligible studies. Grey literature will be searched in ProQuest. Quality assessment will be conducted using the Systematic Review Centre for Laboratory Animal Experimentation risk of bias Tool for animal studies. The findings of this systematic review will shed light on immune-metabolic crosstalk in obesity, and allow the consideration of targeted therapies to modulate muscle macrophages in the treatment and prevention of diabetes. The review will be published in a peer-reviewed journal and presented at conferences.

  7. Probiotic Bacteria Alter Pattern-Recognition Receptor Expression and Cytokine Profile in a Human Macrophage Model Challenged with Candida albicans and Lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Victor H. Matsubara

    2017-11-01

    Full Text Available Probiotics are live microorganisms that confer benefits to the host health. The infection rate of potentially pathogenic organisms such as Candida albicans, the most common agent associated with mucosal candidiasis, can be reduced by probiotics. However, the mechanisms by which the probiotics interfere with the immune system are largely unknown. We evaluated the effect of probiotic bacteria on C. albicans challenged human macrophages. Macrophages were pretreated with lactobacilli alone (Lactobacillus rhamnosus LR32, Lactobacillus casei L324m, or Lactobacillus acidophilus NCFM or associated with Escherichia coli lipopolysaccharide (LPS, followed by the challenge with C. albicans or LPS in a co-culture assay. The expression of pattern-recognition receptors genes (CLE7A, TLR2, and TLR4 was determined by RT-qPCR, and dectin-1 reduced levels were confirmed by flow cytometry. The cytokine profile was determined by ELISA using the macrophage cell supernatant. Overall probiotic lactobacilli down-regulated the transcription of CLEC7A (p < 0.05, resulting in the decreased expression of dectin-1 on probiotic pretreated macrophages. The tested Lactobacillus species down-regulated TLR4, and increased TLR2 mRNA levels in macrophages challenged with C. albicans. The cytokines profile of macrophages challenged with C. albicans or LPS were altered by the probiotics, which generally led to increased levels of IL-10 and IL-1β, and reduction of IL-12 production by macrophages (p < 0.05. Our data suggest that probiotic lactobacilli impair the recognition of PAMPs by macrophages, and alter the production of pro/anti-inflammatory cytokines, thus modulating inflammation.

  8. Studies on the pathogenesis of fever. XV. The production of endogenous pyrogen by peritoneal macrophages.

    Science.gov (United States)

    Hahn, H H; Char, D C; Postel, W B; Wood, W B

    1967-08-01

    Macrophages from oil-induced peritoneal exudates in rabbits produce endogenous pyrogen when first activated by incubation in 4 hr exudate fluid and then stimulated by incubation in potassium-free isotonic sodium chloride solution. The failure of earlier investigators to obtain pyrogen from macrophages is explained, and the relevance of macrophage pyrogen to fevers of agranulocytosis and other diseases, in which mononuclear rather than granulocytic exudates predominate, is discussed.

  9. Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy.

    Directory of Open Access Journals (Sweden)

    Grant R Campbell

    Full Text Available Low vitamin D levels in human immunodeficiency virus type-1 (HIV infected persons are associated with more rapid disease progression and increased risk for Mycobacterium tuberculosis infection. We have previously shown that 1α,25-dihydroxycholecalciferol (1,25D3, the active form of vitamin D, inhibits HIV replication in human macrophages through the induction of autophagy. In this study, we report that physiological concentrations of 1,25D3 induce the production of the human cathelicidin microbial peptide (CAMP and autophagic flux in HIV and M. tuberculosis co-infected human macrophages which inhibits mycobacterial growth and the replication of HIV. Using RNA interference for Beclin-1 and the autophagy-related 5 homologue, combined with the chemical inhibitors of autophagic flux, bafilomycin A₁, an inhibitor of autophagosome-lysosome fusion and subsequent acidification, and SID 26681509 an inhibitor of the lysosome hydrolase cathepsin L, we show that the 1,25D3-mediated inhibition of HIV replication and mycobacterial growth during single infection or dual infection is dependent not only upon the induction of autophagy, but also through phagosomal maturation. Moreover, through the use of RNA interference for CAMP, we demonstrate that cathelicidin is essential for the 1,25D3 induced autophagic flux and inhibition of HIV replication and mycobacterial growth. The present findings provide a biological explanation for the benefits and importance of vitamin D sufficiency in HIV and M. tuberculosis-infected persons, and provide new insights into novel approaches to prevent and treat HIV infection and related opportunistic infections.

  10. Combination Therapy for Radiation-Induced Bone Marrow Aplasia in Nonhuman Primates Using Synthokine SC-55494 and Recombinant Human Granulocyte Colony-Stimulating Factor

    National Research Council Canada - National Science Library

    MacVittle, Thomas J; Farese, Ann M; Herodin, Francis; Grab, Lisa B; Baum, Charles M; McKearn, John P

    1996-01-01

    .... After irradiation on day (d) 0, cohorts of animals subcutaneously received single-agent protocols of either human serum albumin (HSA; every day [OD], 15 microng/kg/d. n = 10). Synthokine (twice daily [BID...

  11. Adherent Human Alveolar Macrophages Exhibit a Transient Pro-Inflammatory Profile That Confounds Responses to Innate Immune Stimulation

    Science.gov (United States)

    Tomlinson, Gillian S.; Booth, Helen; Petit, Sarah J.; Potton, Elspeth; Towers, Greg J.; Miller, Robert F.; Chain, Benjamin M.; Noursadeghi, Mahdad

    2012-01-01

    Alveolar macrophages (AM) are thought to have a key role in the immunopathogenesis of respiratory diseases. We sought to test the hypothesis that human AM exhibit an anti-inflammatory bias by making genome-wide comparisons with monocyte derived macrophages (MDM). Adherent AM obtained by bronchoalveolar lavage of patients under investigation for haemoptysis, but found to have no respiratory pathology, were compared to MDM from healthy volunteers by whole genome transcriptional profiling before and after innate immune stimulation. We found that freshly isolated AM exhibited a marked pro-inflammatory transcriptional signature. High levels of basal pro-inflammatory gene expression gave the impression of attenuated responses to lipopolysaccharide (LPS) and the RNA analogue, poly IC, but in rested cells pro-inflammatory gene expression declined and transcriptional responsiveness to these stimuli was restored. In comparison to MDM, both freshly isolated and rested AM showed upregulation of MHC class II molecules. In most experimental paradigms ex vivo adherent AM are used immediately after isolation. Therefore, the confounding effects of their pro-inflammatory profile at baseline need careful consideration. Moreover, despite the prevailing view that AM have an anti-inflammatory bias, our data clearly show that they can adopt a striking pro-inflammatory phenotype, and may have greater capacity for presentation of exogenous antigens than MDM. PMID:22768282

  12. Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability.

    Directory of Open Access Journals (Sweden)

    Kejie Chen

    Full Text Available Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß. Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3 inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis.

  13. Coxsackievirus B4 Can Infect Human Peripheral Blood-Derived Macrophages

    Directory of Open Access Journals (Sweden)

    Enagnon Kazali Alidjinou

    2015-11-01

    Full Text Available Beyond acute infections, group B coxsackieviruses (CVB are also reported to play a role in the development of chronic diseases, like type 1 diabetes. The viral pathogenesis mainly relies on the interplay between the viruses and innate immune response in genetically-susceptible individuals. We investigated the interaction between CVB4 and macrophages considered as major players in immune response. Monocyte-derived macrophages (MDM generated with either M-CSF or GM-CSF were inoculated with CVB4, and infection, inflammation, viral replication and persistence were assessed. M-CSF-induced MDM, but not GM-CSF-induced MDM, can be infected by CVB4. In addition, enhancing serum was not needed to infect MDM in contrast with parental monocytes. The expression of viral receptor (CAR mRNA was similar in both M-CSF and GM-CSF MDM. CVB4 induced high levels of pro-inflammatory cytokines (IL-6 and TNFα in both MDM populations. CVB4 effectively replicated and persisted in M-CSF MDM, but IFNα was produced in the early phase of infection only. Our results demonstrate that CVB4 can replicate and persist in MDM. Further investigations are required to determine whether the interaction between the virus and MDM plays a role in the pathogenesis of CVB-induced chronic diseases.

  14. Therapeutic Antibody-Like Immunoconjugates against Tissue Factor with the Potential to Treat Angiogenesis-Dependent as Well as Macrophage-Associated Human Diseases

    Directory of Open Access Journals (Sweden)

    Zhiwei Hu

    2018-01-01

    Full Text Available Accumulating evidence suggests that tissue factor (TF is selectively expressed in pathological angiogenesis-dependent as well as macrophage-associated human diseases. Pathological angiogenesis, the formation of neovasculature, is involved in many clinically significant human diseases, notably cancer, age-related macular degeneration (AMD, endometriosis and rheumatoid arthritis (RA. Macrophage is involved in the progression of a variety of human diseases, such as atherosclerosis and viral infections (human immunodeficiency virus, HIV and Ebola. It is well documented that TF is selectively expressed on angiogenic vascular endothelial cells (VECs in these pathological angiogenesis-dependent human diseases and on disease-associated macrophages. Under physiology condition, TF is not expressed by quiescent VECs and monocytes but is solely restricted on some cells (such as pericytes that are located outside of blood circulation and the inner layer of blood vessel walls. Here, we summarize TF expression on angiogenic VECs, macrophages and other diseased cell types in these human diseases. In cancer, for example, the cancer cells also overexpress TF in solid cancers and leukemia. Moreover, our group recently reported that TF is also expressed by cancer-initiating stem cells (CSCs and can serve as a novel oncotarget for eradication of CSCs without drug resistance. Furthermore, we review and discuss two generations of TF-targeting therapeutic antibody-like immunoconjugates (ICON and L-ICON1 and antibody-drug conjugates that are currently being tested in preclinical and clinical studies for the treatment of some of these human diseases. If efficacy and safety are proven in current and future clinical trials, TF-targeting immunoconjugates may provide novel therapeutic approaches with potential to broadly impact the treatment regimen of these significant angiogenesis-dependent, as well as macrophage-associated, human diseases.

  15. HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Kuan-Teh Jeang

    2012-07-01

    Full Text Available A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages.

  16. Subcellular localisation and properties of histone phosphate phosphatase in human polymorphonuclear leukocytes: alterations in pregnancy and chronic granulocytic leukaemia and relationship to alkaline phosphatase

    International Nuclear Information System (INIS)

    Smith, G.P.; Peters, T.J.

    1981-01-01

    Using [ 32 P]histone as substrate, an assay for histone phosphate phosphatase was optimised for human polymorphonuclear leukocytes. Kinetic studies showed that the activity was optimal at pH 6.8, was stimulated by Mn 2+ and Co 2+ , and inhibited by sodium sulphite and zinc chloride. The apparent Ksub(m) of the enzyme for histone phosphate was 0.89 μmol/l. (Auth.)

  17. A STUDY OF INTERMEDIATES INVOLVED IN THE FOLDING PATHWAY FOR RECOMBINANT HUMAN MACROPHAGE COLONY-STIMULATING FACTOR (M-CSF) - EVIDENCE FOR 2 DISTINCT FOLDING PATHWAYS

    NARCIS (Netherlands)

    WILKINS, JA; CONE, J; RANDHAWA, ZI; WOOD, D; WARREN, MK; WITKOWSKA, HE

    The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding

  18. Granulocyte-macrophage colony-stimulating factor does not increase the potency or efficacy of a foot-and-mouth disease virus subunit vaccine Fator estimulante de colônias de granu-lócitos e macrófagos (GM-CSF não aumenta a eficácia ou potência da vacina de subunidades da febre aftosa em suínos

    Directory of Open Access Journals (Sweden)

    Luizinho Caron

    2005-09-01

    Full Text Available Foot-and-mouth disease (FMD is one of the most feared diseases of livestock worldwide. Vaccination has been a very effective weapon in controlling the disease, however a number of concerns with the current vaccine including the inability of approved diagnostic tests to reliably distinguish vaccinated from infected animals and the need for high containment facilities for vaccine production, have limited its use during outbreaks in countries previously free of the disease. A number of FMD vaccine candidates have been tested and a replication-defective human adenovirus type 5 (Ad5 vector containing the FMDV capsid (P1-2A and 3C protease coding regions has been shown to completely protect pigs against challenge with the homologous virus (FMDV A12 and A24. An Ad5-P1-2A+3C vaccine for FMDV O1 Campos (Ad5-O1C, however, only induced a low FMDV-specific neutralizing antibody response in swine potency tests. Granulocyte-macrophage colony-stimulating factor (GM-CSF has been successfully used to stimulate the immune response in vaccine formulations against a number of diseases, including HIV, hepatitis C and B. To attempt to improve the FMDV-specific immune response induced by Ad5-O1C, we inoculated swine with Ad5-O1C and an Ad5 vector containing the gene for porcine GM-CSF (pGM-CSF. However, in the conditions used in this trial, pGM-CSF did not improve the immune response to Ad5-O1C and adversely affected the level of protection of swine challenged with homologous FMDV.A febre aftosa é uma das doenças mais temidas nos rebanhos em todo o mundo. A vacinação tem sido uma arma eficiente no controle da doença, no entanto há preocupações com as vacinas atualmente utilizadas incluindo a necessidade de instalações de alta segurança para a produção dessas vacinas e a falta de um teste de diagnóstico aprovado que faça distinção precisa entre animais vacinados dos infectados. Várias vacinas têm sido testadas contra a febre aftosa e uma dessas

  19. Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Arcangeletti, Maria-Cristina, E-mail: mariacristina.arcangeletti@unipr.it [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Germini, Diego; Rodighiero, Isabella [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Mirandola, Prisco [Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma (Italy); De Conto, Flora; Medici, Maria-Cristina [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Gatti, Rita [Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma (Italy); Chezzi, Carlo; Calderaro, Adriana [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy)

    2013-05-25

    Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promoting cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle. - Highlights: ► We studied HCMV infection impact on THP-1 macrophage cell cycle. ► We analysed the role played by Toll-like receptor (TLR) 4 upon HCMV infection. ► HCMV pushes THP-1 macrophages (i.e. resting cells) to re-enter the cell cycle. ► TLR4 pathway inhibition strongly affects the effectiveness of HCMV replication. ► TLR4 pathway inhibition significantly decreases HCMV-induced cell cycle re-entry.

  20. The Activin A-Peroxisome Proliferator-Activated Receptor Gamma Axis Contributes to the Transcriptome of GM-CSF-Conditioned Human Macrophages.

    Science.gov (United States)

    Nieto, Concha; Bragado, Rafael; Municio, Cristina; Sierra-Filardi, Elena; Alonso, Bárbara; Escribese, María M; Domínguez-Andrés, Jorge; Ardavín, Carlos; Castrillo, Antonio; Vega, Miguel A; Puig-Kröger, Amaya; Corbí, Angel L

    2018-01-01

    GM-CSF promotes the functional maturation of lung alveolar macrophages (A-MØ), whose differentiation is dependent on the peroxisome proliferator-activated receptor gamma (PPARγ) transcription factor. In fact, blockade of GM-CSF-initiated signaling or deletion of the PPARγ-encoding gene PPARG leads to functionally defective A-MØ and the onset of pulmonary alveolar proteinosis. In vitro , macrophages generated in the presence of GM-CSF display potent proinflammatory, immunogenic and tumor growth-limiting activities. Since GM-CSF upregulates PPARγ expression, we hypothesized that PPARγ might contribute to the gene signature and functional profile of human GM-CSF-conditioned macrophages. To verify this hypothesis, PPARγ expression and activity was assessed in human monocyte-derived macrophages generated in the presence of GM-CSF [proinflammatory GM-CSF-conditioned human monocyte-derived macrophages (GM-MØ)] or M-CSF (anti-inflammatory M-MØ), as well as in ex vivo isolated human A-MØ. GM-MØ showed higher PPARγ expression than M-MØ, and the expression of PPARγ in GM-MØ was found to largely depend on activin A. Ligand-induced activation of PPARγ also resulted in distinct transcriptional and functional outcomes in GM-MØ and M-MØ. Moreover, and in the absence of exogenous activating ligands, PPARγ knockdown significantly altered the GM-MØ transcriptome, causing a global upregulation of proinflammatory genes and significantly modulating the expression of genes involved in cell proliferation and migration. Similar effects were observed in ex vivo isolated human A-MØ, where PPARγ silencing led to enhanced expression of genes coding for growth factors and chemokines and downregulation of cell surface pathogen receptors. Therefore, PPARγ shapes the transcriptome of GM-CSF-dependent human macrophages ( in vitro derived GM-MØ and ex vivo isolated A-MØ) in the absence of exogenous activating ligands, and its expression is primarily regulated by activin A

  1. A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages.

    Science.gov (United States)

    Thyer, Lynda; Ward, Emma; Smith, Rodney; Fiore, Maria Giulia; Magherini, Stefano; Branca, Jacopo J V; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco; Pacini, Stefania

    2013-07-08

    The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This allows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

  2. A Novel Role for a Major Component of the Vitamin D Axis: Vitamin D Binding Protein-Derived Macrophage Activating Factor Induces Human Breast Cancer Cell Apoptosis through Stimulation of Macrophages

    Directory of Open Access Journals (Sweden)

    Marco Ruggiero

    2013-07-01

    Full Text Available The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH(2D3, its two binding proteins that are the vitamin D receptor (VDR and the vitamin D-binding protein-derived macrophage activating factor (GcMAF. In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This al1ows 1,25(OH(2D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

  3. Advanced glycation end-product (AGE)-albumin from activated macrophage is critical in human mesenchymal stem cells survival and post-ischemic reperfusion injury.

    Science.gov (United States)

    Son, Myeongjoo; Kang, Woong Chol; Oh, Seyeon; Bayarsaikhan, Delger; Ahn, Hyosang; Lee, Jaesuk; Park, Hyunjin; Lee, Sojung; Choi, Junwon; Lee, Hye Sun; Yang, Phillip C; Byun, Kyunghee; Lee, Bonghee

    2017-09-14

    Post-ischemic reperfusion injury (PIRI) triggers an intense inflammatory response which is essential for repair but is also implicated in pathogenesis of post-ischemic remodeling in several organs in human. Stem cell therapy has recently emerged as a promising method for treatment of PIRI in human. However, satisfactory results have not been reported due to severe loss of injected stem cells in PIRI including critical limb ischemia (CLI). For investigating the advanced glycation end-product-albumin (AGE-albumin) from activated macrophages is critical in both muscle cell and stem cell death, we evaluated the recovery of PIRI-CLI by injection of human bone marrow derived mesenchymal stem cells (hBD-MSCs) with or without soluble receptor for AGEs (sRAGE). Our results showed that activated M1 macrophages synthesize and secrete AGE-albumin, which induced the skeletal muscle cell death and injected hBD-MSCs in PIRI-CLI through RAGE increase. Combined injection of sRAGE and hBD-MSCs resulted in enhanced survival of hBD-MSCs and angiogenesis in PIRI-CLI mice. Taken together, AGE-albumin from activated macrophages is critical for both skeletal muscle cell and hBD-MSCs death in PIRI-CLI. Therefore, the inhibition of AGE-albumin from activated macrophages could be a successful therapeutic strategy for treatment of PIRI including CLI with or without stem cell therapy.

  4. Modulation of Human Macrophage Responses to Mycobacterium tuberculosis by Silver Nanoparticles of Different Size and Surface Modification.

    Directory of Open Access Journals (Sweden)

    Srijata Sarkar

    Full Text Available Exposure to silver nanoparticles (AgNP used in consumer products carries potential health risks including increased susceptibility to infectious pathogens. Systematic assessments of antimicrobial macrophage immune responses in the context of AgNP exposure are important because uptake of AgNP by macrophages may lead to alterations of innate immune cell functions. In this study we examined the effects of exposure to AgNP with different particle sizes (20 and 110 nm diameters and surface chemistry (citrate or polyvinlypyrrolidone capping on cellular toxicity and innate immune responses against Mycobacterium tuberculosis (M.tb by human monocyte-derived macrophages (MDM. Exposures of MDM to AgNP significantly reduced cellular viability, increased IL8 and decreased IL10 mRNA expression. Exposure of M.tb-infected MDM to AgNP suppressed M.tb-induced expression of IL1B, IL10, and TNFA mRNA. Furthermore, M.tb-induced IL-1β, a cytokine critical for host resistance to M.tb, was inhibited by AgNP but not by carbon black particles indicating that the observed immunosuppressive effects of AgNP are particle specific. Suppressive effects of AgNP on the M.tb-induced host immune responses were in part due to AgNP-mediated interferences with the TLR signaling pathways that culminate in the activation of the transcription factor NF-κB. AgNP exposure suppressed M.tb-induced expression of a subset of NF-κB mediated genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A. In addition, AgNP exposure increased the expression of HSPA1A mRNA and the corresponding stress-induced Hsp72 protein. Up-regulation of Hsp72 by AgNP can suppress M.tb-induced NF-κB activation and host immune responses. The observed ability of AgNP to modulate infectious pathogen-induced immune responses has important public health implications.

  5. Cloning the human lysozyme cDNA: Inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells

    International Nuclear Information System (INIS)

    Chung, L.P.; Keshav, S.; Gordon, S.

    1988-01-01

    Lysozyme is a major secretory product of human and rodent macrophages and a useful marker for myelomonocytic cells. Based on the known human lysozyme amino acid sequence, oligonucleotides were synthesized and used as probes to screen a phorbol 12-myristate 13-acetate-treated U937 cDNA library. A full-length human lysozyme cDNA clone, pHL-2, was obtained and characterized. Sequence analysis shows that human lysozyme, like chicken lysozyme, has in 18-amino-acid-long signal peptide, but unlike the chicken lysozyme cDNA, the human lysozyme cDNA has a >1-kilobase-long 3' nontranslated sequence. Interestingly, within this 3' region, an inverted repeat of the Alu family of repetitive sequences was discovered. In RNA blot analyses, DNA probes prepared from pHL-2 can be used to detect lysozyme mRNA not only from human but also from mouse and rat. Moreover, by in situ hybridization, complementary RNA transcripts have been used as probes to detect lysozyme mRNA in mouse macrophages and Paneth cells. This human lysozyme cDNA clone is therefore likely to be a useful molecular probe for studying macrophage distribution and gene expression

  6. Effects of vitamin D-binding protein-derived macrophage-activating factor on human breast cancer cells.

    Science.gov (United States)

    Pacini, Stefania; Punzi, Tiziana; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco

    2012-01-01

    Searching for additional therapeutic tools to fight breast cancer, we investigated the effects of vitamin D-binding protein-derived macrophage activating factor (DBP-MAF, also known as GcMAF) on a human breast cancer cell line (MCF-7). The effects of DBP-MAF on proliferation, morphology, vimentin expression and angiogenesis were studied by cell proliferation assay, phase-contrast microscopy, immunohistochemistry and western blotting, and chorioallantoic membrane (CAM) assay. DBP-MAF inhibited human breast cancer cell proliferation and cancer cell-stimulated angiogenesis. MCF-7 cells treated with DBP-MAF predominantly grew in monolayer and appeared to be well adherent to each other and to the well surface. Exposure to DBP-MAF significantly reduced vimentin expression, indicating a reversal of the epithelial/mesenchymal transition, a hallmark of human breast cancer progression. These results are consistent with the hypothesis that the known anticancer efficacy of DBP-MAF can be ascribed to different biological properties of the molecule that include inhibition of tumour-induced angiogenesis and direct inhibition of cancer cell proliferation, migration and metastatic potential.

  7. 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.

  8. Efficient internalization of silica-coated iron oxide nanoparticles of different sizes by primary human macrophages and dendritic cells

    International Nuclear Information System (INIS)

    Kunzmann, Andrea; Andersson, Britta; Vogt, Carmen; Feliu, Neus; Ye Fei; Gabrielsson, Susanne; Toprak, Muhammet S.; Buerki-Thurnherr, Tina; Laurent, Sophie; Vahter, Marie; Krug, Harald; Muhammed, Mamoun; Scheynius, Annika; Fadeel, Bengt

    2011-01-01

    Engineered nanoparticles are being considered for a wide range of biomedical applications, from magnetic resonance imaging to 'smart' drug delivery systems. The development of novel nanomaterials for biomedical applications must be accompanied by careful scrutiny of their biocompatibility. In this regard, particular attention should be paid to the possible interactions between nanoparticles and cells of the immune system, our primary defense system against foreign invasion. On the other hand, labeling of immune cells serves as an ideal tool for visualization, diagnosis or treatment of inflammatory processes, which requires the efficient internalization of the nanoparticles into the cells of interest. Here, we compare novel monodispersed silica-coated iron oxide nanoparticles with commercially available dextran-coated iron oxide nanoparticles. The silica-coated iron oxide nanoparticles displayed excellent magnetic properties. Furthermore, they were non-toxic to primary human monocyte-derived macrophages at all doses tested whereas dose-dependent toxicity of the smaller silica-coated nanoparticles (30 nm and 50 nm) was observed for primary monocyte-derived dendritic cells, but not for the similarly small dextran-coated iron oxide nanoparticles. No macrophage or dendritic cell secretion of pro-inflammatory cytokines was observed upon administration of nanoparticles. The silica-coated iron oxide nanoparticles were taken up to a significantly higher degree when compared to the dextran-coated nanoparticles, irrespective of size. Cellular internalization of the silica-coated nanoparticles was through an active, actin cytoskeleton-dependent process. We conclude that these novel silica-coated iron oxide nanoparticles are promising materials for medical imaging, cell tracking and other biomedical applications.

  9. Aloe vera downregulates LPS-induced inflammatory cytokine production and expression of NLRP3 inflammasome in human macrophages.

    Science.gov (United States)

    Budai, Marietta M; Varga, Aliz; Milesz, Sándor; Tőzsér, József; Benkő, Szilvia

    2013-12-01

    Aloe vera has been used in traditional herbal medicine as an immunomodulatory agent inducing anti-inflammatory effects. However, its role on the IL-1β inflammatory cytokine production has not been studied. IL-1β production is strictly regulated both at transcriptional and posttranslational levels through the activity of Nlrp3 inflammasome. In this study we aimed to determine the effect of Aloe vera on the molecular mechanisms of Nlrp3 inflammasome-mediated IL-1β production in LPS-activated human THP-1 cells and monocyte-derived macrophages. Our results show that Aloe vera significantly reduced IL-8, TNFα, IL-6 and IL-1β cytokine production in a dose dependent manner. The inhibitory effect was substantially more pronounced in the primary cells. We found that Aloe vera inhibited the expression of pro-IL-1β, Nlrp3, caspase-1 as well as that of the P2X7 receptor in the LPS-induced primary macrophages. Furthermore, LPS-induced activation of signaling pathways like NF-κB, p38, JNK and ERK were inhibited by Aloe vera in these cells. Altogether, we show for the first time that Aloe vera-mediated strong reduction of IL-1β appears to be the consequence of the reduced expression of both pro-IL-1β as well as Nlrp3 inflammasome components via suppressing specific signal transduction pathways. Furthermore, we show that the expression of the ATP sensor P2X7 receptor is also downregulated by Aloe vera that could also contribute to the attenuated IL-1β cytokine secretion. These results may provide a new therapeutic approach to regulate inflammasome-mediated responses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Interaction of Coxiella burnetii Strains of Different Sources and Genotypes with Bovine and Human Monocyte-Derived Macrophages

    Directory of Open Access Journals (Sweden)

    Katharina Sobotta

    2018-01-01

    Full Text Available Most human Q fever infections originate from small ruminants. By contrast, highly prevalent shedding of Coxiella (C. burnetii by bovine milk rarely results in human disease. We hypothesized that primary bovine and human monocyte-derived macrophages (MDM represent a suitable in vitro model for the identification of strain-specific virulence properties at the cellular level. Twelve different C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA genotypes. Infection efficiency and replication of C. burnetii were monitored by cell culture re-titration and qPCR. Expression of immunoregulatory factors after MDM infection was measured by qRT-PCR and flow cytometry. Invasion, replication and MDM response differed between C. burnetii strains but not between MDMs of the two hosts. Strains isolated from ruminants were less well internalized than isolates from humans and rodents. Internalization of MLVA group I strains was lower compared to other genogroups. Replication efficacy of C. burnetii in MDM ranged from low (MLVA group III to high (MLVA group IV. Infected human and bovine MDM responded with a principal up-regulation of pro-inflammatory cytokines such as IL-1β, IL-12, and TNF-α. However, MLVA group IV strains induced a pronounced host response whereas infection with group I strains resulted in a milder response. C. burnetii infection marginally affected polarization of MDM. Only one C. burnetii strain of MLVA group IV caused a substantial up-regulation of activation markers (CD40, CD80 on the surface of bovine and human MDM. The study showed that replication of C. burnetii in MDM and the subsequent host cell response is genotype-specific rather than being determined by the host species pointing to a clear distinction in C. burnetii virulence between the genetic groups.

  11. 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 human... monocytes. PubmedID 11257452 Title LPS induction of gene expression in human monocytes. Authors Guha M, Ma

  12. In an in-vitro model using human fetal membranes, 17-α hydroxyprogesterone caproate is not an optimal progestogen for inhibition of fetal membrane weakening.

    Science.gov (United States)

    Kumar, Deepak; Moore, Robert M; Mercer, Brian M; Mansour, Joseph M; Mesiano, Sam; Schatz, Frederick; Lockwood, Charles J; Moore, John J

    2017-12-01

    The progestogen 17-α hydroxyprogesterone caproate (17-OHPC) is 1 of only 2 agents recommended for clinical use in the prevention of spontaneous preterm delivery, and studies of its efficacy have been conflicting. We have developed an in-vitro model to study the fetal membrane weakening process that leads to rupture in preterm premature rupture of the fetal membranes (pPROM). Inflammation/infection associated with tumor necrosis factor-α (TNF-α) induction and decidual bleeding/abruption associated thrombin release are leading causes of preterm premature rupture of the fetal membranes. Both agents (TNF-α and thrombin) cause fetal membrane weakening in the model system. Furthermore, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both TNF-α and thrombin-induced fetal membrane weakening. In a previous report, we demonstrated that 3 progestogens, progesterone, 17-alpha hydroxyprogesterone (17-OHP), and medroxyprogesterone acetate (MPA), each inhibit both TNF-α- and thrombin-induced fetal membrane weakening at 2 distinct points of the fetal membrane weakening pathway. Each block both the production of and the downstream action of the critical intermediate granulocyte-macrophage colony-stimulating factor. The objective of the study was to characterize the inhibitory effects of 17-OHPC on TNF-α- and thrombin-induced fetal membrane weakening in vitro. Full-thickness human fetal membrane fragments from uncomplicated term repeat cesarean deliveries were mounted in 2.5 cm Transwell inserts and cultured with/without 17-alpha hydroxyprogesterone caproate (10 -9 to 10 -7 M). After 24 hours, medium (supernatant) was removed and replaced with/without the addition of tumor necrosis factor-alpha (20 ng/mL) or thrombin (10 U/mL) or granulocyte-macrophage colony-stimulating factor (200 ng/mL). After 48 hours of culture, medium from the maternal side compartment of the model was assayed for granulocyte-macrophage colony

  13. Tumor necrosis factor-α and receptor activator of nuclear factor-κB ligand augment human macrophage foam-cell destruction of extracellular matrix through protease-mediated processes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Larsen, Lise

    2012-01-01

    By secreting proteases such as cathepsins and matrix metalloproteinases (MMPs), macrophage foam cells may be a major cause of ruptured atherosclerotic plaques. The aims of the present study were to investigate in vitro role of human macrophage foam cells in degrading type I collagen, a major...

  14. Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.

    Science.gov (United States)

    Branca, Jacopo J V; Morucci, Gabriele; Malentacchi, Francesca; Gelmini, Stefania; Ruggiero, Marco; Pacini, Stefania

    2015-09-01

    The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia. © 2015 Wiley Periodicals, Inc.

  15. AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages.

    Directory of Open Access Journals (Sweden)

    Marina Kemmerer

    Full Text Available AMP-activated protein kinase (AMPK maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO. The transcription factor peroxisome proliferator-activated receptor δ (PPARδ also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.

  16. Heme oxygenase-1 induction alters chemokine regulation and ameliorates human immunodeficiency virus-type-1 infection in lipopolysaccharide-stimulated macrophages

    International Nuclear Information System (INIS)

    Zhou, Zhao-Hua; Kumari, Namita; Nekhai, Sergei; Clouse, Kathleen A.; Wahl, Larry M.; Yamada, Kenneth M.; Dhawan, Subhash

    2013-01-01

    Highlights: •Lipopolysaccharide stimulation of heme oxygenase-1 (HO-1) ameliorated HIV-1 infection of primary human macrophages. •The partial protection by HO-1 against HIV infection was associated with induction of chemokines such as MIP1α and MIP1β. •This mechanism explains lipopolysaccharide-stimulated HO-1-mediated inhibition of HIV-1 infection of macrophages. -- Abstract: We have elucidated a putative mechanism for the host resistance against HIV-1 infection of primary human monocyte-derived macrophages (MDM) stimulated with lipopolysaccharide (LPS). We show that LPS-activated MDM both inhibited HIV-1 entry into the cells and were refractory to post-entry productive viral replication. LPS-treated cells were virtually negative for mature virions as revealed by transmission electron microscopy. LPS activation of MDM markedly enhanced the expression of heme oxygenase-1 (HO-1), a potent inducible cytoprotective enzyme. Increased HO-1 expression was accompanied by elevated production of macrophage inflammatory chemokines (MIP1α and MIP1β) by LPS-activated MDM, significantly decreased surface chemokine receptor-5 (CCR-5) expression, and substantially reduced virus replication. Treatment of cells with HO-1 inhibitor SnPP IX (tin protoporphyrin IX) attenuated the LPS-mediated responses, HIV-1 replication and secretion of MIP1α, MIP1β, and LD78β chemokines with little change in surface CCR-5 expression. These results identify a novel role for HO-1 in the modulation of host immune response against HIV infection of MDM

  17. The effects of a cyclooxygenase-2 (COX-2 expression and inhibition on human uveal melanoma cell proliferation and macrophage nitric oxide production

    Directory of Open Access Journals (Sweden)

    Marshall Jean-Claude

    2007-01-01

    Full Text Available Abstract Background Cyclooxygenase-2 (COX-2 expression has previously been identified in uveal melanoma although the biological role of COX-2 in this intraocular malignancy has not been elucidated. This study aimed to investigate the effect of a COX-2 inhibitor on the proliferation rate of human uveal melanoma cells, as well as its effect on the cytotoxic response of macrophages. Methods Human uveal melanoma cell lines were transfected to constitutively express COX-2 and the proliferative rate of these cells using two different methods, with and without the addition of Amfenac, was measured. Nitric oxide production by macrophages was measured after exposure to melanoma-conditioned medium from both groups of cells as well as with and without Amfenac, the active metabolite of Nepafenac. Results Cells transfected to express COX-2 had a higher proliferation rate than those that did not. The addition of Amfenac significantly decreased the proliferation rate of all cell lines. Nitric oxide production by macrophages was inhibited by the addition of melanoma conditioned medium, the addition of Amfenac partially overcame this inhibition. Conclusion Amfenac affected both COX-2 transfected and non-transfected uveal melanoma cells in terms of their proliferation rates as well as their suppressive effects on macrophage cytotoxic activity.

  18. Hemopathologic consequences of protracted gamma irradiation: alterations in granulocyte reserves and granulocyte mobilization

    International Nuclear Information System (INIS)

    Seed, T.M.; Cullen, S.M.; Kaspar, L.V.; Tolle, D.V.; Fritz, T.E.

    1980-01-01

    Aplastic anemia and myelogenous leukemia are prominent pathologic effects in beagles exposed to continuous, daily, low-dose gamma irradiation. In the present work, granulocyte reserves and related mobilization functions have been sequentially assessed by the endotoxin stress assay during the preclinical and clinical phases of these hemopoietic disorders. Characteristic patterns of granulocyte reserve mobilization are described that reflect given stages of pathologic progression. For radiation-induced leukemia, a five-stage pattern has been proposed. In contrast, a simple pattern of progressive, time-dependent contraction of granulocyte reserves and mobilization capacity was noted in the development of terminal aplastic anemia. Early preclinical phases of radiation-induced leukemia appear to involve an extensive depletion of the granulocyte reserves (phase I) during the first approx. 200 days of exposure followed by a partial renewal of the reserves and associated mobilization functions between approx. 200 and 400 days (phase II). Sustained, subnormal granulocyte mobilizations (phase III) following endotoxin stress typify the responses of dogs during the intermediate phase, whereas late preclinical, preleukemic stages (phase IV) are characterized by a further expansion of the reserves and in the mobilization capacities, particularly of the less mature granulocytes. Such late alterations in the pattern of granulocyte mobilization, together with other noted cellular aberrancies in the peripheral blood and marrow, appear to indicate leukemia (phase V) onset

  19. IFI16 is required for DNA sensing in human macrophages by promoting production and function of cGAMP

    NARCIS (Netherlands)

    Jønsson, K L; Laustsen, A; Krapp, C; Skipper, K A; Thavachelvam, K; Hotter, D; Egedal, J H; Kjolby, M; Mohammadi, P; Prabakaran, T; Sørensen, L K; Sun, C; Jensen, S B; Holm, C K; Lebbink, R J; Johannsen, M; Nyegaard, M; Mikkelsen, J G; Kirchhoff, F; Paludan, S R; Jakobsen, M R

    2017-01-01

    Innate immune activation by macrophages is an essential part of host defence against infection. Cytosolic recognition of microbial DNA in macrophages leads to induction of interferons and cytokines through activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Other

  20. UCLA1 aptamer inhibition of human immunodeficiency virus type 1 subtype C primary isolates in macrophages and selection of resistance

    CSIR Research Space (South Africa)

    Mufhandu, Hazel T

    2016-09-01

    Full Text Available isolates in monocyte-derived macrophages (MDMs). Of 4 macrophage-tropic isolates tested, 3 were inhibited by UCLA1 in the low nanomolar range (IC80 <29 nM). One isolate that showed reduced susceptibility (<50 nM) to UCLA1 contained mutations in the a5 helix...

  1. A novel assay system for macrophage-activating factor activity using a human U937 cell line.

    Science.gov (United States)

    Ishikawa, Mami; Inoue, Takahiro; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

    2014-08-01

    Macrophages play important roles in antitumor immunity, and immunotherapy with the group-specific component protein-derived macrophage-activating factor (GcMAF) has been reported to be effective in patients with various types of cancers. However, in macrophage research, it is important to properly evaluate macrophage activity. U937 macrophages were induced by 12-O-tetradecanoyl-13-phorbolacetate (TPA). The phagocytic activity of macrophages was evaluated as the internalized beads ratio. The MAF activity was assessed at 30 min after MAF addition as the activation ratio. We established a novel assay for phagocytic activities using differentiated U937 macrophages. The novel protocol was simple and rapid and was sensitive for GcMAF. This protocol should be useful not only for basic studies, such as those on molecular mechanisms underlying macrophage activation, but also for clinical studies, such as assessment of GcMAF activity prior to clinical use. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  2. The Alveolar Microenvironment of Patients Infected with Human Immunodeficiency Virus Does Not Modify Alveolar Macrophage Interactions with Streptococcus pneumoniae

    Science.gov (United States)

    Jagoe, R. Thomas; Jarman, Elizabeth R.; North, James C.; Pridmore, Alison; Musaya, Janelisa; French, Neil; Zijlstra, Eduard E.; Molyneux, Malcolm E.; Read, Robert C.

    2013-01-01

    We tested the hypothesis that HIV infection results in activation of alveolar macrophages and that this might be associated with impaired defense against pneumococcus. We compared alveolar macrophages and lymphocytes in 131 bronchoalveolar lavage samples from HIV-infected and healthy controls using inflammatory gene microarrays, flow cytometry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA) to determine the pattern of macrophage activation associated with HIV infection and the effect of this activation on defense against pneumococcus. We used gamma interferon (IFN-γ) priming to mimic the cellular milieu in HIV-infected lungs. InnateDB and BioLayout 3D were used to analyze the interactions of the upregulated genes. Alveolar macrophages from HIV-infected adults showed increased gene expression and cytokine production in a classical pattern. Bronchoalveolar lavage from HIV-infected subjects showed excess CD8+ lymphocytes with activated phenotype. Toll-like receptor 4 (TLR4) expression was increased in macrophages from HIV-infected subjects, but function was similar between the groups; lung lavage fluid did not inhibit TLR function in transfected HeLa cells. Alveolar macrophages from HIV-infected subjects showed normal binding and internalization of opsonized pneumococci, with or without IFN-γ priming. Alveolar macrophages from HIV-infected subjects showed classical activation compared to that of healthy controls, but this does not alter macrophage interactions with pneumococci. PMID:23576675

  3. IL-17A influences essential functions of the monocyte/macrophage lineage and is involved in advanced murine and human atherosclerosis.

    Science.gov (United States)

    Erbel, Christian; Akhavanpoor, Mohammadreza; Okuyucu, Deniz; Wangler, Susanne; Dietz, Alex; Zhao, Li; Stellos, Konstantinos; Little, Kristina M; Lasitschka, Felix; Doesch, Andreas; Hakimi, Maani; Dengler, Thomas J; Giese, Thomas; Blessing, Erwin; Katus, Hugo A; Gleissner, Christian A

    2014-11-01

    Atherosclerosis is a chronic inflammatory disease. Lesion progression is primarily mediated by cells of the monocyte/macrophage lineage. IL-17A is a proinflammatory cytokine, which modulates immune cell trafficking and is involved inflammation in (auto)immune and infectious diseases. But the role of IL-17A still remains controversial. In the current study, we investigated effects of IL-17A on advanced murine and human atherosclerosis, the common disease phenotype in clinical care. The 26-wk-old apolipoprotein E-deficient mice were fed a standard chow diet and treated either with IL-17A mAb (n = 15) or irrelevant Ig (n = 10) for 16 wk. Furthermore, essential mechanisms of IL-17A in atherogenesis were studied in vitro. Inhibition of IL-17A markedly prevented atherosclerotic lesion progression (p = 0.001) by reducing inflammatory burden and cellular infiltration (p = 0.01) and improved lesion stability (p = 0.01). In vitro experiments showed that IL-17A plays a role in chemoattractance, monocyte adhesion, and sensitization of APCs toward pathogen-derived TLR4 ligands. Also, IL-17A induced a unique transcriptome pattern in monocyte-derived macrophages distinct from known macrophage types. Stimulation of human carotid plaque tissue ex vivo with IL-17A induced a proinflammatory milieu and upregulation of molecules expressed by the IL-17A-induced macrophage subtype. In this study, we show that functional blockade of IL-17A prevents atherosclerotic lesion progression and induces plaque stabilization in advanced lesions in apolipoprotein E-deficient mice. The underlying mechanisms involve reduced inflammation and distinct effects of IL-17A on monocyte/macrophage lineage. In addition, translational experiments underline the relevance for the human system. Copyright © 2014 by The American Association of Immunologists, Inc.

  4. Tumour-derived GM-CSF promotes granulocyte immunosuppression in mesothelioma patients.

    Science.gov (United States)

    Khanna, Swati; Graef, Suzanne; Mussai, Francis; Thomas, Anish; Wali, Neha; Yenidunya, Bahar Guliz; Yuan, Constance M; Morrow, Betsy; Zhang, Jingli; Korangy, Firouzeh; Greten, Tim F; Steinberg, Seth M; Stetler-Stevenson, Maryalice; Middleton, Gary; De Santo, Carmela; Hassan, Raffit

    2018-03-30

    The cross talk between tumour cells, myeloid cells, and T cells play a critical role in tumour pathogenesis and response to immunotherapies. Although the aetiology of mesothelioma is well understood the impact of mesothelioma on the surrounding immune microenvironment is less well studied. In this study the effect of the mesothelioma microenvironment on circulating and infiltrating granulocytes and T cells is investigated. Tumour and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T cell suppression. Co-cultures of granulocytes, mesothelioma cells, T cells were used to identify the mechanism of T cell inhibition. Analysis of tumours showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T cell proliferation and activation. Characterisation of the blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR- granulocytes at increased frequency compared to healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of Reactive Oxygen Species (ROS) from granulocytes, resulting in T cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumours express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralising antibody, or ROS inhibition, restored T cell proliferation suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients. Our study presents the mechanism behind the cross-talk between mesothelioma and the immune micro-environment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy. Copyright ©2018, American Association for Cancer Research.

  5. Congenital hypogammaglobulinemia associated with granulocyte disorders. A case presentation

    International Nuclear Information System (INIS)

    Sanchez Segura, Miriam C; Marsan Suarez, Vianed; Socarras Ferrer, Bertha B; Ojeda de Leon, Norma

    2009-01-01

    This is the case of a child aged 11 months with a history of systemic sepsis from Pseudomona aeruginosa at 5 months, neutropenia, leucopenia, sepsis-associated anemia and from then, recurrent acute respiratory infections of the high respiratory tract, allergic manifestations and furunculosis from pseudomona. Immunologic study conducted showed a decreased figure of IgG with a light increase of CD4 +c ooperative-IgM of T cells. Also, we found the presence of neutropenia and marked defect of phagocytosis. We made the diagnosis of granulocyte-associate congenital hypogammaglobulinemia. The patient was treated with human gamma globulin by intramuscular route, transference factor and immunoferon, with an obvious improvement

  6. Establishment of a Model of Microencapsulated SGC7901 Human Gastric Carcinoma Cells Cocultured with Tumor-Associated Macrophages

    Directory of Open Access Journals (Sweden)

    Jin-Ming Zhu

    2018-01-01

    Full Text Available The important factors of poor survival of gastric cancer (GC are relapse and metastasis. For further elucidation of the mechanism, a culture system mimicking the microenvironment of the tumor in humans was needed. We established a model of microencapsulated SGC7901 human GC cells and evaluated the effects of coculturing spheres with tumor-associated macrophages (TAMs. SGC7901 cells were encapsulated in alginate-polylysine-sodium alginate (APA microcapsules using an electrostatic droplet generator. MTT assays showed that the numbers of microencapsulated cells were the highest after culturing for 14 days. Metabolic curves showed consumption of glucose and production of lactic acid by day 20. Immunocytochemistry confirmed that Proliferating Cell Nuclear Antigen (PCNA and Vascular Endothelial Growth Factor (VEGF were expressed in microencapsulated SGC7901 cells on days 7 and 14. The expression of PCNA was observed outside spheroids; however, VEGF was found in the entire spheroids. PCNA and VEGF were increased after being cocultured with TAMs. Matrix metalloproteinase-2 (MMP-2 and matrix metalloproteinase-9 (MMP-9 expressions were detected in the supernatant of microencapsulated cells cocultured with TAMs but not in microencapsulated cells. Our study confirms the successful establishment of the microencapsulated GC cells. TAMs can promote PCNA, VEGF, MMP-2, and MMP-9 expressions of the GC cells.

  7. A phase I study of different doses and frequencies of pegylated recombinant human granulocyte-colony stimulating factor (PEG rhG-CSF) in patients with standard-dose chemotherapy-induced neutropenia.

    Science.gov (United States)

    Qin, Yan; Han, Xiaohong; Wang, Lin; Du, Ping; Yao, Jiarui; Wu, Di; Song, Yuanyuan; Zhang, Shuxiang; Tang, Le; Shi, Yuankai

    2017-10-01

    The recommended dose of prophylactic pegylated recombinant human granulocyte-colony stimulating factor (PEG rhG-CSF) is 100 μg/kg once per cycle for patients receiving intense-dose chemotherapy. However, few data are available on the proper dose for patients receiving less-intense chemotherapy. The aim of this phase I study is to explore the proper dose and administration schedule of PEG rhG-CSF for patients receiving standard-dose chemotherapy. Eligible patients received 3-cycle chemotherapy every 3 weeks. No PEG rhG-CSF was given in the first cycle. Patients experienced grade 3 or 4 neutropenia would then enter the cycle 2 and 3. In cycle 2, patients received a single subcutaneous injection of prophylactic PEG rhG-CSF on d 3, and received half-dose subcutaneous injection in cycle 3 on d 3 and d 5, respectively. Escalating doses (30, 60, 100 and 200 μg/kg) of PEG rhG-CSF were investigated. A total of 26 patients were enrolled and received chemotherapy, in which 24 and 18 patients entered cycle 2 and cycle 3 treatment, respectively. In cycle 2, the incidence of grade 3 or 4 neutropenia for patients receiving single-dose PEG rhG-CSF of 30, 60, 100 and 200 μg/kg was 66.67%, 33.33%, 22.22% and 0, respectively, with a median duration less than 1 (0-2) d. No grade 3 or higher neutropenia was noted in cycle 3 in all dose cohorts. The pharmacokinetic and pharmacodynamic profiles of PEG rhG-CSF used in cancer patients were similar to those reported, as well as the safety. Double half dose administration model showed better efficacy result than a single dose model in terms of grade 3 neutropenia and above. The single dose of 60 μg/kg, 100 μg/kg and double half dose of 30 μg/kg were recommended to the phase II study, hoping to find a preferable method for neutropenia treatment.

  8. Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages

    Directory of Open Access Journals (Sweden)

    Meyerhans Andreas

    2010-09-01

    Full Text Available Abstract Background Investigations on pulmonary macrophages (MΦ mostly focus on alveolar MΦ (AM as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM, are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue. Methods Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay. Results IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG. Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6. Conclusion AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.

  9. Purification of human recombinant granulocyte colony stimulating ...

    African Journals Online (AJOL)

    Ramya

    2012-06-21

    Jun 21, 2012 ... Proteins expressed as inclusion bodies are currently solubilized by the use of high ... by dialyzing with buffer containing reducing and oxidizing agents. Renaturation of ... MATERIALS AND METHODS. Expression system and ...

  10. Assessment of macrophage migration inhibitory factor in humans: protocol for accurate and reproducible levels.

    Science.gov (United States)

    Sobierajski, Julia; Hendgen-Cotta, Ulrike B; Luedike, Peter; Stock, Pia; Rammos, Christos; Meyer, Christian; Kraemer, Sandra; Stoppe, Christian; Bernhagen, Jürgen; Kelm, Malte; Rassaf, Tienush

    2013-10-01

    The analytical validation of a possible biomarker is the first step in the long translational process from basic science to clinical routine. Although the chemokine-like cytokine macrophage migration inhibitory factor (MIF) has been investigated intensively in experimental approaches to various disease conditions, its transition into clinical research is just at the very beginning. Because of its presence in preformed storage pools, MIF is the first cytokine to be released under various stimulation conditions. In the first proof-of-concept studies, MIF levels correlated with the severity and outcome of various disease states. In a recent small study with acute coronary syndrome patients, elevation of MIF was described as a new factor for risk assessment. When these studies are compared, not only MIF levels in diseased patients differ, but also MIF levels in healthy control groups are inconsistent. Blood MIF concentrations in control groups vary between 0.56 and 95.6 ng/ml, corresponding to a 170-fold difference. MIF concentrations in blood were analyzed by ELISA. Other than the influence of this approach due to method-based variations, the impact of preanalytical processing on MIF concentrations is unclear and has not been systematically studied yet. Before large randomized studies are performed to determine the impact of circulating MIF on prognosis and outcome and before MIF is characterized as a diagnostic marker, an accurate protocol for the determination of reproducible MIF levels needs to be validated. In this study, the measurement of MIF in the blood of healthy volunteers was investigated focusing on the potential influence of critical preanalytical factors such as anticoagulants, storage conditions, freeze/thaw stability, hemolysis, and dilution. We show how to avoid pitfalls in the measurement of MIF and that MIF concentrations are highly susceptible to preanalytical factors. MIF serum concentrations are higher than plasma concentrations and show broader

  11. The redistribution of granulocytes following E. coli endotoxin induced sepsis

    DEFF Research Database (Denmark)

    Toft, P; Lillevang, S T; Tønnesen, Else Kirstine

    1994-01-01

    Infusion of endotoxin elicits granulocytopenia followed by increased numbers of granulocytes in peripheral blood. The purpose of this study was to investigate the redistribution and sequestration of granulocytes in the tissues following E. coli endotoxin induced sepsis. From 16 rabbits granulocytes...

  12. Macrophages are related to goblet cell hyperplasia and induce MUC5B but not MUC5AC in human bronchus epithelial cells.

    Science.gov (United States)

    Silva, Manuel A; Bercik, Premysl

    2012-06-01

    Airway goblet cell hyperplasia (GCH)--detectable by mucin staining--and abnormal macrophage infiltrate are pathological features present in many chronic respiratory disorders. However, it is unknown if both factors are associated. Using in-vivo and in-vitro models, we investigated whether macrophages are related with GCH and changes in mucin immunophenotypes. Lung sections from Sprague-Dawley rats treated for 48 h with one intra-tracheal dose of PBS or LPS (n=4-6 per group) were immunophenotyped for rat-goblet cells, immune, and proliferation markers. Human monocyte-derived macrophages (MDM) were pre-treated with or without LPS, immunophenotyped, and their supernatant, as well as cytokines at levels equivalent to supernatant were used to challenge primary culture of normal human bronchus epithelial cells (HBEC) in air-liquid interface, followed by MUC5B and MUC5AC mucin immunostaining. An association between increased bronchiolar goblet cells and terminal-bronchiolar proliferative epithelial cells confirmed the presence of GCH in our LPS rat model, which was related with augmented bronchiolar CD68 macrophage infiltration. The in-vitro experiments have shown that MUC5AC phenotype was inhibited when HBEC were challenged with supernatant from MDM pre-treated with or without LPS. In contrast, TNF-α and interleukin-1β at levels equivalent to supernatant from LPS-treated MDM increased MUC5AC. MUC5B was induced by LPS, supernatant from LPS-treated MDM, a mix of cytokines including TNF-α and TNF-α alone at levels present in supernatant from LPS-treated MDM. We demonstrated that macrophages are related with bronchiolar GCH, and that they induced MUC5B and inhibited MUC5AC in HBEC, suggesting a role for them in the pathogenesis of airway MUC5B-related GCH.

  13. Genome-wide identification of hypoxia-inducible factor-1 and -2 binding sites in hypoxic human macrophages alternatively activated by IL-10.

    Science.gov (United States)

    Tausendschön, Michaela; Rehli, Michael; Dehne, Nathalie; Schmidl, Christian; Döring, Claudia; Hansmann, Martin-Leo; Brüne, Bernhard

    2015-01-01

    Macrophages (MΦ) often accumulate in hypoxic areas, where they significantly influence disease progression. Anti-inflammatory cytokines, such as IL-10, generate alternatively activated macrophages that support tumor growth. To understand how alternative activation affects the transcriptional profile of hypoxic macrophages, we globally mapped binding sites of hypoxia-inducible factor (HIF)-1α and HIF-2α in primary human monocyte-derived macrophages prestimulated with IL-10. 713 HIF-1 and 795 HIF-2 binding sites were identified under hypoxia. Pretreatment with IL-10 altered the binding pattern, with 120 new HIF-1 and 188 new HIF-2 binding sites emerging. HIF-1 binding was most prominent in promoters, while HIF-2 binding was more abundant in enhancer regions. Comparison of ChIP-seq data obtained in other cells revealed a highly cell type specific binding of HIF. In MΦ HIF binding occurred preferentially in already active enhancers or promoters. To assess the roles of HIF on gene expression, primary human macrophages were treated with siRNA against HIF-1α or HIF-2α, followed by genome-wide gene expression analysis. Comparing mRNA expression to the HIF binding profile revealed a significant enrichment of hypoxia-inducible genes previously identified by ChIP-seq. Analysis of gene expression under hypoxia alone and hypoxia/IL-10 showed the enhanced induction of a set of genes including PLOD2 and SLC2A3, while another group including KDM3A and ADM remained unaffected or was reduced by IL-10. Taken together IL-10 influences the DNA binding pattern of HIF and the level of gene induction. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Influence of Leishmania RNA Virus 1 on Proinflammatory Biomarker Expression in a Human Macrophage Model of American Tegumentary Leishmaniasis.

    Science.gov (United States)

    Kariyawasam, Ruwandi; Grewal, Jugvinder; Lau, Rachel; Purssell, Andrew; Valencia, Braulio M; Llanos-Cuentas, Alejandro; Boggild, Andrea K

    2017-10-17

    Species of the Leishmania Viannia (L. V.) subgenus harbor the double-stranded Leishmania RNA virus 1 (LRV-1), previously identified in isolates from Brazil and Peru. Higher levels of LRV-1 in metastasizing strains of L. V. guyanensis have been documented in both human and murine models, and correlated to disease severity. Expression of proinflammatory biomarkers, including interleukin (IL) 1β, tumor necrosis factor alpha (TNF-α), CXCL10, CCL5, IL-6, and superoxide dismutase, in human macrophages infected with 3 ATCC and 5 clinical isolates of L. V. braziliensis, L. V. guyanensis, and L. V. panamensis for 24 and 48 hours were measured by commercial enzyme immunoassay. Analyses were performed at 24 and 48 hours, stratified by LRV-1 status and species. LRV-1-positive L. V. braziliensis demonstrated significantly lower expression levels of TNF-α (P = .01), IL-1β (P = .0015), IL-6 (P = .001), and CXCL10 (P = .0004) compared with LRV-1-negative L. V. braziliensis. No differences were observed in strains of L. V. panamensis by LRV-1 status. Compared to LRV-1-negative L. V. braziliensis, LRV-1-positive strains of L. V. braziliensis produced a predominant Th2-biased immune response, correlated in humans to poorer immunologic control of infection and more severe disease, including mucosal leishmaniasis. Effects of LRV-1 on the pathogenesis of American tegumentary leishmaniasis may be species specific. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  15. Differential MicroRNA Expression in Human Macrophages with Mycobacterium tuberculosis Infection of Beijing/W and Non-Beijing/W Strain Types.

    Directory of Open Access Journals (Sweden)

    Lin Zheng

    Full Text Available The role of microRNAs in association with Mycobacterium tuberculosis (MTB infection and the immunology regulated by microRNAs upon MTB infection have not been fully unravelled. We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of Beijing/W and non-Beijing/W clinical strains. We also studied the microRNA profiles of the host macrophages by microarray in a small cohort with active MTB disease, latent infection (LTBI, and from healthy controls.The results revealed that 14 microRNAs differentiated infections of Beijing/W from non-Beijing/W strains (P<0.05. A unique signature of 11 microRNAs in human macrophages was identified to differentiate active MTB disease from LTBI and healthy controls. Pathway analyses of these differentially expressed miRNAs suggest that the immune-regulatory interactions involving TGF-β signalling pathway take part in the dysregulation of critical TB processes in the macrophages, resulting in active expression of both cell communication and signalling transduction systems.We showed for the first time that the Beijing/W TB strains repressed a number of miRNAs expressions which may reflect their virulence characteristics in altering the host response. The unique signatures of 11 microRNAs may deserve further evaluation as candidates for biomarkers in the diagnosis of MTB and Beijing/W infections.

  16. Differential MicroRNA Expression in Human Macrophages with Mycobacterium tuberculosis Infection of Beijing/W and Non-Beijing/W Strain Types.

    Science.gov (United States)

    Zheng, Lin; Leung, Eric; Lee, Nelson; Lui, Grace; To, Ka-Fai; Chan, Raphael C Y; Ip, Margaret

    2015-01-01

    The role of microRNAs in association with Mycobacterium tuberculosis (MTB) infection and the immunology regulated by microRNAs upon MTB infection have not been fully unravelled. We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of Beijing/W and non-Beijing/W clinical strains. We also studied the microRNA profiles of the host macrophages by microarray in a small cohort with active MTB disease, latent infection (LTBI), and from healthy controls. The results revealed that 14 microRNAs differentiated infections of Beijing/W from non-Beijing/W strains (PmicroRNAs in human macrophages was identified to differentiate active MTB disease from LTBI and healthy controls. Pathway analyses of these differentially expressed miRNAs suggest that the immune-regulatory interactions involving TGF-β signalling pathway take part in the dysregulation of critical TB processes in the macrophages, resulting in active expression of both cell communication and signalling transduction systems. We showed for the first time that the Beijing/W TB strains repressed a number of miRNAs expressions which may reflect their virulence characteristics in altering the host response. The unique signatures of 11 microRNAs may deserve further evaluation as candidates for biomarkers in the diagnosis of MTB and Beijing/W infections.

  17. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages

    International Nuclear Information System (INIS)

    Haberland, M.E.; Fogelman, A.M.

    1985-01-01

    Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125 I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. Although ligands recognized by the scavenger receptor typically are anionic, the authors propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). They conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor

  18. Effect of Macrophage Migration Inhibitory Factor (MIF) in Human Placental Explants Infected with Toxoplasma gondii Depends on Gestational Age

    Science.gov (United States)

    de Oliveira Gomes, Angelica; de Oliveira Silva, Deise Aparecida; Silva, Neide Maria; de Freitas Barbosa, Bellisa; Franco, Priscila Silva; Angeloni, Mariana Bodini; Fermino, Marise Lopes; Roque-Barreira, Maria Cristina; Bechi, Nicoletta; Paulesu, Luana Ricci; dos Santos, Maria Célia; Mineo, José Roberto; Ferro, Eloisa Amália Vieira

    2011-01-01

    Because macrophage migration inhibitory factor (MIF) is a key cytokine in pregnancy and has a role in inflammatory response and pathogen defense, the objective of the present study was to investigate the effects of MIF in first- and third-trimester human placental explants infected with Toxoplasma gondii. Explants were treated with recombinant MIF, IL-12, interferon-γ, transforming growth factor-β1, or IL-10, followed by infection with T. gondii RH strain tachyzoites. Supernatants of cultured explants were assessed for MIF production. Explants were processed for morphologic analysis, immunohistochemistry, and real-time PCR analysis. Comparison of infected and stimulated explants versus noninfected control explants demonstrated a significant increase in MIF release in first-trimester but not third-trimester explants. Tissue parasitism was higher in third- than in first-trimester explants. Moreover, T. gondii DNA content was lower in first-trimester explants treated with MIF compared with untreated explants. However, in third-trimester explants, MIF stimulus decreased T. gondii DNA content only at the highest concentration of the cytokine. In addition, high expression of MIF receptor was observed in first-trimester placental explants, whereas MIF receptor expression was low in third-trimester explants. In conclusion, MIF was up-regulated and demonstrated to be important for control of T. gondii infection in first-trimester explants, whereas lack of MIF up-regulation in third-trimester placentas may be involved in higher susceptibility to infection at this gestational age. PMID:21641401

  19. Human Properdin Opsonizes Nanoparticles and Triggers a Potent Pro-inflammatory Response by Macrophages without Involving Complement Activation

    Science.gov (United States)

    Kouser, Lubna; Paudyal, Basudev; Kaur, Anuvinder; Stenbeck, Gudrun; Jones, Lucy A.; Abozaid, Suhair M.; Stover, Cordula M.; Flahaut, Emmanuel; Sim, Robert B.; Kishore, Uday

    2018-01-01

    Development of nanoparticles as tissue-specific drug delivery platforms can be considerably influenced by the complement system because of their inherent pro-inflammatory and tumorigenic consequences. The complement activation pathways, and its recognition subcomponents, can modulate clearance of the nanoparticles and subsequent inflammatory response and thus alter the intended translational applications. Here, we report, for the first time, that human properdin, an upregulator of the complement alternative pathway, can opsonize functionalized carbon nanotubes (CNTs) via its thrombospondin type I repeat (TSR) 4 and 5. Binding of properdin and TSR4+5 is likely to involve charge pattern/polarity recognition of the CNT surface since both carboxymethyl cellulose-coated carbon nanotubes (CMC-CNT) and oxidized (Ox-CNT) bound these proteins well. Properdin enhanced the uptake of CMC-CNTs by a macrophage cell line, THP-1, mounting a robust pro-inflammatory immune response, as revealed by qRT-PCR, multiplex cytokine array, and NF-κB nuclear translocation analyses. Properdin can be locally synthesized by immune cells in an inflammatory microenvironment, and thus, its interaction with nanoparticles is of considerable importance. In addition, recombinant TSR4+5 coated on the CMC-CNTs inhibited complement consumption by CMC-CNTs, suggesting that nanoparticle decoration with TSR4+5, can be potentially used as a complement inhibitor in a number of pathological contexts arising due to exaggerated complement activation. PMID:29483907

  20. Quantification of deposition of neutrophilic granulocytes on vascular grafts in dogs with 111In-labeled granulocytes

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Solis, E.; Mackey, S.T.; Socher, S.; Chowdhury, S.; Wu, F.P.; Kaye, M.P.

    1985-01-01

    A new radioisotopic technique has been developed for quantification of deposition of neutrophilic granulocytes on vascular grafts. Nine healthy mongrel dogs underwent bilateral femoral artery resection and reconstruction with grafts of the femoral vein and Gore-Tex. Pure granulocytes that had been separated from whole blood by centrifugal elutriation were labeled with 111 In-tropolone in plasma. The granulocyte harvesting efficiency was 25 +/- 12%, and the labeling efficiency was 87 +/- 7%. Three hours after injection of labeled granulocytes and 2 hours after reperfusion, the grafts were harvested and cut into several segments for study of areas of anastomoses and midsections. On the basis of the radioactivity in the blood and in anastomotic and graft sections, the area of graft sections, and the neutrophilic granulocyte and differential leukocyte counts, the number of neutrophilic granulocytes adherent to a unit area and the total number of neutrophilic granulocytes on graft sections were calculated. These quantifications of the deposition of neutrophilic granulocytes indicated that the midsections of Gore-Tex grafts retained more neutrophilic granulocytes than did the midsections of vein grafts. Although the anastomotic areas retained more neutrophilic granulocytes than did the midsections of vein grafts, the opposite finding prevailed for the Gore-Tex grafts. A major fraction of neutrophilic granulocytes on Gore-Tex grafts was incorporated into the thrombus. Semiquantitative information obtained by scintigraphy of the deposition of neutrophilic granulocytes on vascular grafts also confirmed this observation

  1. Expression of YKL-40 by peritumoral macrophages in human small cell lung cancer

    DEFF Research Database (Denmark)

    Junker, Nanna; Johansen, Julia S; Andersen, Claus B

    2005-01-01

    YKL-40 is a 40 kDa protein with possible involvement in tissue remodeling, cell proliferation and angiogenesis. Elevated serum YKL-40 levels in patients with metastatic cancers (including small cell lung cancer (SCLC)) are associated with poor prognosis. The aim of this study was to identify...... the cellular source of YKL-40 in SCLC patient biopsies and in a panel of 20 human SCLC lines cultured in vitro and in vivo in nude mice. In general, the SCLC cell lines had no or very limited (human) YKL-40 expression, whereas, by RT-PCR a pronounced murine (i.e., stromal) YKL-40 expression was present in all...

  2. Endothelial lipase is highly expressed in macrophages in advanced human atherosclerotic lesions

    DEFF Research Database (Denmark)

    Bartels, Emil D; Nielsen, John E; Lindegaard, Marie Louise Skakkebæk

    2007-01-01

    Endothelial lipase (EL) is expressed in endothelial cells, and affects plasma lipoprotein metabolism by hydrolyzing phospholipids in HDL. To determine the cellular expression of EL mRNA and protein in human atherosclerotic lesions, we performed in situ hybridization and immunohistochemical studies...

  3. Eicosanoid and cytokine production by human peritoneal macrophages : characterization and modulation

    NARCIS (Netherlands)

    W.M. Pruimboom (Wanda)

    1995-01-01

    textabstractThe human body has an extensive and specialized cellular network to defend against invading microorganisms, and to prevent the development and spread of neoplastic disease. The mononuclear phagocytic cells are important secretory cells that, through their receptors and secretory

  4. In vitro differentiation of human monocytes to macrophages: change of PDE profile and its relationship to suppression of tumour necrosis factor-α release by PDE inhibitors

    Science.gov (United States)

    Gantner, Florian; Kupferschmidt, Rochus; Schudt, Christian; Wendel, Albrecht; Hatzelmann, Armin

    1997-01-01

    During in vitro culture in 10% human AB serum, human peripheral blood monocytes acquire a macrophage-like phenotype. The underlying differentiation was characterized by increased activities of the macrophage marker enzymes unspecific esterase (NaF-insensitive form) and acid phosphatase, as well as by a down-regulation in surface CD14 expression. In parallel, a dramatic change in the phosphodiesterase (PDE) profile became evident within a few days that strongly resembled that previously described for human alveolar macrophages. Whereas PDE1 and PDE3 activities were augmented, PDE4 activity, which represented the major cyclic AMP-hydrolysing activity of peripheral blood monocytes, rapidly declined. Monocytes and monocyte-derived macrophages responded to lipopolysaccharide (LPS) with the release of tumour necrosis factor-α (TNF). In line with the change in CD14 expression, the EC50 value of LPS for induction of TNF release increased from approximately 0.1 ng ml−1 in peripheral blood monocytes to about 2 ng ml−1 in macrophages. Both populations of cells were equally susceptible towards inhibition of TNF release by cyclic AMP elevating agents such as dibutyryl cyclic AMP, prostaglandin E2 (PGE2) or forskolin, which all led to a complete abrogation of TNF production in a concentration-dependent manner and which were more efficient than the glucocorticoid dexamethasone. In monocytes, PDE4 selective inhibitors (rolipram, RP73401) suppressed TNF formation by 80%, whereas motapizone, a PDE3 selective compound, exerted a comparatively weak effect (10–15% inhibition). Combined use of PDE3 plus PDE4 inhibitors resulted in an additive effect and fully abrogated LPS-induced TNF release as did the mixed PDE3/4 inhibitor tolafentrine. In monocyte-derived macrophages, neither PDE3- nor PDE4-selective drugs markedly affected TNF generation when used alone (<15% inhibition), whereas in combination, they led to a maximal inhibition of TNF formation by about 40–50

  5. Combined measurement of growth and differentiation in suspension cultures of purified human CD34-positive cells enables a detailed analysis of myelopoiesis

    NARCIS (Netherlands)

    Kerst, J. M.; Slaper-Cortenbach, I. C.; von dem Borne, A. E.; van der Schoot, C. E.; van Oers, R. H.

    1992-01-01

    In this study we have made a detailed analysis of growth factor (granulocyte-macrophage colony-stimulating factor [GM-CSF], granulocyte colony-stimulating factor [G-CSF], and macrophage colony-stimulating factor [M-CSF])-induced proliferation and differentiation of highly purified CD34+ committed

  6. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    OpenAIRE

    Selleri, Silvia; Bifsha, Panojot; Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Ren?e; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-...

  7. Effect of conjugated linoleic acids on the activity and mRNA expression of 5- and 15-lipoxygenases in human macrophages.

    Science.gov (United States)

    Stachowska, Ewa; Dziedziejko, Violetta; Safranow, Krzysztof; Jakubowska, Katarzyna; Olszewska, Maria; Machaliñski, Bogusław; Chlubek, Dariusz

    2007-06-27

    Lipoxygenases are a family of non-heme enzyme dioxygenases. The role of lipoxygenases is synthesis of hydroperoxides of fatty acids, which perform signaling functions in the body. Studies on conjugated linoleic acids (CLAs) as fatty acids with a potential anti-atherosclerotic function have recently been initiated. The aim of the study was to test the effect of CLAs and linoleic acid on 5- and 15-lipoxygenase (5-LO, 15-LO-1) enzyme activity, their mRNA expression, and concentration in the cells. It was also desired to determine whether the CLAs are substrates for the enzymes. For the experiments monocytic cell line (THP-1) and monocytes obtained from human venous blood were used. Monocytes were differentiated to macrophages: THP-1 (CD14+) by PMA administration (100 nM for 24 h) and monocytes from blood (CD14+) by 7-day cultivation with the autologous serum (10%). After differentiation, macrophages were cultured with 30 microM CLAs or linoleic acid for 48 h. The 15- and 5-lipoxygenase products were measured by HPLC method. mRNA expression and protein content were analyzed by real-time PCR and Western blot analysis. The in vitro studies proved that both CLA isomers are not substrates for 15-LO-1; in ex vivo studies hydroxydecadienoic acid (HODE) concentration was significantly reduced (p = 0.019). The trans-10,cis-12 CLA isomer reduced HODE concentration by 28% (p = 0.046) and the cis-9,trans-11 CLA isomer by 35% (p = 0.028). In macrophages obtained from THP-1 fatty acids did not change significantly mRNA expression of the majority of the investigated genes. CLAs did not change the content of 5-LO and 15-LO-1 proteins in macrophages obtained from peripheral blood. Linoleic acid induced 15-LO-1 expression (2.6 times, p < 0.05). CLAs may perform the function of an inhibitor of lipoxygenase 15-LO-1 activity in macrophages.

  8. Radiation effects on cultured human monocytes and on monocyte-derived macrophages

    International Nuclear Information System (INIS)

    Buescher, E.S.; Gallin, J.I.

    1984-01-01

    Prior to administration, leukocyte transfusions are commonly irradiated with up to 5,000 R to eliminate lymphocytes and thereby prevent graft-versus-host disease in the recipient. It has been widely believed that phagocytes are resistant to this irradiation. In a recent report, it was noted that phagocyte oxidative metabolism was compromised during preparation of white cells for transfusion. As part of the effort to examine the basis for this inhibition of phagocyte function during white cell preparation, an assessment was made of the effects of irradiation on the long-lived monocytes that have been shown to persist at inflammatory foci posttransfusion. Human monocytes were irradiated for up to 3 min, receiving 2,500-5,000 R. This irradiation damaged human monocytes, significantly decreasing their in vitro survival for the first 3 wk of culture, and growth as assessed by two-dimensional cell size measurements during the first 2 wk of culture. Despite smaller cell size, total cell protein was significantly increased over time in irradiated cultures. Extracellular release of lysozyme and beta-glucuronidase per cell was not affected by irradiation, but extracellular lactate dehydrogenase (LDH) release was significantly increased after irradiation. Irradiated monocytes killed Listeria monocytogenes at a slower rate than the nonirradiated controls. Thus, the data indicate that irradiation in doses used to prevent graft-versus-host disease in leukocyte transfusion recipients has a deleterious effect on in vitro human monocyte survival and function

  9. Serum neopterin and soluble CD163 as markers of macrophage activation in paracetamol (acetaminophen)-induced human acute liver injury.

    Science.gov (United States)

    Craig, D G; Lee, P; Pryde, E A; Hayes, P C; Simpson, K J

    2013-12-01

    Macrophage activation is implicated in the pathogenesis of the systemic inflammatory response syndrome (SIRS) following paracetamol (acetaminophen) overdose (POD). Neopterin is synthesised from macrophages and reflects the intensity of monocyte/macrophage activation. Soluble CD163 (sCD163) is a marker of alternatively activated M2 macrophages. To examine neopterin and sCD163 levels in a cohort of acute liver injury patients. Consecutive patients (n = 41, (18 (43.9%) male) with acute liver injury were enrolled. Neopterin and sCD163 levels were measured by ELISA. A total of 24/33 (72.7%) POD patients developed hepatic encephalopathy (HE), and therefore acute liver failure. Both neopterin and sCD163 levels were significantly higher in PODs compared with chronic liver disease (neopterin P paracetamol overdose, and reflect the degree of macrophage activation in this condition. Serum neopterin in particular may have value as an early proxy marker of macrophage activation following paracetamol overdose. © 2013 John Wiley & Sons Ltd.

  10. Impaired IFNγ-Signaling and Mycobacterial Clearance in IFNγR1-Deficient Human iPSC-Derived Macrophages

    Directory of Open Access Journals (Sweden)

    Anna-Lena Neehus

    2018-01-01

    Full Text Available Mendelian susceptibility to mycobacterial disease (MSMD is caused by inborn errors of interferon gamma (IFNγ immunity and is characterized by severe infections by weakly virulent mycobacteria. Although IFNγ is the macrophage-activating factor, macrophages from these patients have never been studied. We demonstrate the generation of heterozygous and compound heterozygous (iMSMD-cohet induced pluripotent stem cells (iPSCs from a single chimeric patient, who suffered from complete autosomal recessive IFNγR1 deficiency and received bone-marrow transplantation. Loss of IFNγR1 expression had no influence on the macrophage differentiation potential of patient-specific iPSCs. In contrast, lack of IFNγR1 in iMSMD-cohet macrophages abolished IFNγ-dependent phosphorylation of STAT1 and induction of IFNγ-downstream targets such as IRF-1, SOCS-3, and IDO. As a consequence, iMSMD-cohet macrophages show impaired upregulation of HLA-DR and reduced intracellular killing of Bacillus Calmette-Guérin. We provide a disease-modeling platform that might be suited to investigate novel treatment options for MSMD and to gain insights into IFNγ signaling in macrophages.

  11. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

    Directory of Open Access Journals (Sweden)

    Prajna Jena

    Full Text Available Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

  12. Impaired IFNγ-Signaling and Mycobacterial Clearance in IFNγR1-Deficient Human iPSC-Derived Macrophages.

    Science.gov (United States)

    Neehus, Anna-Lena; Lam, Jenny; Haake, Kathrin; Merkert, Sylvia; Schmidt, Nico; Mucci, Adele; Ackermann, Mania; Schubert, Madline; Happle, Christine; Kühnel, Mark Philipp; Blank, Patrick; Philipp, Friederike; Goethe, Ralph; Jonigk, Danny; Martin, Ulrich; Kalinke, Ulrich; Baumann, Ulrich; Schambach, Axel; Roesler, Joachim; Lachmann, Nico

    2018-01-09

    Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of interferon gamma (IFNγ) immunity and is characterized by severe infections by weakly virulent mycobacteria. Although IFNγ is the macrophage-activating factor, macrophages from these patients have never been studied. We demonstrate the generation of heterozygous and compound heterozygous (iMSMD-cohet) induced pluripotent stem cells (iPSCs) from a single chimeric patient, who suffered from complete autosomal recessive IFNγR1 deficiency and received bone-marrow transplantation. Loss of IFNγR1 expression had no influence on the macrophage differentiation potential of patient-specific iPSCs. In contrast, lack of IFNγR1 in iMSMD-cohet macrophages abolished IFNγ-dependent phosphorylation of STAT1 and induction of IFNγ-downstream targets such as IRF-1, SOCS-3, and IDO. As a consequence, iMSMD-cohet macrophages show impaired upregulation of HLA-DR and reduced intracellular killing of Bacillus Calmette-Guérin. We provide a disease-modeling platform that might be suited to investigate novel treatment options for MSMD and to gain insights into IFNγ signaling in macrophages. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Long-term persistence of human donor alveolar macrophages in lung transplant recipients

    DEFF Research Database (Denmark)

    Eguíluz-Gracia, Ibon; Schultz, Hans Henrik Lawaetz; Sikkeland, Liv I. B.

    2016-01-01

    and life span of human AMFs is scarce. METHODS: To follow the origin and longevity of AMFs in patients with lung transplantation for more than 100 weeks, we obtained transbronchial biopsies from 10 gender-mismatched patients with lung transplantation. These were subjected to combined in situ hybridisation...... transplantation we found that recipient monocytes seeded the alveoli early after transplantation, and showed subsequent phenotypical changes consistent with differentiation into proliferating mature AMFs. This resulted in a stable mixed chimerism between donor and recipient AMFs throughout the 2-year period...

  14. Defibrotide in combination with granulocyte colony-stimulating factor significantly enhances the mobilization of primitive and committed peripheral blood progenitor cells in mice.

    Science.gov (United States)

    Carlo-Stella, Carmelo; Di Nicola, Massimo; Magni, Michele; Longoni, Paolo; Milanesi, Marco; Stucchi, Claudio; Cleris, Loredana; Formelli, Franca; Gianni, Massimo A

    2002-11-01

    Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (P Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (P Defibrotide plus rhG-CSF resulted in a significant increase (P Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (P Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.

  15. Mitochondrial Fragmentation in Aspergillus fumigatus as Early Marker of Granulocyte Killing Activity

    Science.gov (United States)

    Ruf, Dominik; Brantl, Victor; Wagener, Johannes

    2018-01-01

    The host's defense against invasive mold infections relies on diverse antimicrobial activities of innate immune cells. However, studying these mechanisms in vitro is complicated by the filamentous nature of such pathogens that typically form long, branched, multinucleated and compartmentalized hyphae. Here we describe a novel method that allows for the visualization and quantification of the antifungal killing activity exerted by human granulocytes against hyphae of the opportunistic pathogen Aspergillus fumigatus. The approach relies on the distinct impact of fungal cell death on the morphology of mitochondria that were visualized with green fluorescent protein (GFP). We show that oxidative stress induces complete fragmentation of the tubular mitochondrial network which correlates with cell death of affected hyphae. Live cell microscopy revealed a similar and non-reversible disruption of the mitochondrial morphology followed by fading of fluorescence in Aspergillus hyphae that were killed by human granulocytes. Quantitative microscopic analysis of fixed samples was subsequently used to estimate the antifungal activity. By utilizing this assay, we demonstrate that lipopolysaccharides as well as human serum significantly increase the killing efficacy of the granulocytes. Our results demonstrate that evaluation of the mitochondrial morphology can be utilized to assess the fungicidal activity of granulocytes against A. fumigatus hyphae. PMID:29868488

  16. Melatonin Nanoparticles Adsorbed to Polyethylene Glycol Microspheres as Activators of Human Colostrum Macrophages

    Directory of Open Access Journals (Sweden)

    Cristiane de Castro Pernet Hara

    2013-01-01

    Full Text Available The effectiveness of hormones associated with polymeric matrices has amplified the possibility of obtaining new drugs to activate the immune system. Melatonin has been reported as an important immunomodulatory agent that can improve many cell activation processes. It is possible that the association of melatonin with polymers could influence its effects on cellular function. Thus, this study verified the adsorption of the hormone melatonin to polyethylene glycol (PEG microspheres and analyzed its ability to modulate the functional activity of human colostrum phagocytes. Fluorescence microscopy and flow cytometry analyses revealed that melatonin was able to adsorb to the PEG microspheres. This system increased the release of superoxide and intracellular calcium. There was an increase of phagocytic and microbicidal activity by colostrum phagocytes when in the presence of melatonin adsorbed to PEG microspheres. The modified delivery of melatonin adsorbed to PEG microspheres may be an additional mechanism for its microbicidal activity and represents an important potential treatment for gastrointestinal infections of newborns.

  17. Melatonin Nanoparticles Adsorbed to Polyethylene Glycol Microspheres as Activators of Human Colostrum Macrophages

    International Nuclear Information System (INIS)

    Hara, C.D.C.P.; Honorio-Frana, A.C.; Fagundes, D.L.G.; Guimares, P.C.L.; Franca, E.L.

    2013-01-01

    The effectiveness of hormones associated with polymeric matrices has amplified the possibility of obtaining new drugs to activate the immune system. Melatonin has been reported as an important immunomodulatory agent that can improve many cell activation processes. It is possible that the association of melatonin with polymers could influence its effects on cellular function. Thus, this study verified the adsorption of the hormone melatonin to polyethylene glycol (PEG) microspheres and analyzed its ability to modulate the functional activity of human colostrum phagocytes. Fluorescence microscopy and flow cytometry analyses revealed that melatonin was able to adsorb to the PEG microspheres. This system increased the release of superoxide and intracellular calcium. There was an increase of phagocytic and microbicidal activity by colostrum phagocytes when in the presence of melatonin adsorbed to PEG microspheres. The modified delivery of melatonin adsorbed to PEG microspheres may be an additional mechanism for its microbicidal activity and represents an important potential treatment for gastrointestinal infections of newborns.

  18. Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions.

    Science.gov (United States)

    Trayhurn, Paul; Denyer, Gareth

    2012-01-01

    Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity.

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

    Science.gov (United States)

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

    2012-01-01

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

  20. Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view.

    Science.gov (United States)

    Guehrs, Erik; Schneider, Michael; Günther, Christian M; Hessing, Piet; Heitz, Karen; Wittke, Doreen; López-Serrano Oliver, Ana; Jakubowski, Norbert; Plendl, Johanna; Eisebitt, Stefan; Haase, Andrea

    2017-03-21

    Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches. We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 μg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in