Science.gov

Sample records for alfa-9802 gm-csf study

  1. Phase I dose intensification study of 2-weekly epirubicin with GM-CSF in advanced cancer.

    PubMed

    Michael, M; Toner, G C; Olver, I N; Fenessy, A; Bishop, J F

    1997-06-01

    This study investigated dose intensification of epirubicin administered as a 2-weekly regimen with granulocyte-macrophage colony-stimulating factor (GM-CSF) support. The aim was to define the maximally tolerated dose of epirubicin and to assess the efficacy of GM-CSF to ameliorate its toxicity. Patients with anthracycline-responsive advanced malignancies were eligible. Six dose levels, commencing at 90 mg/m2, of epirubicin administered every 2 weeks for four courses were planned with GM-CSF 10 micrograms/kg/day administered for 10 days from the second day of each course. Six patients were to be entered at each dose level, and escalation to the next level was based upon toxicity criteria. Twelve patients were entered, six at dose level 1 (90 mg/m2) and six at dose level 2 (120 mg/m2). Prospectively defined haematological dose-limiting toxicities were noted in one patient at dose level 1 and in five patients at dose level 2. Further dose escalation was not attempted. Significant nonhaematological toxicities included febrile neutropenia in two and four patients at dose levels 1 and 2, respectively. This study has demonstrated that epirubicin can be safely administered at 2 week intervals with GM-CSF at a dose of 90 mg/m2, equivalent to the previously reported maximum tolerated dose intensity of 45 mg/m2/week. Neutropenia was dose-limiting despite the use of GM-CSF.

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

    PubMed

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

    2014-02-01

    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.

  3. Nontransformed, GM-CSF-dependent macrophage lines are a unique model to study tissue macrophage functions.

    PubMed

    Fejer, György; Wegner, Mareike Dorothee; Györy, Ildiko; Cohen, Idan; Engelhard, Peggy; Voronov, Elena; Manke, Thomas; Ruzsics, Zsolt; Dölken, Lars; Prazeres da Costa, Olivia; Branzk, Nora; Huber, Michael; Prasse, Antje; Schneider, Robert; Apte, Ron N; Galanos, Chris; Freudenberg, Marina A

    2013-06-11

    Macrophages are diverse cell types in the first line of antimicrobial defense. Only a limited number of primary mouse models exist to study their function. Bone marrow-derived, macrophage-CSF-induced cells with a limited life span are the most common source. We report here a simple method yielding self-renewing, nontransformed, GM-CSF/signal transducer and activator of transcription 5-dependent macrophages (Max Planck Institute cells) from mouse fetal liver, which reflect the innate immune characteristics of alveolar macrophages. Max Planck Institute cells are exquisitely sensitive to selected microbial agents, including bacterial LPS, lipopeptide, Mycobacterium tuberculosis, cord factor, and adenovirus and mount highly proinflammatory but no anti-inflammatory IL-10 responses. They show a unique pattern of innate responses not yet observed in other mononuclear phagocytes. This includes differential LPS sensing and an unprecedented regulation of IL-1α production upon LPS exposure, which likely plays a key role in lung inflammation in vivo. In conclusion, Max Planck Institute cells offer an useful tool to study macrophage biology and for biomedical science.

  4. Effect of locally applied GM-CSF on oral mucositis after stem cell transplantation: a prospective placebo-controlled double-blind study.

    PubMed

    van der Lelie, H; Thomas, B L; van Oers, R H; Ek-Post, M; Sjamsoedin, S A; van Dijk-Overtoom, M L; Timmer, J G; von dem Borne, A E

    2001-03-01

    Oral mucositis is a frequent side effect of myeloablative chemo- and radiotherapy preceding stem cell transplantation. It causes pain, poor food intake, and is a port of entry for infection. We studied whether GM-CSF applied topically in the oral cavity can prevent or ameliorate this mucositis. In 36 consecutive patients undergoing a stem cell transplantation, we performed a double-blind placebo-controlled study of 300 micrograms GM-CSF in a 2% methylcellulose gel daily versus a 2% methylcellulose gel alone. Both were locally applied in the oral cavity. The primary end-point was mucositis as measured by the WHO toxicity scale for mucositis, oral assessment scale, and a subjective pain scale, all scored daily. The secondary end-points were need to give parenteral nutrition and morphine, incidence of fever and infections, and duration of neutropenia and hospitalization. No differences were found in the median subjective pain scores, WHO scores, and oral assessment scores between the placebo and the GM-CSF groups. In both groups, nine patients required morphine for pain control. Ten patients in the placebo group and 11 in the GM-CSF group received parenteral nutrition. Documented infections, use of broad-spectrum antibiotics, and number of days with fever were similar in the placebo and the GM-CSF groups. The duration of neutropenia below 0.5 x 10(9)/l (median 14.5 days in the placebo group versus 17 days in the GM-CSF group) and the duration of hospitalization (28.5 versus 29 days) was also not significantly different. We found no beneficial effect of 300 micrograms GM-CSF dissolved in a 2% methylcellulose gel applied locally for chemo- and radiotherapy-induced mucositis in patients undergoing a stem cell transplantation.

  5. Targeting GM-CSF in rheumatoid arthritis.

    PubMed

    Avci, Ali Berkant; Feist, Eugen; Burmester, Gerd-Rüdiger

    2016-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is well-known as a haemopoietic growth factor. However, it is also essential in regulating functions of mature myeloid cells such as macrophages. Preclinical studies and observations of flares of arthritis in patients following GM-CSF treatment supported its important contribution to the pathogenesis of rheumatoid arthritis (RA). As the most advanced compound, mavrilimumab, a monoclonal antibody against GM-CSF receptor, has already completed phase II trials with a long term of follow-up period of 74 weeks. During this exposure period, an acceptable sustained safety and tolerability profile has been observed addressing the concerns of development of cytopenias or pulmonary alveolar proteinosis. Of note, a rapid and sustained efficacy and normalisation of acute phase reactants were consistently shown in studies both targeting GM-CSF and its receptor. Its tumour necrosis factor (TNF) independent mode of action with concurrent blockade of GM-CSF as well as IL-17 signalling reported from preclinical studies supports the assumption that it can be a useful biologic and an alternative agent in TNF inhibitor resistant patients with RA. Therefore, subsequent studies are warranted to investigate the safety and efficacy of GM-CSF blocking agents in different subgroups of RA. PMID:27586802

  6. GM-CSF and phorbol esters modulate GM-CSF receptor expression by independent mechanisms.

    PubMed

    Brizzi, M F; Arduino, C; Avanzi, G C; Bussolino, F; Pegoraro, L

    1991-07-01

    Human granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.1 nM) down-modulates its receptor in IL-3/GM-CSF dependent M-07e cells, in KG-1 cells and normal granulocytes, whereas phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) (2 nM) down-modulates the GM-CSF receptor in M-07e cells and granulocytes but not in KG-1 cells. As data analysis shows by nonlinear regression, the decreased binding ability depends on a reduction of the binding sites with no significant change of their dissociation constant. To gain insight into the mechanisms involved in the GM-CSF receptor regulation, we investigated the role of protein kinase C (PKC). GM-CSF, unlike TPA, was unable to activate PKC in all the cells studied. Moreover, unlike TPA, GM-CSF was still able to down-modulate its receptor in cells where PKC was inhibited by 1-(5-isoquinolonesulphonyl)-2-methylpiperazine (H7) and staurosporine or in cells where PKC was exhausted by prolonged incubation with 1 microM TPA. Finally, the receptor re-expression rate was accelerated by protein kinases inhibitors. These results, taken together, indicate the presence of a PKC-dependent and -independent down-modulation mechanism and a negative role of the endogeneous protein kinases in GM-CSF receptor re-expression.

  7. rhGM-CSF vs placebo following rhGM-CSF-mobilized PBPC transplantation: a phase III double-blind randomized trial.

    PubMed

    Legros, M; Fleury, J; Bay, J O; Choufi, B; Basile, M; Condat, P; Glenat, C; Communal, Y; Tavernier, F; Bons, J M; Chollet, P; Plagne, R; Chassagne, J

    1997-02-01

    In this placebo-controlled randomized trial we evaluated the hematological and clinical effects of r-Hu GM-CSF after high-dose chemotherapy (HDC) followed by GM-CSF-mobilized PBPC transplantation. Fifty patients with poor prognosis malignancies were randomized in a double-blind study to receive either GM-CSF or placebo after HDC followed by PBPC rescue. For all patients, PBPCs were recruited using a combination of VP-16 (300 mg/m2 on days 1 and 2), cytoxan (3 g/m2 on days 3 and 4) and GM-CSF (5 micrograms/kg from day 5). No differences were demonstrated between the two groups in median time to neutrophil or platelet recoveries. There was no significant difference between the GM-CSF group and the placebo group in the median duration of post-transplant hospitalization, in the number of days of antibiotic treatment, in the number of infections and in red blood cell or platelet transfusion requirements. There was a significant difference with an advantage for the placebo group in the mean duration of febrile days (P = 0.01). We conclude that the administration of GM-CSF in patients transplanted with GM-CSF-mobilized PBPC is not associated with a clinical benefit in term of tempo of engraftment, numbers of documented infections, transfusion requirements and mucositis grading.

  8. Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis.

    PubMed

    Piccoli, Luca; Campo, Ilaria; Fregni, Chiara Silacci; Rodriguez, Blanca Maria Fernandez; Minola, Andrea; Sallusto, Federica; Luisetti, Maurizio; Corti, Davide; Lanzavecchia, Antonio

    2015-06-16

    Pulmonary alveolar proteinosis (PAP) is a severe autoimmune disease caused by autoantibodies that neutralize GM-CSF resulting in impaired function of alveolar macrophages. In this study, we characterize 21 GM-CSF autoantibodies from PAP patients and find that somatic mutations critically determine their specificity for the self-antigen. Individual antibodies only partially neutralize GM-CSF activity using an in vitro bioassay, depending on the experimental conditions, while, when injected in mice together with human GM-CSF, they lead to the accumulation of a large pool of circulating GM-CSF that remains partially bioavailable. In contrast, a combination of three non-cross-competing antibodies completely neutralizes GM-CSF activity in vitro by sequestering the cytokine in high-molecular-weight complexes, and in vivo promotes the rapid degradation of GM-CSF-containing immune complexes in an Fc-dependent manner. Taken together, these findings provide a plausible explanation for the severe phenotype of PAP patients and for the safety of treatments based on single anti-GM-CSF monoclonal antibodies.

  9. Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis

    PubMed Central

    Piccoli, Luca; Campo, Ilaria; Fregni, Chiara Silacci; Rodriguez, Blanca Maria Fernandez; Minola, Andrea; Sallusto, Federica; Luisetti, Maurizio; Corti, Davide; Lanzavecchia, Antonio

    2015-01-01

    Pulmonary alveolar proteinosis (PAP) is a severe autoimmune disease caused by autoantibodies that neutralize GM-CSF resulting in impaired function of alveolar macrophages. In this study, we characterize 21 GM-CSF autoantibodies from PAP patients and find that somatic mutations critically determine their specificity for the self-antigen. Individual antibodies only partially neutralize GM-CSF activity using an in vitro bioassay, depending on the experimental conditions, while, when injected in mice together with human GM-CSF, they lead to the accumulation of a large pool of circulating GM-CSF that remains partially bioavailable. In contrast, a combination of three non-cross-competing antibodies completely neutralizes GM-CSF activity in vitro by sequestering the cytokine in high-molecular-weight complexes, and in vivo promotes the rapid degradation of GM-CSF-containing immune complexes in an Fc-dependent manner. Taken together, these findings provide a plausible explanation for the severe phenotype of PAP patients and for the safety of treatments based on single anti-GM-CSF monoclonal antibodies. PMID:26077231

  10. Development of Membrane-Bound GM-CSF and IL-18 as an Effective Tumor Vaccine

    PubMed Central

    Cheng, Ta-Chun; Chuang, Chih-Hung; Kao, Chien-Han; Hsieh, Yuan-Chin; Cheng, Kuang-Hung; Wang, Jaw-Yuan; Cheng, Chiu-Min; Chen, Chien-Shu; Cheng, Tian-Lu

    2015-01-01

    The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cells as a tumor vaccine. In this study, we expressed membrane-bound granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-18 (IL-18) as adjuvants in tumor cells to stimulate immune response. B7 transmembrane domain fused GM-CSF and IL-18 was successfully expressed in the cell membrane and stimulated mouse splenocyte proliferation. Co-expression of GM-CSF and IL-18 reduced tumorigenesis (P<0.05) and enhanced tumor protective efficacy (P<0.05) significantly in comparison with GM-CSF alone. These results indicated that the combination of GM-CSF andIL-18 will enhance the immunogenicity of a cell-based anti-tumor vaccine. This membrane-bound approach can be applied to other cytokines for the development of novel vaccine strategies. PMID:26186692

  11. Biologic Effects of Anti-Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Antibody Formation in Patients Treated with GM-CSF (Sargramostim) as Adjuvant Therapy of Melanoma

    PubMed Central

    Spitler, Lynn E.; Cao, Huynh; Piironen, Timo; Whiteside, Theresa L.; Weber, Robert W.; Cruickshank, Scott

    2014-01-01

    OBJECTIVES We investigated the development of binding and neutralizing antibodies to GM-CSF in patients receiving prolonged therapy with GM-CSF as adjuvant therapy of melanoma and the impact of these antibodies on biologic effects. METHODS Fifty-three patients with high-risk melanoma which had been surgically excised were treated with GM-CSF, 125 µg/m2 daily for 14 days every 28 days for 1 year following surgical resection of disease. Serum samples for antibodies to GM-CSF were measured before treatment and on Study Days 155 and 351. Blood draws for testing biologic effects were keyed to GM-CSF administration: Days 0 (before), 15 (after 14 days on GM-CSF), 29 (after 14 days off GM-CSF), 155, and 351 (after 14 days on GM-CSF in the 6th and 13th cycle of treatment). RESULTS Of 53 patients enrolled, 43 were evaluable for the development of anti-GM-CSF antibodies. Of these, 93% developed binding antibodies and 42% developed both binding and neutralizing antibodies. The increase in the white blood cell (WBC) count, percent eosinophils, or neopterin levels engendered by GM-CSF administration, was abrogated or markedly decreased in patients with neutralizing antibodies but not in patients who developed only binding antibodies. CONCLUSIONS Ninety-three percent of patients with melanoma treated with GM-CSF as adjuvant therapy develop antibodies to GM-CSF. In those with neutralizing antibodies, a diminution of the biologic effects of GM-CSF was observed. The development of neutralizing antibodies might also abrogate the potential clinical benefit of this treatment and should be considered in the design of future clinical trials. PMID:25286079

  12. MafB antagonizes phenotypic alteration induced by GM-CSF in microglia

    SciTech Connect

    Koshida, Ryusuke Oishi, Hisashi Hamada, Michito; Takahashi, Satoru

    2015-07-17

    Microglia are tissue-resident macrophages which are distributed throughout the central nervous system (CNS). Recent studies suggest that microglia are a unique myeloid population distinct from peripheral macrophages in terms of origin and gene expression signature. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a pleiotropic cytokine regulating myeloid development, has been shown to stimulate proliferation and alter phenotype of microglia in vitro. However, how its signaling is modulated in microglia is poorly characterized. MafB, a bZip transcriptional factor, is highly expressed in monocyte-macrophage lineage cells including microglia, although its role in microglia is largely unknown. We investigated the crosstalk between GM-CSF signaling and MafB by analyzing primary microglia. We found that Mafb-deficient microglia grew more rapidly than wild-type microglia in response to GM-CSF. Moreover, the expression of genes associated with microglial differentiation was more downregulated in Mafb-deficient microglia cultured with GM-CSF. Notably, such differences between the genotypes were not observed in the presence of M-CSF. In addition, we found that Mafb-deficient microglia cultured with GM-CSF barely extended their membrane protrusions, probably due to abnormal activation of RhoA, a key regulator of cytoskeletal remodeling. Altogether, our study reveals that MafB is a negative regulator of GM-CSF signaling in microglia. These findings could provide new insight into the modulation of cytokine signaling by transcription factors in microglia. - Highlights: • GM-CSF alters the phenotype of microglia in vitro more potently than M-CSF. • Transcription factor MafB antagonizes the effect of GM-CSF on microglia in vitro. • MafB deficiency leads to RhoA activation in microglia in response to GM-CSF. • We show for the first time the function of MafB in microglia.

  13. Stimulatory versus suppressive effects of GM-CSF on tumor progression in multiple cancer types

    PubMed Central

    Hong, In-Sun

    2016-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF, also called CSF-2) is best known for its critical role in immune modulation and hematopoiesis. A large body of experimental evidence indicates that GM-CSF, which is frequently upregulated in multiple types of human cancers, effectively marks cancer cells with a ‘danger flag' for the immune system. In this context, most studies have focused on its function as an immunomodulator, namely its ability to stimulate dendritic cell (DC) maturation and monocyte/macrophage activity. However, recent studies have suggested that GM-CSF also promotes immune-independent tumor progression by supporting tumor microenvironments and stimulating tumor growth and metastasis. Although some studies have suggested that GM-CSF has inhibitory effects on tumor growth and metastasis, an even greater number of studies show that GM-CSF exerts stimulatory effects on tumor progression. In this review, we summarize a number of findings to provide the currently available information regarding the anticancer immune response of GM-CSG. We then discuss the potential roles of GM-CSF in the progression of multiple types of cancer to provide insights into some of the complexities of its clinical applications. PMID:27364892

  14. MafB antagonizes phenotypic alteration induced by GM-CSF in microglia.

    PubMed

    Koshida, Ryusuke; Oishi, Hisashi; Hamada, Michito; Takahashi, Satoru

    Microglia are tissue-resident macrophages which are distributed throughout the central nervous system (CNS). Recent studies suggest that microglia are a unique myeloid population distinct from peripheral macrophages in terms of origin and gene expression signature. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a pleiotropic cytokine regulating myeloid development, has been shown to stimulate proliferation and alter phenotype of microglia in vitro. However, how its signaling is modulated in microglia is poorly characterized. MafB, a bZip transcriptional factor, is highly expressed in monocyte-macrophage lineage cells including microglia, although its role in microglia is largely unknown. We investigated the crosstalk between GM-CSF signaling and MafB by analyzing primary microglia. We found that Mafb-deficient microglia grew more rapidly than wild-type microglia in response to GM-CSF. Moreover, the expression of genes associated with microglial differentiation was more downregulated in Mafb-deficient microglia cultured with GM-CSF. Notably, such differences between the genotypes were not observed in the presence of M-CSF. In addition, we found that Mafb-deficient microglia cultured with GM-CSF barely extended their membrane protrusions, probably due to abnormal activation of RhoA, a key regulator of cytoskeletal remodeling. Altogether, our study reveals that MafB is a negative regulator of GM-CSF signaling in microglia. These findings could provide new insight into the modulation of cytokine signaling by transcription factors in microglia.

  15. Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs.

    PubMed

    Zhou, Ming; Wang, Lei; Zhou, Songqin; Wang, Zhao; Ruan, Juncheng; Tang, Lijun; Jia, Ziming; Cui, Min; Zhao, Ling; Fu, Zhen F

    2015-11-17

    Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs.

  16. GM-CSF primes cardiac inflammation in a mouse model of Kawasaki disease.

    PubMed

    Stock, Angus T; Hansen, Jacinta A; Sleeman, Matthew A; McKenzie, Brent S; Wicks, Ian P

    2016-09-19

    Kawasaki disease (KD) is the leading cause of pediatric heart disease in developed countries. KD patients develop cardiac inflammation, characterized by an early infiltrate of neutrophils and monocytes that precipitates coronary arteritis. Although the early inflammatory processes are linked to cardiac pathology, the factors that regulate cardiac inflammation and immune cell recruitment to the heart remain obscure. In this study, using a mouse model of KD (induced by a cell wall Candida albicans water-soluble fraction [CAWS]), we identify an essential role for granulocyte/macrophage colony-stimulating factor (GM-CSF) in orchestrating these events. GM-CSF is rapidly produced by cardiac fibroblasts after CAWS challenge, precipitating cardiac inflammation. Mechanistically, GM-CSF acts upon the local macrophage compartment, driving the expression of inflammatory cytokines and chemokines, whereas therapeutically, GM-CSF blockade markedly reduces cardiac disease. Our findings describe a novel role for GM-CSF as an essential initiating cytokine in cardiac inflammation and implicate GM-CSF as a potential target for therapeutic intervention in KD.

  17. Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs.

    PubMed

    Zhou, Ming; Wang, Lei; Zhou, Songqin; Wang, Zhao; Ruan, Juncheng; Tang, Lijun; Jia, Ziming; Cui, Min; Zhao, Ling; Fu, Zhen F

    2015-11-17

    Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs. PMID:26436700

  18. Flow cytometry measurement of GM-CSF receptors in acute leukemic blasts, and normal hemopoietic cells.

    PubMed

    Lanza, F; Castagnari, B; Rigolin, G; Moretti, S; Latorraca, A; Ferrari, L; Bardi, A; Castoldi, G

    1997-10-01

    patients was above the highest values seen in normal samples (>69.2 x 10[3] MESF/cell), allowing the possibility of using this marker for the monitoring of the minimal residual disease (MRD) in a subset of AML. Cell culture studies aimed at evaluating GM-CSF receptor modulation following AML blast exposure to rhGM-CSF showed two distinct patterns of response; in the first group (6/10 cases) rhGM-CSF down-modulated GM-CSF receptors, whereas in the second group (4/10 cases), rhGM-CSF treatment was associated with either an increase or no change in the number of GM-CSF/R. In conclusion, cellular GM-CSF/R expression was variable and ranged from undetectable (ALL and a minority of AML) to very high intensities in M5 AML, and were also documented in some M0 AML, thus suggesting the concept that GM-CSF/R detection may be of help in lineage assignment of undifferentiated forms. Since the number of GM-CSF/R on AML blasts may be modulated after GM-CSF treatment, it can be postulated that the clinical use of GM-CSF in this disease may be optimized by a dynamic analysis of the number and the affinity status of GM-CSF-R in blasts and normal hemopoietic cells. PMID:9324292

  19. The relative balance of GM-CSF and TGF-β1 regulates lung epithelial barrier function.

    PubMed

    Overgaard, Christian E; Schlingmann, Barbara; Dorsainvil White, StevenClaude; Ward, Christina; Fan, Xian; Swarnakar, Snehasikta; Brown, Lou Ann S; Guidot, David M; Koval, Michael

    2015-06-15

    Lung barrier dysfunction is a cardinal feature of the acute respiratory distress syndrome (ARDS). Alcohol abuse, which increases the risk of ARDS two- to fourfold, induces transforming growth factor (TGF)-β1, which increases epithelial permeability and impairs granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent barrier integrity in experimental models. We hypothesized that the relative balance of GM-CSF and TGF-β1 signaling regulates lung epithelial barrier function. GM-CSF and TGF-β1 were tested separately and simultaneously for their effects on lung epithelial cell barrier function in vitro. TGF-β1 alone caused an ∼ 25% decrease in transepithelial resistance (TER), increased paracellular flux, and was associated with projections perpendicular to tight junctions ("spikes") containing claudin-18 that colocalized with F-actin. In contrast, GM-CSF treatment induced an ∼ 20% increase in TER, decreased paracellular flux, and showed decreased colocalization of spike-associated claudin-18 with F-actin. When simultaneously administered to lung epithelial cells, GM-CSF antagonized the effects of TGF-β1 on epithelial barrier function in cultured cells. Given this, GM-CSF and TGF-β1 levels were measured in bronchoalveolar lavage (BAL) fluid from patients with ventilator-associated pneumonia and correlated with markers for pulmonary edema and patient outcome. In patient BAL fluid, protein markers of lung barrier dysfunction, serum α2-macroglobulin, and IgM levels were increased at lower ratios of GM-CSF/TGF-β1. Critically, patients who survived had significantly higher GM-CSF/TGF-β1 ratios than nonsurviving patients. This study provides experimental and clinical evidence that the relative balance between GM-CSF and TGF-β1 signaling is a key regulator of lung epithelial barrier function. The GM-CSF/TGF-β1 ratio in BAL fluid may provide a concentration-independent biomarker that can predict patient outcomes in ARDS.

  20. A Review of GM-CSF Therapy in Sepsis.

    PubMed

    Mathias, Brittany; Szpila, Benjamin E; Moore, Frederick A; Efron, Philip A; Moldawer, Lyle L

    2015-12-01

    Determine what clinical role, if any, GM-CSF may have in the clinical treatment of sepsis in the adult patient. Advancements in the management of sepsis have led to significant decreases in early mortality; however, sepsis remains a significant source of long-term mortality and disability which places strain on healthcare resources with a substantial growing economic impact. Historically, early multiple organ failure (MOF) and death in patients with severe sepsis was thought to result from an exaggerated proinflammatory response called the systemic inflammatory response syndrome (SIRS). Numerous prospective randomized controlled trials (PRCTs) tested therapies aimed at decreasing the organ injury associated with an exaggerated inflammatory response. With few exceptions, the results from these PRCTs have been disappointing, and currently no specific therapeutic agent is approved to counteract the early SIRS response in patients with severe sepsis. It has long been recognized that there is a delayed immunosuppressive state that contributes to long-term morbidity. However, recent findings now support a concurrent proinflammatory and anti-inflammatory response present throughout sepsis. Multiple immunomodulating agents have been studied to combat the immunosuppressive phase of sepsis with the goal of decreasing secondary infection, reducing organ dysfunction, decreasing ICU stays, and improving survival. Granulocyte-macrophage colony stimulating factor (GM-CSF), a myelopoietic growth factor currently used in patients with neutropenia secondary to chemotherapy-induced myelosuppression, has been studied as a potential immune-activating agent. The applicability of GM-CSF as a standard therapy for generalized sepsis is still largely understudied; however, small-scale studies available have demonstrated some improved recovery from infection, decreased hospital length of stay, decreased days requiring mechanical ventilation, and decreased medical costs. PMID:26683913

  1. A Review of GM-CSF Therapy in Sepsis

    PubMed Central

    Mathias, Brittany; Szpila, Benjamin E.; Moore, Frederick A.; Efron, Philip A.; Moldawer, Lyle L.

    2015-01-01

    Abstract Determine what clinical role, if any, GM-CSF may have in the clinical treatment of sepsis in the adult patient. Advancements in the management of sepsis have led to significant decreases in early mortality; however, sepsis remains a significant source of long-term mortality and disability which places strain on healthcare resources with a substantial growing economic impact. Historically, early multiple organ failure (MOF) and death in patients with severe sepsis was thought to result from an exaggerated proinflammatory response called the systemic inflammatory response syndrome (SIRS). Numerous prospective randomized controlled trials (PRCTs) tested therapies aimed at decreasing the organ injury associated with an exaggerated inflammatory response. With few exceptions, the results from these PRCTs have been disappointing, and currently no specific therapeutic agent is approved to counteract the early SIRS response in patients with severe sepsis. It has long been recognized that there is a delayed immunosuppressive state that contributes to long-term morbidity. However, recent findings now support a concurrent proinflammatory and anti-inflammatory response present throughout sepsis. Multiple immunomodulating agents have been studied to combat the immunosuppressive phase of sepsis with the goal of decreasing secondary infection, reducing organ dysfunction, decreasing ICU stays, and improving survival. Granulocyte-macrophage colony stimulating factor (GM-CSF), a myelopoietic growth factor currently used in patients with neutropenia secondary to chemotherapy-induced myelosuppression, has been studied as a potential immune-activating agent. The applicability of GM-CSF as a standard therapy for generalized sepsis is still largely understudied; however, small-scale studies available have demonstrated some improved recovery from infection, decreased hospital length of stay, decreased days requiring mechanical ventilation, and decreased medical costs. PMID

  2. Pivotal Roles of GM-CSF in Autoimmunity and Inflammation

    PubMed Central

    Shiomi, Aoi; Usui, Takashi

    2015-01-01

    Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor, which stimulates the proliferation of granulocytes and macrophages from bone marrow precursor cells. In autoimmune and inflammatory diseases, Th17 cells have been considered as strong inducers of tissue inflammation. However, recent evidence indicates that GM-CSF has prominent proinflammatory functions and that this growth factor (not IL-17) is critical for the pathogenicity of CD4+ T cells. Therefore, the mechanism of GM-CSF-producing CD4+ T cell differentiation and the role of GM-CSF in the development of autoimmune and inflammatory diseases are gaining increasing attention. This review summarizes the latest knowledge of GM-CSF and its relationship with autoimmune and inflammatory diseases. The potential therapies targeting GM-CSF as well as their possible side effects have also been addressed in this review. PMID:25838639

  3. Characterization of pathogenic human monoclonal autoantibodies against GM-CSF

    PubMed Central

    Wang, Yanni; Thomson, Christy A.; Allan, Lenka L.; Jackson, Linda M.; Olson, Melanie; Hercus, Timothy R.; Nero, Tracy L.; Turner, Amanda; Parker, Michael W.; Lopez, Angel L.; Waddell, Thomas K.; Anderson, Gary P.; Hamilton, John A.; Schrader, John W.

    2013-01-01

    The origin of pathogenic autoantibodies remains unknown. Idiopathic pulmonary alveolar proteinosis is caused by autoantibodies against granulocyte–macrophage colony-stimulating factor (GM-CSF). We generated 19 monoclonal autoantibodies against GM-CSF from six patients with idiopathic pulmonary alveolar proteinosis. The autoantibodies used multiple V genes, excluding preferred V-gene use as an etiology, and targeted at least four nonoverlapping epitopes on GM-CSF, suggesting that GM-CSF is driving the autoantibodies and not a B-cell epitope on a pathogen cross-reacting with GM-CSF. The number of somatic mutations in the autoantibodies suggests that the memory B cells have been helped by T cells and re-entered germinal centers. All autoantibodies neutralized GM-CSF bioactivity, with general correlations to affinity and off-rate. The binding of certain autoantibodies was changed by point mutations in GM-CSF that reduced binding to the GM-CSF receptor. Those monoclonal autoantibodies that potently neutralize GM-CSF may be useful in treating inflammatory disease, such as rheumatoid arthritis and multiple sclerosis, cancer, and pain. PMID:23620516

  4. rhGM-CSF ameliorates neutropenia in patients with malignant glioma treated with BCNU.

    PubMed Central

    Rampling, R.; Steward, W.; Paul, J.; Macham, M. A.; Harvey, E.; Eckley, D.

    1994-01-01

    Nitrosoureas are the drugs most effective in the treatment of patients with intracerebral malignant glioma. Their limiting toxicity is delayed myelosuppression. A prospective, randomised crossover study of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was performed in patients receiving BCNU for relapsed glioblastoma, to investigate whether the resulting haematological toxicity profile could be modified by rhGM-CSF. Adequate data for analysis were obtained in 13 patients. Following BCNU, the nadir neutrophil count was higher in 12 out of 13 patients during the rhGM-CSF-protected cycles compared with the unprotected cycles. The median nadir was also significantly higher (1.79, CI 0.76-3.52, P < 0.005). Five episodes of neutropenia (< 2 x 10(9) l-1) occurred during the unprotected cycles compared with none in the rhGM-CSF-protected cycles (P = 0.076). There was no evidence of any effect on platelets. This result shows that the haematological toxicity profile following therapeutic doses of BCNU can be modified. It suggests that rhGM-CSF and other growth factors should be investigated for clinical efficacy in chemotherapy using nitrosoureas. PMID:8123485

  5. Enhancement of natural killer cell-mediated cytotoxicity by coexpression of GM-CSF/B70 in hepatoma.

    PubMed

    Kim, K Y; Kang, M A; Nam, M J

    2001-05-10

    On investigating the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and costimulatory molecule, B70, in antitumor immunity, we have found important effects of GM-CSF/B70 coexpression in the interaction with natural killer (NK) cells. We used the pLSN vector system to contain the neomycin-resistant gene and LTR promoter. The pLSNGM-CSF, pLSNB70 and pLSNB70/GM-CSF, pLSN vectors each containing GM-CSF, B70, and B70/GM-CSF cDNA, respectively, were constructed. They were transfected into human hepatocellular carcinoma cell (SK-HEP1), and stable cells (SK-pLSN, SK-GM, SK-B70 and SK-BG) were selected after neomycin treatment. According to enzyme-linked immunosorbent analysis and FACS, we showed that expression of GM-CSF was increased up to 23-fold in SK-GM and SK-BG cells, and also expression of B70 was induced at least 76-97% in SK-B70 and SK-BG cells. Expression of B70 was remarkably increased by autocrine effect of GM-CSF in SK-BG cells. Primary cytolytic ability of GM-CSF and B70 significantly increased almost 4-fold (effector/target ratio, 100:1) in SK-BG cells. In in vivo studies, SK-BG cells showed much less subcutaneous tumor formation in nude mice accompanying increased NK cell proliferation and cytotoxicity. Therefore, these results suggest that combining expression of GM-CSF and B70 may enhance NK-mediated cytotoxicity, and then induce the antitumor immunity in hepatoma transplanted into nude mice.

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

    PubMed

    Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

    2015-01-01

    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.

  7. Molecular modeling of the GM-CSF and IL-3 receptor complexes.

    PubMed Central

    Lyne, P. D.; Bamborough, P.; Duncan, D.; Richards, W. G.

    1995-01-01

    A model for the structure of the cytokine interleukin-3 (IL-3) is presented based on the structural homology of the hematopoietic cytokines and utilizing the crystal structures of interleukin-5 and granulocyte macrophage colony stimulating factor (GM-CSF). In addition, models of the receptor complexes of GM-CSF and IL-3 are presented based on the structural homology of the hematopoietic receptors to growth hormone. Several key interactions between the ligands and their receptors are discovered, some in agreement with previous mutagenesis studies and others that have not yet been the subject of mutagenesis studies. The models provide insights into the binding of GM-CSF and IL-3 to their receptors. PMID:8535258

  8. Increased in vitro Cell Proliferation by Chitosan/pGM-CSF Complexes

    PubMed Central

    Şalva, E.; Turan, S. O.; Akbuğa, J.

    2011-01-01

    Granulocyte macrophage colony stimulating factor, a potent hematopoietic cytokine, has been shown to stimulate production of white blood cells following chemotherapy. Therefore, the granulocyte macrophage colony stimulating factor gene is a potential candidate for the treatment of different pathological conditions. The purpose of this study is to investigate the suitability of chitosan as carrier for pORF-hGMCSF plasmid encoding granulocyte macrophage colony stimulating factor gene and also to study the effect of complexes on protein production and cell proliferation. Chitosan/pGM-CSF complexes were prepared using different (+/-) ratios (from 0.01/1 to 5/1). Complex formation was checked with agarose gel electrophoresis. The size and zeta potential values were measured. Enzyme and serum stability of complexes were studied. In vitro transfection properties of complexes were studied in HeLa cells. According to agarose gel electrophoresis, full complexation was obtained at 0.1/1 and higher chitosan/pGM-CSF ratios. Complexes having about 132 nm size and +13.7 mV zeta potential value were obtained. Chitosan complexes protected plasmid against enzymatic and serum effects. The gene expression-dependent cell proliferation after transfection of chitosan/pGM-CSF complexes at 72 h was markedly increased in comparision with the level of control group. These results indicate that the effect of chitosan/pGM-CSF complexes on cell proliferation was changed with N/P ratio and time-dependently. For GM-CSF therapy, chitosan/pGM-CSF complexes may be used as alternative to conventional protein treatments. Chitosan may be a good carrier for pORF-hGMCSF. Further, in vivo study is ongoing. PMID:22303054

  9. Interleukin-33 stimulates GM-CSF and M-CSF production by human endothelial cells.

    PubMed

    Montanari, Eliana; Stojkovic, Stefan; Kaun, Christoph; Lemberger, Christof E; de Martin, Rainer; Rauscher, Sabine; Gröger, Marion; Maurer, Gerald; Neumayer, Christoph; Huk, Ihor; Huber, Kurt; Demyanets, Svitlana; Wojta, Johann

    2016-08-01

    Interleukin (IL)-33, a member of the IL-1 family of cytokines, is involved in various inflammatory conditions targeting amongst other cells the endothelium. Besides regulating the maturation and functions of myeloid cells, granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) have been shown to play a role in such pathologies too. It was the aim of our study to investigate a possible influence of IL-33 on GM-CSF and M-CSF production by human endothelial cells. IL-33, but not IL-18 or IL-37, stimulated GM-CSF and M-CSF mRNA expression and protein production by human umbilical vein endothelial cells (HUVECs) and human coronary artery ECs (HCAECs) through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in an IL-1-independent way. This effect was inhibited by the soluble form of ST2 (sST2), which is known to act as a decoy receptor for IL-33. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor fluvastatin could also be shown to moderately reduce the IL-33-mediated effect on M-CSF, but not on GM-CSF expression. In addition, IL-33, IL-1β, GM-CSF and M-CSF were detected in endothelial cells of human carotid atherosclerotic plaques using immunofluorescence. Upregulation of GM-CSF and M-CSF production by human endothelial cells, an effect that appears to be mediated by NF-κB and to be independent of IL-1, may be an additional mechanism through which IL-33 contributes to inflammatory activation of the vessel wall. PMID:27173404

  10. Induction of high-affinity GM-CSF receptors during all-trans retinoic acid treatment of acute promyelocytic leukemia.

    PubMed

    de Gentile, A; Toubert, M E; Dubois, C; Krawice, I; Schlageter, M H; Balitrand, N; Castaigne, S; Degos, L; Rain, J D; Najean, Y

    1994-10-01

    Differentiation of normal myeloid cells is accompanied by the increase of high-affinity GM-CSF receptors necessary for progenitor proliferation/differentiation and mature neutrophil function. All-trans retinoic acid (ATRA) induces terminal differentiation of acute promyelocytic leukemia cells (AML3 subtype). We report in this study that AML3 cells, like other AML subtypes, harbor high-affinity GM-CSF R (n = 138.3 +/- 69.3 sites/cell, Kd = 76.9 +/- 68.8 pM). In all cases, incubation with ATRA induces either an increase in the number of affinity of GM-CSF R (n = 212.7 +/- 116.2 sites/cell, Kd = 43.2 +/- 22.5 pM). The data presented show that modulation of GM-CSF receptors cells is correlated to the degree of ATRA-induced granulocytic differentiation but not to increased cell growth.

  11. Role of defective ERK phosphorylation in the impaired GM-CSF-induced oxidative response of neutrophils in elderly humans.

    PubMed

    Tortorella, Cosimo; Stella, Isabella; Piazzolla, Giuseppina; Simone, Olivia; Cappiello, Valentina; Antonaci, Salvatore

    2004-08-01

    GM-CSF-induced oxidative responses are defective in neutrophils of elderly humans. In the present study we evaluated whether this phenomenon might be related to alterations in cytokine-dependent MAPK signalling. Neutrophils obtained from elderly humans and stimulated with GM-CSF showed a significant reduction in phosphorylated ERK1/2 levels and an even higher decrease in ERK1/2 activation with respect to baseline. No changes in GM-CSF-induced p38 MAPK phosphorylation were observed. Cell pretreatment with the MEK inhibitor PD98059 determined a marked suppression of GM-CSF-induced O2- release. Interestingly, under the above experimental condition, there was no longer any difference in O2- production observed between elderly and young subjects. Furthermore, despite the fact that the p38 MAPK pathway was activated less strongly by GM-CSF, the p38 MAPK inhibitor SB203580 reduced GM-CSF-induced O2- production in the neutrophils of the elderly to levels similar to those obtained with PD98059. TNF-alpha-triggered O2- production was not altered by ageing and in fact, a similar ERK1/2 or p38 MAPK activation was found in TNF-alpha-stimulated neutrophils from elderly and young individuals. In accordance with the different potency of TNF-alpha in activating ERK1/2 and p38 MAPK, the TNF-alpha-induced oxidative responses were more sensitive to the inhibitory effects of SB203580 than to those of PD98059 in young as well as elderly subjects. These results suggest that, along the GM-CSF-dependent ERK signalling pathway, a step proximal to MEK1/2 but distal to the connection with the p38 MAPK module likely becomes defective as a feature of age. The consequent decline in ERK1/2 activation could potentially account for the GM-CSF-dependent impairment of the neutrophil respiratory burst that occurs with ageing.

  12. A phase I/II study of dose and administration of non-glycosylated bacterially synthesized G-M CSF in chemotherapy-induced neutropenia in patients with non-Hodgkin's lymphomas.

    PubMed

    Hovgaard, D; Nissen, N I

    1992-06-01

    Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) derived from E. coli was administered to 24 previously untreated patients with non-Hodgkin's lymphoma following the first cycle of CHOP chemotherapy. Four dose levels were examined, 1.5, 3.0, 5.5 and 11 micrograms/kg and patients were randomized to receive the drug either once or twice daily subcutaneously (s.c.). During rhGM-CSF treatment, the leucocyte counts increased up to 3-4 fold in 20/24 patients, reaching a peak 24-48 (mean 35) hours after initiation of rhGM-CSF. The leukopenic period in cycle one of the CHOP chemotherapy with rhGM-CSF, was shorter than after the course of chemotherapy without rhGM-CSF and also shorter when compared to cycle one of CHOP in the 127 historical controls (p < 0.05 and p < 0.001 respectively). Similar results were observed for neutrophil counts. No effect was seen on platelet counts at nadir but a significant, although moderate increase occurred in the recovery period on days 15 and 22 when compared to control cycles and historical controls. When dose levels were compared, there was only a trend to higher WBC counts at the higher dose groups (5.5 and 11 micrograms/kg) when compared to the two lower dose groups (1.5 and 3.0 micrograms/kg). In the overall evaluation there was no statistical significant difference in results between patients treated s.c. once daily versus twice daily. However when only the two highest dose levels (5.5 + 11 micrograms/kg) were compared, s.c. administration of rhGM-CSF twice daily led to higher leucocyte counts than once daily in the recovery period on day 15 (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

  13. GM-CSF contributes to aortic aneurysms resulting from SMAD3 deficiency

    PubMed Central

    Ye, Ping; Chen, Wenhao; Wu, Jie; Huang, Xiaofan; Li, Jun; Wang, Sihua; Liu, Zheng; Wang, Guohua; Yang, Xiao; Zhang, Peng; Lv, Qiulun; Xia, Jiahong

    2013-01-01

    Heterozygous loss-of-function SMAD3 (Mothers against decapentaplegic homolog 3) mutations lead to aneurysm-osteoarthritis syndrome (AOS). In the present study, we found that mice lacking Smad3 had a vascular phenotype similar to AOS, marked by the progressive development of aneurysms. These aneurysms were associated with various pathological changes in transmural inflammatory cell infiltration. Bone marrow transplants from Smad3–/– mice induced aortitis and aortic root dilation in irradiated WT recipient mice. Transplantation of CD4+ T cells from Smad3–/– mice also induced aortitis in Smad3+/+ recipient mice, while depletion of CD4+ T cells in Smad3–/– mice reduced the infiltration of inflammatory cells in the aortic root. Furthermore, IFN-γ deficiency increased, while IL-17 deficiency decreased, disease severity in Smad3+/– mice. Cytokine secretion was measured using a cytokine quantibody array, and Smad3–/– CD4+ T cells secreted more GM-CSF than Smad3+/+ CD4+ T cells. GM-CSF induced CD11b+Gr-1+Ly-6Chi inflammatory monocyte accumulation in the aortic root, but administration of anti–GM-CSF mAb to Smad3–/– mice resulted in significantly less inflammation and dilation in the aortic root. We also identified a missense mutation (c.985A>G) in a family of thoracic aortic aneurysms. Intense inflammatory infiltration and GM-CSF expression was observed in aortas specimens of these patients, suggesting that GM-CSF is potentially involved in the development of AOS. PMID:23585475

  14. Roles of myeloperoxidase and GAPDH in interferon-gamma production of GM-CSF-dependent macrophages.

    PubMed

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

    2016-02-01

    Interferon (IFN)-gamma is highly expressed in atherosclerotic lesions and may have an important role in atherogenesis. Myeloperoxidase (MPO), the most abundant protein in neutrophils, is a marker of plaque vulnerability and a possible bridge between inflammation and cardiovascular disease. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has also been implicated in the pathogenesis of atherosclerosis. The present study investigated the role of neutrophil activation in atherosclerosis. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IFN-gamma protein by GM-CSF-dependent-macrophages was investigated by enzyme-linked immunosorbent assay after stimulation with MPO. GM-CSF enhanced macrophage expression of the mannose receptor (CD206), which is involved in MPO uptake. MPO increased IFN-gamma production by GM-CSF-dependent macrophages in a concentration-dependent manner. Pretreatment of macrophages with small interfering RNA (siRNA) for CD206 or extracellular signal-regulated kinase (ERK)-2 attenuated IFN-gamma production, while siRNA for ERK-1 did not. GAPDH is known to bind to adenylate/uridylate (AU)-rich elements of RNA and may influence IFN-gamma protein expression by binding to the AU-rich element of IFN-gamma mRNA. Interestingly, pretreatment with siRNA for GAPDH significantly reduced IFN-gamma production by macrophages, while it did not affect TF protein expression. In conclusion, MPO upregulates IFN-gamma production by GM-CSF-dependent-macrophages via the CD206/ERK-2 signaling pathway, while silencing GAPDH reduces IFN-gamma production. PMID:27441256

  15. Tissue localization of GM-CSF receptor in bovine ovarian follicles and its role on glucose uptake by mural granulosa cells.

    PubMed

    Peralta, O A; Bucher, D; Angulo, C; Castro, M A; Ratto, M H; Concha, Il

    2016-07-01

    The granulocyte-macrophage colony stimulating factor (GM-CSF) is a multifunctional cytokine implicated in proliferation, differentiation, and activation of several cell types including those involved in hematopoiesis and reproduction. In the present study, the expression of the α- and β-subunit genes of GM-CSF receptor during follicular development in cattle was assessed. The spatial association of α- and β-subunits of GM-CSF with follicle stimulating hormone receptor (FSHR) and 3β-hydroxysteroid dehydrogenase (3β-HSD), and the temporal associations with gene expression of hexose transporters (GLUTs) in granulosa cells of cattle were also evaluated. The effect of GM-CSF on the functionality of hexose transporters was also determined in an in vitro primary culture of granulosa cells. The spatial association of subunits of the GM-CSF receptor with 3β-HSD and FSHR suggests a potential steroidogenic regulation of GM-CSF in granulosa cells. Immunodetection of GLUTs and uptake kinetic assays confirmed expression and functionality of these genes for hexose transporters in granulosa cells of cattle. Treatment of granulosa cells with GM-CSF, FSH or insulin- like growth factor-I (IGF-I) alone increased 2-deoxyglucose (DOG) or 3-0-methylglucose (OMG) uptake; however, when cells were treated with various combination of these factors there were no additive effect. Unexpectedly, the combination of GM-CSF and FSH decreased DOG uptake compared to FSH treatment alone. Thus, the expression pattern of GM-CSF receptor subunit genes during follicle development in cattle and promotion of DOG and OMG uptake in granulosa cells indicate a role for GM-CSF, FSH and/or IGF-I alone in regulating granulosa cell metabolic activity, specifically by promoting glucose uptake. PMID:27236376

  16. Phase I clinical study with multiple peptide vaccines in combination with tetanus toxoid and GM-CSF in advanced-stage HLA-A*0201-positive melanoma patients.

    PubMed

    Bins, Adriaan; Mallo, Henk; Sein, Johan; van den Bogaard, Colette; Nooijen, Willem; Vyth-Dreese, Florry; Nuijen, Bastiaan; de Gast, Gijsbert C; Haanen, John B A G

    2007-01-01

    Successful induction of functional tumor-specific T cells by peptide vaccination in animal models has resulted in many clinical trials to test this approach in advanced-stage melanoma patients. In this phase I clinical trial, 11 end-stage melanoma patients were vaccinated intradermally with 3 peptides: MART-1(26-35) E27L (ELAGIGILTV), tyrosinase(368-376) N375Q (YMDGTMSQV), and gp100(209-217) T210M (IMQVPFSV), admixed with tetanus toxoid and granulocyte-monocyte colony stimulating factor. The peptide vaccine was well tolerated at all tested doses, and led to grade 1-2 toxicity only. Although all patients did show a rise in antitetanus IgG titers, in only 3 patients peptide-specific CD8 T-cells were induced. In 2 cases, the response was directed against MART-1(26-35) and consisted of 0.2% and 3.3% of the CD8 population; however, in both instances these cells did not produce interferon-gamma on stimulation with the unmodified peptide. The third patient mounted a small (0.1%) response against gp100. In a fourth patient, a nonfunctional tyrosinase-specific response (0.6%) was found that was present before vaccination, but was not affected in size nor in function by the vaccine. None of the 11 patients responded clinically according to response evaluation criteria in solid tumors criteria. Although this study is a small scale phase I clinical trial, the efficacy that was observed was disappointingly low. In accordance with previously published peptide vaccination studies, these results add to the increasing evidence that peptide vaccination in itself is not potent enough as an effective melanoma immunotherapy in advanced-stage patients.

  17. The Ch14.18-GM-CSF fusion protein is effective at mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro.

    PubMed

    Batova, A; Kamps, A; Gillies, S D; Reisfeld, R A; Yu, A L

    1999-12-01

    Granulocyte/macrophage-colony stimulating factor (GM-CSF) is very effective at enhancing antibody-dependent cellular cytotoxicity (ADCC) mediated by granulocytes and monocytes. Recently, a fusion protein consisting of GM-CSF and chimeric human/mouse anti-ganglioside G(D2) antibody Ch14.18 (Ch14.18-GM-CSF) has been generated to improve the effectiveness of immunotherapy by directing GM-CSF to the tumor microenvironment and prolonging its relatively short half-life. In this study, we examined the ability of this fusion protein to enhance the in vitro killing of G(D2)-expressing human neuroblastoma cells by granulocytes and mononuclear cells, as well as by complement. The Ch14.18-GM-CSF fusion protein was equally effective as the combination of equivalent amounts of free Ch14.18 and GM-CSF in mediating the killing of NMB7 neuroblastoma cells by granulocytes from seven of eight neuroblastoma patients. The fusion protein was also equally effective as the combination of Ch14.18 and GM-CSF in mediating ADCC by neuroblastoma patients' mononuclear cells. In addition, the fusion protein was as effective as Ch14.18 alone in directing complement-dependent cytotoxicity against NMB7 cells. Our results demonstrate that the biological activities expressed by ADCC and complement-dependent cytotoxicity of both monoclonal antibody Ch14.18 and GM-CSF are retained by the Ch14.18-GM-CSF fusion protein and lend further support for future clinical trials of this fusion protein in patients with neuroblastoma.

  18. Critical role of AZI2 in GM-CSF-induced dendritic cell differentiation.

    PubMed

    Fukasaka, Masahiro; Ori, Daisuke; Kawagoe, Tatsukata; Uematsu, Satoshi; Maruyama, Kenta; Okazaki, Toshihiko; Kozaki, Tatsuya; Imamura, Tomoko; Tartey, Sarang; Mino, Takashi; Satoh, Takashi; Akira, Shizuo; Takeuchi, Osamu

    2013-06-01

    TNFR-associated factor family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) is critical for the activation of IFN regulatory factor 3 and type I IFN production upon virus infection. A set of TBK1-binding proteins, 5-azacytidine-induced gene 2 (AZI2; also known as NAP1), TANK, and TBK1-binding protein 1 (TBKBP1), have also been implicated in the production of type I IFNs. Among them, TANK was found to be dispensable for the responses against virus infection. However, physiological roles of AZI2 and TBKBP1 have yet to be clarified. In this study, we found that none of these TBK1-binding proteins is critical for type I IFN production in mice. In contrast, AZI2, but not TBKBP1, is critical for the differentiation of conventional dendritic cells (cDCs) from bone marrow cells in response to GM-CSF. AZI2 controls GM-CSF-induced cell cycling of bone marrow cells via TBK1. GM-CSF-derived DCs from AZI2-deficient mice show severe defects in cytokine production and T cell activation both in vitro and in vivo. Reciprocally, overexpression of AZI2 results in efficient generation of cDCs, and the cells show enhanced T cell activation in response to Ag stimulation. Taken together, AZI2 expression is critical for the generation of cDCs by GM-CSF and can potentially be used to increase the efficiency of immunization by cDCs. PMID:23610142

  19. Comparative antitumor effect among GM-CSF, IL-12 and GM-CSF+IL-12 genetically modified tumor cell vaccines.

    PubMed

    Miguel, A; Herrero, M J; Sendra, L; Botella, R; Algás, R; Sánchez, M; Aliño, S F

    2013-10-01

    Genetically modified cells have been shown to be one of the most effective cancer vaccine strategies. An evaluation is made of the efficacy of both preventive and therapeutic antitumor vaccines against murine melanoma, using C57BL/6 mice and irradiated B16 tumor cells expressing granulocyte and macrophage colony-stimulating factor (GM-CSF), interleukin-12 (IL-12) or both. Tumor was transplanted by the injection of wild-type B16 cells. Tumor growth and survival were measured to evaluate the efficacy of vaccination. Specific humoral response and immunoglobulin G (IgG) switch were evaluated measuring total IgG and IgG1 and IgG2a subtypes against tumor membrane proteins of B16 cells. In preventive vaccination, all treated groups showed delayed tumor growth. In addition, the group vaccinated to express only GM-CSF achieved 100% animal survival (P<0.005). Vaccination with GM-CSF+IL-12-producing B16 cells yielded lesser results (60% survival, P<0.005). Furthermore, all surviving animals remained disease-free after second tumor implantation 1 year later. The therapeutic vaccination strategies resulted in significantly delayed tumor growth, mainly using B16 cells producing GM-CSF+IL-12 cytokines, with 70% tumor growth inhibition (P<0.001)-although none of the animals reached overall survival. The results obtained suggest that the GM-CSF+IL-12 combination only increases the efficacy of therapeutic vaccines. No differences in classical regulatory T cells were found among the different groups.

  20. Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury

    PubMed Central

    Han, Xiaonan; Gilbert, Shila; Groschwitz, Katherine; Hogan, Simon; Jurickova, Ingrid; Trapnell, Bruce; Samson, Charles; Gully, Jonathan

    2014-01-01

    Background Administration of granulocyte-macrophage colony stimulating factor (GM-CSF) relieves symptoms in Crohn's disease (CD). It has been reported that reduced GM-CSF bioactivity is associated with more aggressive ileal behaviour and that GM-CSF-null mice exhibit ileal barrier dysfunction and develop a transmural ileitis following exposure to non-steroidal anti-inflammatory drugs (NSAIDs). STAT5 signalling is central to GM-CSF action. It was therefore hypothesised that GM-CSF signalling in non-haematopoietic cells is required for ileal homeostasis. Methods Bone marrow (BM) chimeras were generated by reconstituting irradiated GM-CSF receptor (gm-csfr) β chain or GM-CSF (gm-csf) deficient mice with wild type BM (WTBM→GMRKO and WTBM→GMKO). Intestinal barrier function and the response to NSAID-induced ileal injury were examined. Expression of gm-csf, gm-csfr or stat5 in Caco-2 and HT-29 intestinal epithelial cell (IEC) lines was knocked down and the effect of GM-CSF signalling on IEC survival and proliferation was determined. Results Elevated levels of GM-CSF autoantibodies in ileal CD were found to be associated with dysregulation of IEC survival and proliferation. GM-CSF receptor-deficient mice and WTBM→GMRKO chimeras exhibited ileal hyperpermeability. NSAID exposure induced a transmural ileitis in GM-CSF receptor-deficient mice and WTBM→GMRKO chimeras. Transplantation of wild type BM into GM-CSF-deficient mice prevented NSAID ileal injury and restored ileal barrier function. Ileal crypt IEC proliferation was reduced in WTBM→GMRKO chimeras, while STAT5 activation in ileal IEC following NSAID exposure was abrogated in WTBM→GMRKO chimeras. Following knock down of gm-csf, gm-csfr α or β chain or stat5a/b expression in Caco-2 cells, basal proliferation was suppressed. GM-CSF normalised proliferation of Caco-2 cells exposed to NSAID, which was blocked by stat5a/b RNA interference. Conclusions Loss of GM-CSF signalling in non-haematopoietic cells

  1. Age-related impairment of GM-CSF-induced signalling in neutrophils: role of SHP-1 and SOCS proteins.

    PubMed

    Tortorella, Cosimo; Simone, Olivia; Piazzolla, Giuseppina; Stella, Isabella; Antonaci, Salvatore

    2007-08-01

    Functional activities of mature human neutrophils are strongly influenced by the pro-inflammatory cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). Accordingly, a defective response to GM-CSF might have dramatic consequences for neutrophil functions and the host defence against infections. Such an event is most likely to occur in senescence. A number of studies have, in fact, reported an impairment of the GM-CSF capacity to prime and/or to activate respiratory burst, as well as to delay apoptotic events, in neutrophils from elderly individuals. In the last 2 decades many efforts have been made to explore at molecular levels the mechanism underlying these defects. Recent studies let us depict a scenario in which an increased activity of inhibitory molecules, such as Src homology domain-containing protein tyrosine phosphatase-1 (SHP-1) and suppressors of cytokine signalling (SOCS), is responsible for the age-related failure of GM-CSF to stimulate neutrophil functions via inhibition of Lyn-, phosphoinositide 3-kinase (PI3-K)/extracellular signal-regulated kinase (ERK)- and signal transducers and activators of transcription (STAT)-dependent pathways. The control of SHP-1 and/or SOCS activity might therefore be an important therapeutic target for the restoration of normal immune responses during senescence.

  2. IL-33-Induced Cytokine Secretion and Survival of Mouse Eosinophils Is Promoted by Autocrine GM-CSF

    PubMed Central

    Willebrand, Ralf; Voehringer, David

    2016-01-01

    Eosinophils are major effector cells during allergic responses and helminth infections. Recent studies further highlight eosinophils as important players in many other biological processes. Therefore it is important to understand how these cells can be modulated in terms of survival and effector function. In the present study we investigated how eosinophils respond to the alarmin IL-33. We show that IL-33 promotes eosinophil survival in a ST2- and MyD88-dependent manner. IL-33-mediated protection from apoptosis was dependent on autocrine GM-CSF release. In addition, GM-CSF increased the IL-33-induced secretion of IL-4 and IL-13 from eosinophils. Unexpectedly, this effect was further enhanced by cross-linking of Siglec-F, a proposed inhibitory and apopotosis-inducing receptor on eosinophils. Co-culture experiments with eosinophils and macrophages revealed that the IL-33-induced release of IL-4 and IL-13 from eosinophils was required for differentiation of alternatively activated macrophages (AAMs). The differentiation of AAMs could be further increased in the presence of GM-CSF. These results indicate that cross-talk between Siglec-F and the receptors for IL-33, LPS and GM-CSF plays an important role for efficient secretion of IL-4 and IL-13. Deciphering the molecular details of this cross-talk could lead to the development of new therapeutic option to treat eosinophil-associated diseases. PMID:27690378

  3. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    SciTech Connect

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W.

    2008-08-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

  4. Specific Contributions of CSF-1 and GM-CSF to the Dynamics of the Mononuclear Phagocyte System.

    PubMed

    Louis, Cynthia; Cook, Andrew D; Lacey, Derek; Fleetwood, Andrew J; Vlahos, Ross; Anderson, Gary P; Hamilton, John A

    2015-07-01

    M-CSF (or CSF-1) and GM-CSF can regulate the development and function of the mononuclear phagocyte system (MPS). To address some of the outstanding and sometimes conflicting issues surrounding this biology, we undertook a comparative analysis of the effects of neutralizing mAbs to these CSFs on murine MPS populations in the steady-state and during acute inflammatory reactions. CSF-1 neutralization, but not of GM-CSF, in normal mice rapidly reduced the numbers of more mature Ly6C(-) monocytes in blood and bone marrow, without any effect on proliferating precursors, and also the numbers of the resident peritoneal macrophages, observations consistent with CSF-1 signaling being essential only at a relatively late state in steady-state MPS development; in contrast, GM-CSF neutralization had no effect on the numbers of these particular populations. In Ag-induced peritonitis (AIP), thioglycolate-induced peritonitis, and LPS-induced lung inflammation, CSF-1 neutralization lowered inflammatory macrophage number; in the AIP model, this reduced number was not due to suppressed proliferation. More detailed studies with the convenient AIP model indicated that CSF-1 neutralization led to a relatively uniform reduction in all inflammatory cell populations; GM-CSF neutralization, in contrast, was more selective, resulting in the preferential loss among the MPS populations of a cycling, monocyte-derived inflammatory dendritic cell population. Some mechanistic options for the specific CSF-dependent biologies enumerated are discussed.

  5. Expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene under control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice.

    PubMed

    Burkov, I A; Serova, I A; Battulin, N R; Smirnov, A V; Babkin, I V; Andreeva, L E; Dvoryanchikov, G A; Serov, O L

    2013-10-01

    Expression of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected "protective or enhancer effect" from the MAR element on the hGM-CSF gene expression was not observed.

  6. GM-CSF Promotes Macrophage Alternative Activation after Renal Ischemia/Reperfusion Injury

    PubMed Central

    Huynh, Larry; Marlier, Arnaud; Lee, Yashang; Moeckel, Gilbert W.; Cantley, Lloyd G.

    2015-01-01

    After kidney ischemia/reperfusion (I/R) injury, monocytes home to the kidney and differentiate into activated macrophages. Whereas proinflammatory macrophages contribute to the initial kidney damage, an alternatively activated phenotype can promote normal renal repair. The microenvironment of the kidney during the repair phase mediates the transition of macrophage activation from a proinflammatory to a reparative phenotype. In this study, we show that macrophages isolated from murine kidneys during the tubular repair phase after I/R exhibit an alternative activation gene profile that differs from the canonical alternative activation induced by IL-4–stimulated STAT6 signaling. This unique activation profile can be reproduced in vitro by stimulation of bone marrow-derived macrophages with conditioned media from serum-starved mouse proximal tubule cells. Secreted tubular factors were found to activate macrophage STAT3 and STAT5 but not STAT6, leading to induction of the unique alternative activation pattern. Using STAT3-deficient bone marrow-derived macrophages and pharmacologic inhibition of STAT5, we found that tubular cell-mediated macrophage alternative activation is regulated by STAT5 activation. Both in vitro and after renal I/R, tubular cells expressed GM-CSF, a known STAT5 activator, and this pathway was required for in vitro alternative activation of macrophages by tubular cells. Furthermore, administration of a neutralizing antibody against GM-CSF after renal I/R attenuated kidney macrophage alternative activation and suppressed tubular proliferation. Taken together, these data show that tubular cells can instruct macrophage activation by secreting GM-CSF, leading to a unique macrophage reparative phenotype that supports tubular proliferation after sterile ischemic injury. PMID:25388222

  7. Regulation of GM-CSF and IL-3 production from the murine keratinocyte cell line PAM 212 following exposure to ultraviolet radiation

    SciTech Connect

    Gallo, R.L.; Staszewski, R.; Sauder, D.N.; Knisely, T.L.; Granstein, R.D. )

    1991-08-01

    Ultraviolet radiation (UVR) exposure induces profound changes in the synthesis and secretion of various cytokines both in vivo and in vitro. Little is known regarding the mechanism of these responses. This investigation evaluated the effects of UVR on the ability of a murine keratinocyte line (PAM 212) to produce interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF). Subconfluent rapidly dividing PAM 212 cells were shown by RNA slot-blot hybridization studies to have increased levels of mRNA for both IL-3 and GM-CSF within 1 h of UVR exposure. However, only GM-CSF-specific bioactivity, as determined by antibody neutralization studies, was shown to increase above baseline in cell supernatants. Cells grown to confluence responded differently to UVR. Under these culture conditions an apparent decrease in bioactivity was detected after UVR exposure for both growth factors, and no change in mRNA levels was detected. In addition to culture density, removal of extracellular calcium or sodium during irradiation, treatment with amiloride, or inhibition of new mRNA synthesis with cordycepin was shown to influence the UVR-induced alteration in release of IL-3 or GM-CSF bioactivity from both confluent and subconfluent PAM 212 cells. These results demonstrate that UVR influences the release of the colony stimulating factors GM-CSF and IL-3 from keratinocyte, and suggests that the state of cell growth and conditions of membrane ion transport influence the mechanisms regulating secretion of those factors.

  8. Proteomic analysis of rice endosperm cells in response to expression of hGM-CSF.

    PubMed

    Luo, Junling; Ning, Tingting; Sun, Yunfang; Zhu, Jinghua; Zhu, Yingguo; Lin, Qishan; Yang, Daichang

    2009-02-01

    The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm.

  9. Proteomic analysis of rice endosperm cells in response to expression of hGM-CSF.

    PubMed

    Luo, Junling; Ning, Tingting; Sun, Yunfang; Zhu, Jinghua; Zhu, Yingguo; Lin, Qishan; Yang, Daichang

    2009-02-01

    The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm. PMID:18778094

  10. In vivo kinetics of 111indium-labelled autologous granulocytes following i.v. administration of granulocyte-macrophage colony-stimulating factor (GM-CSF).

    PubMed

    Hovgaard, D; Schifter, S; Rabøl, A; Mortensen, B T; Nissen, N I

    1992-04-01

    Administration of both glycosylated and non-glycosylated recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) induces an immediate transient granulocytopenia of 1-3 hours' duration. In order to explore this phenomenon, granulocytes were labelled with 111Indium and the effect on the kinetics of granulocytes after administration of rhGM-CSF was studied in 10 previously untreated patients with malignant lymphoma. For both types and doses of rhGM-CSF, a significant and dramatic accumulation of the 111Indium-labelled granulocytes was observed in the lung within a few minutes after i.v. injection of rhGM-CSF. The accumulation of radioactivity coincided with the pronounced and transient granulocytopenia in peripheral blood. The 111Indium-labelled granulocytes later reappeared in the peripheral blood, indicating reversible pulmonary vascular margination of the granulocytes. Half-life of labelled granulocytes after reappearance was comparable to half-life values under normal conditions. The transient accumulation of granulocytes in the pulmonary vessels seems not to be of clinical importance in the management of patients, but it may to some degree explain previously described side-effects, such as transient hypoxemia ("first-dose" reaction) following administration of rhGM-CSF.

  11. Rabbit M1 and M2 macrophages can be induced by human recombinant GM-CSF and M-CSF.

    PubMed

    Yamane, Kazuyoshi; Leung, Kai-Poon

    2016-09-01

    Macrophages can change their phenotype in response to environmental cues. Polarized macrophages are broadly classified into two groups: classical activated M1 and alternative activated M2. Characterization of human macrophages has been widely studied, but polarized macrophages in rabbits have not been characterized. We characterized rabbit macrophages that were polarized using human recombinant GM-CSF and M-CSF. GM-CSF-treated macrophages had higher mRNA expression of proinflammatory cytokines (M1 phenotype) than did the M-CSF-treated counterpart. By contrast, high levels of TGF-β and IL-10 expression (M2 phenotype) were found in M-CSF-treated macrophages. The present study may be useful to understand roles of polarized macrophages in rabbit disease models. PMID:27642558

  12. GM-CSF augments the immunosuppressive capacity of neonatal spleen cells in vitro

    SciTech Connect

    Morrissey, P.J.; Ireland, R. )

    1991-09-01

    Addition of exogenous granulocyte-macrophage colony stimulating factor (GM-CSF) to cultures of adult murine spleen cells with sheep red blood cells (SRBC) results in an augmented plaque forming cell (PFC) response. The influence of GM-CSF on the ability of neonatal spleen cells to suppress the anti-SRBC plaque forming response of adult spleen cells was tested by adding GM-CSF to cultures of neonatal and adult spleen cells. The suppressive capacity of the neonatal spleen cells was augmented by exogenous GM-CSF. The augmented suppression of the neonatal spleen cells was dependent on a G-10 adherent population since the addition of GM-CSF to cultures containing G-10 passed neonatal spleen cells resulted in an augmented PFC response and not suppression. Neonatal splenic glass adherent cells were also capable of suppressing the response. Neonatal spleen cells or purified neonatal glass adherent spleen cells cultured in the presence of GM-CSF had markedly increased levels of PGE2 in the culture supernatant. Neonatal spleen cells cultured with GM-CSF had increased numbers of morphologically identifiable macrophages after 48 hr of culture. Both irradiation and G-10 passage of the neonatal spleen diminished the numbers of macrophages formed in response to GM-CSF, and both of these manipulations resulted in reversal of suppression in response to GM-CSF. Thus, the augmented suppressive capacity of neonatal spleen cells in response to GM-CSF is probably mediated by its ability to drive monocyte to macrophage differentiation as well as increase the suppressive capacity of the existing neonatal splenic macrophages by increasing their production of PGE2.

  13. Conformational Changes in the GM-CSF Receptor Suggest a Molecular Mechanism for Affinity Conversion and Receptor Signaling.

    PubMed

    Broughton, Sophie E; Hercus, Timothy R; Nero, Tracy L; Dottore, Mara; McClure, Barbara J; Dhagat, Urmi; Taing, Houng; Gorman, Michael A; King-Scott, Jack; Lopez, Angel F; Parker, Michael W

    2016-08-01

    The GM-CSF, IL-3, and IL-5 receptors constitute the βc family, playing important roles in inflammation, autoimmunity, and cancer. Typical of heterodimeric type I cytokine receptors, signaling requires recruitment of the shared subunit to the initial cytokine:α subunit binary complex through an affinity conversion mechanism. This critical process is poorly understood due to the paucity of crystal structures of both binary and ternary receptor complexes for the same cytokine. We have now solved the structure of the binary GM-CSF:GMRα complex at 2.8-Å resolution and compared it with the structure of the ternary complex, revealing distinct conformational changes. Guided by these differences we performed mutational and functional studies that, importantly, show GMRα interactions playing a major role in receptor signaling while βc interactions control high-affinity binding. These results support the notion that conformational changes underlie the mechanism of GM-CSF receptor activation and also suggest how related type I cytokine receptors signal. PMID:27396825

  14. Oncolytic and immunologic cancer therapy with GM-CSF-armed vaccinia virus of Tian Tan strain Guang9.

    PubMed

    Deng, Lili; Fan, Jun; Guo, Mingming; Huang, Biao

    2016-03-28

    Targeted oncolytic vaccinia viruses are being developed as a novel strategy in cancer therapy. Arming vaccinia viruses with immunostimulatory cytokines can enhance antitumor efficacy. Such engineered oncolytic viruses, like JX-594, a Wyeth strain vaccinia virus modified with human granulocyte-macrophage colony-stimulating factor (GM-CSF), have shown promising results and have proceeded rapidly in clinical trials. However, the oncolytic potential of the Chinese vaccine strain Tian Tan (VTT) has not been explored. In this study, we constructed a targeted oncolytic vaccinia virus of Tian Tan strain Guang9 (VG9) expressing murine GM-CSF (VG9-GMCSF) and evaluated the antitumor effect of this recombinant vaccinia virus in a murine melanoma model. In vitro, viral replication and cytotoxicity of VG9-GMCSF was as potent as VG9; in vivo, VG9-GMCSF significantly inhibited the growth of subcutaneously implanted melanoma tumors, prolonged the survival of tumor-bearing mice, and produced an antitumor cytotoxic response. Such antitumor effect may be due to the lytic nature of virus as well as the stimulation of immune activity by GM-CSF production. Our results indicate that VG9-GMCSF induces strong tumoricidal activity, providing a potential therapeutic strategy for combating cancer.

  15. Characterization of GM-CSF-inhibitory factor and Uracil DNA glycosylase encoding genes from camel pseudocowpoxvirus.

    PubMed

    Nagarajan, G; Swami, Shelesh Kumar; Dahiya, Shyam Singh; Narnaware, S D; Mehta, S C; Singh, P K; Singh, Raghvendar; Tuteja, F C; Patil, N V

    2015-06-01

    The present study describes the PCR amplification of GM-CSF-inhibitory factor (GIF) and Uracil DNA glycosylase (UDG) encoding genes of pseudocowpoxvirus (PCPV) from the Indian Dromedaries (Camelus dromedarius) infected with contagious ecthyma using the primers based on the corresponding gene sequences of human PCPV and reindeer PCPV, respectively. The length of GIF gene of PCPV obtained from camel is 795 bp and due to the addition of one cytosine residue at position 374 and one adenine residue at position 516, the open reading frame (ORF) got altered, resulting in the production of truncated polypeptide. The ORF of UDG encoding gene of camel PCPV is 696 bp encoding a polypeptide of 26.0 kDa. Comparison of amino acid sequence homologies of GIF and UDG of camel PCPV revealed that the camel PCPV is closer to ORFV and PCPV (reference stains of both human and reindeer), respectively. PMID:25816930

  16. IFN-gamma enhances killing of methicillin-resistant Staphylococcus aureus by human monocytes more effectively than GM-CSF in the presence of daptomycin and other antibiotics.

    PubMed

    Smith, Raymond P; Baltch, Aldona L; Ritz, William J; Michelsen, Phyllis B; Bopp, Lawrence H

    2010-09-01

    Because cytokines have been utilized in treatment of sepsis in neonates, we studied the effects of interferon-gamma (IFN-gamma) and GM-CSF on killing of intracellular methicilin-resistant Staphylococcus aureus (MRSA) by human monocyte derived macrophages (MDM) in the presence of daptomycin (Dap), rifampin (Rif), gentamicin (Gen), and combinations of these drugs. MDM infected with MRSA were treated with Dap (1 x MIC), Gen (0.5 x MIC), or Rif (1 x MIC), singly or in combination, with or without cytokines. MDM were lysed and viable bacteria counted. With antibiotics, MDM activated by IFN-gamma had a more rapid and prolonged bacterial killing effect than MDM activated by GM-CSF. This effect was most obvious with the triple-drug combination. In contrast, GM-CSF reduced intracellular killing under most experimental conditions compared to the effect of antibiotics alone. Dap alone and two- and three-drug combinations demonstrated significant killing effect for the 48 h of the assay. IFN-gamma enhanced rapid intracellular killing of MRSA in the presence of triple-drug treatment or Dap alone. GM-CSF in combination with the antibiotics reduced killing under most conditions studied. Further studies to confirm these observations with IFN-gamma-activated MDM and other MRSA strains are needed to support clinical trials for difficult-to-treat MRSA infections.

  17. Identification of a fourth ancient member of the IL-3/IL-5/GM-CSF cytokine family, KK34, in many mammals.

    PubMed

    Yamaguchi, Takuya; Schares, Susann; Fischer, Uwe; Dijkstra, Johannes M

    2016-12-01

    The related cytokine genes IL-3, IL-5 and GM-CSF map to the (extended) TH2 cytokine locus of the mammalian genome. For chicken an additional related cytokine gene, KK34, was reported downstream of the IL-3 plus GM-CSF cluster, but hitherto it was believed that mammalian genomes lack this gene. However, the present study identifies an intact orthologue of chicken KK34 gene in many mammals like cattle and pig, while remnants of KK34 can be found in human and mouse. Bovine KK34 was found to be transcribed, and its recombinant protein could induce STAT5 phosphorylation and proliferation of lymphocytes upon incubation with bovine PBMCs. This concludes that KK34 is a fourth functional cytokine of the IL-3/IL-5/GM-CSF/KK34-family (alias IL-5 family) in mammals. While analyzing KK34, the present study also made new identifications of cytokine genes in the extended TH2 cytokine loci for reptiles, birds and marsupials. This includes a hitherto unknown cytokine gene in birds and reptiles which we designated "IL-5famE". Other newly identified genes are KK34, GM-CSF(-like), IL-5, and IL-13 in reptiles, and IL-3 in marsupials. PMID:27492645

  18. The Expression of GM-CSF and Osteopontin in Immunocompetent Cells Precedes the Odontoblast Differentiation Following Allogenic Tooth Transplantation in Mice

    PubMed Central

    Saito, Kotaro; Nakatomi, Mitsushiro; Ida-Yonemochi, Hiroko; Kenmotsu, Shin-ichi; Ohshima, Hayato

    2011-01-01

    Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the expression of granulocyte macrophage colony-stimulating factor (GM-CSF) and osteopontin (OPN) in the process of reparative dentin formation by allogenic tooth transplantation using in situ hybridization for OPN and immunohistochemistry for GM-CSF and OPN at both levels of light and electron microscopes. Following the extraction of the mouse molar, the roots and pulp floor were resected and immediately allografted into the sublingual region. On days 1 to 3, immunocompetent cells such as macrophages and dendritic cells expressed both GM-CSF and OPN, and some of them were arranged along the pulp-dentin border and extended their cellular processes into the dentinal tubules. On days 5 to 7, tubular dentin formation commenced next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until day 14, bone-like tissue formation occurred in the pulp chamber, where OPN-positive osteoblasts surrounded the bone matrix. These results suggest that the secretion of GM-CSF and OPN by immunocompetent cells such as macrophages and dendritic cells plays a role in the maturation of dendritic cells and the differentiation of odontoblasts, respectively, in the regenerated pulp tissue following tooth transplantation. PMID:21430263

  19. Enhancing immune responses of EV71 VP1 DNA vaccine by co-inoculating plasmid IL-12 or GM-CSF expressing vector in mice.

    PubMed

    Peng, X; Fang, X; Li, J; Kong, L; Li, B; Ding, X

    2016-01-01

    Enterovirus 71 (EV71) is a major causative viral agent for large outbreaks of hand, foot, and mouth disease in children and infants, yet there is no vaccine or effective antiviral treatment for severe EV71 infection. The immunogenicity of EV71 VP1 DNA vaccine and the immunoregulatory activity of interleukin-12 (IL-12) or granulocyte-monocyte colony stimulating factor (GM-CSF) were investigated. DNA vaccine plasmids, pcDNA-VP1, pcDNA-IL-12 and pcDNA-GM-CSF were constructed and inoculated into BALB/c mice with or without pcDNA-IL-12 or pcDNA-GM-CSF by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, cytokine release assay and FACS. The VP1 DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 or GM-CSF plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against EV71. PMID:27188732

  20. Enhancing immune responses of EV71 VP1 DNA vaccine by co-inoculating plasmid IL-12 or GM-CSF expressing vector in mice.

    PubMed

    Peng, X; Fang, X; Li, J; Kong, L; Li, B; Ding, X

    2016-01-01

    Enterovirus 71 (EV71) is a major causative viral agent for large outbreaks of hand, foot, and mouth disease in children and infants, yet there is no vaccine or effective antiviral treatment for severe EV71 infection. The immunogenicity of EV71 VP1 DNA vaccine and the immunoregulatory activity of interleukin-12 (IL-12) or granulocyte-monocyte colony stimulating factor (GM-CSF) were investigated. DNA vaccine plasmids, pcDNA-VP1, pcDNA-IL-12 and pcDNA-GM-CSF were constructed and inoculated into BALB/c mice with or without pcDNA-IL-12 or pcDNA-GM-CSF by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, cytokine release assay and FACS. The VP1 DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 or GM-CSF plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against EV71.

  1. Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) regulates f Met-Leu-Phe receptors on human neutrophils.

    PubMed Central

    Atkinson, Y H; Lopez, A F; Marasco, W A; Lucas, C M; Wong, G G; Burns, G F; Vadas, M A

    1988-01-01

    The regulation of mature human neutrophil function by recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was studied. Preincubation of neutrophils with this CSF did not stimulate superoxide anion directly but enhanced the subsequent release of superoxide anion in response to stimulation with the bacterial product formylmethionylleucyl-phenylalanine (f Met-Leu-Phe). Enhanced superoxide anion production was evident by 5 min and reached a plateau at 30 min. In contrast, neutrophils preincubated with rH GM-CSF exhibited reduced chemotaxis under agarose in response to a gradient of f Met-Leu-Phe. The inhibition of neutrophil migration was dependent on the dose of rH GM-CSF and exhibited a time-course similar to the effect on superoxide production. Binding studies of f Met-Leu-[3H]Phe to purified human neutrophils revealed heterogeneous binding to unstimulated cells. Two affinity components were identified. The high-affinity component consisted of approximately 2000 sites/cell and had an average Kd of 4 +/- 2 nM (n = 6). The low-affinity component consisted of approximately 40,000 sites/cell and had an average Kd of 220 +/- 130 nM (n = 6). rH GM-CSF caused conversion to a linear Scatchard plot showing no significant change in total binding sites but a single Kd of 30 +/- 10 nM. These data indicate that rH GM-CSF may influence neutrophil responses to f Met-Leu-Phe by regulating the affinity of f Met-Leu-Phe receptors. PMID:2842255

  2. p38 MAP kinase and MKK-1 co-operate in the generation of GM-CSF from LPS-stimulated human monocytes by an NF-κB-independent mechanism

    PubMed Central

    Meja, Koremu K; Seldon, Paul M; Nasuhara, Yasuyuki; Ito, Kazuhiro; Barnes, Peter J; Lindsay, Mark A; Giembycz, Mark A

    2000-01-01

    The extent to which the p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK)-1-signalling pathways regulate the expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) from LPS-stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor-α (TNFα).Lipopolysaccharide (LPS) evoked a concentration-dependent generation of GM-CSF from human monocytes. Temporally, this effect was preceded by an increase in GM-CSF mRNA transcripts and abolished by actinomycin D and cycloheximide.LPS-induced GM-CSF release and mRNA expression were associated with a rapid and time-dependent activation of p38 MAP kinase, ERK-1 and ERK-2.The respective MKK-1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS-induced GM-CSF generation by >90%, indicating that both of these signalling cascades co-operate in the generation of this cytokine.Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor κB (NF-κB) : DNA binding. SN50, an inhibitor of NF-κB translocation, abolished LPS-induced NF-κB : DNA binding and the elaboration of TNFα, a cytokine known to be regulated by NF-κB in monocytes. In contrast, SN50 failed to affect the release of GM-CSF from the same monocyte cultures.Collectively, these results suggest that the generation of GM-CSF by LPS-stimulated human monocytes is regulated in a co-operative fashion by p38 MAP kinase- and MKK-1-dependent signalling pathways independently of the activation of NF-κB. PMID:11082122

  3. Effect of AcHERV-GmCSF as an Influenza Virus Vaccine Adjuvant

    PubMed Central

    Jang, Yuyeon; Cho, Yeondong; Heo, Yoon-Ki; Lee, Hee-Jung; Kim, Kang Chang; Choi, Jiwon; Lee, Joong Bok; Kim, Young Bong

    2015-01-01

    Introduction The first identification of swine-originated influenza A/CA/04/2009 (pH1N1) as the cause of an outbreak of human influenza accelerated efforts to develop vaccines to prevent and control influenza viruses. The current norm in many countries is to prepare influenza vaccines using cell-based or egg-based killed vaccines, but it is difficult to elicit a sufficient immune response using this approach. To improve immune responses, researchers have examined the use of cytokines as vaccine adjuvants, and extensively investigated their functions as chemoattractants of immune cells and boosters of vaccine-mediated protection. Here, we evaluated the effect of Granulocyte-macrophage Colony-Stimulating Factor (GmCSF) as an influenza vaccine adjuvant in BALB/c mice. Method and Results Female BALB/c mice were immunized with killed vaccine together with a murine GmCSF gene delivered by human endogenous retrovirus (HERV) envelope coated baculovirus (1×107 FFU AcHERV-GmCSF, i.m.) and were compared with mice immunized with the killed vaccine alone. On day 14, immunized mice were challenged with 10 median lethal dose of mouse adapted pH1N1 virus. The vaccination together with GmCSF treatment exerted a strong adjuvant effect on humoral and cellular immune responses. In addition, the vaccinated mice together with GmCSF were fully protected against infection by the lethal influenza pH1N1 virus. Conclusion Thus, these results indicate that AcHERV-GmCSF is an effective molecular adjuvant that augments immune responses against influenza virus. PMID:26090848

  4. Proinflammatory GM-CSF-producing B cells in multiple sclerosis and B cell depletion therapy.

    PubMed

    Li, Rui; Rezk, Ayman; Miyazaki, Yusei; Hilgenberg, Ellen; Touil, Hanane; Shen, Ping; Moore, Craig S; Michel, Laure; Althekair, Faisal; Rajasekharan, Sathy; Gommerman, Jennifer L; Prat, Alexandre; Fillatreau, Simon; Bar-Or, Amit

    2015-10-21

    B cells are not limited to producing protective antibodies; they also perform additional functions relevant to both health and disease. However, the relative contribution of functionally distinct B cell subsets in human disease, the signals that regulate the balance between such subsets, and which of these subsets underlie the benefits of B cell depletion therapy (BCDT) are only partially elucidated. We describe a proinflammatory, granulocyte macrophage-colony stimulating factor (GM-CSF)-expressing human memory B cell subset that is increased in frequency and more readily induced in multiple sclerosis (MS) patients compared to healthy controls. In vitro, GM-CSF-expressing B cells efficiently activated myeloid cells in a GM-CSF-dependent manner, and in vivo, BCDT resulted in a GM-CSF-dependent decrease in proinflammatory myeloid responses of MS patients. A signal transducer and activator of transcription 5 (STAT5)- and STAT6-dependent mechanism was required for B cell GM-CSF production and reciprocally regulated the generation of regulatory IL-10-expressing B cells. STAT5/6 signaling was enhanced in B cells of untreated MS patients compared with healthy controls, and B cells reemerging in patients after BCDT normalized their STAT5/6 signaling as well as their GM-CSF/IL-10 cytokine secretion ratios. The diminished proinflammatory myeloid cell responses observed after BCDT persisted even as new B cells reconstituted. These data implicate a proinflammatory B cell/myeloid cell axis in disease and underscore the rationale for selective targeting of distinct B cell populations in MS and other human autoimmune diseases. PMID:26491076

  5. IL-17 attenuates the anti-apoptotic effects of GM-CSF in human neutrophils.

    PubMed

    Dragon, Stéphane; Saffar, Arash Shoja; Shan, Lianyu; Gounni, Abdelilah Soussi

    2008-01-01

    Interleukin (IL)-17A is a pleiotropic, pro-inflammatory cytokine that is implicated in chronic inflammatory and degenerative disorders. IL-17 has been demonstrated to link activated T-lymphocyte with the recruitment of neutrophils at sites of inflammation, however whether IL-17 can mediate neutrophil survival and subsequently affect inflammatory responses has not fully been elucidated. In our study, we demonstrate that human peripheral blood and HL-60 differentiated neutrophils express mRNA and cell surface IL-17A receptor. IL-17A does not affect the rate of spontaneous neutrophil apoptosis, however significantly decreased granulocyte macrophage-colony stimulating factor (GM-CSF)-mediated survival by antagonizing the signal transduction pathways of p38, Erk1/2 and signal transducer and activator of transcription (STAT) 5B. These events were associated with reduced myeloid cell lymphoma-1 (Mcl-1) protein levels, increased translocation and aggregation of Bax to mitochondria, decreased mitochondrial transmembrane potential and in an increase in caspase-3/7 activity. These events were independent of increased Fas or soluble Fas ligand expression levels. Taken together, our findings suggest that IL-17 may regulate neutrophil homeostasis and favor the resolution of inflamed tissues by attenuating the delay in neutrophil apoptosis induced by inflammatory cytokines.

  6. GM-CSF reduces expression of chondroitin sulfate proteoglycan (CSPG) core proteins in TGF-β-treated primary astrocytes.

    PubMed

    Choi, Jung-Kyoung; Park, Sang-Yoon; Kim, Kil Hwan; Park, So Ra; Lee, Seok-Geun; Choi, Byung Hyune

    2014-12-01

    GM-CSF plays a role in the nervous system, particularly in cases of injury. A therapeutic effect of GM-CSF has been reported in rat models of various central nervous system injuries. We previously showed that GM-CSF could enhance long-term recovery in a rat spinal cord injury model, inhibiting glial scar formation and increasing the integrity of axonal structure. Here, we investigated molecular the mechanism(s) by which GM-CSF suppressed glial scar formation in an in vitro system using primary astrocytes treated with TGF-β. GM-CSF repressed the expression of chondroitin sulfate proteoglycan (CSPG) core proteins in astrocytes treated with TGF-β. GM-CSF also inhibited the TGF-β-induced Rho-ROCK pathway, which is important in CSPG expression. Finally, the inhibitory effect of GM-CSF was blocked by a JAK inhibitor. These results may provide the basis for GM-CSF's effects in glial scar inhibition and ultimately for its therapeutic effect on neural cell injuries.

  7. Effects of recombinant human GM-CSF on proliferation of clonogenic cells in acute myeloblastic leukemia.

    PubMed

    Griffin, J D; Young, D; Herrmann, F; Wiper, D; Wagner, K; Sabbath, K D

    1986-05-01

    Proliferation of acute myeloblastic leukemia (AML) cells in vitro is limited in most cases to a small subset of blasts that have several properties of stem cells. These leukemic colony-forming cells (AML-CFU) generally require addition of exogenous growth factors for proliferation in agar or methylcellulose. These factors can be supplied by media conditioned by phytohemagglutinin-stimulated normal leukocytes or by CSF-secreting tumor cell lines. However, the exact factor or factors required for stimulation of AML-CFU growth have not been defined. We compared the AML-CFU stimulatory activity of a human recombinant GM-CSF with that of GCT-CM, Mo-CM, and the PHA-leukocyte feeder system in 15 cases of AML. In each of the 12 cases that required exogenous growth factors for maximum AML-CFU growth, recombinant GM-CSF could replace either GM-CSF or Mo-CM, and could partially replace the PHA-leukocyte feeder system. These results indicate that this GM-CSF is a growth promoter of AML-CFU in these culture systems.

  8. Systematic review: new serological markers (anti-glycan, anti-GP2, anti-GM-CSF Ab) in the prediction of IBD patient outcomes.

    PubMed

    Bonneau, J; Dumestre-Perard, C; Rinaudo-Gaujous, M; Genin, C; Sparrow, M; Roblin, X; Paul, S

    2015-03-01

    Traditionally, IBD diagnosis is based on clinical, radiological, endoscopic, and histological criteria. Biomarkers are needed in cases of uncertain diagnosis, or to predict disease course and therapeutic response. No guideline recommends the detection of antibodies (including ASCA and ANCA) for diagnosis or prognosis of IBD to date. However, many recent data suggest the potential role of new serological markers (anti-glycan (ACCA, ALCA, AMCA, anti-L and anti-C), anti-GP2 and anti-GM-CSF Ab). This review focuses on clinical utility of these new serological markers in diagnosis, prognosis and therapeutic monitoring of IBD. Literature review of anti-glycan, anti-GP2 and anti-GM-CSF Ab and their impact on diagnosis, prognosis and prediction of therapeutic response was performed in PubMed/MEDLINE up to June 2014. Anti-glycan, anti-GP2 and anti-GM-CSF Ab are especially associated with CD and seem to be correlated with complicated disease phenotypes even if results differ between studies. Although anti-glycan Ab and anti-GP2 Ab have low sensitivity in diagnosis of IBD, they could identify a small number of CD patients not detected by other tests such as ASCA. Anti-glycan Abs are associated with a progression to a more severe disease course and a higher risk for IBD-related surgery. Anti-GP2 Ab could particularly contribute to better stratify cases of pouchitis. Anti-GM-CSF Ab seems to be correlated with disease activity and could help predict relapses. These new promising biomarkers could particularly be useful in stratification of patients according to disease phenotype and risk of complications. They could be a valuable aid in prediction of disease course and therapeutic response but more prospective studies are needed.

  9. Dual Role of GM-CSF as a Pro-Inflammatory and a Regulatory Cytokine: Implications for Immune Therapy

    PubMed Central

    Bhattacharya, Palash; Budnick, Isadore; Singh, Medha; Thiruppathi, Muthusamy; Alharshawi, Khaled; Elshabrawy, Hatem; Holterman, Mark J.

    2015-01-01

    Granulocyte macrophage colony stimulating factor (GM-CSF) is generally recognized as an inflammatory cytokine. Its inflammatory activity is primarily due its role as a growth and differentiation factor for granulocyte and macrophage populations. In this capacity, among other clinical applications, it has been used to bolster anti-tumor immune responses. GM-CSF-mediated inflammation has also been implicated in certain types of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Thus, agents that can block GM-CSF or its receptor have been used as anti-inflammatory therapies. However, a review of literature reveals that in many situations GM-CSF can act as an anti-inflammatory/regulatory cytokine. We and others have shown that GM-CSF can modulate dendritic cell differentiation to render them “tolerogenic,” which, in turn, can increase regulatory T-cell numbers and function. Therefore, the pro-inflammatory and regulatory effects of GM-CSF appear to depend on the dose and the presence of other relevant cytokines in the context of an immune response. A thorough understanding of the various immunomodulatory effects of GM-CSF will facilitate more appropriate use and thus further enhance its clinical utility. PMID:25803788

  10. Age-associated differential production of IFN-γ, IL-10 and GM-CSF by porcine alveolar macrophages in response to lipopolysaccharide.

    PubMed

    Islam, Mohammad Ariful; Uddin, Muhammad Jasim; Tholen, Ernst; Tesfaye, Dawit; Looft, Christian; Schellander, Karl; Cinar, Mehmet Ulas

    2013-10-01

    The aim of the present study was to investigate the age-related production variation of T helper (Th)-type cytokines (IL-2, IL-4, IFN-γ and IL-10), granulocyte macrophage-colony stimulating factor (GM-CSF) and nitric oxide (NO) by lipopolysaccharide (LPS)-stimulated porcine alveolar macrophages (AMs) in a time-dependent manner. For this purpose, AMs were isolated from 5-days (newborn), 40-days (post-weaned) and 120-days (young) old pigs. Cells were incubated for 24h in the absence or presence of increasing concentrations of LPS (0.0, 0.01, 1.0, 5.0 and 10.0 μg/mL). IL-10, IFN-γ and GM-CSF mRNA expression was upregulated in a dose-dependent manner for all age groups (P<0.05). Age-related differences included a significantly increased IL-10 mRNA and protein production in newborn piglets compared to post-weaned and young pigs. IL-10 production pattern was similar with a higher peak between 12 and 36 h post-induction in all age groups. In contrast, IFN-γ mRNA and protein level was significantly elevated in young pigs 12h and 24h post-induction, respectively, while the time course production of IFN-γ was mostly consistent in newborn and post-weaned piglets. GM-CSF mRNA expression was significantly lower in newborn piglets than in post-weaned and young pigs. The kinetic of GM-CSF expression peaked at 12h in young and post-weaned pigs and at 24h in newborn piglets. IL-4 mRNA levels were very low and no apparent change of IL-2 expression was observed following LPS stimulation in all age groups. Only very low levels of NO were detected in the cell supernatants of young pigs. Collectively, these studies suggest age-related differences in time-dependent production of IL-10, IFN-γ and GM-CSF by porcine AMs with potential immunoregulatory consequences to be explored further.

  11. GM-CSF production from human airway smooth muscle cells is potentiated by human serum.

    PubMed Central

    Sukkar, M B; Hughes, J M; Johnson, P R; Armour, C L

    2000-01-01

    Recent evidence suggests that airway smooth muscle cells (ASMC) actively participate in the airway inflammatory process in asthma. Interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) induce ASMC to release inflammatory mediators in vitro. ASMC mediator release in vivo, however, may be influenced by features of the allergic asthmatic phenotype. We determined whether; (1) allergic asthmatic serum (AAS) modulates ASMC mediator release in response to IL-1beta and TNF-alpha, and (2) IL-1beta/TNF-alpha prime ASMC to release mediators in response to AAS. IL-5 and GM-CSF were quantified by ELISA in culture supernatants of; (1) ASMC pre-incubated with either AAS, nonallergic non-asthmatic serum (NAS) or Monomed (a serum substitute) and subsequently stimulated with IL-1beta and TNF-alpha and (2) ASMC stimulated with IL-1beta/TNF-alpha and subsequently exposed to either AAS, NAS or Monomed. IL-1beta and TNF-alpha induced GM-CSF release in ASMC pre-incubated with AAS was not greater than that in ASMC pre-incubated with NAS or Monomed. IL-1beta and TNF-alpha, however, primed ASMC to release GM-CSF in response to human serum. GM-CSF production following IL-1beta/TNF-alpha and serum exposure (AAS or NAS) was significantly greater than that following IL-1beta/TNF-alpha and Monomed exposure or IL-1beta/TNF-alpha exposure only. Whilst the potentiating effects of human serum were not specific to allergic asthma, these findings suggest that the secretory capacity of ASMC may be up-regulated during exacerbations of asthma, where there is evidence of vascular leakage. PMID:11132773

  12. ILC3 GM-CSF production and mobilisation orchestrate acute intestinal inflammation

    PubMed Central

    Pearson, Claire; Thornton, Emily E; McKenzie, Brent; Schaupp, Anna-Lena; Huskens, Nicky; Griseri, Thibault; West, Nathaniel; Tung, Sim; Seddon, Benedict P; Uhlig, Holm H; Powrie, Fiona

    2016-01-01

    Innate lymphoid cells (ILCs) contribute to host defence and tissue repair but can induce immunopathology. Recent work has revealed tissue-specific roles for ILCs; however, the question of how a small population has large effects on immune homeostasis remains unclear. We identify two mechanisms that ILC3s utilise to exert their effects within intestinal tissue. ILC-driven colitis depends on production of granulocyte macrophage-colony stimulating factor (GM-CSF), which recruits and maintains intestinal inflammatory monocytes. ILCs present in the intestine also enter and exit cryptopatches in a highly dynamic process. During colitis, ILC3s mobilize from cryptopatches, a process that can be inhibited by blocking GM-CSF, and mobilization precedes inflammatory foci elsewhere in the tissue. Together these data identify the IL-23R/GM-CSF axis within ILC3 as a key control point in the accumulation of innate effector cells in the intestine and in the spatio-temporal dynamics of ILCs in the intestinal inflammatory response. DOI: http://dx.doi.org/10.7554/eLife.10066.001 PMID:26780670

  13. ILC3 GM-CSF production and mobilisation orchestrate acute intestinal inflammation.

    PubMed

    Pearson, Claire; Thornton, Emily E; McKenzie, Brent; Schaupp, Anna-Lena; Huskens, Nicky; Griseri, Thibault; West, Nathaniel; Tung, Sim; Seddon, Benedict P; Uhlig, Holm H; Powrie, Fiona

    2016-01-01

    Innate lymphoid cells (ILCs) contribute to host defence and tissue repair but can induce immunopathology. Recent work has revealed tissue-specific roles for ILCs; however, the question of how a small population has large effects on immune homeostasis remains unclear. We identify two mechanisms that ILC3s utilise to exert their effects within intestinal tissue. ILC-driven colitis depends on production of granulocyte macrophage-colony stimulating factor (GM-CSF), which recruits and maintains intestinal inflammatory monocytes. ILCs present in the intestine also enter and exit cryptopatches in a highly dynamic process. During colitis, ILC3s mobilize from cryptopatches, a process that can be inhibited by blocking GM-CSF, and mobilization precedes inflammatory foci elsewhere in the tissue. Together these data identify the IL-23R/GM-CSF axis within ILC3 as a key control point in the accumulation of innate effector cells in the intestine and in the spatio-temporal dynamics of ILCs in the intestinal inflammatory response. PMID:26780670

  14. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces antiapoptotic and proapoptotic signals in acute myeloid leukemia.

    PubMed

    Faderl, Stefan; Harris, David; Van, Quin; Kantarjian, Hagop M; Talpaz, Moshe; Estrov, Zeev

    2003-07-15

    High levels of cytokines are associated with a poor prognosis in acute myeloid leukemia (AML). However, cytokines may induce, on one hand, survival factor expression and cell proliferation and, on the other hand, expression of inhibitory signals such as up-regulation of suppressors of cytokine signaling (SOCS) and induce apoptotic cell death. Because blasts from patients with AML express high procaspase protein levels, we asked whether granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances procaspase protein production in AML cells. In the GM-CSF-responsive OCIM2 AML cell line, GM-CSF induced signal transducer and activator of transcription 5 (Stat 5) phosphorylation, up-regulated cyclin D2, and stimulated cell cycle progression. Concurrently, GM-CSF stimulated expression of SOCS-2 and -3 and of procaspases 2 and 3 and induced caspase 3 activation, poly(ADP[adenosine 5'-diphosphate]-ribose) polymerase (PARP) cleavage, and apoptotic cell death. The Janus kinase (Jak)-Stat inhibitor AG490 abrogated GM-CSF-induced expression of procaspase 3 and activation of caspase 3. Under the same conditions GM-CSF up-regulated production of BAX as well as Bcl-2, Bcl-XL, survivin, and XIAP. GM-CSF also increased procaspase 3 protein levels in OCI/AML3 and Mo7e cells, suggesting that this phenomenon is not restricted to a single leukemia cell line. Our data suggest that GM-CSF exerts a dual effect: it stimulates cell division but contemporaneously up-regulates Jak-Stat-dependent proapoptotic proteins. Up-regulation of procaspase levels in AML is thus a beacon for an ongoing growth-stimulatory signal.

  15. Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by cDNA-transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF.

    PubMed

    Shi, F S; Weber, S; Gan, J; Rakhmilevich, A L; Mahvi, D M

    1999-01-01

    Clinical cancer gene therapy trials have generally focused on the transfer of cytokine cDNA to tumor cells ex vivo and with the subsequent vaccination of the patient with these genetically altered tumor cells. This approach results in high local cytokine concentrations that may account for the efficacy of this technique in animal models. We hypothesized that the expression of certain cytokines by tumor cells would be a superior immune stimulant when compared with local delivery of exogenous cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a nonviral expression vector was inserted into MDA-MB-231 (human breast cancer), M21 (human melanoma), B16 (murine melanoma), and P815 (mastocytoma) cells by particle-mediated gene transfer. The ability of transfected tumor cells to generate a tumor-specific immune response was evaluated in an in vitro mixed lymphocyte-tumor cell assay and in an in vivo murine tumor protection model. Peripheral blood lymphocytes cocultured with human GM-CSF-transfected tumor cells were 3- to 5-fold more effective at lysis of the parental tumor cells than were peripheral blood lymphocytes incubated with irradiated tumor cells and exogenous human GM-CSF. Mice immunized with murine GM-CSF-transfected irradiated B16 murine melanoma cells or P815 mastocytoma cells were protected from subsequent tumor challenge, whereas mice immunized with the nontransfected tumors and cutaneous transfection of murine GM-CSF cDNA at the vaccination site developed tumors more frequently. The results indicate that GM-CSF protein expressed in human and murine tumor cells is a superior antitumor immune stimulant compared with exogenous GM-CSF in the tumor microenvironment. PMID:10078967

  16. Properties of bcr-abl-transformed mouse 12B1 cells secreting interleukin-2 and granulocyte-macrophage colony stimulating factor (GM-CSF): II. Adverse effects of GM-CSF.

    PubMed

    Petráčková, Martina; Staněk, Libor; Mandys, Václav; Dundr, Pavel; Vonka, Vladimír

    2012-06-01

    Granulocyte-macrophage colony stimulating factor (GM-CSF) is considered to be the most effective immunostimulating factor for the construction of gene-engineered anti-cancer vaccines. In some tumour cells, this type of genetic modification has resulted in the loss of the oncogenic potential. This was not the case with bcr-abl-transformed mouse 12B1 cells. A cell line, designated 12B1/GM-CSF/cl-5 producing more than 100 ng/106 cells/24 h, displayed higher pathogenicity than the parental, non-transduced cells. Although the tumours induced by the parental 12B1 cells and 12B1/GM-CSF/cl-5 cells appeared nearly at the same time and then grew at an approximately equal rate, the latter mice were in a much poorer clinical condition. In these animals the growth of the tumours was associated with gradually increasing blood levels of GM-CSF. In both groups of animals splenomegaly was observed; it was much more pronounced in the case of 12B1/GM-CSF/cl-5-inoculated animals. While in the case of animals inoculated with the parental cells the splenomegaly was probably mainly due to infiltration with tumour cells, in the animals inoculated with the GM-CSF-secreting cells splenomegaly and derangement of parenchymal organs, such as lungs, liver and kidneys, were more complex, including congestion and infiltration with hemopoietic cells, predominantly immature cells of myeloid lineage. The most conspicuous of these changes was the hyperaemia of the lungs. No such alterations were seen in animals inoculated with the parental cells. On the other hand, the contents of T regulatory cells were comparable in both groups and they increased in parallel at the end of the observation period. When GM-CSF neutralizing antibody was administered to animals inoculated with the 12B1/GM-CSF/cl-5 cells, the pathological changes observed within the organs were suppressed, this proving that the overproduced GM-CSF and not any other substance, played the key role in their induction.

  17. Chimaeric Lym-1 monoclonal antibody-mediated cytolysis by neutrophils from G-CSF-treated patients: stimulation by GM-CSF and role of Fcγ-receptors

    PubMed Central

    Ottonello, L; Epstein, A L; Mancini, M; Tortolina, G; Dapino, P; Dallegri, F

    2001-01-01

    Chimaeric Lym-1 (chLym-1) is a monoclonal antibody generated by fusing the variable region genes of murine Lym-1 to human γ1 and κ constant regions. Owing to its selectivity and avidity for human malignant B cells, it is an attractive candidate for developing immune-interventions in B-lymphomas. In the attempt to identify rational bases for optimizing potential chLym-1 related therapeutic approaches, we studied the ability of this ch-mAb to trigger neutrophil-mediated Raji cell cytolysis in cooperation with two neutrophil-related cytokines, G-CSF and GM-CSF. ChLym-1 triggered low levels of cytolysis by normal neutrophils but induced consistent cytolysis in neutrophils from individuals treated with G-CSF. When exposed to GM-CSF, neutrophils from subjects treated with G-CSF became potent effectors, also leading to 75% lysis. By using mAbs specific for distinct FcγRs, normal neutrophils were inhibited by mAb IV.3, suggesting the intervention of FcγRII, constitutively expressed on the cells. On the other hand, neutrophils from patients treated with G-CSF were inhibited by mAb IV.3 plus mAb 197, a finding consistent with a cooperative intervention of FCγRII and G-CSF-induced FcγRI. The anti-FcγRIII mAb 3G8 promoted significant enhancement of the neutrophil cytolytic efficiency. Therefore, neutrophil FcγRIII behaves as a down-regulator of the cytolytic potential. The present findings suggest new attempts to develop mAb-based and G-CSF/GM-CSF combined immune-interventions in B lymphomas. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11487281

  18. Coadministration of cruzipain and GM-CSF DNAs, a new immunotherapeutic vaccine against Trypanosoma cruzi infection

    PubMed Central

    Cerny, Natacha; Sánchez Alberti, Andrés; Bivona, Augusto E; De Marzi, Mauricio C; Frank, Fernanda M; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-01-01

    Therapeutic vaccine research and development are especially important in Chagas disease considering the characteristics of the chronic infection and the number of people in the Americas living with a parasite infection for decades. We have previously reported the efficacy of attenuated Salmonella enterica (S) carrying plasmid encoding cruzipain (SCz) to protect against Trypanosoma cruzi infection. In the present work we investigated whether Cz DNA vaccine immunotherapy could be effective in controlling an ongoing T. cruzi infection in mice. We here report the intramuscular administration of naked Cz DNA or the oral administration of Salmonella as Cz DNA delivery system as therapeutic vaccines in mice during acute or chronic infection. The coadministration of a plasmid encoding GM-CSF improved vaccine performance, indicating that the stimulation of innate immune cells is needed in the event of an ongoing infection. These therapeutic vaccines were able to address the response to a protective and sustained Th1 biased profile not only against Cz but also against a variety of parasite antigens. The combined therapeutic vaccine during the chronic phase of infection prevents tissue pathology as shown by a reduced level of enzyme activity characteristic of tissue damage and a tissue status compatible with normal tissue. The obtained results suggest that immunotherapy with Cz and GM-CSF DNAs, either alone or in combination with other drug treatments, may represent a promising alternative for Chagas disease therapy. PMID:26312947

  19. Coadministration of cruzipain and GM-CSF DNAs, a new immunotherapeutic vaccine against Trypanosoma cruzi infection.

    PubMed

    Cerny, Natacha; Sánchez Alberti, Andrés; Bivona, Augusto E; De Marzi, Mauricio C; Frank, Fernanda M; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-01-01

    Therapeutic vaccine research and development are especially important in Chagas disease considering the characteristics of the chronic infection and the number of people in the Americas living with a parasite infection for decades. We have previously reported the efficacy of attenuated Salmonella enterica (S) carrying plasmid encoding cruzipain (SCz) to protect against Trypanosoma cruzi infection. In the present work we investigated whether Cz DNA vaccine immunotherapy could be effective in controlling an ongoing T. cruzi infection in mice. We here report the intramuscular administration of naked Cz DNA or the oral administration of Salmonella as Cz DNA delivery system as therapeutic vaccines in mice during acute or chronic infection. The coadministration of a plasmid encoding GM-CSF improved vaccine performance, indicating that the stimulation of innate immune cells is needed in the event of an ongoing infection. These therapeutic vaccines were able to address the response to a protective and sustained Th1 biased profile not only against Cz but also against a variety of parasite antigens. The combined therapeutic vaccine during the chronic phase of infection prevents tissue pathology as shown by a reduced level of enzyme activity characteristic of tissue damage and a tissue status compatible with normal tissue. The obtained results suggest that immunotherapy with Cz and GM-CSF DNAs, either alone or in combination with other drug treatments, may represent a promising alternative for Chagas disease therapy. PMID:26312947

  20. Coadministration of cruzipain and GM-CSF DNAs, a new immunotherapeutic vaccine against Trypanosoma cruzi infection.

    PubMed

    Cerny, Natacha; Sánchez Alberti, Andrés; Bivona, Augusto E; De Marzi, Mauricio C; Frank, Fernanda M; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-01-01

    Therapeutic vaccine research and development are especially important in Chagas disease considering the characteristics of the chronic infection and the number of people in the Americas living with a parasite infection for decades. We have previously reported the efficacy of attenuated Salmonella enterica (S) carrying plasmid encoding cruzipain (SCz) to protect against Trypanosoma cruzi infection. In the present work we investigated whether Cz DNA vaccine immunotherapy could be effective in controlling an ongoing T. cruzi infection in mice. We here report the intramuscular administration of naked Cz DNA or the oral administration of Salmonella as Cz DNA delivery system as therapeutic vaccines in mice during acute or chronic infection. The coadministration of a plasmid encoding GM-CSF improved vaccine performance, indicating that the stimulation of innate immune cells is needed in the event of an ongoing infection. These therapeutic vaccines were able to address the response to a protective and sustained Th1 biased profile not only against Cz but also against a variety of parasite antigens. The combined therapeutic vaccine during the chronic phase of infection prevents tissue pathology as shown by a reduced level of enzyme activity characteristic of tissue damage and a tissue status compatible with normal tissue. The obtained results suggest that immunotherapy with Cz and GM-CSF DNAs, either alone or in combination with other drug treatments, may represent a promising alternative for Chagas disease therapy.

  1. GM-CSF Down-Regulates TLR Expression via the Transcription Factor PU.1 in Human Monocytes

    PubMed Central

    Sadeghi, Kambis; Wisgrill, Lukas; Wessely, Isabelle; Diesner, Susanne C.; Schüller, Simone; Dürr, Celia; Heinle, Armando; Sachet, Monika; Pollak, Arnold; Förster-Waldl, Elisabeth; Spittler, Andreas

    2016-01-01

    Toll-like receptors (TLR) are crucial sensors of microbial agents such as bacterial or viral compounds. These receptors constitute key players in the induction of inflammation, e.g. in septic or chronic inflammatory diseases. Colony-stimulating factors (CSFs) such as granulocyte-macrophage-CSF (GM-CSF) or granulocyte-CSF (G-CSF) have been extensively investigated in their capacity to promote myelopoiesis in febrile neutropenia or to overcome immunosuppression in patients suffering from sepsis-associated neutropenia or from monocytic immunoincompetence. We report here that GM-CSF, downregulates TLR1, TLR2 and TLR4 in a time- and dose-dependent fashion in human monocytes. Diminished pathogen recognition receptor expression was accompanied by reduced downstream p38 and extracellular-signal-regulated kinase (ERK) signaling upon lipoteichoic acid (LTA) and lipopolysaccharide (LPS) binding—and accordingly led to impaired proinflammatory cytokine production. Knockdown experiments of the transcription factors PU.1 and VentX showed that GM-CSF driven effects on TLR regulation is entirely PU.1 but not VentX dependent. We further analysed monocyte TLR and CD14 expression upon exposure to the IMID® immunomodulatory drug Pomalidomide (CC-4047), a Thalidomide analogue known to downregulate PU.1. Indeed, Pomalidomide in part reversed the GM-CSF-mediated effects. Our data indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. The PU.1 mediated immunomodulatory properties of GM-CSF should be taken into consideration upon usage of GM-CSF in inflammatory or infection-related conditions. PMID:27695085

  2. Lentivirus-ABCG1 instillation reduces lipid accumulation and improves lung compliance in GM-CSF knock-out mice

    SciTech Connect

    Malur, Anagha; Huizar, Isham; Wells, Greg; Barna, Barbara P.; Malur, Achut G.; Thomassen, Mary Jane

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Lentivirus-ABCG1 reduces lipid accumulation in lungs of GM-CSF knock-out mice. Black-Right-Pointing-Pointer Up-regulation of ABCG1 improves lung function. Black-Right-Pointing-Pointer Upregulation of ABCG1 improves surfactant metabolism. -- Abstract: We have shown decreased expression of the nuclear transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR{gamma}) and the PPAR{gamma}-regulated ATP-binding cassette transporter G1 (ABCG1) in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP). PAP patients also exhibit neutralizing antibodies to granulocyte-macrophage colony stimulating factor (GM-CSF), an upregulator of PPAR{gamma}. In association with functional GM-CSF deficiency, PAP lung is characterized by surfactant-filled alveolar spaces and lipid-filled alveolar macrophages. Similar pathology characterizes GM-CSF knock-out (KO) mice. We reported previously that intratracheal instillation of a lentivirus (lenti)-PPAR{gamma} plasmid into GM-CSF KO animals elevated ABCG1 and reduced alveolar macrophage lipid accumulation. Here, we hypothesized that instillation of lenti-ABCG1 might be sufficient to decrease lipid accumulation and improve pulmonary function in GM-CSF KO mice. Animals received intratracheal instillation of lenti-ABCG1 or control lenti-enhanced Green Fluorescent Protein (eGFP) plasmids and alveolar macrophages were harvested 10 days later. Alveolar macrophage transduction efficiency was 79% as shown by lenti-eGFP fluorescence. Quantitative PCR analyses indicated a threefold (p = 0.0005) increase in ABCG1 expression with no change of PPAR{gamma} or ABCA1 in alveolar macrophages of lenti-ABCG1 treated mice. ABCG1 was unchanged in control lenti-eGFP and PBS-instilled groups. Oil Red O staining detected reduced intracellular neutral lipid in alveolar macrophages from lenti-ABCG1 treated mice. Extracellular cholesterol and phospholipids were also decreased as shown by

  3. IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

    SciTech Connect

    Li, Yan; Ohms, Stephen J.; Shannon, Frances M.; Sun, Chao; Fan, Jun Y.

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer DNA methylation is dynamic and flexible and changes rapidly upon cell activation. Black-Right-Pointing-Pointer DNA methylation controls the inducible gene expression in a given cell type. Black-Right-Pointing-Pointer Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

  4. Interleukin-4 enhances trafficking and functional activities of GM-CSF-stimulated mouse myeloid-derived dendritic cells at late differentiation stage

    SciTech Connect

    Yin, Shu-Yi; Wang, Chien-Yu; Yang, Ning-Sun

    2011-09-10

    Mouse bone marrow-derived dendritic cells (BMDCs) are being employed as an important model for translational research into the development of DC-based therapeutics. For such use, the localization and specialized mobility of injected BMDCs within specific immune tissues are known to define their immunity and usefulness in vivo. In this study, we demonstrate that IL-4, a key driving factor for in vitro propagation and differentiation of BMDCs, when added during a late culture stage can enhance the in vivo trafficking activity of granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced BMDCs. It suggests that the temporal control of IL-4 stimulation during the in vitro generation of DCs drastically affects the DC trafficking efficiency in vivo. With this modification of IL-4 stimulation, we also show that much less cytokine was needed to generate BMDCs with high purity and yield that secrete a high level of cytokines and possess a good capacity to induce proliferation of allogeneic CD4{sup +}T cells, as compared to the conventional method that uses a continuous supplement of GM-CSF and IL-4 throughout cultivation. These results provide us with an important know-how for differentiation of BMDCs from myeloid stem cells, and for use of other immune cells in related medical or stem cell applications.

  5. NF-κB and BRG1 bind a distal regulatory element in the IL-3/GM-CSF locus.

    PubMed

    Wurster, Andrea L; Precht, Patricia; Pazin, Michael J

    2011-09-01

    We investigated gene regulation at the IL-3/GM-CSF gene cluster. We found BRG1, a SWI/SNF remodeling ATPase, bound a distal element, CNSa. BRG1 binding was strongest in differentiated, stimulated T helper cells, paralleling IL-3 and GM-CSF expression. Depletion of BRG1 reduced IL-3 and GM-CSF transcription. BAF-specific SWI/SNF subunits bound to this locus and regulated IL-3 expression. CNSa was in closed chromatin in fibroblasts, open chromatin in differentiated T helper cells, and moderately open chromatin in naïve (undifferentiated) T helper cells; BRG1 was required for the most open state. CNSa increased transcription of a reporter in an episomal expression system, in a BRG1-dependent manner. The NF-κB subunit RelA/p65 bound CNSa in activated T helper cells. Inhibition of NF-κB blocked BRG1 binding to CNSa, chromatin opening at CNSa, and activation of IL-3 and GM-CSF. Together, these findings suggest CNSa is a distal enhancer that binds BRG1 and NF-κB.

  6. NF-κB and BRG1 bind a distal regulatory element in the IL-3/GM-CSF locus

    PubMed Central

    Wurster, Andrea L.; Precht, Patricia; Pazin, Michael J.

    2011-01-01

    We investigated gene regulation at the IL-3/GM-CSF gene cluster. We found BRG1, a SWI/SNF remodeling ATPase, bound a distal element, CNSa. BRG1 binding was strongest in differentiated, stimulated T helper cells, paralleling IL-3 and GM-CSF expression. Depletion of BRG1 reduced IL-3 and GM-CSF transcription. BAF-specific SWI/SNF subunits bound to this locus and regulated IL-3 expression. CNSa was in closed chromatin in fibroblasts, open chromatin in differentiated T helper cells, and moderately open chromatin in naïve (undifferentiated) T helper cells; BRG1 was required for the most open state. CNSa increased transcription of a reporter in an episomal expression system, in a BRG1-dependent manner. The NF-κB subunit RelA/p65 bound CNSa in activated T helper cells. Inhibition of NF-κB blocked BRG1 binding to CNSa, chromatin opening at CNSa, and activation of IL-3 and GM-CSF. Together, these findings suggest CNSa is a distal enhancer that binds BRG1 and NF-κB. PMID:21831442

  7. Comparative Antitumor Effect of Preventive versus Therapeutic Vaccines Employing B16 Melanoma Cells Genetically Modified to Express GM-CSF and B7.2 in a Murine Model

    PubMed Central

    Miguel, Antonio; Herrero, María José; Sendra, Luis; Botella, Rafael; Algás, Rosa; Sánchez, Maria; Aliño, Salvador F.

    2012-01-01

    Cancer vaccines have always been a subject of gene therapy research. One of the most successful approaches has been working with genetically modified tumor cells. In this study, we describe our approach to achieving an immune response against a murine melanoma model, employing B16 tumor cells expressing GM-CSF and B7.2. Wild B16 cells were injected in C57BL6 mice to cause the tumor. Irradiated B16 cells transfected with GM-CSF, B7.2, or both, were processed as a preventive and therapeutic vaccination. Tumor volumes were measured and survival curves were obtained. Blood samples were taken from mice, and IgGs of each treatment group were also measured. The regulatory T cells (Treg) of selected groups were quantified using counts of images taken by confocal microscopy. Results: one hundred percent survival was achieved by preventive vaccination with the group of cells transfected with p2F_GM-CSF. Therapeutic vaccination achieved initial inhibition of tumor growth but did not secure overall survival of the animals. Classical Treg cells did not vary among the different groups in this therapeutic vaccination model. PMID:23202306

  8. Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); confirmation of the GM-CSF amino acid sequence by mass spectrometry.

    PubMed Central

    Tsarbopoulos, A.; Pramanik, B. N.; Labdon, J. E.; Reichert, P.; Gitlin, G.; Patel, S.; Sardana, V.; Nagabhushan, T. L.; Trotta, P. P.

    1993-01-01

    A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol. PMID:8268804

  9. Alternaria Fungus Induces the Production of GM-CSF, Interleukin-6 and Interleukin-8 and Calcium Signaling in Human Airway Epithelium through Protease-Activated Receptor 2

    PubMed Central

    Matsuwaki, Yoshinori; Wada, Kota; White, Thomas; Moriyama, Hiroshi; Kita, Hirohito

    2012-01-01

    Rationale Recent studies suggest that host immune responses to environmental fungi may play an important role in the development of allergic diseases, such as human asthma. Epithelium is considered an active participant in allergic inflammation. We previously reported that aspartate protease from Alternaria induces the activation and degranulation of human eosinophils that are mediated through protease-activated receptor 2 (PAR-2). However, our current knowledge on the innate immune responses of epithelium to environmental fungi is very limited. We investigated the responses of epithelium to fungi and the mechanisms of these responses. Methods Human airway epithelial cell line BEAS-2B and Calu-3 (both from American Type Culture Collection) were incubated with PAR-2 peptides and extracts of various fungi. The cellular responses, including GM-CSF, interleukin (IL)-6, IL-8, eotaxin, eotaxin-2 and RANTES production as well as increases in intracellular calcium concentration ([Ca2+]i), were examined. To characterize the proteases involved in these responses, protease inhibitors such as pepstatin A and alkalo-thermophilic Bacillus inhibitor (ATBI), HIV protease inhibitors and 4-amidinophenylmethanesulfonyl fluoride hydrochloride were used. To investigate the role of PAR-2, PAR-2-agonistic and PAR-2-antagonistic peptides were used. Results PAR-2-activating peptide, but not the control peptide, induced GM-CSF, IL-6 and IL-8 production; these cellular responses were accompanied by a quick and marked increase in [Ca2+]i. Among 7 common environmental fungi, only Alternaria induced GM-CSF, IL-6 and IL-8 production and increased [Ca2+]i response. Both cytokine production and increased [Ca2+]i were significantly inhibited by PAR-2 antagonist peptide and by aspartate protease inhibitors (pepstatin A, ritonavir, nelfinavir and ATBI), but not by the PAR-2 control peptide or by other protease inhibitors. Conclusions Aspartate proteases from Alternaria induce cytokine production and

  10. Crystallization and preliminary X-ray diffraction analysis of the ternary human GM-CSF receptor complex

    SciTech Connect

    Hansen, Guido; Hercus, Timothy R.; Xu, Yibin; Lopez, Angel F.; Parker, Michael W.; McKinstry, William J.

    2008-07-28

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a haemopoietic growth factor that acts through a ternary receptor signalling complex containing specific {alpha} (GMR{alpha}) and common {beta} ({beta}c) receptor subunits. Human GM-CSF is encoded by the gene csf2, while the genes for GMR{alpha} and {beta}c are csf2ra and csf2rb, respectively. Crystals of the ternary ectodomain complex comprising GM-CSF and the soluble extracellular regions of both the GMR{alpha} subunit and either {beta}c or its glutamine-substitution mutant N346Q were obtained using the hanging-drop vapour-diffusion method. The best diffracting crystals of the ternary complex were obtained using the N346Q mutation of the {beta}c subunit. These crystals grew using polyethylene glycol 3350 with a high concentration of proline, belonged to space group P6{sub 3}22 and diffracted to 3.3 {angstrom} resolution.

  11. CD1d(hi)CD5+ B cells expanded by GM-CSF in vivo suppress experimental autoimmune myasthenia gravis.

    PubMed

    Sheng, Jian Rong; Quan, Songhua; Soliven, Betty

    2014-09-15

    IL-10-competent subset within CD1d(hi)CD5(+) B cells, also known as B10 cells, has been shown to regulate autoimmune diseases. Whether B10 cells can prevent or suppress the development of experimental autoimmune myasthenia gravis (EAMG) has not been studied. In this study, we investigated whether low-dose GM-CSF, which suppresses EAMG, can expand B10 cells in vivo, and whether adoptive transfer of CD1d(hi)CD5(+) B cells would prevent or suppress EAMG. We found that treatment of EAMG mice with low-dose GM-CSF increased the proportion of CD1d(hi)CD5(+) B cells and B10 cells. In vitro coculture studies revealed that CD1d(hi)CD5(+) B cells altered T cell cytokine profile but did not directly inhibit T cell proliferation. In contrast, CD1d(hi)CD5(+) B cells inhibited B cell proliferation and its autoantibody production in an IL-10-dependent manner. Adoptive transfer of CD1d(hi)CD5(+) B cells to mice could prevent disease, as well as suppress EAMG after disease onset. This was associated with downregulation of mature dendritic cell markers and expansion of regulatory T cells resulting in the suppression of acetylcholine receptor-specific T cell and B cell responses. Thus, our data have provided significant insight into the mechanisms underlying the tolerogenic effects of B10 cells in EAMG. These observations suggest that in vivo or in vitro expansion of CD1d(hi)CD5(+) B cells or B10 cells may represent an effective strategy in the treatment of human myasthenia gravis.

  12. Dissecting the roles of endothelin, TGF-beta and GM-CSF on myofibroblast differentiation by keratinocytes.

    PubMed

    Shephard, Pierre; Hinz, Boris; Smola-Hess, Sigrun; Meister, Jean-Jacques; Krieg, Thomas; Smola, Hans

    2004-08-01

    Myofibroblasts are specialized fibroblasts that contribute to wound healing by producing extracellular matrix and by contracting the granulation tissue. They appear in a phase of wound healing when the dermis strongly interacts with activated epidermal keratinocytes. Direct co-culture with keratinocytes upregulates TGFbeta activity and also induces fibroblast to differentiate into alpha-smooth muscle actin (alphaSMA)-positive myofibroblasts. TGF-beta activity alone cannot completely account for alphaSMA induction in these co-cultures, and here we analyze mechanical force generation, another potent inducer of myofibroblast differentiation in this model. Using deformable silicone substrates, we show that contractile activity of fibroblasts is already induced after 1-2-days of co-culture, when fibroblasts are generally alphaSMA negative. Endothelin-1 (ET-1), the most potent inducer of smooth muscle cell contraction, was up-regulated in co-cultures, while blocking ET-1 with the ET receptor inhibitor PD156252 inhibited contraction in these early co-cultures. In 4-5 days of co-culture, however, fibroblast contractile activity correlated with an increased expression of alphaSMA expression. Stimulation of fibroblast mono-cultures with ET-1 in a low serum medium did not induce alphaSMA expression; however, ET-1 did synergize with TGF-beta. Surprisingly, GM-CSF, another mediatorstimulating myofibroblast differentiation in granulation tissue, inhibited alphaSMA expression in fibroblasts, costimulated with TGF-beta and ET-1. GM-CSF activated NFkappaB, thus interfering with TGF-beta signaling. Blocking TGFbeta and ET-1 largely impaired alphaSMA induction in co-cultures at day 7 and, in combination, almost completely prevented alphaSMA induction. Our results dissect the roles of TGF-beta and ET-1 on mechanical force generation in keratinocyte-fibroblast co-cultures, and identify GM-CSF as an inducer of myofibroblasts acting indirectly.

  13. Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells

    PubMed Central

    Ohoka, Yoshiharu; Yokota-Nakatsuma, Aya; Maeda, Naoko; Takeuchi, Hajime; Iwata, Makoto

    2014-01-01

    Retinoic acid (RA)-producing dendritic cells (DCs) play critical roles in gut immunity. Retinal dehydrogenase 2 (RALDH2) encoded by Aldh1a2 is a key enzyme for generating RA in DCs. Granulocyte–macrophage colony-stimulating factor (GM-CSF) potently induces RALDH2 expression in DCs in an RA-dependent manner, and RA alone weakly induces the expression. However, how GM-CSF and RA induce RALDH2 expression remains unclear. Here, we show that GM-CSF-induced activation of the transcription factor Sp1 and RA-dependent signaling via the RA receptor (RAR)/retinoid X receptor (RXR) complex contribute to Aldh1a2 expression. The RAR antagonist LE540 and the Sp1 inhibitor mithramycin A inhibited GM-CSF-induced Aldh1a2 expression in fms-related tyrosine kinase 3 ligand-generated bone marrow-derived DCs (BM-DCs). ERK and p38 MAPK inhibitors suppressed GM-CSF-induced nuclear translocation of Sp1 and Aldh1a2 expression. Sp1 and the RARα/RXRα complex bound to GC-rich Sp1-binding sites and an RA response element (RARE) half-site, respectively, near the TATA box in the mouse Aldh1a2 promoter. The DNA sequences around these sites were highly conserved among different species. In the presence of RA, ectopic expression of RARα/RXRα and Sp1 synergistically enhanced Aldh1a2 promoter-reporter activity. GM-CSF did not significantly induce Aldh1a2 expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the Aldh1a2 promoter in these cells was mostly unmethylated. These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the Aldh1a2 promoter, and can be regulated by a DNA methylation-independent mechanism. PMID:24788806

  14. Synovial CD4+ T-cell-derived GM-CSF supports the differentiation of an inflammatory dendritic cell population in rheumatoid arthritis

    PubMed Central

    Reynolds, G; Gibbon, J R; Pratt, A G; Wood, M J; Coady, D; Raftery, G; Lorenzi, A R; Gray, A; Filer, A; Buckley, C D; Haniffa, M A; Isaacs, J D; Hilkens, C M U

    2016-01-01

    Objective A population of synovial inflammatory dendritic cells (infDCs) has recently been identified in rheumatoid arthritis (RA) and is thought to be monocyte-derived. Here, we investigated the role and source of granulocyte macrophage-colony-stimulating factor (GM-CSF) in the differentiation of synovial infDC in RA. Methods Production of GM-CSF by peripheral blood (PB) and synovial fluid (SF) CD4+ T cells was assessed by ELISA and flow cytometry. In vitro CD4+ T-cell polarisation experiments were performed with T-cell activating CD2/CD3/CD28-coated beads in the absence or presence of pro-Th1 or pro-Th17 cytokines. CD1c+ DC and CD16+ macrophage subsets were flow-sorted and analysed morphologically and functionally (T-cell stimulatory/polarising capacity). Results RA-SF CD4+ T cells produced abundant GM-CSF upon stimulation and significantly more than RA-SF mononuclear cells depleted of CD4+ T cells. GM-CSF-producing T cells were significantly increased in RA-SF compared with non-RA inflammatory arthritis SF, active RA PB and healthy donor PB. GM-CSF-producing CD4+ T cells were expanded by Th1-promoting but not Th17-promoting conditions. Following coculture with RA-SF CD4+ T cells, but not healthy donor PB CD4+ T cells, a subpopulation of monocytes differentiated into CD1c+ infDC; a process dependent on GM-CSF. These infDC displayed potent alloproliferative capacity and enhanced GM-CSF, interleukin-17 and interferon-γ production by CD4+ T cells. InfDC with an identical phenotype to in vitro generated cells were significantly enriched in RA-SF compared with non-RA-SF/tissue/PB. Conclusions We demonstrate a therapeutically tractable feedback loop of GM-CSF secreted by RA synovial CD4+ T cells promoting the differentiation of infDC with potent capacity to induce GM-CSF-producing CD4+ T cells. PMID:25923217

  15. Summary of bi-shRNA/GM-CSF augmented autologous tumor cell immunotherapy (FANG™) in advanced cancer of the liver.

    PubMed

    Nemunaitis, John; Barve, Minal; Orr, Douglas; Kuhn, Joseph; Magee, Mitchell; Lamont, Jeffrey; Bedell, Cynthia; Wallraven, Gladice; Pappen, Beena O; Roth, Alyssa; Horvath, Staci; Nemunaitis, Derek; Kumar, Padmasini; Maples, Phillip B; Senzer, Neil

    2014-01-01

    Therapies for advanced hepatocellular carcinoma (HCC) are limited. We carried out a phase I trial of a novel autologous whole-cell tumor cell immunotherapy (FANG™), which incorporates a dual granulocyte macrophage colony-stimulating factor (GM-CSF) expressive/bifunctional small hairpin RNA interference (bi-shRNAi) vector. The bi-shRNAi DNA targets furin, which is a proconvertase of transforming growth factors beta (TGFβ) 1 and 2. Safety, mechanism, immunoeffectiveness, and suggested benefit were previously shown [Senzer et al.: Mol Ther 2012;20:679-689; Senzer et al.: J Vaccines Vaccin 2013;4:209]. We now provide further follow-up of a subset of 8 HCC patients. FANG manufacturing was successful in 7 of 8 attempts (one failure due to insufficient cell yield). Median GM-CSF expression was 144 pg/10(6) cells, TGFβ1 knockdown was 100%, and TGFβ2 knockdown was 93% of the vector-transported cells. Five patients were vaccinated (1 or 2.5×10(7) cells/intradermal injection, 6-11 vaccinations). No FANG toxicity was observed. Three of these patients demonstrated evidence of an immune response to the autologous tumor cell sample. Long-term follow-up demonstrated survival of 319, 729, 784, 931+, and 1,043+ days of the FANG-treated patients. In conclusion, evidence supports further assessment of the FANG immunotherapy in HCC. PMID:24968881

  16. Summary of bi-shRNA/GM-CSF augmented autologous tumor cell immunotherapy (FANG™) in advanced cancer of the liver.

    PubMed

    Nemunaitis, John; Barve, Minal; Orr, Douglas; Kuhn, Joseph; Magee, Mitchell; Lamont, Jeffrey; Bedell, Cynthia; Wallraven, Gladice; Pappen, Beena O; Roth, Alyssa; Horvath, Staci; Nemunaitis, Derek; Kumar, Padmasini; Maples, Phillip B; Senzer, Neil

    2014-01-01

    Therapies for advanced hepatocellular carcinoma (HCC) are limited. We carried out a phase I trial of a novel autologous whole-cell tumor cell immunotherapy (FANG™), which incorporates a dual granulocyte macrophage colony-stimulating factor (GM-CSF) expressive/bifunctional small hairpin RNA interference (bi-shRNAi) vector. The bi-shRNAi DNA targets furin, which is a proconvertase of transforming growth factors beta (TGFβ) 1 and 2. Safety, mechanism, immunoeffectiveness, and suggested benefit were previously shown [Senzer et al.: Mol Ther 2012;20:679-689; Senzer et al.: J Vaccines Vaccin 2013;4:209]. We now provide further follow-up of a subset of 8 HCC patients. FANG manufacturing was successful in 7 of 8 attempts (one failure due to insufficient cell yield). Median GM-CSF expression was 144 pg/10(6) cells, TGFβ1 knockdown was 100%, and TGFβ2 knockdown was 93% of the vector-transported cells. Five patients were vaccinated (1 or 2.5×10(7) cells/intradermal injection, 6-11 vaccinations). No FANG toxicity was observed. Three of these patients demonstrated evidence of an immune response to the autologous tumor cell sample. Long-term follow-up demonstrated survival of 319, 729, 784, 931+, and 1,043+ days of the FANG-treated patients. In conclusion, evidence supports further assessment of the FANG immunotherapy in HCC.

  17. Activation of adenosine A(3) receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells.

    PubMed

    Hofer, M; Vacek, A; Pospísil, M; Holá, J; Streitová, D; Znojil, V

    2009-01-01

    Adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been tested from the point of view of potentiating the effects of hematopoietic growth factors interleukin-3 (IL-3), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) on the growth of hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in suspension of normal mouse bone marrow cells in vitro. IB-MECA alone induced no GM-CFC growth. Significant elevation of numbers of GM-CFC evoked by the combinations of IB-MECA with IL-3, SCF, or GM-CSF as compared with these growth factors alone has been noted. Combination of IB-MECA with G-CSF did not induce significantly higher numbers of GM-CFC in comparison with G-CSF alone. Joint action of three drugs, namely of IB-MECA + IL-3 + GM-CSF, produced significantly higher numbers of GM-CFC in comparison with the combinations of IB-MECA + IL-3, IB-MECA + GM-CSF, or IL-3 + GM-CSF. These results give evidence of a significant role of selective activation of adenosine A(3) receptors in stimulation of the growth of granulocyte/ macrophage hematopoietic progenitor cells.

  18. Cyclic AMP-elevating agents down-regulate the oxidative burst induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in adherent neutrophils.

    PubMed Central

    Ottonello, L; Morone, M P; Dapino, P; Dallegri, F

    1995-01-01

    Human neutrophils, plated on fibronectin-precoated wells, were found to release large quantities of superoxide anion (O2-) in response to GM-CSF. O2- production was reduced by prostaglandin E2 (PGE2) and the phosphodiesterase type IV (PDE IV) inhibitor RO 20-1724. Both agents are known to increase intracellular cyclic AMP (cAMP) levels by inducing its production (PGE2) or blocking its catabolism (RO 20-1724). When added in combination, PGE2 and RO 20-1724 had a marked synergistic inhibitory effect, which was reproduced by replacing PGE2 with a direct activator of adenylate cyclase, i.e. forskolin (FK). Moreover, the neutrophil response to GM-CSF was inhibited by a membrane-permeable analogue of cAMP in a dose-dependent manner. As GM-CSF and PGE2 are known to be generated at tissue sites of inflammation, the results suggest the existence of a PGE2-dependent regulatory pathway potentially capable of controlling the neutrophil response to GM-CSF, in turn limiting the risk of local oxidative tissue injury. Moreover, owing to its susceptibility to amplification by RO 20-1724, the PGE2-dependent pathway and in particular PDE-IV may represent a pharmacological target to reduce the generation of histotoxic oxidants by GM-CSF-responding neutrophils. PMID:7664497

  19. Melanoma exosome induction of endothelial cell GM-CSF in pre-metastatic lymph nodes may result in different M1 and M2 macrophage mediated angiogenic processes.

    PubMed

    Hood, Joshua L

    2016-09-01

    Angiogenesis is a key process in the preparation of lymph nodes for melanoma metastasis. Granulocyte macrophage colony stimulating factor (GM-CSF) induces hypoxia inducible factor 1 alpha (HIF-1α) in M1 or HIF-2α in M2 polarized macrophages. HIF-1α promotes neoangiogenesis while HIF-2α facilitates morphogenic normalization of neovasculature. Melanoma exosomes induce GM-CSF expression by endothelial cells in vitro and HIF-1α expression in pre-metastatic lymph nodes in vivo. This suggest a relationship between melanoma exosome induced endothelial GM-CSF and macrophage mediated angiogenesis in lymph nodes. Theoretically, induction of endothelial cell derived GM-CSF by melanoma exosomes mediates different angiogenic functions in pre-metastatic lymph nodes depending on subcapsular sinus (SCS) macrophage polarity. To explore this hypothesis, experiments utilizing melanoma exosomes in a lymph node model are outlined. Despite their opposing immune functions, indirect melanoma exosome stimulation of M1 or M2 SCS macrophages via endothelial derived GM-CSF in lymph nodes may induce different although complementary pro-tumor angiogenic processes. PMID:27515216

  20. A randomized, placebo-controlled trial of subcutaneous administration of GM-CSF as a vaccine adjuvant: effect on cellular and humoral immune responses.

    PubMed

    Somani, Jyoti; Lonial, Sagar; Rosenthal, Hilary; Resnick, Suzanne; Kakhniashvili, Irina; Waller, Edmund K

    2002-12-13

    Thirty healthy volunteers were randomly assigned to receive either a single subcutaneous injection of GM-CSF or placebo at the time of vaccination with tetanus and diptheria toxoid (Td), influenza and hepatitis A vaccines. Humoral response was measured by weekly serum samples assayed for antibodies to tetanus toxoid (TT), influenza and hepatitis A; while cellular response to TT was determined by measuring IL-2 expression in T-cells following in vitro exposure to TT antigen using a flow cytometric assay. It was hypothesized that (1). GM-CSF would augment immune response and (2). that the frequencies of TT responsive T-cells in the blood would predict humoral responses. The administration of subcutaneous GM-CSF as an adjuvant at the time of vaccination did not augment the antibody responses to influenza or hepatitis A in normal volunteers when compared to placebo. Subjects who received GM-CSF had statistically significant lower increases in anti-tetanus antibodies than placebo recipients. Immunization with TT resulted in an increase in the frequency of antigen responsive T-cells in the blood over time. The frequencies of TT responsive T-cells in baseline blood samples were correlated with baseline anti-tetanus antibody titers, but humoral and cellular responses were not correlated following vaccination. Recipients of GM-CSF did not develop significantly higher numbers of TT responsive T-cells after vaccination compared to recipients who received placebo.

  1. Expression of IL-1β, IL-2, IL-10, TNF-β and GM-CSF in peripheral blood leukocytes of rabbits experimentally infected with rabbit haemorrhagic disease virus.

    PubMed

    Trzeciak-Ryczek, Alicja; Tokarz-Deptuła, Beata; Deptuła, Wiesław

    2016-04-15

    Rabbit haemorrhagic disease (RHD) is a highly morbid and mortal viral infection of European rabbits. This disease is one of the main causes of death in wild rabbits, and results in large economic losses in farms of rabbits worldwide. Although the first outbreak of this disease was noted in 1984, the pathogenesis of RHD and mechanisms of RHDV (rabbit haemorrhagic disease virus) pathogenecity have still not been fully elucidated. Recent studies indicate a role of the immune response, especially peripheral blood leukocytes (PBL), in the pathogenesis of this disease. Thus, in the present study we investigated the expression of IL-1β, IL-2, IL-10, TNF-β and GM-CSF genes in PBL of RHDV-infected rabbits. We also compared the expression of genes encoding these cytokines in rabbits with different course of RHDV infection (in animals that died 36h postinfection or survived until 60th h after infection). The study revealed that three (IL-10, TNF-β and GM-CSF) out of five investigated genes encoding cytokines showed increased expression in PBL of RHDV-infected rabbits, and the level of expression depended on the course of RHD. The results indicate the potential role of these cytokines in RHDV infection and their influence on the survival time of infected rabbits.

  2. Expression, crystallization and derivatization of the complete extracellular domain of the beta(c) subunit of the human IL-5, IL-3 and GM-CSF receptors.

    PubMed

    Gustin, S E; Church, A P; Ford, S C; Mann, D A; Carr, P D; Ollis, D L; Young, I G

    2001-05-01

    The major signalling entity of the receptors for the haemopoietic cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is the shared beta(c) receptor, which is activated by ligand-specific alpha receptors. The beta(c) subunit is a stable homodimer whose extracellular region consists of four fibronectin domains and appears to be a duplication of the cytokine receptor homology module. No four domain structure has been determined for this receptor family and the structure of the beta(c) subunit remains unknown. We have expressed the extracellular domain in insect cells using the baculovirus system, purified it to homogeneity and determined its N-terminal sequence. N-glycosylation at two sites was demonstrated. Crystals of the complete domain have been obtained that are suitable for X-ray crystallographic studies, following mutagenesis to remove one of the N-glycosylation sites. The rhombohedral crystals of space group R3, with unit cell dimensions 186.1 A and 103.5 A, diffracted to a resolution of 2.9 A using synchrotron radiation. Mutagenesis was also used to engineer cysteine substitution mutants which formed isomorphous Hg derivatives in order to solve the crystallographic phase problem. The crystal structure will help to elucidate how the beta(c) receptor is activated by heterodimerization with the respective alpha/ligand complexes.

  3. Absence of LTB4/BLT1 axis facilitates generation of mouse GM-CSF-induced long-lasting antitumor immunologic memory by enhancing innate and adaptive immune systems.

    PubMed

    Yokota, Yosuke; Inoue, Hiroyuki; Matsumura, Yumiko; Nabeta, Haruka; Narusawa, Megumi; Watanabe, Ayumi; Sakamoto, Chika; Hijikata, Yasuki; Iga-Murahashi, Mutsunori; Takayama, Koichi; Sasaki, Fumiyuki; Nakanishi, Yoichi; Yokomizo, Takehiko; Tani, Kenzaburo

    2012-10-25

    BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF-induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4(+) T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF-sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4(+) T subsets and increasing numbers of Th17 and memory CD44(hi)CD4(+) T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4(+) T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF-induced antitumor memory CD4(+) T cells.

  4. Rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3L exhibit distinct phenotypical and functional characteristics.

    PubMed

    N'diaye, Marie; Warnecke, Andreas; Flytzani, Sevasti; Abdelmagid, Nada; Ruhrmann, Sabrina; Olsson, Tomas; Jagodic, Maja; Harris, Robert A; Guerreiro-Cacais, Andre Ortlieb

    2016-03-01

    Dendritic cells are professional APCs that play a central role in the initiation of immune responses. The limited ex vivo availability of dendritic cells inspires the widespread use of bone marrow-derived dendritic cells as an alternative in research. However, the functional characteristics of bone marrow-derived dendritic cells are incompletely understood. Therefore, we compared functional and phenotypic characteristics of rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3 ligand bone marrow-derived dendritic cells. A comparison of surface markers revealed that FLT3 ligand-bone marrow-derived dendritic cells expressed signal regulatory protein α, CD103, and CD4 and baseline levels of MHC class II, CD40, and CD86, which were highly up-regulated upon stimulation. Conversely, GM-CSF/IL-4-bone marrow-derived dendritic cells constitutively expressed signal regulatory protein α, CD11c, and CD11b but only mildly up-regulated MHC class II, CD40, or CD86 following stimulation. Expression of dendritic cell-associated core transcripts was restricted to FLT3 ligand-bone marrow-derived dendritic cells . GM-CSF/IL-4-bone marrow-derived dendritic cells were superior at phagocytosis but were outperformed by FLT3 ligand-bone marrow-derived dendritic cells at antigen presentation and T cell stimulation in vitro. Stimulated GM-CSF/IL-4-bone marrow-derived dendritic cells secreted more TNF, CCL5, CCL20, and NO, whereas FLT3 ligand-bone marrow-derived dendritic cells secreted more IL-6 and IL-12. Finally, whereas GM-CSF/IL-4-bone marrow-derived dendritic cell culture supernatants added to resting T cell cultures promoted forkhead box p3(+) regulatory T cell populations, FLT3 ligand-bone marrow-derived dendritic cell culture supernatants drove Th17 differentiation. We conclude that rat GM-CSF/IL-4-bone marrow-derived dendritic cells and FLT3 ligand-bone marrow-derived dendritic cells are functionally distinct. Our data support the current rationale that FLT3

  5. Synergistic anti-tumor efficacy of immunogenic adenovirus ONCOS-102 (Ad5/3-D24-GM-CSF) and standard of care chemotherapy in preclinical mesothelioma model.

    PubMed

    Kuryk, Lukasz; Haavisto, Elina; Garofalo, Mariangela; Capasso, Cristian; Hirvinen, Mari; Pesonen, Sari; Ranki, Tuuli; Vassilev, Lotta; Cerullo, Vincenzo

    2016-10-15

    Malignant mesothelioma (MM) is a rare cancer type caused mainly by asbestos exposure. The median overall survival time of a mesothelioma cancer patient is less than 1-year from diagnosis. Currently there are no curative treatment modalities for malignant mesothelioma, however treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase life expectancy. Pemetrexed-Cisplatin is the only standard of care (SoC) chemotherapy for malignant mesothelioma, but the median PFS/OS (progression-free survival/overall survival) from the initiation of treatment is only up to 12 months. Therefore, new treatment strategies against malignant mesothelioma are in high demand. ONCOS-102 is a dual targeting, chimeric oncolytic adenovirus, coding for human GM-CSF. The safety and immune activating properties of ONCOS-102 have already been assessed in phase 1 study (NCT01598129). In this preclinical study, we evaluated the antineoplastic activity of combination treatment with SoC chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and ONCOS-102 in xenograft BALB/c model of human malignant mesothelioma. We demonstrated that ONCOS-102 is able to induce immunogenic cell death of human mesothelioma cell lines in vitro and showed anti-tumor activity in the treatment of refractory H226 malignant pleural mesothelioma (MPM) xenograft model. While chemotherapy alone showed no anti-tumor activity in the mesothelioma mouse model, ONCOS-102 was able to slow down tumor growth. Interestingly, a synergistic anti-tumor effect was seen when ONCOS-102 was combined with chemotherapy regimens. These findings give a rationale for the clinical testing of ONCOS-102 in combination with first-line chemotherapy in patients suffering from malignant mesothelioma. PMID:27287512

  6. Fusion Protein of Mutant B7-DC and Fc Enhances the Antitumor Immune Effect of GM-CSF-secreting Whole-cell Vaccine

    PubMed Central

    Kojima, Masatsugu; Murata, Satoshi; Mekata, Eiji; Takebayashi, Katsushi; Jaffee, Elizabeth M.; Tani, Tohru

    2015-01-01

    Summary B7-DC [also known as programmed death ligand 2 (PD-L2)] is a costimulatory molecule expressed predominantly on dendritic cells (DCs) and macrophages. In addition to its coinhibitory receptor, programmed death receptor 1 (PD-1), evidence suggests that B7-DC interacts with an unidentified costimulatory receptor on T cells. B7-DC mutants with selective binding capacity for the costimulatory receptor may be effective in stimulating antitumor immune responses, while avoiding the inhibitory effects of PD-1. In this study, we concomitantly administered a GM-CSF-secreting whole cell vaccine together with a fusion protein of mutant B7-DC and Fc portion (mB7-DC-Fc), which binds selectively to the costimulatory receptor. This lead to an increased number of tumor antigen-specific cytotoxic T lymphocytes both in the spleen and at the tumor site and complete elimination of established tumors in vivo. In addition, mB7-DC-Fc increased IFN-γ and IL-2 production and decreased IL-4 and IL-10 production in vitro, indicating that mB7-DC-Fc tips the Th1/Th2 balance toward Th1 dominance, which is more favorable for antitumor immunity. Furthermore, mB7-DC-Fc decreased the PD-1 + proportion of CD8 + T cells in vitro and tumor-infiltrating CD8 + T cells in vivo, suggesting that mB7-DC-Fc may maintain tumor-infiltrating CD8 + T cells in a nonexhausted state. In conclusion, mB7-DC-Fc administration during the T-cell priming phase enhances antitumor effects of vaccine by generating more tumor antigen-specific cytotoxic T lymphocytes and leading to their accumulation at the tumor site. We suggest that this combination approach may be a promising strategy for antitumor immunotherapy. PMID:24598447

  7. Replication of human immunodeficiency virus in monocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) potentiates viral production yet enhances the antiviral effect mediated by 3'-azido- 2'3'-dideoxythymidine (AZT) and other dideoxynucleoside congeners of thymidine

    PubMed Central

    1989-01-01

    We have investigated the influence of granulocyte-macrophage CSF (GM- CSF) on the replication of HIV-1 in cells of monocyte/macrophage (M/M) lineage, and its effect on the anti-HIV activity of several 2'3'- dideoxynucleoside congeners of thymidine in these cells in vitro. We found that replication of both HTLV-IIIBa-L (a monocytotropic strain of HIV-1) and HTLV-IIIB (a lymphocytotropic strain) is markedly enhanced in M/M, but not in lymphocytes exposed to GM-CSF in culture. Moreover, GM-CSF reduced the dose of HIV required to obtain productive infection in M/M. Even in the face of this increased infection, GM-CSF also enhanced the net anti-HIV activity of 3'-azido-2'3'-dideoxythymidine (AZT) and several related congeners: 2'3'-dideoxythymidine (ddT), 2'3'- dideoxy-2'3'-didehydrothymidine (D4T), and 3'-azido-2'3'-dideoxyuridine (AZddU). Inhibition of viral replication in GM-CSF-exposed M/M was achieved with concentrations of AZT and related drugs, which were 10- 100 times lower than those inhibitory for HIV-1 in monocytes in the absence of GM-CSF. Other dideoxynucleosides not related to AZT showed unchanged or decreased anti-HIV activity in GM-CSF-exposed M/M. To investigate the possible biochemical basis for these effects, we evaluated the metabolism of several drugs in M/M exposed to GM-CSF. We observed in these cells markedly increased levels of both parent and mono-, di-, and triphosphate anabolites of AZT and D4T compared with M/M not exposed to GM-CSF. By contrast, only limited increases of endogenous competing 2'-deoxynucleoside-5'-triphosphate pools were observed after GM-CSF exposure. Thus, the ratio of AZT-5'- triphosphate/2'-deoxythymidine-5'-triphosphate and 2'3'-dideoxy-2'3'- didehydrothymidine-5'-triphosphate/2'-deoxythymi dine- 5'-triphosphate is several-fold higher in GM-CSF-exposed M/M, and this may account for the enhanced activity of such drugs in these cells. Taken together, these findings suggest that GM-CSF increases HIV-1 replication in M

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

    PubMed

    Ushach, Irina; Zlotnik, Albert

    2016-09-01

    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

  9. Serotype chimeric oncolytic adenovirus coding for GM-CSF for treatment of sarcoma in rodents and humans.

    PubMed

    Bramante, Simona; Koski, Anniina; Kipar, Anja; Diaconu, Iulia; Liikanen, Ilkka; Hemminki, Otto; Vassilev, Lotta; Parviainen, Suvi; Cerullo, Vincenzo; Pesonen, Saila K; Oksanen, Minna; Heiskanen, Raita; Rouvinen-Lagerström, Noora; Merisalo-Soikkeli, Maiju; Hakonen, Tiina; Joensuu, Timo; Kanerva, Anna; Pesonen, Sari; Hemminki, Akseli

    2014-08-01

    Sarcomas are a relatively rare cancer, but often incurable at the late metastatic stage. Oncolytic immunotherapy has gained attention over the past years, and a wide range of oncolytic viruses have been delivered via intratumoral injection with positive safety and promising efficacy data. Here, we report preclinical and clinical results from treatment of sarcoma with oncolytic adenovirus Ad5/3-D24-GMCSF (CGTG-102). Ad5/3-D24-GMCSF is a serotype chimeric oncolytic adenovirus coding for human granulocyte-macrophage colony-stimulating factor (GM-CSF). The efficacy of Ad5/3-D24-GMCSF was evaluated on a panel of soft-tissue sarcoma (STS) cell lines and in two animal models. Sarcoma specific human data were also collected from the Advanced Therapy Access Program (ATAP), in preparation for further clinical development. Efficacy was seen in both in vitro and in vivo STS models. Fifteen patients with treatment-refractory STS (13/15) or primary bone sarcoma (2/15) were treated in ATAP, and treatments appeared safe and well-tolerated. A total of 12 radiological RECIST response evaluations were performed, and two cases of minor response, six cases of stable disease and four cases of progressive disease were detected in patients progressing prior to virus treatment. Overall, the median survival time post treatment was 170 days. One patient is still alive at 1,459 days post virus treatment. In summary, Ad5/3-D24-GMCSF appears promising for the treatment of advanced STS; a clinical trial for treatment of refractory injectable solid tumors including STS is ongoing.

  10. Characterization of cis-regulatory elements of the c-myc promoter responding to human GM-CSF or mouse interleukin 3 in mouse proB cell line BA/F3 cells expressing the human GM-CSF receptor.

    PubMed Central

    Watanabe, S; Ishida, S; Koike, K; Arai, K

    1995-01-01

    Interleukin 3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) activates c-fos, c-jun, and c-myc genes and proliferation in both hematopoietic and nonhematopoietic cells. Using a series of deletion mutants of the beta subunit of human GM-CSF receptor (hGMR) and inhibitors of tyrosine kinase, two distinct signaling pathways, one for activation of c-fos and c-jun genes, and the other for cell proliferation and activation of c-myc gene have been elucidated. In contrast to wealth of information on the pathway leading to activation of c-fos/c-jun genes, knowledge of the latter is scanty. To clarify the mechanisms of activation of c-myc gene by cytokines, we established a transient transfection assay in mouse proB cell line BA/F3 cells expressing hGMR. Analyses of hGMR beta subunit mutants revealed two cytoplasmic regions involved in activation of the c-myc promoter, one is essential and the other is dispensable but enhances the activity. These regions are located at the membrane proximal and the distal regions covering amino acid positions 455-544 and 544-589, respectively. Characterization of cis-acting regulatory elements of the c-myc gene showed that the region containing the P2 promoter initiation site is sufficient to mediate the response to mIL-3 or hGM-CSF. Electrophoretic mobility shift assay using an oligonucleotide corresponding to the distal putative E2F binding site revealed that p107/E2F complex, the negative regulator of E2F, decreased, and free E2F increased after mIL-3 stimulation. These results support the thesis that mIL-3 or hGM-CSF regulates the c-myc promoter by altering composition of the E2F complexes at E2F binding site. Images PMID:7579683

  11. A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells.

    PubMed

    Cardona, Maria E; Simonson, Oscar E; Oprea, Iulian I; Moreno, Pedro M D; Silva-Lara, Maria F; Mohamed, Abdalla J; Christensson, Birger; Gahrton, Gösta; Dilber, M Sirac; Smith, C I Edvard; Arteaga, H Jose

    2016-01-01

    The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1(+)DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

  12. Doxycycline up-regulates the expression of IL-6 and GM-CSF via MAPK/ERK and NF-κB pathways in mouse thymic epithelial cells.

    PubMed

    Huang, Ying; Li, Rong; Chen, Xun; Zhuo, Ya; Jin, Rong; Qian, Xiao Ping; Jiang, Yan Qiu; Zeng, Zi Han; Zhang, Yu; Shao, Qi Xiang

    2011-09-01

    Thymic epithelial cells (TECs) constitute a major component of the thymic stroma which provides a microenvironment critical for developing thymocytes. We have previously demonstrated that doxycycline (Dox), a tetracycline derivative, enhances the proliferation of the mouse thymic epithelial cell line 1 (MTEC1) via MAPK/ERK signal pathway. Herein we provide evidence that Dox also has profound impact on the cytokine production by MTEC1. Specifically, the expression of IL-6 and GM-CSF, both at mRNA and protein levels, was found to be increased in a time- and dose-dependent manner with the addition of Dox. Western blotting analysis revealed that treatment with Dox-induced phosphorylation of the p65 subunit of NF-κB and ERK. Notably, Dox-induced up-regulation of IL-6 and GM-CSF was largely abolished after pretreatment of MTEC1 with either NF-κB inhibitor BAY11-7082 or MEK1/2 inhibitor U0126, supporting the involvement of the two pathways in the process. These findings warrant further investigation into the potential application of Dox in T-cell reconstitution in such situations as chemotherapy, radiotherapy, bone marrow transplantation and HIV infection.

  13. CCL2 shapes macrophage polarization by GM-CSF and M-CSF: identification of CCL2/CCR2-dependent gene expression profile.

    PubMed

    Sierra-Filardi, Elena; Nieto, Concha; Domínguez-Soto, Angeles; Barroso, Rubén; Sánchez-Mateos, Paloma; Puig-Kroger, Amaya; López-Bravo, María; Joven, Jorge; Ardavín, Carlos; Rodríguez-Fernández, José L; Sánchez-Torres, Carmen; Mellado, Mario; Corbí, Angel L

    2014-04-15

    The CCL2 chemokine mediates monocyte egress from bone marrow and recruitment into inflamed tissues through interaction with the CCR2 chemokine receptor, and its expression is upregulated by proinflammatory cytokines. Analysis of the gene expression profile in GM-CSF- and M-CSF-polarized macrophages revealed that a high CCL2 expression characterizes macrophages generated under the influence of M-CSF, whereas CCR2 is expressed only by GM-CSF-polarized macrophages. Analysis of the factors responsible for this differential expression identified activin A as a critical factor controlling the expression of the CCL2/CCR2 pair in macrophages, as activin A increased CCR2 expression but inhibited the acquisition of CCL2 expression by M-CSF-polarized macrophages. CCL2 and CCR2 were found to determine the extent of macrophage polarization because CCL2 enhances the LPS-induced production of IL-10, whereas CCL2 blockade leads to enhanced expression of M1 polarization-associated genes and cytokines, and diminished expression of M2-associated markers in human macrophages. Along the same line, Ccr2-deficient bone marrow-derived murine macrophages displayed an M1-skewed polarization profile at the transcriptomic level and exhibited a significantly higher expression of proinflammatory cytokines (TNF-α, IL-6) in response to LPS. Therefore, the CCL2-CCR2 axis regulates macrophage polarization by influencing the expression of functionally relevant and polarization-associated genes and downmodulating proinflammatory cytokine production.

  14. In vivo electroporation of plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas combined with electrochemotherapy induces long-term antitumour immunity.

    PubMed

    Heller, L; Pottinger, C; Jaroszeski, M J; Gilbert, R; Heller, R

    2000-12-01

    When cancer cells, including melanoma cells, are genetically altered to secrete cytokines, irradiated and injected into subjects, long-term antitumour immunity is induced. Optimally, existing melanomas induced to produce cytokines in vivo could stimulate this same immune response. Although in vivo electroporation enhances plasmid expression, electroporation of plasmids encoding granulocyte-monocyte colony stimulating factor (GM-CSF) and interleukin-2 (IL2) into B16 mouse melanomas did not significantly alter tumour growth at the concentration tested. Electrochemotherapy, which causes short-term, complete regressions of treated tumour but no resistance to challenge, was combined with plasmid delivery. The combination treatment resulted in the induction of long-term immunity to recurrence and resistance to challenge in up to 25% of mice. PMID:11198480

  15. Adenosine potentiates stimulatory effects on granulocyte-macrophage hematopoietic progenitor cells in vitro of IL-3 and SCF, but not those of G-CSF, GM-CSF and IL-11.

    PubMed

    Hofer, M; Vacek, A; Pospísil, M; Weiterová, L; Holá, J; Streitová, D; Znojil, V

    2006-01-01

    The aim of the studies was to ascertain if adenosine is able to co-operate with selected hematopoietic growth factors and cytokines, namely with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-11 (IL-11), in inducing the growth of colonies from hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) from normal bone marrow cells in vitro. Adenosine was found not to produce any colonies when present in the cultures as the only potential stimulator. All the tested cytokines and growth factors were observed to induce the growth of distinct numbers of GM-CFC colonies, with the exception of IL-11. When suboptimal concentrations of the evaluated cytokines and growth factors were tested in the cultures in which various concentrations of adenosine were concomitantly present, mutually potentiating effects were found in the case of IL-3 and SCF. These results confirm the role of adenosine in regulation of granulopoiesis and predict IL-3 and SCF as candidates for further in vivo studies of their combined administration with adenosine.

  16. 3,3'-Diindolylmethane Inhibits Flt3L/GM-CSF-induced-bone Marrow-derived CD103+ Dendritic Cell Differentiation Regulating Phosphorylation of STAT3 and STAT5

    PubMed Central

    Choi, Ah-Jeong; Kim, Soo-Ji; Jeong, So-Yeon

    2015-01-01

    The intestinal immune system maintains oral tolerance to harmless antigens or nutrients. One mechanism of oral tolerance is mediated by regulatory T cell (Treg)s, of which differentiation is regulated by a subset of dendritic cell (DC)s, primarily CD103+ DCs. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, plays an important role in regulating immunity. The intestines are exposed to various AhR ligands, including endogenous metabolites and phytochemicals. It was previously reported that AhR activation induced tolerogenic DCs in mice or in cultures of bone marrow-derived DCs. However, given the variety of tolerogenic DCs, which type of tolerogenic DCs is regulated by AhR remains unknown. In this study, we found that AhR ligand 3,3'-diindolylmethane (DIM) inhibited the development of CD103+ DCs from mouse bone marrow cells stimulated with Flt3L and GM-CSF. DIM interfered with phosphorylation of STAT3 and STAT5 inhibiting the expression of genes, including Id2, E2-2, IDO-1, and Aldh1a2, which are associated with DC differentiation and functions. Finally, DIM suppressed the ability of CD103+ DCs to induce Foxp3+ Tregs. PMID:26770182

  17. Acacia ferruginea inhibits tumor progression by regulating inflammatory mediators-(TNF-a, iNOS, COX-2, IL-1β, IL-6, IFN-γ, IL-2, GM-CSF) and pro-angiogenic growth factor- VEGF.

    PubMed

    Sakthivel, Kunnathur Murugesan; Guruvayoorappan, Chandrasekaran

    2013-01-01

    The aim of the present investigation was to evaluate the effect of A ferruginea extract on Dalton's lymphoma ascites (DLA) induced tumours in BALB/c mice. Experimental animals received A ferruginea extract (10 mg/ kg.b.wt) intraperitoneally for 14 consecutive days after DLA tumor challenge. Treatment with extract significantly increased the life span, total white blood cell (WBC) count and haemoglobin (Hb) content and decreased the level of serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (γ-GT) and nitric oxide (NO) in DLA bearing ascites tumor models. In addition, administration of extract significantly decreased the tumour volume and body weight in a DLA bearing solid tumor model. The levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and granulocyte monocyte-colony stimulating factor (GM-CSF), as well as pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) were elevated in solid tumour controls, but significantly reduced by A ferruginea administration. On the other hand, the extract stimulated the production of interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in animals with DLA induced solid tumours. Increase in CD4+ T-cell population suggested strong immunostimulant activity for this extract. GC/MS and LC/MS analysis showed quinone, quinoline, imidazolidine, pyrrolidine, cyclopentenone, thiazole, pyrazole, catechin and coumarin derivatives as major compounds present in the A ferruginea methanolic extract. Thus, the outcome of the present study suggests that A ferruginea extract has immunomodulatory and tumor inhibitory activities and has the potential to be developed as a natural anticancer agent. PMID:23886206

  18. PyNTTTTGT and CpG immunostimulatory oligonucleotides: effect on granulocyte/monocyte colony-stimulating factor (GM-CSF) secretion by human CD56+ (NK and NKT) cells.

    PubMed

    Rodriguez, Juan M; Marchicio, José; López, Mariela; Ziblat, Andrea; Elias, Fernanda; Fló, Juan; López, Ricardo A; Horn, David; Zorzopulos, Jorge; Montaner, Alejandro D

    2015-01-01

    CD56+ cells have been recognized as being involved in bridging the innate and acquired immune systems. Herein, we assessed the effect of two major classes of immunostimulatory oligonucleotides (ODNs), PyNTTTTGT and CpG, on CD56+ cells. Incubation of human peripheral blood mononuclear cells (hPBMC) with some of these ODNs led to secretion of significant amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and granulocyte/monocyte colony-stimulating factor (GM-CSF), but only if interleukin 2 (IL2) was present. IMT504, the prototype of the PyNTTTTGT ODN class, was the most active. GM-CSF secretion was very efficient when non-CpG ODNs with high T content and PyNTTTTGT motifs lacking CpGs were used. On the other hand, CpG ODNs and IFNα inhibited this GM-CSF secretion. Selective cell type removal from hPBMC indicated that CD56+ cells were responsible for GM-CSF secretion and that plasmacytoid dendritic cells (PDCs) regulate this process. In addition, PyNTTTTGT ODNs inhibited the IFNα secretion induced by CpG ODNs in PDCs by interference with the TLR9 signaling pathway. Since IFNα is essential for CD56+ stimulation by CpG ODNs, there is a reciprocal interference of CpG and PyNTTTTGT ODNs when acting on this cell population. This suggests that these synthetic ODNs mimic different natural alarm signals for activation of the immune system. PMID:25706946

  19. PyNTTTTGT and CpG Immunostimulatory Oligonucleotides: Effect on Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF) Secretion by Human CD56+ (NK and NKT) Cells

    PubMed Central

    Rodriguez, Juan M.; Marchicio, José; López, Mariela; Ziblat, Andrea; Elias, Fernanda; Fló, Juan; López, Ricardo A.; Horn, David; Zorzopulos, Jorge; Montaner, Alejandro D.

    2015-01-01

    CD56+ cells have been recognized as being involved in bridging the innate and acquired immune systems. Herein, we assessed the effect of two major classes of immunostimulatory oligonucleotides (ODNs), PyNTTTTGT and CpG, on CD56+ cells. Incubation of human peripheral blood mononuclear cells (hPBMC) with some of these ODNs led to secretion of significant amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and granulocyte/monocyte colony-stimulating factor (GM-CSF), but only if interleukin 2 (IL2) was present. IMT504, the prototype of the PyNTTTTGT ODN class, was the most active. GM-CSF secretion was very efficient when non-CpG ODNs with high T content and PyNTTTTGT motifs lacking CpGs were used. On the other hand, CpG ODNs and IFNα inhibited this GM-CSF secretion. Selective cell type removal from hPBMC indicated that CD56+ cells were responsible for GM-CSF secretion and that plasmacytoid dendritic cells (PDCs) regulate this process. In addition, PyNTTTTGT ODNs inhibited the IFNα secretion induced by CpG ODNs in PDCs by interference with the TLR9 signaling pathway. Since IFNα is essential for CD56+ stimulation by CpG ODNs, there is a reciprocal interference of CpG and PyNTTTTGT ODNs when acting on this cell population. This suggests that these synthetic ODNs mimic different natural alarm signals for activation of the immune system. PMID:25706946

  20. PyNTTTTGT and CpG immunostimulatory oligonucleotides: effect on granulocyte/monocyte colony-stimulating factor (GM-CSF) secretion by human CD56+ (NK and NKT) cells.

    PubMed

    Rodriguez, Juan M; Marchicio, José; López, Mariela; Ziblat, Andrea; Elias, Fernanda; Fló, Juan; López, Ricardo A; Horn, David; Zorzopulos, Jorge; Montaner, Alejandro D

    2015-01-01

    CD56+ cells have been recognized as being involved in bridging the innate and acquired immune systems. Herein, we assessed the effect of two major classes of immunostimulatory oligonucleotides (ODNs), PyNTTTTGT and CpG, on CD56+ cells. Incubation of human peripheral blood mononuclear cells (hPBMC) with some of these ODNs led to secretion of significant amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and granulocyte/monocyte colony-stimulating factor (GM-CSF), but only if interleukin 2 (IL2) was present. IMT504, the prototype of the PyNTTTTGT ODN class, was the most active. GM-CSF secretion was very efficient when non-CpG ODNs with high T content and PyNTTTTGT motifs lacking CpGs were used. On the other hand, CpG ODNs and IFNα inhibited this GM-CSF secretion. Selective cell type removal from hPBMC indicated that CD56+ cells were responsible for GM-CSF secretion and that plasmacytoid dendritic cells (PDCs) regulate this process. In addition, PyNTTTTGT ODNs inhibited the IFNα secretion induced by CpG ODNs in PDCs by interference with the TLR9 signaling pathway. Since IFNα is essential for CD56+ stimulation by CpG ODNs, there is a reciprocal interference of CpG and PyNTTTTGT ODNs when acting on this cell population. This suggests that these synthetic ODNs mimic different natural alarm signals for activation of the immune system.

  1. Suitable in vitro culture of Eimeria bovis meront II stages in bovine colonic epithelial cells and parasite-induced upregulation of CXCL10 and GM-CSF gene transcription.

    PubMed

    Hermosilla, Carlos; Stamm, Ivonne; Menge, Christian; Taubert, Anja

    2015-08-01

    We here established a suitable in vitro cell culture system based on bovine colonic epithelial cells (BCEC) for the development of Eimeria bovis merozoites I and the characterization of early parasite-induced innate epithelial host cell reactions as gene transcription of proinflammatory molecules. Both primary and permanent BCEC (BCEC (rim) and BCEC(perm)) were suitable for E. bovis merozoite I invasion and subsequent development of meronts II leading to the release of viable merozoites II. E. bovis merozoite II failed to develop any further neither into gamont nor oocyst stages in BCEC in vitro. E. bovis merozoite I induced innate epithelial host cell reactions at the level of CXC/CCL chemokines (CXCL1, CXCL8, CXCL10, CCL2), IL-6, and GM-CSF gene transcription. Overall, both BCEC types were activated by merozoite I infections since they showed significantly enhanced gene transcript levels of the immunomodulatory molecules CXCL10 and GM-CSF. However, gene transcription profiles of BCEC(prim) and BCEC(perm) revealed different reaction patterns in response to merozoite I infection with regard to quality and kinetics of chemokine/cytokine gene transcription. Although both BCEC types equally showed most prominent responses for CXCL10 and GM-CSF, the induction of CXCL1, CXCL8, CCL2, and IL-6 gene transcripts varied qualitatively and quantitatively. Our results demonstrate that BCEC seem capable to respond to E. bovis merozoite I infection by the upregulation of CXCL10 and GM-CSF gene transcription and therefore probably contribute to host innate effector mechanisms against E. bovis.

  2. Effect of intramammary injection of rboGM-CSF on milk levels of chemiluminescence activity, somatic cell count, and Staphylococcus aureus count in Holstein cows with S. aureus subclinical mastitis

    PubMed Central

    2004-01-01

    Abstract The effect of intramammary injection of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF, 400 μg/10 mL) on quarter milk levels of chemiluminescence (CL) activity, and somatic cell count (SCC) and shedding pattern of Staphylococcus aureus was investigated. Ten Holstein cows, naturally infected with S. aureus were used, with either early-stage or late-stage subclinical mastitis. Injection of rboGM-CSF caused a remarkable increase in milk CL activity with a peak at 6 h after the cytokine injection in the early- and late-stage groups. In the early-stage group, milk SCC stayed around preinjection level at 6 h, rose significantly on days 1 and 2, and was followed by a smooth and significant decline to an under preinjection level (below 200 000 cells/mL) on day 7 postinjection. Alternatively, in the late-stage group, milk SCC rose significantly at 6 h after the cytokine injection and maintained high levels thereafter. The milk S. aureus count decreased drastically by the cytokine injection in the early-stage group. The bacterial count was moderately decreased in the late-stage group, but increased back to preinoculation levels on day 7 after the cytokine injection. The results suggest that the rboGM-CSF has a potential as a therapeutic agent for S. aureus infection causing subclinical mastitis of dairy cows, if the cytokine is applied at the initial stage of infection. PMID:15352542

  3. Synergistic neutralizing antibody response to a dengue virus type 2 DNA vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing GM-CSF.

    PubMed

    Raviprakash, K; Marques, E; Ewing, D; Lu, Y; Phillips, I; Porter, K R; Kochel, T J; August, T J; Hayes, C G; Murphy, G S

    2001-11-10

    We have previously shown that a dengue virus type 1 DNA vaccine expressing premembrane (prM) and envelope (E) genes was immunogenic in mice and monkeys and that rhesus monkeys vaccinated with this construct were completely to partially protected from virus challenge. In order to improve the immunogenicity of dengue DNA vaccines, we have evaluated the effect of lysosome targeting of antigens and coimmunization with a plasmid expressing GM-CSF on antibody responses. A dengue virus type 2 candidate vaccine containing prM and E genes was constructed in which the transmembrane and cytoplasmic regions of E were replaced by those of the lysosome-associated membrane protein (LAMP). The modified vaccine construct expressed antigen that was colocalized with endogenous LAMP in lysosomal vesicles of transfected cells, whereas the antigen expressed from the unmodified construct was not. It was hypothesized that targeting of antigen to the lysosomal compartment will increase antigen presentation by MHC class II, leading to stronger CD4-mediated immune responses. Mice immunized with the modified construct responded with significantly higher levels of virus neutralizing antibodies compared to those immunized with the unmodified construct. Coimmunization of mice with a plasmid expressing murine GM-CSF enhanced the antibody response obtained with either the unmodified or the modified construct alone. The highest antibody responses were noted when the modified construct was coinjected with plasmid expressing the GM-CSF gene. These results could form the basis for an effective tetravalent dengue virus DNA vaccine. PMID:11883007

  4. Yeast (Saccharomyces cerevisiae) Polarizes Both M-CSF- and GM-CSF-Differentiated Macrophages Toward an M1-Like Phenotype.

    PubMed

    Seif, Michelle; Philippi, Anja; Breinig, Frank; Kiemer, Alexandra K; Hoppstädter, Jessica

    2016-10-01

    Macrophages are a heterogeneous and plastic cell population with two main phenotypes: pro-inflammatory classically activated macrophages (M1) and anti-inflammatory alternatively activated macrophages (M2). Saccharomyces cerevisiae is a promising vehicle for the delivery of vaccines. It is well established that S. cerevisiae is taken up by professional phagocytic cells. However, the response of human macrophages to S. cerevisiae is ill-defined. In this study, we characterized the interaction between S. cerevisiae and M1- or M2-like macrophages. M1-like macrophages had a higher yeast uptake capacity than M2-like macrophages, but both cell types internalized opsonized yeast to the same extent. The M1 surface markers HLAII and CD86 were upregulated after yeast uptake in M1- and M2-like macrophages. Moreover, mRNA expression levels of pro-inflammatory cytokines, such as TNF-α, IL-12, and IL-6, increased, whereas the expression of anti-inflammatory mediators did not change. These results demonstrate that S. cerevisiae can target both M1 and M2 macrophages, paralleled by skewing toward an M1 phenotype. Thus, the use of yeast-based delivery systems might be a promising approach for the treatment of pathologic conditions that would benefit from the presence of M1-polarized macrophages, such as cancer.

  5. Standardized serum GM-CSF autoantibody testing for the routine clinical diagnosis of autoimmune pulmonary alveolar proteinosis.

    PubMed

    Uchida, Kanji; Nakata, Koh; Carey, Brenna; Chalk, Claudia; Suzuki, Takuji; Sakagami, Takuro; Koch, Diana E; Stevens, Carrie; Inoue, Yoshikazu; Yamada, Yoshitsugu; Trapnell, Bruce C

    2014-01-15

    Autoantibodies against granulocyte/macrophage colony-stimulating factor (GMAbs) cause autoimmune pulmonary alveolar proteinosis (PAP) and measurement of the GMAb level in serum is now commonly used to identify this disease, albeit, in a clinical research setting. The present study was undertaken to optimize and standardize serum GMAb concentration testing using a GMAb enzyme-linked immunosorbent assay (GMAb ELISA) to prepare for its introduction into routine clinical use. The GMAb ELISA was evaluated using serum specimens from autoimmune PAP patients, healthy people, and GMAb-spiked serum from healthy people. After optimizing assay components and procedures, its accuracy, precision, reliability, sensitivity, specificity, and ruggedness were evaluated. The coefficient of variation in repeated measurements was acceptable (<15%) for well-to-well, plate-to-plate, day-to-day, and inter-operator variation, and was not affected by repeated freeze-thaw cycles of serum specimens or the reference standards, or by storage of serum samples at -80°C. The lower limit of quantification (LLOQ) of the PAP patient-derived polyclonal GMAb reference standard (PCRS) was 0.78ng/ml. Receiver operating characteristic curve analysis identified a serum GMAb level of 5μg/ml (based on PCRS) as the optimal cut off value for distinguishing autoimmune PAP serum from normal serum. A pharmaceutical-grade, monoclonal GMAb reference standard (MCRS) was developed as the basis of a new unit of measure for GMAb concentration: one International Unit (IU) of GMAb is equivalent to 1μg/ml of MCRS. The median [interquartile range] serum GMAb level was markedly higher in autoimmune PAP patients than in healthy people (21.54 [12.83-36.38] versus 0.08 [0.05-0.14] IU; n=56, 38; respectively; P<0.0001). Results demonstrate that serum GMAb measurement using the GMAb ELISA was accurate, precise, reliable, had an acceptable LLOQ, and could be accurately expressed in standardized units. These findings support the

  6. Definition of a GC-rich motif as regulatory sequence of the human IL-3 gene: coordinate regulation of the IL-3 gene by CLE2/GC box of the GM-CSF gene in T cell activation.

    PubMed

    Nishida, J; Yoshida, M; Arai, K; Yokota, T

    1991-03-01

    The human IL-3 gene, located on chromosome 5, contains several cis-acting DNA sequences, i.e. CLE (conserved lymphokine element) and a GC-rich region, similar to the GM-CSF gene. To investigate the role of these elements, the 5' flanking region of the IL-3 gene was attached to a bacterial chloramphenicol acetyltransferase (CAT) gene. The fusion plasmids were analyzed by an in vitro transcription system using Jurkat cell nuclear extract prepared from cells stimulated with phorbol-12-myristate-13-acetate and calcium ionophore (PMA/A23187), introduced into Jurkat cells, expressed transiently, and stimulated by co-transfection of human T cell leukemia virus type I (HTLV-I) encoded transactivator, p40tax. The GC-rich region enhanced TATA-dependent transcription in the in vitro transcription system and also strongly responded to p40tax stimulation in the in vivo cotransfection assay. Using this GC-rich region as a probe, we identified a constitutive DNA-protein complex, alpha, whose binding specificity correlates with transcription activity. However, this element is not sufficient for the expression of the IL-3 gene in response to T cell activation signals (PMA/A23187) and no sequence was found within the IL-3 gene which mediates the response to PMA/A23187. The enhancer sequence which responds to T cell activation signals may be located outside the IL-3 gene and may be shared by other lymphokines, possibly by GM-CSF. We propose that the GM-CSF enhancer (CLE2/GC box) which mediates the response to T cell activation signals may stimulate the expression of the IL-3 gene. PMID:2049340

  7. CSL311, a novel, potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common β chain of the IL-3, GM-CSF and IL-5 receptors

    PubMed Central

    Panousis, Con; Dhagat, Urmi; Edwards, Kirsten M.; Rayzman, Veronika; Hardy, Matthew P.; Braley, Hal; Gauvreau, Gail M.; Hercus, Timothy R.; Smith, Steven; Sehmi, Roma; McMillan, Laura; Dottore, Mara; McClure, Barbara J.; Fabri, Louis J.; Vairo, Gino; Lopez, Angel F; Parker, Michael W.; Nash, Andrew D.; Wilson, Nicholas J.; Wilson, Michael J.; Owczarek, Catherine M.

    2016-01-01

    ABSTRACT The β common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared β common (βc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human βc receptor. The binding epitope of CSL311 on the βc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human βc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 β common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human βc receptor is central to pathogenesis. The coordinates for the βc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU). PMID:26651396

  8. Accessory cells with a veiled morphology and movement pattern generated from monocytes after avoidance of plastic adherence and of NADPH oxidase activation. A comparison with GM-CSF/IL-4-induced monocyte-derived dendritic cells.

    PubMed

    Ruwhof, Cindy; Canning, Martha O; Grotenhuis, Kristel; de Wit, Harm J; Florencia, Zenovia Z; de Haan-Meulman, Meeny; Drexhage, Hemmo A

    2002-07-01

    Veiled cells (VC) present in afferent lymph transport antigen from the periphery to the draining lymph nodes. Although VC in lymph form a heterogeneous population, some of the cells clearly belong on morphological grounds to the Langerhans cell (LC)/ dendritic cell (DC) series. Here we show that culturing monocytes for 24 hrs while avoiding plastic adherence (polypropylene tubes) and avoiding the activation of NADPH oxidase (blocking agents) results in the generation of a population of veiled accessory cells. The generated VC were actively moving cells like lymph-borne VC in vivo. The monocyte (mo)-derived VC population existed of CD14(dim/-) and CD14(brighT) cells. Of these the CD14(dim/-) VC were as good in stimulating allogeneic T cell proliferation as immature DC (iDC) obtained after one week of adherent culture of monocytes in granulocyte-macrophage-colony stimulating factor (GM-CSF)/interleukin (IL)-4. This underscores the accessory cell function of the mo-derived CD14(dim/-) VC. Although the CD14(dim/-)VC had a modest expression of the DC-specific marker CD83 and were positive for S100, expression of the DC-specific markers CD1a, Langerin, DC-SIGN, and DC-LAMP were absent. This indicates that the here generated CD14(dim/-) VC can not be considered as classical LC/DC. It was also impossible to turn the CD14(dim/-) mo-derived VC population into typical DC by culture for one week in GM-CSF/IL-4 or LPS. In fact the cells died tinder such circumstances, gaining some macrophage characteristics before dying. The IL-12 production from mo-derived CD14(dim/-) VC was lower, whereas the production of IL-10 was higher as compared to iDC. Consequently the T cells that were stimulated by these mo-derived VC produced less IFN-gamma as compared with T cells stimulated by iDC. Our data indicate that it is possible to rapidly generate a population of CD14(dim/-) veiled accessory cells from monocytes. The marker pattern and cytokine production of these VC indicate that this

  9. Establishment of a retinoic acid-resistant human acute promyelocytic leukaemia (APL) model in human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic severe combined immunodeficiency (SCID) mice.

    PubMed Central

    Fukuchi, Y.; Kizaki, M.; Kinjo, K.; Awaya, N.; Muto, A.; Ito, M.; Kawai, Y.; Umezawa, A.; Hata, J.; Ueyama, Y.; Ikeda, Y.

    1998-01-01

    To understand the mechanisms and identify novel approaches to overcoming retinoic acid (RA) resistance in acute promyelocytic leukaemia (APL), we established the first human RA-resistant APL model in severe combined immunodeficiency (SCID) mice. UF-1 cells, an RA-resistant APL cell line established in our laboratory, were transplanted into human granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing SCID (hGMTg SCID) mice and inoculated cells formed subcutaneous tumours in all hGMTg SCID mice, but not in the non-transgenic control SCID mice. Single-cell suspensions (UF-1/GMTg SCID cells) were similar in morphological, immunological, cytogenetic and molecular genetic features to parental UF-1 cells. All-trans RA did not change the morphological features of cells or their expression of CD11b. RA did not alter the growth curve of cells as determined by MTT assay, suggesting that UF-1/GMTg SCID cells are resistant to RA. These results demonstrate that this is the first RA-resistant APL animal model that may be useful for investigating the biology of this myeloid leukaemia in vivo, as well as for evaluating novel therapeutic approaches including patients with RA-resistant APL. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9764578

  10. Activating point mutations in the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors suggest the involvement of beta subunit dimerization and cell type-specific molecules in signalling.

    PubMed Central

    Jenkins, B J; D'Andrea, R; Gonda, T J

    1995-01-01

    We have combined retroviral expression cloning with random mutagenesis to identify two activating point mutations in the common signal-transducing subunit (h beta c) of the receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 by virtue of their ability to confer factor independence on the haemopoietic cell line, FDC-P1. One mutation (V449E) is located within the transmembrane domain and, by analogy with a similar mutation in the neu oncogene, may act by inducing dimerization of h beta c. The other mutation (I374N) lies in the extracellular, membrane-proximal portion of h beta c. Neither of these mutants, nor a previously described mutant of h beta c (FI delta, which has a small duplication in the extracellular region), was capable of inducing factor independence in CTLL-2 cells, while only V449E could induce factor independence in BAF-B03 cells. These results imply that the extracellular and transmembrane mutations act by different mechanisms. Furthermore, they imply that the mutants, and hence also wild-type h beta c, interact with cell type-specific signalling molecules. Models are presented which illustrate how these mutations may act and predict some of the characteristics of the putative receptor-associated signalling molecules. Images PMID:7556069

  11. High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighboring Stromal Fibroblasts.

    PubMed

    Rudisch, Albin; Dewhurst, Matthew Richard; Horga, Luminita Gabriela; Kramer, Nina; Harrer, Nathalie; Dong, Meng; van der Kuip, Heiko; Wernitznig, Andreas; Bernthaler, Andreas; Dolznig, Helmut; Sommergruber, Wolfgang

    2015-01-01

    We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the "cytokine fingerprints" identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay. PMID:25919140

  12. Clinical trial in healthy malaria-naïve adults to evaluate the safety, tolerability, immunogenicity and efficacy of MuStDO5, a five-gene, sporozoite/hepatic stage Plasmodium falciparum DNA vaccine combined with escalating dose human GM-CSF DNA

    PubMed Central

    Richie, Thomas L.; Charoenvit, Yupin; Wang, Ruobing; Epstein, Judith E.; Hedstrom, Richard C.; Kumar, Sanjai; Luke, Thomas C.; Freilich, Daniel A.; Aguiar, Joao C.; Sacci, Jr., John B.; Sedegah, Martha; Nosek, Jr., Ronald A.; De La Vega, Patricia; Berzins, Mara P.; Majam, Victoria F.; Abot, Esteban N.; Ganeshan, Harini; Richie, Nancy O.; Banania, Jo Glenna; Baraceros, Maria Fe B.; Geter, Tanya G.; Mere, Robin; Bebris, Lolita; Limbach, Keith; Hickey, Bradley W.; Lanar, David E.; Ng, Jennifer; Shi, Meng; Hobart, Peter M.; Norman, Jon A.; Soisson, Lorraine A.; Hollingdale, Michael R.; Rogers, William O.; Doolan, Denise L.; Hoffman, Stephen L.

    2012-01-01

    When introduced in the 1990s, immunization with DNA plasmids was considered potentially revolutionary for vaccine development, particularly for vaccines intended to induce protective CD8 T cell responses against multiple antigens. We conducted, in 1997−1998, the first clinical trial in healthy humans of a DNA vaccine, a single plasmid encoding Plasmodium falciparum circumsporozoite protein (PfCSP), as an initial step toward developing a multi-antigen malaria vaccine targeting the liver stages of the parasite. As the next step, we conducted in 2000–2001 a clinical trial of a five-plasmid mixture called MuStDO5 encoding pre-erythrocytic antigens PfCSP, PfSSP2/TRAP, PfEXP1, PfLSA1 and PfLSA3. Thirty-two, malaria-naïve, adult volunteers were enrolled sequentially into four cohorts receiving a mixture of 500 μg of each plasmid plus escalating doses (0, 20, 100 or 500 μg) of a sixth plasmid encoding human granulocyte macrophage-colony stimulating factor (hGM-CSF). Three doses of each formulation were administered intramuscularly by needle-less jet injection at 0, 4 and 8 weeks, and each cohort had controlled human malaria infection administered by five mosquito bites 18 d later. The vaccine was safe and well-tolerated, inducing moderate antigen-specific, MHC-restricted T cell interferon-γ responses but no antibodies. Although no volunteers were protected, T cell responses were boosted post malaria challenge. This trial demonstrated the MuStDO5 DNA and hGM-CSF plasmids to be safe and modestly immunogenic for T cell responses. It also laid the foundation for priming with DNA plasmids and boosting with recombinant viruses, an approach known for nearly 15 y to enhance the immunogenicity and protective efficacy of DNA vaccines. PMID:23151451

  13. 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

    SciTech Connect

    Ryu, Janice K. . E-mail: janice.ryu@ucdmc.ucdavis.edu; Swann, Suzanne; LeVeque, Francis; Johnson, Darlene J.; Chen, Allan; Fortin, Andre; Kim, Harold; Ang, Kian K.

    2007-03-01

    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 {mu}g/m{sup 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.

  14. Effect of Granulocyte-Macrophage Colony-Stimulating Factor on Chemotherapy-Related Neutropenia in Patients with Non-Hodgkin's Lymphomas-A Phase I/II Study of Dose and Mode of Administration.

    PubMed

    Hovgaard, D J; Nissen, N I

    1991-01-01

    The effect of mammalian glycosylated recombinant granulocyte-macrophage colony-stimulating factor was investigated in 24 patients with newly diagnosed non-Hodgkin's lymphoma in a phase I/II study. All patients received standard chemotherapy with CHOP. RhGM-CSF was administered after the first cycle for 5 days, and at one of four dose levels (2, 4, 8 and 16 μg/kg). Patients were randomized to receive the drug either by continuous intravenous infusion or twice daily as subcutaneous injection. No significant difference in results was observed between subcutaneous administration of rhGM-CSF and continuous i.v. infusion and these patient groups could therefore be combined in the analysis. Administration of rhGM-CSF resulted in a significant dose-dependent increase of total WBC, mainly neutrophils, eosinophils and monocytes. The increase was observed in 18/24 patients, reaching a peak 24-72 (median 24) hours after the start of rhGM-CSF. The CHOP chemotherapy-induced leucocyte nadir occurred on day 12 (mean) compared to day 14 for the 127 historical controls. The WBC nadir values were higher (2.4 ± 1.4) than for historical controls (1.8 ± 1.1) and the leucopenic/neutropenic period was of shorter duration. Following the chemotherapy nadir a more rapid recovery of WBC was seen than in controls. GM-CSF was well tolerated, the side effects were mild and transient, and included myalgias, low grade fever, headache, chest/bone discomfort, nausea, erythema at injection site and superficial phlebitis. The encouraging results of this phase I/II study indicate the need for a prospective controlled study of GM-CSF in chemotherapy of malignant lymphoma.

  15. Progenitor cell release plus exercise to improve functional performance in peripheral artery disease: the PROPEL Study.

    PubMed

    Domanchuk, Kathryn; Ferrucci, Luigi; Guralnik, Jack M; Criqui, Michael H; Tian, Lu; Liu, Kiang; Losordo, Douglas; Stein, James; Green, David; Kibbe, Melina; Zhao, Lihui; Annex, Brian; Perlman, Harris; Lloyd-Jones, Donald; Pearce, William; Taylor, Doris; McDermott, Mary M

    2013-11-01

    Functional impairment, functional decline, and mobility loss are major public health problems in people with lower extremity peripheral artery disease (PAD). Few medical therapies significantly improve walking performance in PAD. We describe methods for the PROgenitor cell release Plus Exercise to improve functionaL performance in PAD (PROPEL) Study, a randomized controlled clinical trial designed to determine whether granulocyte-macrophage colony stimulating factor (GM-CSF) combined with supervised treadmill walking exercise improves six-minute walk distance more than GM-CSF alone, more than supervised treadmill exercise alone, and more than placebo plus attention control in participants with PAD, respectively. PROPEL Study participants are randomized to one of four arms in a 2 by 2 factorial design. The four study arms are GM-CSF plus supervised treadmill exercise, GM-CSF plus attention control, placebo plus supervised exercise therapy, or placebo plus attention control. The primary outcome is change in six-minute walk distance at 12-week follow-up. Secondary outcomes include change in brachial artery flow-mediated dilation (FMD), change in maximal treadmill walking time, and change in circulating CD34+ cells at 12-week follow-up. Outcomes are also measured at six-week and six-month follow-up. Results of the PROPEL Study will have important implications for understanding mechanisms of improving walking performance and preventing mobility loss in the large and growing number of men and women with PAD.

  16. Cosmos 2229 immunology study (Experiment K-8-07)

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, Gerald

    1993-01-01

    The purpose of the current study was to further validate use of the rhesus monkey as a model for humans in future space flight testing. The areas of immunological importance examined in the Cosmos 2229 flight were represented by two sets of studies. The first set of studies determined the effect of space flight on the ability of bone marrow cells to respond to granulocyte/monocyte colony stimulating factor (GM-CSF). GM-CSF is an important regulator in the differentiation of bone marrow cells of both monocyte/macrophage and granulocyte lineages and any change in the ability of these cells to respond to GM-CSF can result in altered immune function. A second set of studies determined space flight effects on the expression of cell surface markers on both spleen and bone marrow cells. Immune cell markers included in this study were those for T-cell, B-cell, natural killer cell, and interleukin-2 populations. Variations from a normal cell population percentage, as represented by these markers, can be correlated with alterations in immunological function. Cells were stained with fluorescein-labelled antibodies directed against the appropriate antigens, and then analyzed using a flow cytometer.

  17. Cosmos: 1989 immunology studies

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, Gerald

    1991-01-01

    The effects of flight on Cosmos mission 2044 on leukocyte subset distribution and the sensitivity of bone marrow cells to colony stimulating factor-GM were determined. A parallel study with antiorthostatic suspension was also carried out. The study involved repetition and expansion of studies performed on Cosmos 1887. Spleen and bone marrow cells were obtained from flown, vivarium control, synchronous control, and suspended rats. The cells were stained with a series of monoclonal antibodies directed against rat leukocyte cell surface antigens. Control cells were stained with a monoclonal antibody directed against an irrelevant species or were unstained. Cells were then analyzed for fluorescence using a FACSCAN flow cytometer. Bone marrow cells were placed in culture with GM-CSF in McCoy's 5a medium and incubated for 5 days. Cultures were then evaluated for the number of colonies of 50 cells or greater.

  18. Synthesis of granulocyte–macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material

    PubMed Central

    Zhang, Qiang; Johnston, Eric V.; Shieh, Jae-Hung; Moore, Malcolm A. S.; Danishefsky, Samuel J.

    2014-01-01

    Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a medicinally important glycoprotein, used as an immunostimulant following bone-marrow transplant. On the basis of reports of its potential utility as an anticancer vaccine adjuvant, we undertook to develop a synthetic route toward single-glycoform GM-CSF. We describe herein a convergent total synthesis of GM-CSF aglycone and two homogeneous glycoforms. Analytical and biological studies confirm the structure and activity of these synthetic congeners. PMID:24516138

  19. Chronic ethanol ingestion in rats decreases granulocyte-macrophage colony-stimulating factor receptor expression and downstream signaling in the alveolar macrophage.

    PubMed

    Joshi, Pratibha C; Applewhite, Lisa; Ritzenthaler, Jeffrey D; Roman, Jesse; Fernandez, Alberto L; Eaton, Douglas C; Brown, Lou Ann S; Guidot, David M

    2005-11-15

    Although it is well recognized that alcohol abuse impairs alveolar macrophage immune function and renders patients susceptible to pneumonia, the mechanisms are incompletely understood. Alveolar macrophage maturation and function requires priming by GM-CSF, which is produced and secreted into the alveolar space by the alveolar epithelium. In this study, we determined that although chronic ethanol ingestion (6 wk) in rats had no effect on GM-CSF expression within the alveolar space, it significantly decreased membrane expression of the GM-CSF receptor in alveolar macrophages. In parallel, ethanol ingestion decreased cellular expression and nuclear binding of PU.1, the master transcription factor that activates GM-CSF-dependent macrophage functions. Furthermore, treatment of ethanol-fed rats in vivo with rGM-CSF via the upper airway restored GM-CSF receptor membrane expression as well as PU.1 protein expression and nuclear binding in alveolar macrophages. Importantly, GM-CSF treatment also restored alveolar macrophage function in ethanol-fed rats, as reflected by endotoxin-stimulated release of TNF-alpha and bacterial phagocytosis. We conclude that ethanol ingestion dampens alveolar macrophage immune function by decreasing GM-CSF receptor expression and downstream PU.1 nuclear binding and that these chronic defects can be reversed relatively quickly with rGM-CSF treatment in vivo.

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

    PubMed

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

    1991-04-01

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

  1. Comparative pharmacokinetics of single-dose administration of mammalian and bacterially-derived recombinant human granulocyte-macrophage colony-stimulating factor.

    PubMed

    Hovgaard, D; Mortensen, B T; Schifter, S; Nissen, N I

    1993-01-01

    Pharmacokinetics of recombinant human non-glycosylated bacterially-synthesized (E. coli) granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied following single intravenous (i.v.) and subcutaneous (s.c.) bolus injection, and compared to equivalent doses of glycosylated mammalian-derived CHO-GM-CSF. Each route of administration gave a different GM-CSF concentration-time profile. The highest peak serum concentrations (Cmax) were observed following i.v. bolus injection. After i.v. administration, a two-phase decline in concentration was noted for both types of GM-CSF with a significantly shorter t1/2 alpha of 7.8 minutes for the E. coli GM-CSF versus 20.0 min for the CHO-GM-CSF, while no significant difference was observed for the terminal phase. Following s.c. administration of equivalent doses, a higher peak serum concentration was observed in the E. coli-treated patients and, again, a faster elimination where pretreatment serum levels were reached after 16-20 h, versus more than 48 h after administration of CHO-GM-CSF. Although the non-glycosylated E. coli GM-CSF thus seems to undergo a faster elimination that the glycosylated CHO-GM-CSF no significant difference could be demonstrated in the in vivo effect of corresponding doses of the two compounds with respect to stimulation of granulopoiesis--with reservation for small patient numbers and a large individual variations in response.

  2. M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.

    PubMed

    Van Overmeire, Eva; Stijlemans, Benoît; Heymann, Felix; Keirsse, Jiri; Morias, Yannick; Elkrim, Yvon; Brys, Lea; Abels, Chloé; Lahmar, Qods; Ergen, Can; Vereecke, Lars; Tacke, Frank; De Baetselier, Patrick; Van Ginderachter, Jo A; Laoui, Damya

    2016-01-01

    Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade. PMID:26573801

  3. M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.

    PubMed

    Van Overmeire, Eva; Stijlemans, Benoît; Heymann, Felix; Keirsse, Jiri; Morias, Yannick; Elkrim, Yvon; Brys, Lea; Abels, Chloé; Lahmar, Qods; Ergen, Can; Vereecke, Lars; Tacke, Frank; De Baetselier, Patrick; Van Ginderachter, Jo A; Laoui, Damya

    2016-01-01

    Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.

  4. Immunization With AFP + GM CSF Plasmid Prime and AFP Adenoviral Vector Boost in Patients With Hepatocellular Carcinoma

    ClinicalTrials.gov

    2015-12-01

    Hepatocellular Carcinoma; Hepatoma; Liver Cancer, Adult; Liver Cell Carcinoma; Liver Cell Carcinoma, Adult; Cancer of Liver; Cancer of the Liver; Cancer, Hepatocellular; Hepatic Cancer; Hepatic Neoplasms; Hepatocellular Cancer; Liver Cancer; Neoplasms, Hepatic; Neoplasms, Liver

  5. Granulocyte-Macrophage Colony-Stimulating Factor Auto-Antibodies: A Marker of Aggressive Crohn’s Disease

    PubMed Central

    Gathungu, Grace; Kim, Mi-Ok; Ferguson, John P.; Sharma, Yashoda; Zhang, Wei; Ng, Sok Meng E.; Bonkowski, Erin; Ning, Kaida; Simms, Lisa A.; Croft, Anthony R.; Stempak, Joanne M.; Walker, Nicole; Huang, Ning; Xiao, Yang; Silverberg, Mark S.; Trapnell, Bruce C.; Cho, Judy H.; Radford-Smith, Graham L.; Denson, Lee A.

    2013-01-01

    Background & Aims Neutralizing auto-antibodies (Ab) against granulocyte-macrophage colony-stimulating factor (GM-CSF Ab) have been associated with stricturing ileal Crohn’s disease (CD) in a largely pediatric patient cohort (total 394, adult CD 57). The aim of this study was to examine this association in two independent predominantly adult inflammatory bowel disease patient cohorts. Methods Serum samples from 745 subjects from the NIDDK IBD Genetics Consortium and 737 patients from Australia were analyzed for GM-CSF Ab and genetic markers. We conducted multiple regression analysis with backwards elimination to assess the contribution of GM-CSF Ab levels, established CD risk alleles and smoking on ileal disease location in the 477 combined CD subjects from both cohorts. We also determined associations of GM-CSF Ab levels with complications requiring surgical intervention in combined CD subjects in both cohorts. Results Serum samples from CD patients expressed significantly higher concentrations of GM-CSF Ab when compared to Ulcerative Colitis or controls in each cohort. Non-smokers with ileal CD expressed significantly higher GM-CSF Ab concentrations in the Australian cohort (p= 0.002). Elevated GM-CSF Ab, ileal disease location and disease duration greater than 3 years were independently associated with stricturing/penetrating behavior and intestinal resection for CD. Conclusions The expression of high GM-CSF Ab is a risk marker for aggressive CD behavior and complications including surgery. Modifying factors include environmental exposure to smoking and genetic risk markers. PMID:23749272

  6. Granulocyte-macrophage colony-stimulating factor dependent monocyte-mediated cytotoxicity post-autologous bone marrow transplantation.

    PubMed

    Nagler, A; Shur, I; Barak, V; Fabian, I

    1996-08-01

    We investigated the in vitro antitumor activity of monocytes derived from autologous bone marrow transplanted (ABMT) patients treated in vivo with granulocyte-macrophage colony-stimulating factor (GM-CSF). Thirty-four patients (17 female, 17 male), median age 42 (range 3-57) years, were enrolled in the study. Fourteen patients were diagnosed with non-Hodgkin's lymphoma (NHL), eight with Hodgkin's disease (HD), nine with breast cancer and three with neuroblastoma. Six patients who did not receive GM-CSF post-ABMT served as controls. We assessed cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), expression of the activation antigen CD16, and cytokine production by an enriched population of monocytes (> 90% CD+14) pre-, during and post-GM-CSF administration. Within the group of patients receiving treatment, ADCC was significantly higher during in vivo GM-CSF administration than post-therapy (P < 0.05) and in 50% of these patients, ADCC increased during in vivo GM-CSF administration over pretreatment values. In addition, in vivo GM-CSF administration caused the monocytes to secrete elevated levels of tumor necrosis factor-alpha (TNF-alpha) and GM-CSF (P < 0.05). We conclude that GM-CSF augments monocyte-mediated cytotoxicity post-ABMT, and therefore may have a role in controlling minimal residual disease post-transplant.

  7. Increased Prevalence of Luminal Narrowing and Stricturing Identified by Enterography in Pediatric Crohn Disease Patients with Elevated Granulocyte-Macrophage Colony Stimulating Factor Auto-antibodies

    PubMed Central

    Dykes, Dana M.H.; Towbin, Alexander J.; Bonkowski, Erin; Chalk, Claudia; Bezold, Ramona; Lake, Kathleen; Kim, Mi-Ok; Heubi, James E.; Trapnell, Bruce C.; Podberesky, Daniel J.; Denson, Lee A.

    2013-01-01

    Background Crohn disease (CD) patients with elevated Granulocyte-Macrophage Colony-Stimulating Factor auto-antibodies (GM-CSF Ab) are more likely to develop stricturing behavior requiring surgery. Computed Tomography or Magnetic Resonance Enterography (CTE or MRE) may detect luminal narrowing (LN) prior to stricture development. Objective To determine whether CD patients with elevated GM-CSF Ab (≥ 1.6 mcg/mL) have a higher prevalence of LN and stricturing on CTE or MRE. Methods A single center, cross-sectional study of 153 pediatric CD patients and controls undergoing CTE or MRE. A novel scoring system evaluated for disease activity, presence of LN, stricture, intra-abdominal abscess, or fistulae Ouutcomes were compared with respect to antibody status using Fisher's exact test, logistic regression, and the unpaired t-test. Results GM-CSF Ab were elevated in CD patients (n=114) with a median (IQR) GM-CSF Ab level of 2.3 mcg/mL (0.5, 6.6) compared with healthy and disease controls, p=0.001. Ileal disease location was more common in CD patients with high GM-CSF Ab, p<0.001. Luminal narrowing increased from 39% in CD patients with low GM-CSF Ab to 71% in those with high levels (p=0.004). High GM-CSF Ab remained significantly associated with LN in a multivariate logistic model. Stricturing increased from 4% in CD patients with low GM-CSF Ab to 19% in those with high GM-CSF Ab (p=0.03). Conclusions Pediatric CD patients with high GM-CSF Ab levels have a higher prevalence of LN on CTE or MRE. Further study will be needed to determine whether medical therapy will reduce progression to stricturing behavior in these patients. PMID:23893081

  8. Granulocyte/macrophage colony-stimulating factor stimulates the expression of the 5-lipoxygenase-activating protein (FLAP) in human neutrophils

    PubMed Central

    1994-01-01

    The synthesis of leukotrienes in human blood neutrophils chiefly relies on the activity of two enzymes, phospholipase A2 and 5-lipoxygenase (5- LO). In turn, the activation of the 5-LO requires the participation of a recently characterized membrane-bound protein, the 5-LO-activating protein (FLAP). In this study, we have investigated conditions under which FLAP expression in neutrophils may be modulated. Of several cytokines tested, only granulocyte/macrophage colony-stimulating factor (GM-CSF) (and to a lesser extent tumor necrosis factor alpha) significantly increased expression of FLAP. GM-CSF increased FLAP mRNA steady-state levels in a time- and dose-dependent manner. The stimulatory effect of GM-CSF on FLAP mRNA was inhibited by prior treatment of the cells with the transcription inhibitor, actinomycin D, and pretreatment of the cells with the protein synthesis inhibitor, cycloheximide, failed to prevent the increase in FLAP mRNA induced by GM-CSF. The accumulation of newly synthesized FLAP, as determined by immunoprecipitation after incorporation of 35S-labeled amino acids, was also increased after incubation of neutrophils with GM-CSF. In addition, the total level of FLAP protein was increased in GM-CSF- treated neutrophils, as determined by two-dimensional gel electrophoresis, followed by Western blot. GM-CSF did not alter the stability of the FLAP protein, indicating that the effect of GM-CSF on FLAP accumulation was the consequence of increased de novo synthesis as opposed to decreased degradation of FLAP. Finally, incubation of neutrophils with the synthetic glucocorticoid dexamethasone directly stimulated the upregulation of FLAP mRNA and protein, and enhanced the effect of GM-CSF. Taken together, these data demonstrate that FLAP expression may be upmodulated after appropriate stimulation of neutrophils. The increase in FLAP expression induced by GM-CSF in inflammatory conditions could confer upon neutrophils a prolonged capacity to synthesize

  9. Engineering superactive granulocyte macrophage colony-stimulating factor transferrin fusion proteins as orally-delivered candidate agents for treating neurodegenerative disease.

    PubMed

    Heinzelman, Pete; Priebe, Molly C

    2015-01-01

    Intravenously injected granulocyte macrophage colony-stimulating factor (GM-CSF) has shown efficacy in Alzheimer's Disease (AD) and Parkinson's Disease (PD) animal studies and is undergoing clinical evaluation. The likely need for dosing of GM-CSF to patients over months or years motivates pursuit of avenues for delivering GM-CSF to circulation via oral administration. Flow cytometric screening of 37 yeast-displayed GM-CSF saturation mutant libraries revealed residues P12, H15, R23, R24, and K72 as key determinants of GM-CSF's CD116 and CD131 GM-CSF receptor (GM-CSFR) subunit binding affinity. Screening combinatorial GM-CSF libraries mutated at positions P12, H15, and R23 yielded variants with increased affinities toward both CD116 and CD131. Genetic fusion of GM-CSF to human transferrin (Trf), a strategy that enables oral delivery of other biopharmaceuticals in animals, yielded bioactive wild type and variant cytokines upon secretion from cultured Human Embryonic Kidney cells. Surface plasmon resonance (SPR) measurements showed that all evaluated variants possess decreases in CD116 and CD131 binding KD values of up to 2.5-fold relative to wild type. Improved affinity led to increased in vitro bioactivity; the most bioactive variant, P12D/H15L/R23L, had a leukocyte proliferation assay EC50 value 3.5-fold lower than the wild type GM-CSF/Trf fusion. These outcomes are important first steps toward our goal of developing GM-CSF/Trf fusions as orally available AD and PD therapeutics. PMID:25737095

  10. Differential regulation of spontaneous and immune complex-induced neutrophil apoptosis by proinflammatory cytokines. Role of oxidants, Bax and caspase-3.

    PubMed

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

    2002-07-01

    Neutrophil apoptosis represents a crucial step in the mechanisms governing the resolution of neutrophilic inflammation. Several soluble mediators of inflammation modulate neutrophil survival, retarding their apoptosis, whereas neutrophil activation by immune complexes (IC) results in the acceleration of apoptosis. To investigate neutrophil fate at the site of inflammation, we studied the effects of interleukin (IL)-2, IL-6, IL-8, IL-15, GM-CSF, and fMLP on spontaneous and IC-induced neutrophil apoptosis and the mechanisms regulating the survival of these cells. Spontaneous apoptosis was inhibited by GM-CSF, IL-6, and IL-15, but only GM-CSF overturned IC-induced apoptosis. No role of oxidants on the modulation of IC-dependent apoptosis was found. Indeed, fMLP or GM-CSF augmented the IC-dependent oxidative response, whereas the other compounds were ineffective. CGD neutrophils showed low levels of spontaneous apoptosis, but when exposed to IC, underwent a sharp increment of the apoptotic rate in a GM-CSF-inhibitable manner. Conversely, the expression of the proapoptotic protein Bax in 18-h aged neutrophils was down-regulated by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a nearly threefold Bax up-regulation, which was completely reversed only by GM-CSF. Accordingly, the spontaneous activity of caspase-3 was inhibited by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a sharp increment of enzymatic activity, and only GM-CSF inhibited the IC-dependent acceleration. Our results show that apoptosis of resting and IC-activated neutrophils is regulated differently, GM-CSF being the most potent neutrophil antiapoptotic factor. The results also unveil the existence of an oxidant-independent, Bax- and caspase-3-dependent, intracellular pathway regulating neutrophil apoptosis.

  11. Hereditary Pulmonary Alveolar Proteinosis

    PubMed Central

    Suzuki, Takuji; Sakagami, Takuro; Young, Lisa R.; Carey, Brenna C.; Wood, Robert E.; Luisetti, Maurizio; Wert, Susan E.; Rubin, Bruce K.; Kevill, Katharine; Chalk, Claudia; Whitsett, Jeffrey A.; Stevens, Carrie; Nogee, Lawrence M.; Campo, Ilaria; Trapnell, Bruce C.

    2010-01-01

    Rationale: We identified a 6-year-old girl with pulmonary alveolar proteinosis (PAP), impaired granulocyte-macrophage colony–stimulating factor (GM-CSF) receptor function, and increased GM-CSF. Objectives: Increased serum GM-CSF may be useful to identify individuals with PAP caused by GM-CSF receptor dysfunction. Methods: We screened 187 patients referred to us for measurement of GM-CSF autoantibodies to diagnose autoimmune PAP. Five were children with PAP and increased serum GM-CSF but without GM-CSF autoantibodies or any disease causing secondary PAP; all were studied with family members, subsequently identified patients, and controls. Measurement and Main Results: Eight children (seven female, one male) were identified with PAP caused by recessive CSF2RA mutations. Six presented with progressive dyspnea of insidious onset at 4.8 ± 1.6 years and two were asymptomatic at ages 5 and 8 years. Radiologic and histopathologic manifestations were similar to those of autoimmune PAP. Molecular analysis demonstrated that GM-CSF signaling was absent in six and severely reduced in two patients. The GM-CSF receptor β chain was detected in all patients, whereas the α chain was absent in six and abnormal in two, paralleling the GM-CSF signaling defects. Genetic analysis revealed multiple distinct CSF2RA abnormalities, including missense, duplication, frameshift, and nonsense mutations; exon and gene deletion; and cryptic alternative splicing. All symptomatic patients responded well to whole-lung lavage therapy. Conclusions: CSF2RA mutations cause a genetic form of PAP presenting as insidious, progressive dyspnea in children that can be diagnosed by a combination of characteristic radiologic findings and blood tests and treated successfully by whole-lung lavage. PMID:20622029

  12. Effects of free amino acids on cytokine secretion and proliferative activity of feline T cells in an in vitro study using the cell line MYA-1.

    PubMed

    Paßlack, Nadine; Doherr, Marcus G; Zentek, Jürgen

    2016-10-01

    In vitro studies might be an interesting screening method for targeted in vivo studies in the field of immunonutrition and help to reduce and refine animal studies. As the role of amino acids for immune function of cats has not been evaluated in detail so far, the present study aimed at investigating the effects of eight different amino acids (arginine, leucine, isoleucine, valine, glutamine, lysine, threonine and tryptophan) in six concentrations each (0, 0.25, 0.5, 1, 2 and 8x the cat blood level) on cytokine secretion and proliferative activity of feline T cells (MYA-1) in vitro. The results demonstrated that high doses of arginine increased IL-4, IL-10 and TNF-α secretion of T cells, while increasing concentrations of lysine increased IL-10 secretion and proliferative activity of the T cells. High doses of leucine enhanced GM-CSF and IL-10 secretion, while concentrations of threonine in the cell culture media greater than blood concentration also increased GM-CSF and additionally TNF-α secretion of the cells. The effects of glutamine and isoleucine on T cell function were only small. In conclusion, the present in vitro study could evaluate the immunomodulating potential of specific amino acids for feline T cell function. High doses of arginine, lysine, leucine and threonine had a significant impact on cytokine secretion and proliferative activity of the T cells. Targeted in vivo studies should investigate the clinical relevance of dietary supplementation of those amino acids in healthy and diseased cats as a next step. PMID:27510653

  13. Effects of free amino acids on cytokine secretion and proliferative activity of feline T cells in an in vitro study using the cell line MYA-1.

    PubMed

    Paßlack, Nadine; Doherr, Marcus G; Zentek, Jürgen

    2016-10-01

    In vitro studies might be an interesting screening method for targeted in vivo studies in the field of immunonutrition and help to reduce and refine animal studies. As the role of amino acids for immune function of cats has not been evaluated in detail so far, the present study aimed at investigating the effects of eight different amino acids (arginine, leucine, isoleucine, valine, glutamine, lysine, threonine and tryptophan) in six concentrations each (0, 0.25, 0.5, 1, 2 and 8x the cat blood level) on cytokine secretion and proliferative activity of feline T cells (MYA-1) in vitro. The results demonstrated that high doses of arginine increased IL-4, IL-10 and TNF-α secretion of T cells, while increasing concentrations of lysine increased IL-10 secretion and proliferative activity of the T cells. High doses of leucine enhanced GM-CSF and IL-10 secretion, while concentrations of threonine in the cell culture media greater than blood concentration also increased GM-CSF and additionally TNF-α secretion of the cells. The effects of glutamine and isoleucine on T cell function were only small. In conclusion, the present in vitro study could evaluate the immunomodulating potential of specific amino acids for feline T cell function. High doses of arginine, lysine, leucine and threonine had a significant impact on cytokine secretion and proliferative activity of the T cells. Targeted in vivo studies should investigate the clinical relevance of dietary supplementation of those amino acids in healthy and diseased cats as a next step.

  14. The effects of granulocyte-macrophage colony-stimulating factor on tumour-infiltrating lymphocytes from renal cell carcinoma.

    PubMed Central

    Steger, G. G.; Kaboo, R.; deKernion, J. B.; Figlin, R.; Belldegrun, A.

    1995-01-01

    It has been shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce specific and non-specific anti-tumour cytotoxicity and also stimulates the proliferation and function of peripheral lymphocytes and thymocytes. GM-CSF and interleukin 2 (IL-2) act synergistically on peripheral lymphocytes for the induction of a highly effective cytotoxic cell population. Thus, the goal of our investigation was to study the effects of GM-CSF upon expansion, proliferation and in vitro killing activity of tumour-infiltrating lymphocytes (TILs) from renal cell carcinoma (RCC). TILs from seven consecutive tumours were cultured with GM-CSF (500 or 1000 nmol ml-1) without IL-2 supplementation, with suboptimal doses of IL-2 (8 and 40 U ml-1) plus GM-CSF (1000 nmol ml-1), and with a dose of IL-2 (400 U ml-1) which sufficed alone to induce TIL development plus GM-CSF (500 or 1000 nmol ml-1). GM-CSF alone or together with suboptimal doses of IL-2 was not able to induce or facilitate TIL development in these cultures. When GM-CSF at both concentrations studied was added to optimal doses of IL-2 the resulting TIL populations proliferated significantly better and faster (+66%), resulting in a higher cell yield (+24%) at the time of maximal expansion of the TIL cultures. The length of the culture periods of TILs was not affected by GM-CSF when compared with the control cultures supplemented with IL-2 alone. In vitro killing activity of TIL populations stimulated with IL-2 and GM-CSF remained unspecific, but lysis of the autologous tumour targets as well as the allogeneic renal tumour targets was significantly enhanced (+138%) as compared with the corresponding control TILs stimulated with IL-2 alone. Lysis of the natural killer (NK)-sensitive control cell line K562 and the NK-resistant Daudi cell line remained unchanged even though FACS analysis of TILs cultured with IL-2 and 1000 nmol of GM-CSF demonstrated a significantly higher proportion of cells expressing the CD56

  15. Antigens and cytokine genes in antitumor vaccines: the importance of the temporal delivery sequence in antitumor signals.

    PubMed

    Herrero, María José; Botella, Rafael; Dasí, Francisco; Algás, Rosa; Sánchez, María; Aliño, Salvador F

    2006-12-01

    Studies against cancer, including clinical trials, have shown that a correct activation of the immune system can lead to tumor rejection whereas incorrect signaling results in no positive effects or even anergy. We have worked assuming that two signals, GM-CSF (granulocyte and macrophage colony-stimulating factor) and tumor antigens are necessary to mediate an antitumor effective response. To study which is the ideal temporal sequence for their administration, we have used a murine model of antimelanoma vaccine employing whole B16 tumor cells or their membrane protein antigens (TMPs) in combination with gm-csf transfer before or after the antigen delivery. Our results show that: (i) When gm-csf tisular transfection is performed before TMP delivery, a tumor growth inhibition is observed, but with a limit effect when administering high antigen doses; in contrast, when signals are inverted, the limited effect is lost and greater antitumor efficacy is obtained. (ii) A similar behavior, but with stronger positive results, is observed employing gm-csf transfection and whole tumor cells as antigens. While negative results are obtained with gm-csf before cells, the best results (total survival of treated mice) are obtained when GM-CSF is administered in transfected cells. We conclude that optimal antitumoral response can be obtained when the antigen signal is given before (or simultaneous with) GM-CSF production, while the inversion of the signals could result in the undesired inhibition or anergy of the immune response.

  16. A randomized, placebo-controlled trial of granulocyte-macrophage colony-stimulating factor and nucleoside analogue therapy in AIDS.

    PubMed

    Brites, C; Gilbert, M J; Pedral-Sampaio, D; Bahia, F; Pedroso, C; Alcantara, A P; Sasaki, M D; Matos, J; Renjifo, B; Essex, M; Whitmore, J B; Agosti, J M; Badaro, R

    2000-11-01

    Preliminary preclinical and clinical data suggest that granulocyte-macrophage colony-stimulating factor (GM-CSF) may decrease viral replication. Therefore, 105 individuals with AIDS who were receiving nucleoside analogue therapy were enrolled in a placebo-controlled, double-blind study and were randomized to receive either 125 microgram/m(2) of yeast-derived, GM-CSF (sargramostim) or placebo subcutaneously twice weekly for 6 months. Subjects were evaluated for toxicity and disease progression. A significant decrease in mean virus load (VL) was observed for the GM-CSF treatment group at 6 months (-0.07 log(10) vs. -0.60 log(10); P=.02). More subjects achieved human immunodeficiency virus (HIV)-RNA levels <500 copies/mL at >/=2 evaluations (2% on placebo vs. 11% on GM-CSF; P=.04). Genotypic analysis of 46 subjects demonstrated a lower frequency of zidovudine-resistant mutations among those receiving GM-CSF (80% vs. 50%; P=.04). No difference was observed in the incidence of opportunistic infections (OIs) through 6 months or survival, despite a higher risk for OI among GM-CSF recipients. GM-CSF reduced VL and limited the evolution of zidovudine-resistant genotypes, potentially providing adjunctive therapy in HIV disease.

  17. Cross-talk between ICAM-1 and granulocyte-macrophage colony-stimulating factor receptor signaling modulates eosinophil survival and activation.

    PubMed

    Pazdrak, Konrad; Young, Travis W; Stafford, Susan; Olszewska-Pazdrak, Barbara; Straub, Christof; Starosta, Vitaliy; Brasier, Allan; Kurosky, Alexander

    2008-03-15

    Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor beta-chain (GMRbeta) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480-488) showed binding to Shp2 phosphatase and GMRbeta. However, the interaction of GMRbeta with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMRbeta with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMRbeta. Importantly, we found that inhibition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-alpha. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking Ab effectively inhibited ERK activation and eosinophil survival. We concluded that the interaction between ICAM-1 and the GM-CSF receptor was essential for GM-CSF-induced eosinophil activation and survival. Taken together, these results provide novel mechanistic insights defining the interaction between ICAM-1 and the GM-CSF receptor and highlight the importance of targeting ICAM-1 and GM-CSF/IL-5/IL-3 receptor systems as a therapeutic strategy to counter eosinophilia in asthma.

  18. Delivery of granulocyte-macrophage colony-stimulating factor in bioadhesive hydrogel stimulates migration of dendritic cells in models of human papillomavirus-associated (pre)neoplastic epithelial lesions.

    PubMed

    Hubert, Pascale; Evrard, Brigitte; Maillard, Catherine; Franzen-Detrooz, Elizabeth; Delattre, Luc; Foidart, Jean-Michel; Noël, Agnes; Boniver, Jacques; Delvenne, Philippe

    2004-11-01

    Because of the central role of dendritic cells and/or Langerhans cells(DC/LC) in the induction of cellular immune responses, pharmacological agents that modulate the recruitment of these cells might have a clinical interest. The present study was designed to evaluate the capacity of several pharmaceutical formulations to topically deliver granulocyte-macrophage colony-stimulating factor (GM-CSF) on human papillomavirus (HPV)-associated genital (pre)neoplastic lesions. The formulations were evaluated for their bioactivity and for their potential to recruit DC in organotypic cultures of HPV-transformed keratinocytes. We found that a bioadhesive polycarbophil gel (Noveon) at pH 5.5 is able to maintain the bioactivity of GM-CSF at 4 or 37 degrees C for at least 7 days, whereas a decreased activity of GM-CSF was observed when the molecule is included in other polymer gels. GM-CSF incorporated in the polycarbophil gel was also a potent factor in enhancing the colonization of DC into organotypic cultures of HPV-transformed keratinocytes since the infiltration of DC in the in vitro-formed (pre)neoplastic epithelium was very low under basal conditions and dramatically increased in the presence of GM-CSF gel. We next demonstrated that GM-CSF incorporated in polycarbophil gel induces the recruitment of human DC in a human (pre)neoplastic epithelium grafted into NOD/SCID mice. The efficacy of GM-CSF in this formulation was equivalent to that observed with liquid GM-CSF. These results suggest that GM-CSF incorporated in polycarbophil gel could play an important role in the recruitment of DC/LC in mucosal surfaces and be useful as a new immunotherapeutic approach for genital HPV-associated (pre)neoplastic lesions.

  19. Regulation of Wound Healing by Granulocyte-Macrophage Colony-Stimulating Factor after Vocal Fold Injury

    PubMed Central

    Lim, Jae-Yol; Choi, Byung Hyune; Lee, Songyi; Jang, Yun Ho; Choi, Jeong-Seok; Kim, Young-Mo

    2013-01-01

    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 regeneration

  20. GM-CSF as successful salvage therapy of metamizole (dipyrone)-induced agranulocytosis with Fournier's gangrene and severe septic shock in an adolescent.

    PubMed

    Winkler, Annegret; Kietz, Silke; Bahlmann, Hagen; Jafarzade, Gunel; Lode, Holger N; Heckmann, Matthias

    2016-08-01

    This case report describes the successful use of granulocyte and macrophage colony-stimulating factor as salvage therapy and an alternative to hematopoietic stem cell transplantation in a 14-year-old adolescent with metamizole (dipyrone)-induced agranulocytosis and severe septic shock.

  1. In vitro expression of IL-1α, GM-CSF, and TNF-α by multinucleated macrophages from BCG-infected mice.

    PubMed

    Iljine, D A; Arkhipov, S A; Shkurupy, V A

    2013-09-01

    Peritoneal cells from intact and BCG-infected mice were explanted in vitro. In these cultures, multinucleated macrophages in different number of nuclei were formed. The intensity of multinucleated cell formation was higher in cultures from BCG-infected mice. Increasing role of amitosis in the formation of multinucleated macrophages with relatively high number of nuclei was noted with presumable domination of cell fusion mechanism. Relatively high level of IL-1α expression was noted only in the population of binucleated macrophages of BCG-infected mice in comparison with mononuclear cells. It was found macrophages from BCG-infected mice demonstrate a kind of "lineage commitment" towards multinucleated cells, which manifested in culture in initially high and increasing (with increasing the number of nuclei in cells) expression of granulocyte-macrophage CSF and TNF-α as well as initially high amitotic activity of macrophages.

  2. Vaccine Therapy With Sargramostim (GM-CSF) in Treating Patients With Her-2 Positive Stage III-IV Breast Cancer or Ovarian Cancer

    ClinicalTrials.gov

    2016-05-02

    HER2-positive Breast Cancer; Stage III Ovarian Epithelial Cancer; Stage III Ovarian Germ Cell Tumor; Stage IIIA Breast Cancer; Stage IIIB Breast Cancer; Stage IIIC Breast Cancer; Stage IV Breast Cancer; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor

  3. GM-CSF as successful salvage therapy of metamizole (dipyrone)-induced agranulocytosis with Fournier's gangrene and severe septic shock in an adolescent.

    PubMed

    Winkler, Annegret; Kietz, Silke; Bahlmann, Hagen; Jafarzade, Gunel; Lode, Holger N; Heckmann, Matthias

    2016-08-01

    This case report describes the successful use of granulocyte and macrophage colony-stimulating factor as salvage therapy and an alternative to hematopoietic stem cell transplantation in a 14-year-old adolescent with metamizole (dipyrone)-induced agranulocytosis and severe septic shock. PMID:27525093

  4. Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor

    SciTech Connect

    Williams, D.E.; Bicknell, D.C.; Park, L.S.; Straneva, J.E.; Cooper, S.; Broxmeyer, H.E.

    1988-01-01

    Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with /sup 125/I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with /sup 125/I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor. Affinity crosslinking studies demonstrated that /sup 125/I-labeled GM-CSF bound specifically to two species of M/sub r/ 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The M/sub r/ 70,000 species is thought to be a proteolytic fragment of the intact M/sub r/ 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.

  5. Interferon-γ and granulocyte/monocyte colony-stimulating factor production by natural killer cells involves different signaling pathways and the adaptor stimulator of interferon genes (STING).

    PubMed

    Souza-Fonseca-Guimaraes, Fernando; Parlato, Marianna; de Oliveira, Rosane B; Golenbock, Douglas; Fitzgerald, Katherine; Shalova, Irina N; Biswas, Subhra K; Cavaillon, Jean-Marc; Adib-Conquy, Minou

    2013-04-12

    Natural killer (NK) cells are important for innate immunity in particular through the production of IFN-γ and GM-CSF. Both cytokines are important in restoration of immune function of tolerized leukocytes under inflammatory events. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors, rarely seeking their protein expression. We previously showed that murine spleen NK cells express TLR9 intracellularly and respond to CpG oligodeoxynucleotide (CpG-ODN) by producing IFN-γ and GM-CSF. However, to get such production the presence of accessory cytokines (such as IL-15 and IL-18) was required, whereas CpG-ODN or accessory cytokines alone did not induce IFN-γ or GM-CSF. We show here that TLR9 overlaps with the Golgi apparatus in NK cells. Furthermore, CpG-ODN stimulation in the presence of accessory cytokines induces the phosphorylation of c-Jun, STAT3, and IκBα. IFN-γ and GM-CSF production requires NF-κB and STAT3 activation as well as Erk-dependent mechanisms for IFN-γ and p38 signaling for GM-CSF. Using knock-out-mice, we show that UNC93b1 and IL-12 (produced by NK cells themselves) are also necessary for IFN-γ and GM-CSF production. IFN-γ production was found to be MyD88- and TLR9-dependent, whereas GM-CSF was TLR9-independent but dependent on STING (stimulator of interferon genes), a cytosolic adaptor recently described for DNA sensing. Our study thereby allows us to gain insight into the mechanisms of synergy between accessory cytokines and CpG-ODN in NK cells. It also identifies a new and alternative signaling pathway for CpG-ODN in murine NK cells.

  6. Cytokine amplification and macrophage effector functions in aortic inflammation and abdominal aortic aneurysm formation.

    PubMed

    Ijaz, Talha; Tilton, Ronald G; Brasier, Allan R

    2016-08-01

    On April 29, 2015, Son and colleagues published an article entitled "Granulocyte macrophage colony-stimulating factor (GM-CSF) is required for aortic dissection/intramural haematoma" in Nature Communications. The authors observed that the heterozygous Kruppel-like transcription factor 6 (KLF6) deficiency or absence of myeloid-specific KLF6 led to upregulation of macrophage GM-CSF expression, promoted the development of aortic hematoma/dissection, and stimulated abdominal aortic aneurysm (AAA) formation when the vessel wall was subjected to an inflammatory stimulus. The additional findings of increased adventitial fibrotic deposition, marked infiltration of macrophages, and increased expression of matrix metalloprotease-9 (MMP-9) and IL-6 were blocked with neutralizing GM-CSF antibodies, or recapitulated in normal mice with excess GM-CSF administration. The authors concluded that GM-CSF is a key regulatory molecule in the development of AAA and further suggested that activation of GM-CSF is independent of the transforming growth factor β (TGFβ)-Smad pathway associated with the Marfan aortic pathology. In this perspective, we expand on this mechanism, drawing from previous studies implicating a similar essential role for IL-6 signaling in macrophage activation, Th17 expansion and aortic dissections. We propose a sequential "two-hit" model of vascular inflammation involving initial vascular injury followed by recruitment of Ly6C(hi) macrophages. Aided by fibroblast interactions inflammatory macrophages produce amplification of IL-6 and GM-CSF expression that converge on a common, pathogenic Janus kinase (JAK)-signal transducers and activations of transcription 3 (STAT3) signaling pathway. This pathway stimulates effector functions of macrophages, promotes differentiation of Th17 lymphocytes and enhances matrix metalloproteinase expression, ultimately resulting in deterioration of vascular wall structural integrity. Further research evaluating the impact of

  7. Cytokine amplification and macrophage effector functions in aortic inflammation and abdominal aortic aneurysm formation

    PubMed Central

    Ijaz, Talha; Tilton, Ronald G.

    2016-01-01

    On April 29, 2015, Son and colleagues published an article entitled “Granulocyte macrophage colony-stimulating factor (GM-CSF) is required for aortic dissection/intramural haematoma” in Nature Communications. The authors observed that the heterozygous Kruppel-like transcription factor 6 (KLF6) deficiency or absence of myeloid-specific KLF6 led to upregulation of macrophage GM-CSF expression, promoted the development of aortic hematoma/dissection, and stimulated abdominal aortic aneurysm (AAA) formation when the vessel wall was subjected to an inflammatory stimulus. The additional findings of increased adventitial fibrotic deposition, marked infiltration of macrophages, and increased expression of matrix metalloprotease-9 (MMP-9) and IL-6 were blocked with neutralizing GM-CSF antibodies, or recapitulated in normal mice with excess GM-CSF administration. The authors concluded that GM-CSF is a key regulatory molecule in the development of AAA and further suggested that activation of GM-CSF is independent of the transforming growth factor β (TGFβ)-Smad pathway associated with the Marfan aortic pathology. In this perspective, we expand on this mechanism, drawing from previous studies implicating a similar essential role for IL-6 signaling in macrophage activation, Th17 expansion and aortic dissections. We propose a sequential “two-hit” model of vascular inflammation involving initial vascular injury followed by recruitment of Ly6Chi macrophages. Aided by fibroblast interactions inflammatory macrophages produce amplification of IL-6 and GM-CSF expression that converge on a common, pathogenic Janus kinase (JAK)-signal transducers and activations of transcription 3 (STAT3) signaling pathway. This pathway stimulates effector functions of macrophages, promotes differentiation of Th17 lymphocytes and enhances matrix metalloproteinase expression, ultimately resulting in deterioration of vascular wall structural integrity. Further research evaluating the impact of

  8. Cytokine amplification and macrophage effector functions in aortic inflammation and abdominal aortic aneurysm formation

    PubMed Central

    Ijaz, Talha; Tilton, Ronald G.

    2016-01-01

    On April 29, 2015, Son and colleagues published an article entitled “Granulocyte macrophage colony-stimulating factor (GM-CSF) is required for aortic dissection/intramural haematoma” in Nature Communications. The authors observed that the heterozygous Kruppel-like transcription factor 6 (KLF6) deficiency or absence of myeloid-specific KLF6 led to upregulation of macrophage GM-CSF expression, promoted the development of aortic hematoma/dissection, and stimulated abdominal aortic aneurysm (AAA) formation when the vessel wall was subjected to an inflammatory stimulus. The additional findings of increased adventitial fibrotic deposition, marked infiltration of macrophages, and increased expression of matrix metalloprotease-9 (MMP-9) and IL-6 were blocked with neutralizing GM-CSF antibodies, or recapitulated in normal mice with excess GM-CSF administration. The authors concluded that GM-CSF is a key regulatory molecule in the development of AAA and further suggested that activation of GM-CSF is independent of the transforming growth factor β (TGFβ)-Smad pathway associated with the Marfan aortic pathology. In this perspective, we expand on this mechanism, drawing from previous studies implicating a similar essential role for IL-6 signaling in macrophage activation, Th17 expansion and aortic dissections. We propose a sequential “two-hit” model of vascular inflammation involving initial vascular injury followed by recruitment of Ly6Chi macrophages. Aided by fibroblast interactions inflammatory macrophages produce amplification of IL-6 and GM-CSF expression that converge on a common, pathogenic Janus kinase (JAK)-signal transducers and activations of transcription 3 (STAT3) signaling pathway. This pathway stimulates effector functions of macrophages, promotes differentiation of Th17 lymphocytes and enhances matrix metalloproteinase expression, ultimately resulting in deterioration of vascular wall structural integrity. Further research evaluating the impact of

  9. Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor-beta 1 and myofibroblast accumulation.

    PubMed Central

    Xing, Z.; Tremblay, G. M.; Sime, P. J.; Gauldie, J.

    1997-01-01

    We have previously reported that transfer to rat lung of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene leads to high expression of GM-CSF between days 1 and 4 and granulation tissue formation followed by an irreversible fibrotic response starting from day 12 onward. In the current study, we investigated the underlying mechanisms. We found that GM-CSF overexpression did not enhance production of tumor necrosis factor-alpha in a significant manner at any time after GM-CSF gene transfer. However, the content of transforming growth factor-beta 1 in bronchoalveolar lavage fluid was markedly induced at day 4 and appeared to be maximal around day 7 and remained high at day 12. Macrophages purified from bronchoalveolar lavage fluid 7 days after GM-CSF gene transfer spontaneously released significant quantities of transforming growth factor-beta 1 protein in vitro. After peak transforming growth factor-beta 1 production was the emergence of alpha-smooth muscle actin-rich myofibroblasts. Accumulation of these cells was most prominent at day 12 within the granulation tissues and they were still present in fibrotic areas between days 12 and 24 and diminished markedly afterward. Thus, we provide the first in vivo evidence that tumor necrosis factor-alpha may be dissociated from participation in a fibrotic process in the lung and GM-CSF may play a more direct role in pulmonary fibrogenesis at least in part through its capability to induce transforming growth factor-beta 1 in macrophages and the subsequent emergence of myofibroblast phenotypes. This GM-CSF transgene lung model is useful for a stepwise dissection of both cellular and molecular events involved in pulmonary fibrosis. Images Figure 2 Figure 5 Figure 6 PMID:9006322

  10. Phase 1 study of intratumoral Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus, in pediatric cancer patients.

    PubMed

    Cripe, Timothy P; Ngo, Minhtran C; Geller, James I; Louis, Chrystal U; Currier, Mark A; Racadio, John M; Towbin, Alexander J; Rooney, Cliona M; Pelusio, Adina; Moon, Anne; Hwang, Tae-Ho; Burke, James M; Bell, John C; Kirn, David H; Breitbach, Caroline J

    2015-03-01

    Pexa-Vec (pexastimogene devacirepvec, JX-594) is an oncolytic and immunotherapeutic vaccinia virus designed to destroy cancer cells through viral lysis and induction of granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven tumor-specific immunity. Pexa-Vec has undergone phase 1 and 2 testing alone and in combination with other therapies in adult patients, via both intratumoral and intravenous administration routes. We sought to determine the safety of intratumoral administration in pediatric patients. In a dose-escalation study using either 10(6) or 10(7) plaque-forming units per kilogram, we performed one-time injections in up to three tumor sites in five pediatric patients and two injections in one patient. Ages at study entry ranged from 4 to 21 years, and their cancer diagnoses included neuroblastoma, hepatocellular carcinoma, and Ewing sarcoma. All toxicities were ≤ grade 3. The most common side effects were sinus fever and sinus tachycardia. All three patients at the higher dose developed asymptomatic grade 1 treatment-related skin pustules that resolved within 3-4 weeks. One patient showed imaging evidence suggestive of antitumor biological activity. The two patients tested for cellular immunoreactivity to vaccinia antigens showed strong responses. Overall, our study suggests Pexa-Vec is safe to administer to pediatric patients by intratumoral administration and could be studied further in this patient population.

  11. Therapeutic Potential of Dental Pulp Stem Cell Secretome for Alzheimer's Disease Treatment: An In Vitro Study.

    PubMed

    Ahmed, Nermeen El-Moataz Bellah; Murakami, Masashi; Hirose, Yujiro; Nakashima, Misako

    2016-01-01

    The secretome obtained from stem cell cultures contains an array of neurotrophic factors and cytokines that might have the potential to treat neurodegenerative conditions. Alzheimer's disease (AD) is one of the most common human late onset and sporadic neurodegenerative disorders. Here, we investigated the therapeutic potential of secretome derived from dental pulp stem cells (DPSCs) to reduce cytotoxicity and apoptosis caused by amyloid beta (Aβ) peptide. We determined whether DPSCs can secrete the Aβ-degrading enzyme, neprilysin (NEP), and evaluated the effects of NEP expression in vitro by quantitating Aβ-degrading activity. The results showed that DPSC secretome contains higher concentrations of VEGF, Fractalkine, RANTES, MCP-1, and GM-CSF compared to those of bone marrow and adipose stem cells. Moreover, treatment with DPSC secretome significantly decreased the cytotoxicity of Aβ peptide by increasing cell viability compared to nontreated cells. In addition, DPSC secretome stimulated the endogenous survival factor Bcl-2 and decreased the apoptotic regulator Bax. Furthermore, neprilysin enzyme was detected in DPSC secretome and succeeded in degrading Aβ 1-42 in vitro in 12 hours. In conclusion, our study demonstrates that DPSCs may serve as a promising source for secretome-based treatment of Alzheimer's disease. PMID:27403169

  12. Therapeutic Potential of Dental Pulp Stem Cell Secretome for Alzheimer's Disease Treatment: An In Vitro Study

    PubMed Central

    Ahmed, Nermeen El-Moataz Bellah; Murakami, Masashi; Hirose, Yujiro; Nakashima, Misako

    2016-01-01

    The secretome obtained from stem cell cultures contains an array of neurotrophic factors and cytokines that might have the potential to treat neurodegenerative conditions. Alzheimer's disease (AD) is one of the most common human late onset and sporadic neurodegenerative disorders. Here, we investigated the therapeutic potential of secretome derived from dental pulp stem cells (DPSCs) to reduce cytotoxicity and apoptosis caused by amyloid beta (Aβ) peptide. We determined whether DPSCs can secrete the Aβ-degrading enzyme, neprilysin (NEP), and evaluated the effects of NEP expression in vitro by quantitating Aβ-degrading activity. The results showed that DPSC secretome contains higher concentrations of VEGF, Fractalkine, RANTES, MCP-1, and GM-CSF compared to those of bone marrow and adipose stem cells. Moreover, treatment with DPSC secretome significantly decreased the cytotoxicity of Aβ peptide by increasing cell viability compared to nontreated cells. In addition, DPSC secretome stimulated the endogenous survival factor Bcl-2 and decreased the apoptotic regulator Bax. Furthermore, neprilysin enzyme was detected in DPSC secretome and succeeded in degrading Aβ1–42 in vitro in 12 hours. In conclusion, our study demonstrates that DPSCs may serve as a promising source for secretome-based treatment of Alzheimer's disease. PMID:27403169

  13. Primary in vitro culture of porcine tracheal epithelial cells in an air-liquid interface as a model to study airway epithelium and Aspergillus fumigatus interactions.

    PubMed

    Khoufache, Khaled; Cabaret, Odile; Farrugia, Cécile; Rivollet, Danièle; Alliot, Annie; Allaire, Eric; Cordonnier, Catherine; Bretagne, Stéphane; Botterel, Françoise

    2010-12-01

    Since the airway epithelium is the first tissue encountered by airborne fungal spores, specific models are needed to study this interaction. We developed such a model using primary porcine tracheal epithelial cells (PTEC) as a possible alternative to the use of primary human cells. PTEC were obtained from pigs and were cultivated in an air-liquid interface. Fluorescent brightener was employed to quantify the internalization of Aspergillus fumigatus conidia. Potential differences (Vt) and transepithelial resistances (Rt) after challenge with the mycotoxin, verruculogen, were studied. Primers for porcine inflammatory mediator genes IL-8, TNF-alpha, and GM-CSF were designed for a quantitative real-time PCR procedure to study cellular responses to challenges with A. fumigatus conidia. TEM showed the differentiation of ciliated cells and the PTEC ability to internalize conidia. The internalization rate was 21.9 ± 1.4% after 8 h of incubation. Verruculogen (10(-6) M) significantly increased Vt without having an effect on the Rt. Exposure of PTEC to live A. fumigatus conidia for 24 h induced a 10- to 40-fold increase in the mRNA levels of inflammatory mediator genes. PTEC behave similarly to human cells and are therefore a suitable alternative to human cells for studying interaction between airway epithelium and A. fumigatus. PMID:20608777

  14. Key role for myeloid cells: phase II results of anti-G(D2) antibody 3F8 plus granulocyte-macrophage colony-stimulating factor for chemoresistant osteomedullary neuroblastoma.

    PubMed

    Cheung, Nai-Kong V; Cheung, Irene Y; Kramer, Kim; Modak, Shakeel; Kuk, Deborah; Pandit-Taskar, Neeta; Chamberlain, Elizabeth; Ostrovnaya, Irina; Kushner, Brian H

    2014-11-01

    Anti-G(D2) murine antibody 3F8 plus subcutaneously (sc) administered granulocyte-macrophage colony-stimulating factor (GM-CSF) was used against primary refractory neuroblastoma in metastatic osteomedullary sites. Large study size and long follow-up allowed assessment of prognostic factors in a multivariate analysis not reported with other anti-G(D2) antibodies. In a phase II trial, 79 patients without prior progressive disease were treated for persistent osteomedullary neuroblastoma documented by histology and/or metaiodobenzyl-guanidine (MIBG) scan. In the absence of human antimouse antibody, 3F8 + scGM-CSF cycles were repeated up to 24 months. Minimal residual disease (MRD) in bone marrow was measured by quantitative reverse transcription-polymerase chain reaction pre-enrollment and post-cycle #2, before initiation of 13-cis-retinoic acid. Study endpoints were: (i) progression-free survival (PFS) compared with the predecessor trial of 3F8 plus intravenously administered (iv) GM-CSF (26 patients) and (ii) impact of MRD on PFS. Using all 105 patients from the two consecutive 3F8 + GM-CSF trials, prognostic factors were analyzed by multivariate Cox regression model. Complete response rates to 3F8 + scGM-CSF were 87% by histology and 38% by MIBG. Five-year PFS was 24 ± 6%, which was significantly superior to 11 ± 7% with 3F8 + ivGM-CSF (p = 0.002). In the multivariate analysis, significantly better PFS was associated with R/R or H/R FCGR2A polymorphism, sc route of GM-CSF and early MRD response. MYCN amplification was not prognostic. Complement consumption was similar with either route of GM-CSF. Toxicities were manageable, allowing outpatient treatment. 3F8 + scGM-CSF is highly active against chemoresistant osteomedullary neuroblastoma. MRD response may be an indicator of tumor sensitivity to anti-G(D2) immunotherapy. Correlative studies highlight the antineoplastic potency of myeloid effectors.

  15. Induction of Specific Cellular and Humoral Responses against Renal Cell Carcinoma after Combination Therapy with Cryoablation and Granulocyte-Macrophage Colony Stimulating Factor: A Pilot Study

    PubMed Central

    Thakur, Archana; Littrup, Peter; Paul, Elyse N.; Adam, Barbara; Heilbrun, Lance K.; Lum, Lawrence G.

    2013-01-01

    Cryotherapy offers a minimally invasive treatment option for the management of both irresectable and localized prostate, liver, pulmonary and renal tumors. The anti-neoplastic effects of cryotherapy are mediated by direct tumor lysis and by indirect effects such as intracellular dehydration, pH changes, and microvascular damage resulting in ischemic necrosis. In this study, we investigated whether percutaneous cryoablation of lung metastasis from renal cell carcinoma (RCC) in combination with aerosolized granulocyte-macrophage colony stimulating factor (GM-CSF) can induce systemic cellular and humoral immune responses in 6 RCC patients. Peripheral blood mononuclear cells (PBMC) were sequentially studied up to 63 days post cryoimmunotherapy (CI). PBMC from pre and post CI were phenotyped for lymphocyte subsets and tested for cytotoxicity and IFNγ Elispots directed at RCC cells. Humoral responses were measured by in vitro antibody synthesis assay directed at RCC cells. The immune monitoring data showed that CI induced tumor specific CTL, specific in vitro anti-tumor antibody responses, and enhanced Th1 cytokine production in 4 out of 6 patients. More importantly, the magnitude of cellular and humoral anti-tumor response appears to be associated with clinical responses. These pilot data show that CI can induce robust and brisk cellular and humoral immune responses in metastatic RCC patients, but requires further evaluation in optimized protocols. PMID:21577139

  16. Role of Granulocyte-Macrophage Colony-Stimulating Factor Signaling in Regulating Neutrophil Antifungal Activity and the Oxidative Burst During Respiratory Fungal Challenge.

    PubMed

    Kasahara, Shinji; Jhingran, Anupam; Dhingra, Sourabh; Salem, Anand; Cramer, Robert A; Hohl, Tobias M

    2016-04-15

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that plays a critical role in regulating myeloid cell host defense. In this study, we demonstrated that GM-CSF signaling plays an essential role in antifungal defense against Aspergillus fumigatus. Mice that lack the GM-CSF receptor β chain (GM-CSFRβ) developed invasive hyphal growth and exhibited impaired survival after pulmonary challenge with A. fumigatus conidia. GM-CSFRβ signaling regulated the recruitment of inflammatory monocytes to infected lungs, but not the recruitment of effector neutrophils. Cell-intrinsic GM-CSFRβ signaling mediated neutrophil and inflammatory monocyte antifungal activity, because lung GM-CSFRβ(-/-) leukocytes exhibited impaired conidial killing compared with GM-CSFRβ(+/+) counterparts in mixed bone marrow chimeric mice. GM-CSFRβ(-/-) neutrophils exhibited reduced (hydrogenated) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in vivo. Conversely, administration of recombinant GM-CSF enhanced neutrophil NADPH oxidase function, conidiacidal activity, and lung fungal clearance in A. fumigatus-challenged mice. Thus, our study illustrates the functional role of GM-CSFRβ signaling on lung myeloid cell responses against inhaled A. fumigatus conidia and demonstrates a benefit for systemic GM-CSF administration. PMID:26908736

  17. Granulocyte/macrophage colony-stimulating factor attenuates endothelial hyperpermeability after thermal injury.

    PubMed

    Zhao, Jingling; Chen, Lei; Shu, Bin; Tang, Jinming; Zhang, Lijun; Xie, Julin; Liu, Xusheng; Xu, Yingbin; Qi, Shaohai

    2015-01-01

    Microvascular hyperpermeability followed by burn injury is the main cause of shock, and cardiovascular collapse can result if the condition is treated improperly. Our previous studies demonstrated that granulocyte/macrophage colony-stimulating factor (GM-CSF) clearly reduces microvascular permeability and protects microvessels against burn injury. However, the mechanism underlying the protective function of GM-CSF on burn-injured microvessels remains unknown. This study aimed to investigate the effect and mechanism of GM-CSF on endothelial cells after exposure to burn serum. We demonstrated that GM-CSF reduced post-burn endothelial "capillary leak" by inhibiting the activity of RhoA and maintaining the membrane localization of VE-cadherin. Membranous VE-cadherin enhances adherens junctions between endothelial cells and co-localizes with and activates VEGFR2, which protect cells from burn serum-induced apoptosis. Our findings suggest that the protective mechanism of GM-CSF on burn serum-injured endothelial monolayer hyperpermeability is achieved by strengthening cell adherens junctions and improving cell viability.

  18. Comparative effect of recombinant IL-1, -2, -3, -4, and -6, IFN-gamma, granulocyte-macrophage-colony-stimulating factor, tumor necrosis factor-alpha, and histamine-releasing factors on the secretion of histamine from basophils

    SciTech Connect

    Alam, R.; Welter, J.B.; Forsythe, P.A.; Lett-Brown, M.A.; Grant, J.A. )

    1989-05-15

    Most cytokines possess multiple biologic activities. This study was undertaken to investigate the effect of rIL-1 beta, -2, -3, -4 and -6, IFN-gamma, TNF-alpha, and granulocyte-macrophage (GM)-CSF on basophils from 16 donors and the amount of histamine released was compared with that by partially purified mononuclear cell-derived histamine-releasing factor (HRF) and anti-IgE. We found that only IL-3 and GM-CSF at relatively high doses (50 to 500 ng/ml) released small amounts of histamine (3 to 14%) from two allergic donors. In contrast, both HRF and anti-IgE released significant amounts of histamine from all donors. Other cytokines did not release any measurable quantity of histamine. Simultaneous addition of several cytokines to the basophils also failed to release histamine. IL-3, GM-CSF, and IL-1 can also release histamine at lower concentrations (less than 5 ng/ml) when incubated with basophils in the presence of D{sub 2}O. Basophils from 6 out of 13 allergic donors released histamine in response to IL-3, whereas three donors responded to IL-1 beta and two responded to GM-CSF. The results of this study demonstrated that although IL-3 and GM-CSF release small amounts of histamine only from a select group of allergic patients, mononuclear cell-derived HRF is more potent in their action and release histamine from normals as well as allergic patients.

  19. Deficiency of β Common Receptor Moderately Attenuates the Progression of Myeloproliferative Neoplasm in NrasG12D/+ Mice*

    PubMed Central

    Zhang, Jingfang; Ranheim, Erik A.; Du, Juan; Liu, Yangang; Wang, Jinyong; Kong, Guangyao; Zhang, Jing

    2015-01-01

    Activating Ras signaling is a major driver in juvenile and the myeloproliferative variant of chronic myelomonocytic leukemia (JMML/MP-CMML). Numerous studies suggest that GM-CSF signaling plays a central role in establishing and maintaining JMML/MP-CMML phenotypes in human and mouse. However, it remains elusive how GM-CSF signaling impacts on JMML/MP-CMML initiation and progression. Here, we investigate this issue in a well characterized MP-CMML model induced by endogenous NrasG12D/+ mutation. In this model, NrasG12D/+ hematopoietic stem cells (HSCs) are required to initiate and maintain CMML phenotypes and serve as CMML-initiating cells. We show that the common β chain of the GM-CSF receptor (βc) is dispensable for NrasG12D/+ HSC function; loss of βc does not affect the expansion, increased self-renewal, or myeloid differentiation bias in NrasG12D/+ HSCs. Therefore, βc−/− does not abrogate CMML in NrasG12D/+ mice. However, βc deficiency indeed significantly reduces NrasG12D/+-induced splenomegaly and spontaneous colony formation and prolongs the survival of CMML-bearing mice, suggesting that GM-CSF signaling plays an important role in promoting CMML progression. Together, our results suggest that inhibiting GM-CSF signaling in JMML/MP-CMML patients might alleviate disease symptoms but would not eradicate the disease. PMID:26082490

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

    PubMed

    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.

  1. Meningeal mast cell-T cell crosstalk regulates T cell encephalitogenicity.

    PubMed

    Russi, Abigail E; Walker-Caulfield, Margaret E; Guo, Yong; Lucchinetti, Claudia F; Brown, Melissa A

    2016-09-01

    GM-CSF is a cytokine produced by T helper (Th) cells that plays an essential role in orchestrating neuroinflammation in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. Yet where and how Th cells acquire GM-CSF expression is unknown. In this study we identify mast cells in the meninges, tripartite tissues surrounding the brain and spinal cord, as important contributors to antigen-specific Th cell accumulation and GM-CSF expression. In the absence of mast cells, Th cells do not accumulate in the meninges nor produce GM-CSF. Mast cell-T cell co-culture experiments and selective mast cell reconstitution of the meninges of mast cell-deficient mice reveal that resident meningeal mast cells are an early source of caspase-1-dependent IL-1β that licenses Th cells to produce GM-CSF and become encephalitogenic. We also provide evidence of mast cell-T cell co-localization in the meninges and CNS of recently diagnosed acute MS patients indicating similar interactions may occur in human demyelinating disease. PMID:27396526

  2. Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide

    PubMed Central

    Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

    2015-01-01

    Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%–92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp. PMID:26473722

  3. Haemorrhagic shock in mice--intracellular signalling and immunomodulation of peritoneal macrophages' LPS response.

    PubMed

    Rani, Meenakshi; Husain, Baher; Lendemans, Sven; Schade, Fritz U; Flohé, Sascha

    2006-01-01

    Haemorrhagic shock leads to decreased proinflammatory cytokine response which is associated with an increased susceptibility to bacterial infections. In the present study, the effect of GM-CSF on lipopolysaccharide (LPS)-induced TNF-alpha release and MAPkinase activation was analysed on the background of a possible immunostimulating activity of this substance. Male BALB/c mice were bled to a mean arterial blood pressure of 50 mmHg for 45 min followed by resuscitation. Peritoneal macrophages were isolated 20 h after haemorrhage and incubated with 10 ng/ml GM-CSF for 6h before LPS stimulation. TNF-alpha synthesis was studied in the culture supernatants using ELISA. Phosphorylation of ERK, p38MAPK and IkappaBalpha was detected by Western blotting. LPS-induced TNF-alpha production of peritoneal macrophages was significantly decreased 20 h after haemorrhage in comparison to the corresponding cells of sham-operated mice. In parallel the phosphorylation of IkappaBalpha was less in LPS-stimulated peritoneal macrophages from haemorrhagic mice. LPS-induced phosphorylation of ERK1/2 was also decreased in peritoneal macrophages isolated after haemorrhagic shock. In contrast, p38MAPK was phosphorylated more intensely after LPS-stimulation in macrophages collected from shocked mice. GM-CSF incubation elevated LPS-induced TNF-alpha response of macrophages from both sham-operated and shocked mice which was accompanied by an elevated IkappaB and ERK phosphorylation. In general, GM-CSF treatment in vitro enhanced peritoneal macrophages LPS-response both in terms of TNF-alpha synthesis and IkappaB and MAPK signalling, but the levels always stayed lower than those of GM-CSF-treated cells from sham-operated animals. In conclusion, GM-CSF preincubation could partly reactivate the depressed functions of peritoneal macrophages and may therefore exert immunostimulating properties after shock or trauma.

  4. Antibiotics and production of granulocyte-macrophage colony-stimulating factor by human bronchial epithelial cells in vitro. A comparison of cefodizime and ceftriaxone.

    PubMed

    Pacheco, Y; Hosni, R; Dagrosa, E E; Gormand, F; Guibert, B; Chabannes, B; Lagarde, M; Perrin-Fayolle, M

    1994-04-01

    Cultured human bronchial epithelial cells (HBEC) produce both granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 8 (IL-8). The influence of cefodizime (CAS 69739-16-8), a new broad spectrum cephalosporin with immunostimulatory effects, and ceftriaxone on the production of GM-CSF and IL-8 in HBEC primary cultures was investigated. HBEC were isolated from biopsy specimens obtained during fibreoptic bronchoscopy in 12 patients (most frequent diagnosis: chronic bronchitis). Confluent monolayers of HBEC cultured on collagen were incubated for 24 h in a medium without study drugs (spontaneous production) or containing cefodizime or ceftriaxone at the clinically relevant concentrations of 1, 10 and 100 mg/l, with or without tumor necrosis factor alpha (TNF alpha, 100 U/ml). GM-CSF and IL-8 were measured in supernatant by ELISA technique. TNF alpha alone led to a significant (p < 0.005) increase in both GM-CSF and IL-8 production. Cefodizime induced a significant (p < 0.05), dose-dependent increase in GM-CSF release. No additive effect of cefodizime with TNF alpha was observed. Cefodizime did not affect IL-8 production and ceftriaxone had no influence on cytokine production. This is the first report of a stimulatory effect of a beta-lactam antibiotic on cytokine production by epithelial cells. GM-CSF production by epithelial cells is an important immunological step for neutrophil and monocyte recruitment and cell priming during lung defence. Previous studies with cefodizime in immunodepressed subjects have shown activation of phagocytosis and phagocytosis-related functions in non-lung phagocytes. An indirect mechanism of action, similar to that indicated by our results, may have been responsible for these stimulatory effects.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Influence of Granulocyte-Macrophage Colony-Stimulating Factor or Influenza Vaccination on HLA-DR, Infection and Delirium Days in Immunosuppressed Surgical Patients: Double Blind, Randomised Controlled Trial

    PubMed Central

    Lachmann, Gunnar; Renius, Markus; von Haefen, Clarissa; Wernecke, Klaus-Dieter; Bahra, Marcus; Schiemann, Alexander; Paupers, Marco; Meisel, Christian

    2015-01-01

    Purpose Surgical patients are at high risk for developing infectious complications and postoperative delirium. Prolonged infections and delirium result in worse outcome. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and influenza vaccination are known to increase HLA-DR on monocytes and improve immune reactivity. This study aimed to investigate whether GM-CSF or vaccination reverses monocyte deactivation. Secondary aims were whether it decreases infection and delirium days after esophageal or pancreatic resection over time. Methods In this prospective, randomized, placebo-controlled, double-blind, double dummy trial setting on an interdisciplinary ICU of a university hospital 61 patients with immunosuppression (monocytic HLA-DR [mHLA-DR] <10,000 monoclonal antibodies [mAb] per cell) on the first day after esophageal or pancreatic resection were treated with either GM-CSF (250 μg/m2/d), influenza vaccination (Mutagrip 0.5 ml/d) or placebo for a maximum of 3 consecutive days if mHLA-DR remained below 10,000 mAb per cell. HLA-DR on monocytes was measured daily until day 5 after surgery. Infections and delirium were followed up for 9 days after surgery. Primary outcome was HLA-DR on monocytes, and secondary outcomes were duration of infection and delirium. Results mHLA-DR was significantly increased compared to placebo (p < 0.001) and influenza vaccination (p < 0.001) on the second postoperative day. Compared with placebo, GM-CSF-treated patients revealed shorter duration of infection (p < 0.001); the duration of delirium was increased after vaccination (p = 0.003). Conclusion Treatment with GM-CSF in patients with postoperative immune suppression was safe and effective in restoring monocytic immune competence. Furthermore, therapy with GM-CSF reduced duration of infection in immune compromised patients. However, influenza vaccination increased duration of delirium after major surgery. Trial Registration www.controlled-trials.com ISRCTN27114642 PMID

  6. Induction of cytokine granulocyte-macrophage colony-stimulating factor and chemokine macrophage inflammatory protein 2 mRNAs in macrophages by Legionella pneumophila or Salmonella typhimurium attachment requires different ligand-receptor systems.

    PubMed Central

    Yamamoto, Y; Klein, T W; Friedman, H

    1996-01-01

    The attachment of bacteria to macrophages is mediated by different ligands and receptors and induces various intracellular molecular responses. In the present study, induction of cytokines and chemokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage inflammatory protein 2 (MIP-2), was examined, following bacterial attachment, with regard to the ligand-receptor systems involved. Attachment of Legionella pneumophila or Salmonella typhimurium to cultured mouse peritoneal macrophages increased the steady-state levels of cellular mRNAs for the cytokines interleukin 1beta (IL-1beta), IL-6, and GM-CSF as well as the chemokines MIP-1beta, MIP-2, and KC. However, when macrophages were treated with alpha-methyl-D-mannoside (alphaMM), a competitor of glycopeptide ligands, induction of cytokine mRNAs was inhibited, but the levels of chemokine mRNAs were not. Pretreatment of the bacteria with fresh mouse serum enhanced the level of GM-CSF mRNA but not the level of MIP-2 mRNA. In addition, serum treatment reduced the inhibitory effect of alphaMM on GM-CSF mRNA. These results indicate that bacterial attachment increases the steady-state levels of the cytokine and chemokine mRNAs tested by at least two distinct receptor-ligand systems, namely, one linked to cytokine induction and involving mannose or other sugar residues and the other linked to chemokine induction and relatively alphaMM insensitive. Furthermore, opsonization with serum engages other pathways in the cytokine response which are relatively independent of the alphaMM-sensitive system. Regarding bacterial surface ligands involved in cytokine mRNA induction, evidence is presented that the flagellum may be important in stimulating cytokine GM-CSF message but not chemokine MIP-2 message. Analysis of cytokine GM-CSF and chemokine MIP-2 signaling pathways with protein kinase inhibitors revealed the involvement of calmodulin and myosin light-chain kinase in GM-CSF but not MIP-2 m

  7. Online Haemodiafiltration Improves Inflammatory State in Dialysis Patients: A Longitudinal Study

    PubMed Central

    Rama, Ines; Llaudó, Ines; Fontova, Pere; Cerezo, Gema; Soto, Carlos; Javierre, Casimiro; Hueso, Miguel; Montero, Nuria; Martínez-Castelao, Alberto; Torras, Juan; Grinyó, Josep M.; Cruzado, Josep M.; Lloberas, Nuria

    2016-01-01

    Background Patients undergoing conventional hemodialysis (C-HD) present a greater immuno-inflammatory state probably related to uremia, sympathetic nervous system (SNS) activation and /or membrane bioincompatibility, which could improve with a technique-switching to online hemodiafiltration (OL-HD). The antigen-independent pathway activation of this modified immunologic state turns dendritic cells (DC) into an accurate cell model to study these patients. The aim of this study is to further evaluate the immune-inflammatory state of patients in C-HD assessed by DC maturation. Methods 31 patients were submitted to C-HD and after 4 months switched to the OL-HD technique. Monocytes-derived DCs from HD patients were cultured in the presence of IL-4/GM-CSF. DC-maturation was evaluated by assessing the maturation phenotype by flow cytometry (FACs). DCs-functional capacity to elicit T-cell alloresponse was studied by mixed leucocyte reaction. Cytokine release was assessed by FACs and SNS was evaluated measuring renalase levels by ELISA. Results An up-regulation of maturation markers was observed in C-HD DCs which induced two fold more T cells proliferation than OL-HD DCs. Also, C-HD-mDCs presented with over-production of pro-inflammatory cytokines (IL-6, IL-1β, IL-8, IL-10 and TNF-α) compared with OL-HD-mDC (P<0·05). Results were correlated with clinical data. When SNS was evaluated, hypotension events and blood pressure were significantly lower and renalase levels were significantly higher after conversion to OL-HD. Diabetes mellitus type 2 patients also found beneficial reduction of mDC when converted to OL-HD compared to non-diabetics. Conclusions OL-HD could interfere with immuno-inflammatory state in HD patients with an improvement of renalase levels as potential key mediators in the mechanistic pathway of down-regulation of DC maturation. PMID:27783636

  8. Circulating biomarkers in acute myofascial pain: A case-control study.

    PubMed

    Grosman-Rimon, Liza; Parkinson, William; Upadhye, Suneel; Clarke, Hance; Katz, Joel; Flannery, John; Peng, Philip; Kumbhare, Dinesh

    2016-09-01

    The aims of the present study were to compare levels of circulating inflammatory biomarkers and growth factors between patients with myofascial pain syndrome (MPS) and healthy control participants, and to assess the relationship among inflammatory markers and growth factors in the two groups.Biomarkers levels were assessed in patients (n = 37) with myofascial pain complaints recruited from the hospital emergency department and non-MPS controls (n = 21), recruited via advertisements in the hospital and community.Blood levels of the cytokines, namely, interleukin-6 (IL-6), tumor necrosis factor (TNF), and interleukin-12 (IL-12), and the chemokine, namely, monocyte chemoattractant protein-1 (MCP-1), macrophage-derived chemokine (MDC), eotaxin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-8 (IL-8), and macrophage inflammatory proteins-1β (MIP-1β) were significantly higher in patients with MPS than controls. The results of the growth factor analyses revealed significantly higher levels of fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) in MPS patients versus controls. The pattern of correlation coefficients between cytokines and growth factors differed considerably for MPS patients and controls with far fewer significant positive coefficients observed in the controls. Serum inflammatory and growth factor biomarkers were elevated in MPS patients.Inflammatory biomarkers and growth factor levels may play an important role in the onset and maintenance of MPS and therefore may be useful in the diagnosis and treatment of MPS. Understanding the mechanisms of inflammation in MPS necessitates future research. PMID:27631214

  9. Circulating biomarkers in acute myofascial pain: A case-control study.

    PubMed

    Grosman-Rimon, Liza; Parkinson, William; Upadhye, Suneel; Clarke, Hance; Katz, Joel; Flannery, John; Peng, Philip; Kumbhare, Dinesh

    2016-09-01

    The aims of the present study were to compare levels of circulating inflammatory biomarkers and growth factors between patients with myofascial pain syndrome (MPS) and healthy control participants, and to assess the relationship among inflammatory markers and growth factors in the two groups.Biomarkers levels were assessed in patients (n = 37) with myofascial pain complaints recruited from the hospital emergency department and non-MPS controls (n = 21), recruited via advertisements in the hospital and community.Blood levels of the cytokines, namely, interleukin-6 (IL-6), tumor necrosis factor (TNF), and interleukin-12 (IL-12), and the chemokine, namely, monocyte chemoattractant protein-1 (MCP-1), macrophage-derived chemokine (MDC), eotaxin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-8 (IL-8), and macrophage inflammatory proteins-1β (MIP-1β) were significantly higher in patients with MPS than controls. The results of the growth factor analyses revealed significantly higher levels of fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) in MPS patients versus controls. The pattern of correlation coefficients between cytokines and growth factors differed considerably for MPS patients and controls with far fewer significant positive coefficients observed in the controls. Serum inflammatory and growth factor biomarkers were elevated in MPS patients.Inflammatory biomarkers and growth factor levels may play an important role in the onset and maintenance of MPS and therefore may be useful in the diagnosis and treatment of MPS. Understanding the mechanisms of inflammation in MPS necessitates future research.

  10. Establishing Porcine Monocyte-Derived Macrophage and Dendritic Cell Systems for Studying the Interaction with PRRSV-1

    PubMed Central

    Singleton, Helen; Graham, Simon P.; Bodman-Smith, Katherine B.; Frossard, Jean-Pierre; Steinbach, Falko

    2016-01-01

    Monocyte-derived macrophages (MoMØ) and monocyte-derived dendritic cells (MoDC) are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect myeloid cells, such as macrophages (MØ) and dendritic cells (DC). Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated MoMØ were stimulated with activators for classical (M1) or alternative (M2) activation. GM-CSF and IL-4 generated MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype, and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells toward PRRSV-1 infection. PMID:27313573

  11. Establishing Porcine Monocyte-Derived Macrophage and Dendritic Cell Systems for Studying the Interaction with PRRSV-1.

    PubMed

    Singleton, Helen; Graham, Simon P; Bodman-Smith, Katherine B; Frossard, Jean-Pierre; Steinbach, Falko

    2016-01-01

    Monocyte-derived macrophages (MoMØ) and monocyte-derived dendritic cells (MoDC) are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect myeloid cells, such as macrophages (MØ) and dendritic cells (DC). Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated MoMØ were stimulated with activators for classical (M1) or alternative (M2) activation. GM-CSF and IL-4 generated MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype, and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells toward PRRSV-1 infection.

  12. Viral vectored granulocyte-macrophage colony stimulating factor inhibits vaccine protection in an SIV challenge model: protection correlates with neutralizing antibody

    PubMed Central

    Schell, John B.; Bahl, Kapil; Rose, Nina F.; Buonocore, Linda; Hunter, Meredith; Marx, Preston A.; LaBranche, Celia C.; Montefiori, David C.; Rose, John K.

    2012-01-01

    In a previous vaccine study, we reported significant and apparently sterilizing immunity to high-dose, mucosal, simian immunodeficiency virus (SIV) quasispecies challenge (27). The vaccine consisted of vectors based on vesicular stomatitis virus (VSV) expressing simian immunodeficiency virus (SIV) gag and env genes, a boost with propagating replicon particles expressing the same SIV genes, and a second boost with VSV-based vectors. Concurrent with that published study we had a parallel group of macaques given the same doses of vaccine vectors, but in addition, we included a third VSV vector expressing rhesus macaque GM-CSF in the priming immunization only. We report here that addition of the vector expressing GM-CSF did not enhance CD8 T cell or antibody responses to SIV antigens, and almost completely abolished the vaccine protection against high-dose mucosal challenge with SIV. Expression of GM-CSF may have limited vector replication excessively in the macaque model. Our results suggest caution in the use of GM-CSF as a vaccine adjuvant, especially when expressed by a viral vector. Combining vaccine group animals from this study and the previous study we found that there was a marginal but significant positive correlation between the neutralizing antibody to a neutralization resistant SIV Env and protection from infection. PMID:22537983

  13. Effects of interleukin-3 following chemotherapy of non-Hodgkin's lymphoma. A prospective, controlled phase I/II study.

    PubMed

    Hovgaard, D J; Nissen, N I

    1995-02-01

    The effect of rhIL-3 was investigated in 32 patients with newly diagnosed non-Hodgkin lymphoma in a phase I/II trial. All patients received 6 cycles of standard CHOP chemotherapy, and each patient was his own control where rhIL-3 was given as a daily s.c. injection for 14 days (day 2-15) in cycle 2 and 4, while cycle 1 and 3 were control cycles. Five dose levels were examined (0.5 - 1 - 5 - 7.5 - 10 micrograms/kg). Compared to the other more lineage-specific hemopoietic growth factors G- and GM-CSF, the effect of rhIL-3 on the hemopoiesis was less dramatic and more delayed, i.e. the most apparent effect was observed in the 2 weeks of treatment. Thus, the neutrophil counts from days 15 to 22 following CHOP were significantly raised and the duration of neutropenia was shorter (significantly only at 10 micrograms/kg), while the nadir values were unaffected. Platelet recovery from days 12-22 was significantly increased and nadir values occurred earlier compared to control cycles, but were only increased in some subsets. Other cell populations affected moderately in the recovery period were eosinophils and monocytes. Reticulocytes increased, but no effect on hemoglobin or RBC transfusion requirement was noted. Only moderate adverse reactions occurred such as fever, chills, flushing of the face and flu-like symptoms. There was no evidence of stimulation of tumor growth. Most significant, the rhIL-3 treatment at all but the lowest dose levels led to an improved tolerance to chemotherapy, as indicated by a decline in number of delayed cycles. A conclusion concerning the role of rhIL-3 as post-chemotherapy adjuvant should await studies using rhIL-3 in combination with more lineage-restricted hemopoietic growth factors.

  14. In vivo transformation of factor-dependent hemopoietic cells: role of intracisternal A-particle transposition for growth factor gene activation.

    PubMed Central

    Dührsen, U; Stahl, J; Gough, N M

    1990-01-01

    Cells of the granulocyte-macrophage colony stimulating factor (GM-CSF) or multi-lineage colony stimulating factor (Multi-CSF) dependent line FDC-P1 undergo leukemic transformation after injection into irradiated DBA/2 mice. About one third of factor-independent FDC-P1 variants isolated from leukemic animals express GM-CSF or Multi-CSF, assessed either by bioassay or by sensitive RNA detection using the polymerase chain reaction. All of the GM-CSF-secreting lines studied had a rearrangement in one allele of the GM-CSF gene, three of four Multi-CSF-secreting lines had Multi-CSF gene rearrangements, while factor-independent lines lacking evidence of growth factor production had no demonstrable CSF gene alterations. All rearrangements were characterized by insertions of novel DNA in the 5'-flanking regions of the CSF genes. The inserted segments of DNA varied in size between 0.35 and 6.5 kb and displayed restriction enzyme cleavage maps reminiscent of intracisternal A-particle (IAP) genomes. This was confirmed in two cases by molecular cloning and nucleotide sequence analysis. In these instances, the insertion consisted of solitary IAP long terminal repeats. The transformation system described provides a model for the study of IAP transpositions and their effects on gene activation. Images Fig. 2. Fig. 3. Fig. 4. Fig. 6. Fig. 9. PMID:2108861

  15. Targeted cytokine delivery to neuroblastoma.

    PubMed

    Dehal, P K; Embleton, M J; Kemshead, J T; Hawkins, R E

    2002-08-01

    The aim of this study was to construct a fusion protein from the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) and a single-chain Fv fragment (scFv D29) and to investigate its potential to activate cells of the immune system against neuroblastoma cells expressing neural cell adhesion molecule (NCAM). Mammalian cell expression of the scFv D29-GM-CSF fusion protein was compared using a number of vectors, including retroviral and adenoviral vectors. The resultant fusion protein, expressed by HeLa cells, was found by ELISA to bind immobilized recombinant NCAM. Moreover, FACS analysis confirmed binding to the human neuroblastoma cell line SKNBE and a murine neuroblastoma cell line engineered to express the glycosylphosphatidylinositol form of human NCAM (N2A-rKNIE). The fusion protein was also found to stimulate the proliferation of the FDC-P1 haemopoietic cell line, which is dependent on GM-CSF (or interleukin 3) for continued growth. In vitro clonogenic assays indicated that scFv-GM-CSF could selectively induce growth inhibition of SKNBE cells by murine lymphoid cells.

  16. Interactions of dimethyl sulfoxide and granulocyte-macrophage colony-stimulating factor on the cell cycle kinetics and phosphoproteins of G1-enriched HL-60 cells: evidence of early effects on lamin B phosphorylation.

    PubMed

    Brennan, J K; Lee, K S; Frazel, M A; Keng, P C; Young, D A

    1991-03-01

    We have found that GM-CSF and DMSO have antagonistic effects on the proliferation but not maturation of asynchronously growing HL-60 cells such that growth in the presence of both more closely resembles normal hematopoiesis (Brennan et al., J. Cell Physiol. 132:246, 1987). Studies were undertaken to determine whether or not the agents affected the same mitogenic pathway and locus in the cell cycle. HL-60 populations containing at least 90% G1 cells were obtained by centrifugal elutriation, exposed to 100 u/ml recombinant human GM-CSF and/or 0-1.25% DMSO, and phosphoprotein changes quantified on autoradiograms of [32P]-orthophosphate-labeled cell proteins separated by giant 2-D gel electrophoresis. Results were correlated with 1) intracellular pH, determined by measurement of BCECF fluorescence; 2) [32P]-orthophosphate uptake; 3) cell cycle progression, determined by flow quantitation of DNA content in mithramycin or propidium iodide-stained cells; and 4) growth, determined by cell volume and concentration. GM-CSF stimulated and DMSO inhibited the GM-CSF-stimulated phosphorylation of 1 protein (approximately 65 kDa, p.i. 5.6) within 2 min of exposure. These effects were sustained through G1, not associated with changes in intracellular pH, and preceded similar antagonistic effects on phosphate uptake (15-30 minutes), cell volume change (16-24 hr), and cell concentration increase (28-32 hr). GM-CSF accelerated and DMSO inhibited G1 to S transit with the most marked antagonism observed in the second cycle following synchronization (28 to 40 hrs). Cell maturation (morphology, NBT reduction) was dominated by DMSO and not antagonized by GM-CSF. We have identified p65 as the nuclear intermediate filament protein, lamin B, on the basis of its locus on gels and its binding of a monoclonal antibody to intermediate filaments and antiserum to human lamin B on immunoblots. These studies suggest that at least part of the GM-CSF-DMSO antagonism is exerted through the same

  17. Protective effects of paeoniflorin and albiflorin on chemotherapy-induced myelosuppression in mice.

    PubMed

    Zhu, Ying-Li; Wang, Lin-Yuan; Wang, Jing-Xia; Wang, Chun; Wang, Cheng-Long; Zhao, Dan-Ping; Wang, Zi-Chen; Zhang, Jian-Jun

    2016-08-01

    Paeonia lactiflora root (baishao in Chinese) is a commonly used herb in traditional Chinese medicines (TCM). Two isomers, paeoniflorin (PF) and albiflorin (AF), are isolated from P. lactiflora. The present study aimed to investigate the protective effects of PF and AF on myelosuppression induced by chemotherapy in mice and to explore the underlying mechanisms. The mouse myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CP, 200 mg·kg(-1)). The blood cell counts were performed. The thymus index and spleen index were also determined and bone morrow histological examination was performed. The levels of tumor necrosis factor-α (TNF-α) in serum and colony-stimulating factor (G-CSF) in plasma were measured by Enzyme-Linked Immunosorbent Assays (ELISA) and the serum levels of interleukin-3 (IL-3), granulocyte-macrophagecolony-stimulatingfactor (GM-CSF), and interleukin-6 (IL-6) were measured by radioimmunoassay (RIA). The levels of mRNA expression protein of IL-3, GM-CSF and G-CSF in spleen and bone marrow cells were determined respectively. PF and AF significantly increased the white blood cell (WBC) counts and reversed the atrophy of thymus. They also increased the serum levels of GM-CSF and IL-3 and the plasma level of G-CSF and reduced the level of TNF-α in serum. PF enhanced the mRNA level of IL-3 and AF enhanced the mRNA levels of GM-CSF and G-CSF in the spleen. PF and AF both increased the protein levels of GM-CSF and G-CSF in bone marrow cells. In conclusion, our results demonstrated that PF and AF promoted the recovery of bone marrow hemopoietic function in the mouse myelosuppression model. PMID:27608949

  18. Recombinant immunocytokines targeting the mouse transferrin receptor: construction and biological activities.

    PubMed

    Dreier, T; Lode, H N; Xiang, R; Dolman, C S; Reisfeld, R A; Kang, A S

    1998-01-01

    Localized cytokine therapies with recombinant monoclonal antibody-cytokine fusion proteins, designated immunocytokines, have become of increasing interest for tumor immunotherapy, since they direct immunomodulatory cytokines into the tumor microenvironment. To investigate their mechanisms of action in a variety of syngeneic tumor models, recombinant mouse cytokines IL2 and GM-CSF were engineered as fusion proteins to the carboxyl terminus of a chimeric rat/mouse antitransferrin receptor antibody, ch17217 and expressed in stable-transfected Chinese hamster ovary cells. The recombinant immunocytokines were readily purified by affinity chromatography and their binding characteristics were identical to those shown for the ch17217 antibody. The IL2 immunocytokine had an activity similar to recombinant mouse IL2, whereas the GM-CSF immunocytokine had enhanced cytokine activity relative to recombinant mouse GM-CSF. The clearance rates of ch17217 and the GM-CSF and IL2 immunocytokines were relatively similar with elimination phases (t1/2alpha) of 1.8 h and distribution phases (t1/2beta) of 83, 88, and 91 h, respectively. Both immunocytokines demonstrated effective antitumor activity by suppressing the growth of hepatic metastases of mouse neuroblastoma and pulmonary metastases of mouse colon carcinoma in syngeneic A/J and BALB/c mice, respectively. These results indicate that biologically effective IL2 and GM-CSF immunocytokines combine the targeting ability of an antitransferrin receptor monoclonal antibody with the immunomodulatory functions of each cytokine. Because of the universal expression of the transferrin receptor on mouse tumor cell lines, these constructs should prove useful to determine their efficacy in a wide variety of syngeneic mouse tumor models and to perform detailed studies of their modes of action.

  19. A phase 1 study of a heterologous prime-boost vaccination involving a truncated HER2 sequence in patients with HER2-expressing breast cancer

    PubMed Central

    Kim, Sung-Bae; Ahn, Jin-Hee; Kim, Jeongeun; Jung, Kyung Hae

    2015-01-01

    A phase 1 clinical trial was conducted to assess the safety, tolerability, and preliminary efficacy of a heterologous prime-boost strategy involving plasmid DNA (pHM-GM-CSF, expressing truncated human epidermal growth factor receptor 2 (HER2) and granulocyte macrophage colony-stimulation factor (GM-CSF) as a bicistronic message) and an adenoviral vector (Ad-HM, containing the same modified HER2 sequence only), in patients with stage III–IV metastatic breast cancer expressing HER2. Nine eligible subjects were divided into three cohorts based on the dosages (2, 4, and 8 mg/patient/visit) of pHM-GM-CSF used as the primer, which was intramuscularly injected three times at weeks 0, 2, and 4. It was followed by a single injection of Ad-HM (3 × 109 virus particles), used as a booster, at week 6. During the 6-month follow-up period, adverse events (AEs), pharmacokinetics and pharmacodynamics, and HER2-specific cellular and humoral immune responses were evaluated. Seven cases of minor grade 1 toxicities in four of nine subjects and no serious drug-related AEs were reported. HER2-specific cell-mediated or humoral immunity was produced in all (100%) or three subjects (33%), respectively. One subject showed a partial response, and seven subjects had stable diseases. However, there were no differences in clinical tumor response and HER2-specific immune responses among the cohorts. These results showed that intramuscular injections of pHM-GM-CSF and Ad-HM were well tolerated and safe. PMID:26445724

  20. A phase 1 study of a heterologous prime-boost vaccination involving a truncated HER2 sequence in patients with HER2-expressing breast cancer.

    PubMed

    Kim, Sung-Bae; Ahn, Jin-Hee; Kim, Jeongeun; Jung, Kyung Hae

    2015-01-01

    A phase 1 clinical trial was conducted to assess the safety, tolerability, and preliminary efficacy of a heterologous prime-boost strategy involving plasmid DNA (pHM-GM-CSF, expressing truncated human epidermal growth factor receptor 2 (HER2) and granulocyte macrophage colony-stimulation factor (GM-CSF) as a bicistronic message) and an adenoviral vector (Ad-HM, containing the same modified HER2 sequence only), in patients with stage III-IV metastatic breast cancer expressing HER2. Nine eligible subjects were divided into three cohorts based on the dosages (2, 4, and 8 mg/patient/visit) of pHM-GM-CSF used as the primer, which was intramuscularly injected three times at weeks 0, 2, and 4. It was followed by a single injection of Ad-HM (3 × 10(9) virus particles), used as a booster, at week 6. During the 6-month follow-up period, adverse events (AEs), pharmacokinetics and pharmacodynamics, and HER2-specific cellular and humoral immune responses were evaluated. Seven cases of minor grade 1 toxicities in four of nine subjects and no serious drug-related AEs were reported. HER2-specific cell-mediated or humoral immunity was produced in all (100%) or three subjects (33%), respectively. One subject showed a partial response, and seven subjects had stable diseases. However, there were no differences in clinical tumor response and HER2-specific immune responses among the cohorts. These results showed that intramuscular injections of pHM-GM-CSF and Ad-HM were well tolerated and safe. PMID:26445724

  1. TNF-α alters the inflammatory secretion profile of human first trimester placenta.

    PubMed

    Siwetz, Monika; Blaschitz, Astrid; El-Heliebi, Amin; Hiden, Ursula; Desoye, Gernot; Huppertz, Berthold; Gauster, Martin

    2016-04-01

    Implantation and subsequent placental development depend on a well-orchestrated interaction between fetal and maternal tissues, involving a fine balanced synergistic cross-talk of inflammatory and immune-modulating factors. Tumor necrosis factor (TNF)-α has been increasingly recognized as pivotal factor for successful pregnancy, although high maternal TNF-α levels are associated with a number of adverse pregnancy conditions including gestational hypertension and gestational diabetes mellitus. This study describes effects of exogenously applied TNF-α, mimicking increased maternal TNF-α levels, on the secretion profile of inflammation associated factors in human first trimester villous placenta. Conditioned culture media from first trimester villous placental explants were analyzed by inflammation antibody arrays and ELISA after 48 h culture in the presence or absence of TNF-α. Inflammation antibody arrays identified interleukin (IL)-6, IL-8, chemokine (C-C motif) ligand 2 (CCL2), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as the most abundantly secreted inflammation-associated factors under basal culture conditions. In the presence of TNF-α, secretion of GM-CSF, CCL5, and IL-10 increased, whereas IL-4 and macrophage CSF levels decreased compared with controls. ELISA analysis verified antibody arrays by showing significantly increased synthesis and release of GM-CSF and CCL5 by placental explants in response to TNF-α. Immunohistochemistry localized GM-CSF in the villous trophoblast compartment, whereas CCL5 was detected in maternal platelets adhering to perivillous fibrin deposits on the villous surface. mRNA-based in situ padlock probe approach localized GM-CSF and CCL5 transcripts in the villous trophoblast layer and the villous stroma. Results from this study suggest that the inflammatory secretion profile of human first trimester placenta shifts towards increased levels of GM-CSF, CCL5, and IL10 in response to elevated maternal

  2. In vitro and in vivo activation of endothelial cells by colony-stimulating factors.

    PubMed Central

    Bussolino, F; Ziche, M; Wang, J M; Alessi, D; Morbidelli, L; Cremona, O; Bosia, A; Marchisio, P C; Mantovani, A

    1991-01-01

    This study was designed to identify the set of functions activated in cultured endothelial cells by the hematopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage-colony-stimulating factor (GM-CSF), and to compare them with those elicited by prototypic cytokines active on these cells. Moreover, indications as to the in vivo relevance of in vitro effects were obtained. G-CSF and GM-CSF induced endothelial cells to proliferate and migrate. In contrast, unlike appropriate reference cytokines (IL-1 and tumor necrosis factor, IFN-gamma), G-CSF and GM-CSF did not modulate endothelial cell functions related to hemostasis-thrombosis (production of procoagulant activity and of platelet activating factor), inflammation (expression of leukocyte adhesion molecule-1 and production of platelet activating factor), and accessory function (expression of class II antigens of MHC). Other colony-stimulating factors (IL-3 and macrophage-colony-stimulating factor) were inactive on all functions tested. In comparison to basic fibroblast growth factor (bFGF), G-CSF and GM-CSF induced lower maximal proliferation of endothelial cells, whereas migration was of the same order of magnitude. G-CSF and GM-CSF stimulated repair of mechanically wounded endothelial monolayers. Exposure to both cytokines induced shape changes and cytoskeletal reorganization consistent with a migratory phenotype. To explore the in vivo relevance of the in vitro effects of these cytokines on endothelium, we studied the angiogenic activity of human G-CSF in the rabbit cornea. G-CSF, but not the heat-inactivated molecule, had definite angiogenic activity, without any sign of inflammatory reactions. G-CSF was less active than bFGF. However, the combination of a nonangiogenic dose of bFGF with G-CSF resulted in an angiogenic response higher than that elicited by either individual cytokines. Thus, G-CSF and GM-CSF induce endothelial cells to express an activation

  3. Comparison of WTC Dust Size on Macrophage Inflammatory Cytokine Release In vivo and In vitro

    PubMed Central

    Weiden, Michael D.; Naveed, Bushra; Kwon, Sophia; Segal, Leopoldo N.; Cho, Soo Jung; Tsukiji, Jun; Kulkarni, Rohan; Comfort, Ashley L.; Kasturiarachchi, Kusali J.; Prophete, Colette; Cohen, Mitchell D.; Chen, Lung-Chi; Rom, William N.; Prezant, David J.; Nolan, Anna

    2012-01-01

    Background The WTC collapse exposed over 300,000 people to high concentrations of WTC-PM; particulates up to ∼50 mm were recovered from rescue workers’ lungs. Elevated MDC and GM-CSF independently predicted subsequent lung injury in WTC-PM-exposed workers. Our hypotheses are that components of WTC dust strongly induce GM-CSF and MDC in AM; and that these two risk factors are in separate inflammatory pathways. Methodology/Principal Findings Normal adherent AM from 15 subjects without WTC-exposure were incubated in media alone, LPS 40 ng/mL, or suspensions of WTC-PM10–53 or WTC-PM2.5 at concentrations of 10, 50 or 100 µg/mL for 24 hours; supernatants assayed for 39 chemokines/cytokines. In addition, sera from WTC-exposed subjects who developed lung injury were assayed for the same cytokines. In the in vitro studies, cytokines formed two clusters with GM-CSF and MDC as a result of PM10–53 and PM2.5. GM-CSF clustered with IL-6 and IL-12(p70) at baseline, after exposure to WTC-PM10–53 and in sera of WTC dust-exposed subjects (n = 70) with WTC lung injury. Similarly, MDC clustered with GRO and MCP-1. WTC-PM10–53 consistently induced more cytokine release than WTC-PM2.5 at 100 µg/mL. Individual baseline expression correlated with WTC-PM-induced GM-CSF and MDC. Conclusions WTC-PM10–53 induced a stronger inflammatory response by human AM than WTC-PM2.5. This large particle exposure may have contributed to the high incidence of lung injury in those exposed to particles at the WTC site. GM-CSF and MDC consistently cluster separately, suggesting a role for differential cytokine release in WTC-PM injury. Subject-specific response to WTC-PM may underlie individual susceptibility to lung injury after irritant dust exposure. PMID:22815721

  4. In vitro effects of nanosized diamond particles on macrophages.

    PubMed

    Shkurupy, V A; Arkhipov, S A; Neshchadim, D V; Akhramenko, E S; Troitskii, A V

    2015-02-01

    The effects of synthetic diamond nanoparticles (4-6 nm) on mouse macrophage biotropism and biocompatibility and the modulation of the macrophage functions (expression of IL-1α, TNF-α, GM-CSF, bFGF, and TGF-β) by nanoparticles in different concentrations were studied in vitro during exposure of different duration. Macrophage endocytosis of nanodiamonds increased with increasing the concentration of nanoparticles in culture and incubation time. Nanodiamonds exhibited high biotropism and biocompatibility towards macrophages; in doses of 10-20 μg/ml, they induced expression of GM-CSF and TGF-β, inhibited expression of bFGF, and did not stimulate IL-1α and TNF-α. These data indicate that nanodiamond capture by macrophages in the studied experimental model led to modulation of the functional status of macrophages that determine their capacity to stimulate reparative processes without increasing proinflammatory and profibrogenic status.

  5. In vitro effects of nanosized diamond particles on macrophages.

    PubMed

    Shkurupy, V A; Arkhipov, S A; Neshchadim, D V; Akhramenko, E S; Troitskii, A V

    2015-02-01

    The effects of synthetic diamond nanoparticles (4-6 nm) on mouse macrophage biotropism and biocompatibility and the modulation of the macrophage functions (expression of IL-1α, TNF-α, GM-CSF, bFGF, and TGF-β) by nanoparticles in different concentrations were studied in vitro during exposure of different duration. Macrophage endocytosis of nanodiamonds increased with increasing the concentration of nanoparticles in culture and incubation time. Nanodiamonds exhibited high biotropism and biocompatibility towards macrophages; in doses of 10-20 μg/ml, they induced expression of GM-CSF and TGF-β, inhibited expression of bFGF, and did not stimulate IL-1α and TNF-α. These data indicate that nanodiamond capture by macrophages in the studied experimental model led to modulation of the functional status of macrophages that determine their capacity to stimulate reparative processes without increasing proinflammatory and profibrogenic status. PMID:25705036

  6. A pilot study evaluating non-contact low-frequency ultrasound and underlying molecular mechanism on diabetic foot ulcers.

    PubMed

    Yao, Min; Hasturk, Hatice; Kantarci, Alpdogan; Gu, Guosheng; Garcia-Lavin, Silvia; Fabbi, Matteo; Park, Nanjin; Hayashi, Hisae; Attala, Khaled; French, Michael A; Driver, Vickie R

    2014-12-01

    Non-contact low-frequency ultrasound (NCLF-US) devices have been increasingly used for the treatment of chronic non-healing wounds. The appropriate dose for NCLF-US is still in debate. The aims of this pilot study were to evaluate the relationship between dose and duration of treatment for subjects with non-healing diabetic foot ulcers (DFUs) and to explore the correlation between wound healing and change of cytokine/proteinase/growth factor profile. This was a prospective randomised clinical study designed to evaluate subjects with non-healing DFUs for 5 weeks receiving standard of care and/or NCLF-US treatment. Subjects were randomly assigned to one of the three groups: application of NCLF-US thrice per week (Group 1), NCLF-US once per week (Group 2) and the control (Group 3) that received no NCLF-US. All subjects received standard wound care plus offloading for a total of 4 weeks. Percent area reduction (PAR) of each wound compared with baseline was evaluated weekly. Profiles of cytokines/proteinase/growth factors in wound fluid and biopsied tissue were quantified to explore the correlation between wound healing and cytokines/growth factor expression. Twelve DFU patients, 2 (16·7%) type 1 and 10 (83·3%) type 2 diabetics, with an average age of 58 ± 10 years and a total of 12 foot ulcers were enrolled. Average ulcer duration was 36·44 ± 24·78 weeks and the average ABI was 0·91 ± 0·06. Group 1 showed significant wound area reduction at weeks 3, 4 and 5 compared with baseline, with the greatest PAR, 86% (P < 0·05); Groups 2 and 3 showed 25% PAR and 39% PAR, respectively, but there were no statistically significant differences between Groups 2 and 3 over time. Biochemical and histological analyses indicated a trend towards reduction of pro-inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α and GM-CSF), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and macrophages in response to NCLF-US consistent with wound reduction, when

  7. A pilot study evaluating non-contact low-frequency ultrasound and underlying molecular mechanism on diabetic foot ulcers.

    PubMed

    Yao, Min; Hasturk, Hatice; Kantarci, Alpdogan; Gu, Guosheng; Garcia-Lavin, Silvia; Fabbi, Matteo; Park, Nanjin; Hayashi, Hisae; Attala, Khaled; French, Michael A; Driver, Vickie R

    2014-12-01

    Non-contact low-frequency ultrasound (NCLF-US) devices have been increasingly used for the treatment of chronic non-healing wounds. The appropriate dose for NCLF-US is still in debate. The aims of this pilot study were to evaluate the relationship between dose and duration of treatment for subjects with non-healing diabetic foot ulcers (DFUs) and to explore the correlation between wound healing and change of cytokine/proteinase/growth factor profile. This was a prospective randomised clinical study designed to evaluate subjects with non-healing DFUs for 5 weeks receiving standard of care and/or NCLF-US treatment. Subjects were randomly assigned to one of the three groups: application of NCLF-US thrice per week (Group 1), NCLF-US once per week (Group 2) and the control (Group 3) that received no NCLF-US. All subjects received standard wound care plus offloading for a total of 4 weeks. Percent area reduction (PAR) of each wound compared with baseline was evaluated weekly. Profiles of cytokines/proteinase/growth factors in wound fluid and biopsied tissue were quantified to explore the correlation between wound healing and cytokines/growth factor expression. Twelve DFU patients, 2 (16·7%) type 1 and 10 (83·3%) type 2 diabetics, with an average age of 58 ± 10 years and a total of 12 foot ulcers were enrolled. Average ulcer duration was 36·44 ± 24·78 weeks and the average ABI was 0·91 ± 0·06. Group 1 showed significant wound area reduction at weeks 3, 4 and 5 compared with baseline, with the greatest PAR, 86% (P < 0·05); Groups 2 and 3 showed 25% PAR and 39% PAR, respectively, but there were no statistically significant differences between Groups 2 and 3 over time. Biochemical and histological analyses indicated a trend towards reduction of pro-inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α and GM-CSF), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and macrophages in response to NCLF-US consistent with wound reduction, when

  8. The βc receptor family - Structural insights and their functional implications.

    PubMed

    Broughton, Sophie E; Nero, Tracy L; Dhagat, Urmi; Kan, Winnie L; Hercus, Timothy R; Tvorogov, Denis; Lopez, Angel F; Parker, Michael W

    2015-08-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5 are members of a small family of cytokines that share a beta receptor subunit (βc). These cytokines regulate the growth, differentiation, migration and effector function activities of many hematopoietic cells in bone marrow, blood and sites of inflammation. Excessive or aberrant signaling can result in chronic inflammatory conditions and myeloid leukemias. The crystal structures of the GM-CSF ternary complex, the IL-5 binary complex and the very recent IL-3 receptor alpha subunit build upon decades of structure-function studies, giving new insights into cytokine-receptor specificity and signal transduction. Selective modulation of receptor function is now a real possibility and the structures of the βc receptor family are being used to discover novel and disease-specific therapeutics. PMID:25982846

  9. Vaccination of prostate cancer patients with modified vaccinia ankara delivering the tumor antigen 5T4 (TroVax): a phase 2 trial.

    PubMed

    Amato, Robert J; Drury, Noel; Naylor, Stuart; Jac, Jaroslaw; Saxena, Somya; Cao, Amy; Hernandez-McClain, Joan; Harrop, Richard

    2008-01-01

    The attenuated vaccinia virus, modified vaccinia Ankara, has been engineered to deliver the tumor antigen 5T4 (TroVax). TroVax has been evaluated in an open-label phase 2 trial in hormone refractory prostate cancer patients in which the vaccine was administered either alone or in combination with granulocyte macrophage-colony stimulating factor (GM-CSF). The comparative safety and immunologic and clinical efficacy of TroVax alone or in combination with GM-CSF was determined. Twenty-seven patients with metastatic hormone refractory prostate cancer were treated with TroVax alone (n=14) or TroVax+GM-CSF (n=13). 5T4-specific cellular and humoral responses were monitored throughout the study. Clinical responses were assessed by quantifying prostate-specific antigen concentrations and measuring changes in tumor burden by computer-assisted tomography scan. TroVax was well tolerated in all patients with no serious adverse events attributed to vaccination. Of 24 immunologically evaluable patients, all mounted 5T4-specific antibody responses. Periods of disease stabilization from 2 to >10 months were observed. Time to progression was significantly greater in patients who mounted 5T4-specific cellular responses compared with those who did not (5.6 vs. 2.3 mo, respectively). There were no objective clinical responses seen in this study. In this study, the combination of GM-CSF with TroVax showed similar clinical and immunologic responses to TroVax alone. The high frequency of 5T4-specific immune responses and relationship with enhanced time to progression is encouraging and warrants further investigation.

  10. Effect of Pore Structure of Macroporous Poly(Lactide-co-Glycolide) Scaffolds on the in Vivo Enrichment of Dendritic Cells

    PubMed Central

    2015-01-01

    The in vivo enrichment of dendritic cells (DCs) in implanted macroporous scaffolds is an emerging strategy to modulate the adaptive immune system. The pore architecture is potentially one of the key factors in controlling enrichment of DCs. However, there have been few studies examining the effects of scaffold pore structure on in vivo DC enrichment. Here we present the effects of surface porosity, pore size, and pore volume of macroporous poly(lactide-co-glycolide) (PLG) scaffolds encapsulating granulocyte macrophage colony-stimulating factor (GM-CSF), an inflammatory chemoattractant, on the in vivo enrichment of DCs. Although in vitro cell seeding studies using PLG scaffolds without GM-CSF showed higher cell infiltration in scaffolds with higher surface porosity, in vivo results revealed higher DC enrichment in GM-CSF loaded PLG scaffolds with lower surface porosity despite a similar level of GM-CSF released. The diminished compressive modulus of high surface porosity scaffolds compared to low surface porosity scaffolds lead to the significant shrinkage of these scaffolds in vivo, suggesting that the mechanical strength of scaffolds was critical to maintain a porous structure in vivo for accumulating DCs. The pore volume was also found to be important in total number of recruited cells and DCs in vivo. Varying the pore size significantly impacted the total number of cells, but similar numbers of DCs were found as long as the pore size was above 10–32 μm. Collectively, these results suggested that one can modulate in vivo enrichment of DCs by altering the pore architecture and mechanical properties of PLG scaffolds. PMID:24844318

  11. The Ability of Precursory Monocytes (MO) to Differentiate Varies Among Individuals But Is Stable Over Time

    PubMed Central

    Laudanski, Krzysztof; Zawadka, Mateusz; Lapko, Natalia

    2016-01-01

    Background The ability to generate dendritic cells (DCs) from precursory monocytes (MOs) was a breakthrough in the field of immunology. However, it is unknown whether the ability of MOs to differentiate into immature DCs (iDCs) differs across subjects or is time dependent. Given that the study of immune system function is gaining recognition in the field of clinical medicine, it is important to know how certain immunologic features vary over time. Material/Methods This study investigates how much individuals’ MO-to-iDC differentiation potential changes over time. We estimated this potential by measuring the expression of an iDC marker (CD1a), cytokine secretion (interleukin [IL]-12p70), and the ability of IL-4 and granulocyte macrophage colony-stimulating factor (GM-CSF) differentiation MOs to stimulate T cells. We collected MOs obtained from different subjects (n=17) at least 1 month apart. Furthermore, we investigated several variables (expression for cytokine receptors, timing, and emergence of DC-related transcriptional factor PU.1). Results The ability of MOs to become DCs under the influence of IL-4 and GM-CSF varied greatly between individuals (range of CD1a expression, 20–80%) but was stable over time (change of CD1a expression between sampling, ~5%). A similar pattern emerged when production of IL-12p70 was analyzed. The ability to stimulate T cells was variable and depended on the T-cell source. The ability of MOs to become iDCs was not linked to the surface expression of receptors for IL-4 and GM-CSF but rather to the activation of PU.1 in the precursory MO. It took 5 days for all committed MOs to become iDCs under in vitro influence of IL-4 and GM-CSF. Conclusions We concluded that the potential of MO to become iDC is an individual feature and depends on activation of PU.1. PMID:27415582

  12. Comparative analysis of CD80 and CD86 on human Langerhans cells: expression and function.

    PubMed

    Yokozeki, H; Takayama, K; Ohki, O; Satoh, T; Umeda, T; Katayama, I; Nishioka, K

    1998-10-01

    Although both CD80 (B7-1) and CD86 (B7-2/B70) have been recently identified in cultured human Langerhans cells (LC), little is known of the role and regulatory properties of CD80 and CD86 on human LC. We present here the results of a study comparing the expression and function of CD80 and CD86 in human LC using the T-helper type-1 cytokines IL-2 and interferon gamma (IFN)-gamma, and the T-helper type-2 cytokines IL-10, IL-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Freshly isolated human LC expressed little CD80 and CD86 in vitro, but the expression of both molecules was rapidly induced during a 72-h incubation with cytokines and the expression of CD86 occurred much earlier and more strongly than that of CD80. The expression of both CD80 and CD86 was upregulated by GM-CSF and downregulated by IL-10, and the expression of CD86, but not that of CD80, was upregulated by both IL-4 and IFN-gamma. Finally, pretreatment of LC with GM-CSF and IFN-gamma, but not with IL-4, enhanced the alloreactive T-cell proliferation induced by the LC, and IL-10 pretreatment of LC decreased their capacity for alloreaction. These results indicate that the expression of both CD80 and CD86 on human LC may be regulated by these cytokines (IL-2, IL-4, GM-CSF, IFN-gamma and IL-10) secreted from helper T cells infiltrating into the inflammatory microenvironment.

  13. Colony-stimulating factors for the treatment of the hematopoietic component of the acute radiation syndrome (H-ARS): a review.

    PubMed

    Singh, Vijay K; Newman, Victoria L; Seed, Thomas M

    2015-01-01

    One of the greatest national security threats to the United States is the detonation of an improvised nuclear device or a radiological dispersal device in a heavily populated area. As such, this type of security threat is considered to be of relatively low risk, but one that would have an extraordinary high impact on health and well-being of the US citizenry. Psychological counseling and medical assessments would be necessary for all those significantly impacted by the nuclear/radiological event. Direct medical interventions would be necessary for all those individuals who had received substantial radiation exposures (e.g., >1 Gy). Although no drugs or products have yet been specifically approved by the United States Food and Drug Administration (US FDA) to treat the effects of acute radiation syndrome (ARS), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and pegylated G-CSF have been used off label for treating radiation accident victims. Recent threats of terrorist attacks using nuclear or radiologic devices makes it imperative that the medical community have up-to-date information and a clear understanding of treatment protocols using therapeutically effective recombinant growth factors and cytokines such as G-CSF and GM-CSF for patients exposed to injurious doses of ionizing radiation. Based on limited human studies with underlying biology, we see that the recombinants, G-CSF and GM-CSF appear to have modest, but significant medicinal value in treating radiation accident victims. In the near future, the US FDA may approve G-CSF and GM-CSF as ‘Emergency Use Authorization’ (EUA) for managing radiation-induced aplasia, an ARS-related pathology. In this article, we review the status of growth factors for the treatment of radiological/nuclear accident victims. PMID:25215458

  14. In vitro production of functional immune cells derived from human hematopoietic stem cells

    PubMed Central

    Payuhakrit, Witchuda; Panichakul, Tasanee; Charoenphon, Natthawut; Chalermsaenyakorn, Panus; Jaovisidha, Adithep; Wongborisuth, Chokdee; Udomsangpetch, Rachanee

    2015-01-01

    Hematopoietic stem cells (HSC) from cord blood are potentially high sources for transplantation due to their low immunogenicity and the presence of the multipotent cells. These cells are capable of differentiating to produce various lineages of blood cells under specific conditions. We have enriched highly purified CD34+ cells from cord blood, determined in vitro growth of the cells in culture systems in the absence (condition A) or presence of GM-CSF and G-CSF (condition B), and determined the profile of immune cells during the period of cultivation by using flow cytometry. PhytohemagglutininA (PHA) was used as a mitogen to stimulate T lymphocytes derived from hematopoietic stem cells. GM-CSF and G-CSF prolonged the survival of the growing cells and also maintained expansion of cells in blastic stage. By day 12 of cultivation, when cell numbers peaked, various types of immune cells had appeared (CD14+ cells, CD40+HLA-DR+ cells, CD3+CD56+ cells, CD19+ cells, CD3+CD4+ cells, CD3+CD8+cells and CD3-CD56+). A significantly higher percentage of monocytes (p = 0.002) were observed under culture with GM-CSF, G-CSF when compared with culture without GM-CSF, G-CSF. In addition, T lymphocytes derived from HSC responded to 50 µg/ml of PHA. This is the first report showing the complete differentiation and proliferation of immune cells derived from CD34+ HSC under in vitro culture conditions. Lymphocytes, monocytes, dendritic cells and polymorph nuclear cells derived from HSC in vitro are unique, and thus may benefit various studies such as innate immunity and pathophysiology of immune disorders. PMID:26933404

  15. The Addition of Recombinant Vaccinia HER2/neu to Oncolytic Vaccinia-GMCSF Given into the Tumor Microenvironment Overcomes MDSC-Mediated Immune Escape and Systemic Anergy

    PubMed Central

    de Vries, Christiaan R.; Monken, Claude E.; Lattime, Edmund C.

    2015-01-01

    Effective immunotherapeutic strategies require the ability to generate a systemic antigen-specific response capable of impacting both primary and metastatic disease. We have built on our oncolytic vaccinia GM-CSF strategy by adding recombinant tumor antigen to increase the response in the tumor microenvironment and systemically. In the present study, orthotopic growth of a syngeneic HER2/neu-overexpressing mammary carcinoma in FVB/N mice (NBT1) was associated with increased Gr1+CD11b+ myeloid derived suppressor cells (MDSCs) both systemically and in the tumor microenvironment. This MDSC population had inhibitory effects on the HER2/neu specific Th1 immune response. VVneu and VVGMCSF are recombinant oncolytic vaccinia viruses that encode HER2/neu and GM-CSF, respectively. Naïve FVB mice vaccinated with combined VVneu and VVGMCSF given systemically developed systemic HER2/neu-specific immunity. NBT1 bearing mice became anergic to systemic immunization with combined VVneu and VVGMCSF. Intratumoral VVGMCSF failed to result in systemic antitumor immunity until combined with intratumoral VVneu. Infection/transfection of the tumor microenvironment with combined VVGMCSF and VVneu resulted in development of systemic tumor-specific immunity, reduction in splenic and tumor MDSC, and therapeutic efficacy against tumor. These studies demonstrate the enhanced efficacy of oncolytic vaccinia virus recombinants encoding combined tumor antigen and GM-CSF in modulating the microenvironment of MDSC-rich tumors. PMID:25633483

  16. Pretreatment plasma levels and diagnostic utility of hematopoietic cytokines in cervical cancer or cervical intraepithelial neoplasia patients.

    PubMed

    Ławicki, Sławomir; Będkowska, Grażyna E; Gacuta-Szumarska, Ewa; Knapp, Paweł; Szmitkowski, Maciej

    2012-07-04

    In this study, we compared plasma levels and the diagnostic utility of hematopoietic growth factors (HGFs) with SCC-Ag in cervical cancer patients in relation to control groups and cervical intraepithelial neoplasia (CIN) patients and healthy subjects. Pretreatment plasma levels of HGFs (SCF, GM-CSF, G-CSF and M-CSF) were determined by the use of immunoenzyme assay (ELISA), and SCC-Ag by chemiluminescent microparticle immunoassay (CMIA). Significantly different concentrations of GM-CSF, G-CSF and M-CSF were observed in the group of patients with cervical cancer and CIN compared to the healthy controls. Significant differences in plasma levels of GM-CSF and M-CSF between cervical cancer and benign lesions patients were also found. The HGFs and SCC-Ag diagnostic specificities received high values. The diagnostic sensitivity and the predictive value of a positive and negative test result were higher for M-CSF than for antigen SCC in the cancer group. The M-CSF area under the ROC curve (AUC) was the largest from hematopoietic cytokines and SCC-Ag. These results suggest the potential utility of M-CSF as a good candidate for a marker of cervical cancer as well as benign lesions of this organ (CIN).

  17. Culture perfusion schedules influence the metabolic activity and granulocyte-macrophage colony-stimulating factor production rates of human bone marrow stromal cells.

    PubMed

    Caldwell, J; Palsson, B O; Locey, B; Emerson, S G

    1991-05-01

    The metabolic function and GM-CSF production rates of adherent human bone marrow stromal cells were investigated as functions of medium and serum feeding rates. A range of medium exchange schedules was studied, ranging from a typical Dexter culture protocol of one weekly medium exchange to a full media exchange daily, which more closely approximates what bone marrow cells experience in situ. Glucose consumption was found to be significantly higher at full daily exchange rate than at any other exchange schedule examined. However, the lactate yield on glucose was a constant, at 1.8 mol/mol, under all conditions considered. Differential serum vs. medium exchange experiment showed that both serum supply and medium nutrients were responsible for the altered behavior at high exchange rates. Glutamine consumption was found to be insignificant under all culture conditions examined. A change in exchange schedule from 50% daily medium exchange to full daily medium exchange after 14 days of culture was found to result in a transient production of GM-CSF and a change in metabolic behavior to resemble that of cultures which had full daily exchange from day one. These results suggest that both stromal cell metabolism and GM-CSF production are sensitive to medium exchange schedules. Taken together, the data presented indicate that attempts to model the function of human bone marrow in vitro may be well served by beginning with medium exchange schedules that more closely mimic the in vivo physiologic state of bone marrow. PMID:2040665

  18. Biological mechanisms underlying the ultraviolet radiation-induced formation of skin wrinkling and sagging II: over-expression of neprilysin plays an essential role.

    PubMed

    Imokawa, Genji; Nakajima, Hiroaki; Ishida, Koichi

    2015-04-08

    Our previous studies strongly indicated that the up-regulated activity of skin fibroblast-derived elastase plays a pivotal role in wrinkling and/or sagging of the skin via the impairment of elastic fiber configuration and the subsequent loss of skin elasticity. Fortunately, we succeeded in identifying human skin fibroblast-derived elastase as a previously known enzyme, neprilysin or neutral endopeptidase (NEP). We have also characterized epithelial-mesenchymal paracrine cytokine interactions between UVB-exposed-keratinocytes and dermal fibroblasts and found that interleukin-1α and granulocyte macrophage colony stimulatory factor (GM-CSF) are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by fibroblasts. On the other hand, direct UVA exposure of human fibroblasts significantly stimulates the secretion of IL-6 and also elicits a significant increase in the gene expression of matrix metallo-protease(MMP)-1 as well as neprilysin (to a lesser extent), which is followed by distinct increases in their protein and enzymatic activity levels. Direct UVA exposure of human keratinocytes also stimulates the secretion of IL-6, IL-8 and GM-CSF but not of IL-1 and endothelin-1. These findings suggest that GM-CSF secreted by UVA-exposed keratinocytes as well as IL-6 secreted by UVA-exposed dermal fibroblasts play important and additional roles in UVA-induced sagging and wrinkling by up-regulation of neprilysin and MMP-1, respectively, in dermal fibroblasts.

  19. Cytokines derived from cultured skeletal muscle cells after mechanical strain promote neutrophil chemotaxis in vitro.

    PubMed

    Peterson, Jennifer M; Pizza, Francis X

    2009-01-01

    We tested the hypothesis that cytokines derived from differentiated skeletal muscle cells in culture induce neutrophil chemotaxis after mechanical strain. Flexible-bottom plates with cultured human muscle cells attached were exposed to mechanical strain regimens (ST) of 0, 10, 30, 50, or 70 kPa of negative pressure. Conditioned media were tested for the ability to induce chemotaxis of human blood neutrophils in vitro and for a marker of muscle cell injury (lactate dehydrogenase). Conditioned media promoted neutrophil chemotaxis in a manner that was related both to the degree of strain and to the magnitude of muscle cell injury (ST 70 > ST 50 > ST 30). Protein profiling using a multiplex cytokine assay revealed that mechanical strain increased the presence of IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor, monocyte chemotactic protein (MCP)-1, and IL-6 in conditioned media. We also detected 14 other cytokines in conditioned media from control cultures that did not respond to mechanical strain. Neutralization of IL-8 and GM-CSF completely inhibited the chemotactic response for ST 30 and ST 50 and reduced the chemotactic response for ST 70 by 40% and 47%, respectively. Neutralization of MCP-1 or IL-6 did not reduce chemotaxis after ST 70. This study enhances our understanding of the immunobiology of skeletal muscle by revealing that skeletal muscle cell-derived IL-8 and GM-CSF promote neutrophil chemotaxis after injurious mechanical strain.

  20. Uptake of 12-HETE by human bronchial epithelial cells (HBEC): effects on HBEC cytokine production.

    PubMed

    Gormand, F; Chabannes, B; Moliere, P; Perrin-Fayolle, M; Lagarde, M; Pacheco, Y

    1996-04-01

    12-HETE, the major lipoxygenase end-product of platelets and macrophages, may be released in contact of bronchial epithelium in inflammatory diseases of the lung. We have studied the outcome of 12-HETE in presence of human bronchial epithelial cells (HBEC). When HBEC were incubated with [3H]12-HETE for 30 minutes, 27.5% of total radioactivity was found in HBEC and 72.5% in supernatants. Unesterified 12-HETE accounted for 22.4% of total radioactivity, 4.5% being recovered in phospholipids, preferentially in phosphatidylcholine and phosphatidylethanolamine. No incorporation in neutral lipids was detected. 72.9% of the incubated radioactivity was recovered in un identified metabolites. As 12-HETE has been shown to modulate the expression and production of various proteins, the consequence of the 12-HETE uptake on the release of GM-CSF and IL8 by HBEC was assessed. HBEC from control subjects were cultured for 24 hours with 12-HETE (10(-9) to 10(-7)M) in the presence or absence of TNF alpha. Detectable amounts of both cytokines were released in the supernatant in basal conditions at 24hr, and TNF alpha increased significantly the release of GM-CSF. 12-HETE at 10(-7)M weakly but significantly decreased the TNF-induced release of GM-CSF from HBEC. Thus the uptake of 12-HETE could affect the epithelial cell function in some situations.

  1. Lentinan mitigates therarubicin-induced myelosuppression by activating bone marrow-derived macrophages in an MAPK/NF-κB-dependent manner.

    PubMed

    Liu, Qiang; Dong, Lei; Li, Hong; Yuan, Jia; Peng, Yuping; Dai, Shejiao

    2016-07-01

    Bone marrow (BM) suppression (also known as myelosuppression) is the most common and most severe side-effect of therarubicin (THP) and thereby limits the clinical application of this anticancer agent. Lentinan (LNT), a glucan extracted from dried shiitake mushrooms (Lentinula edodes), exhibits a variety of pharmacological activities. The objectives of the present study were to determine the effect of LNT on the myelosuppression of THP-treated mice and to examine the pharmacological mechanism of these effects. In vivo experiments indicated that non-cytotoxic levels of LNT strongly increased blood myeloperoxidase (MPO) activity; improved BM structural injuries; increased the numbers of leukocytes and neutrophils in the blood and BM; elevated the blood levels of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF); and reduced the self-healing period in THP-treated mice. In vitro experiments indicated that LNT increased the viability of BM-derived macrophages (BMDMs) in a time- and dose-dependent manner without toxic side-effects and markedly increased the release of G-CSF, GM-CSF and M-CSF by BMDMs. Further analyses revealed that LNT activated the NF-κB and MAPK signalling pathways and promoted the nuclear import of p65 and that BAY 11-7082 (a specific inhibitor of NF-κB) suppressed the release of G-CSF, GM-CSF and M-CSF. Furthermore, we found that U0126, SB203580 and SP600125 (specific inhibitors of ERK, p38 and JNK, respectively) markedly inhibited the IKK/IκB/NF-κB-dependent release of G-CSF, GM-CSF and M-CSF. In conclusion, LNT induces the production of G-CSF, GM-CSF and M-CSF by activating the MAPK/NF-κB signalling pathway in BM cells, thereby mitigating THP-induced myelosuppression. PMID:27121155

  2. Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells.

    PubMed

    Iwai, Y; Bickel, M; Pluznik, D H; Cohen, R B

    1991-09-25

    Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region. PMID:1917935

  3. Murine Anti-GD2 Monoclonal Antibody 3F8 Combined With Granulocyte-Macrophage Colony-Stimulating Factor and 13-Cis-Retinoic Acid in High-Risk Patients With Stage 4 Neuroblastoma in First Remission

    PubMed Central

    Cheung, Nai-Kong V.; Cheung, Irene Y.; Kushner, Brian H.; Ostrovnaya, Irina; Chamberlain, Elizabeth; Kramer, Kim; Modak, Shakeel

    2012-01-01

    Purpose Anti-GD2 monoclonal antibody (MoAb) combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown efficacy against neuroblastoma (NB). Prognostic variables that could influence clinical outcome were explored. Patients and Methods One hundred sixty-nine children diagnosed with stage 4 NB (1988 to 2008) were enrolled onto consecutive anti-GD2 murine MoAb 3F8 ± GM-CSF ± 13-cis-retinoic acid (CRA) protocols after achieving first remission (complete remission/very good partial remission). Patients enrolled in regimen A (n = 43 high-risk [HR] patients) received 3F8 alone; regimen B (n = 41 HR patients), 3F8 + intravenous GM-CSF + CRA, after stem-cell transplantation (SCT); and regimen C (n = 85), 3F8 + subcutaneous GM-CSF + CRA, 46 of 85 after SCT, whereas 28 of 85 required additional induction therapy and were deemed ultra high risk (UHR). Marrow minimal residual disease (MRD) was measured by quantitative reverse transcription polymerase chain reaction. Survival probability was calculated by the Kaplan-Meier method, and prognostic variables were analyzed by multivariate Cox regression model. Results At 5 years from the start of immunotherapy, progression-free survival (PFS) improved from 44% for HR patients receiving regimen A to 56% and 62% for those receiving regimens B and C, respectively. Overall survival (OS) was 49%, 61%, and 81%, respectively. PFS and OS of UHR patients were 36% and 75%, respectively. Relapse was mostly at isolated sites. Independent adverse prognostic factors included UHR (PFS) and post–cycle two MRD (PFS and OS), whereas the prognostic factors for improved outcome were missing killer immunoglobulin-like receptor ligand (PFS and OS), human antimouse antibody response (OS), and regimen C (OS). Conclusion Retrospective analysis of consecutive trials from a single center demonstrated that MoAb 3F8 + GM-CSF + CRA is effective against chemotherapy-resistant marrow MRD. Its positive impact on long-term survival can only

  4. Lack of survival advantage with autologous stem-cell transplantation in high-risk neuroblastoma consolidated by anti-GD2 immunotherapy and isotretinoin.

    PubMed

    Kushner, Brian H; Ostrovnaya, Irina; Cheung, Irene Y; Kuk, Deborah; Modak, Shakeel; Kramer, Kim; Roberts, Stephen S; Basu, Ellen M; Yataghene, Karima; Cheung, Nai-Kong V

    2016-01-26

    Since 2003, high-risk neuroblastoma (HR-NB) patients at our center received anti-GD2 antibody 3F8/GM-CSF + isotretinoin - but not myeloablative therapy with autologous stem-cell transplantation (ASCT). Post-ASCT patients referred from elsewhere also received 3F8/GM-CSF + isotretinoin. We therefore accrued a study population of two groups treated during the same period and whose consolidative therapy, aside from ASCT, was identical. We analyzed patients enrolled in 1st complete/very good partial remission (CR/VGPR). Their event-free survival (EFS) and overall survival (OS) were calculated from study entry. Large study size allowed robust statistical analyses of key prognosticators including MYCN amplification, minimal residual disease (MRD), FCGR2A polymorphisms, and killer immunoglobulin-like receptor genotypes of natural killer cells. The 170 study patients included 60 enrolled following ASCT and 110 following conventional chemotherapy. The two cohorts had similar clinical and biological features. Five-year rates for ASCT and non-ASCT patients were, respectively: EFS 65% vs. 51% (p = .128), and OS 76% vs. 75% (p = .975). In multivariate analysis, ASCT was not prognostic and only MRD-negativity after two cycles of 3F8/GM-CSF correlated with significantly improved EFS and OS. Although a trend towards better EFS is seen with ASCT, OS is near identical. Cure rates may be similar, as close surveillance detects localized relapse and effective salvage treatments are applied. ASCT may not be needed to improve outcome when anti-GD2 immunotherapy is used for consolidation after dose-intensive conventional chemotherapy.

  5. Lack of survival advantage with autologous stem-cell transplantation in high-risk neuroblastoma consolidated by anti-GD2 immunotherapy and isotretinoin.

    PubMed

    Kushner, Brian H; Ostrovnaya, Irina; Cheung, Irene Y; Kuk, Deborah; Modak, Shakeel; Kramer, Kim; Roberts, Stephen S; Basu, Ellen M; Yataghene, Karima; Cheung, Nai-Kong V

    2016-01-26

    Since 2003, high-risk neuroblastoma (HR-NB) patients at our center received anti-GD2 antibody 3F8/GM-CSF + isotretinoin - but not myeloablative therapy with autologous stem-cell transplantation (ASCT). Post-ASCT patients referred from elsewhere also received 3F8/GM-CSF + isotretinoin. We therefore accrued a study population of two groups treated during the same period and whose consolidative therapy, aside from ASCT, was identical. We analyzed patients enrolled in 1st complete/very good partial remission (CR/VGPR). Their event-free survival (EFS) and overall survival (OS) were calculated from study entry. Large study size allowed robust statistical analyses of key prognosticators including MYCN amplification, minimal residual disease (MRD), FCGR2A polymorphisms, and killer immunoglobulin-like receptor genotypes of natural killer cells. The 170 study patients included 60 enrolled following ASCT and 110 following conventional chemotherapy. The two cohorts had similar clinical and biological features. Five-year rates for ASCT and non-ASCT patients were, respectively: EFS 65% vs. 51% (p = .128), and OS 76% vs. 75% (p = .975). In multivariate analysis, ASCT was not prognostic and only MRD-negativity after two cycles of 3F8/GM-CSF correlated with significantly improved EFS and OS. Although a trend towards better EFS is seen with ASCT, OS is near identical. Cure rates may be similar, as close surveillance detects localized relapse and effective salvage treatments are applied. ASCT may not be needed to improve outcome when anti-GD2 immunotherapy is used for consolidation after dose-intensive conventional chemotherapy. PMID:26623730

  6. Increased mRNA expression of manganese superoxide dismutase in psoriasis skin lesions and in cultured human keratinocytes exposed to IL-1 beta and TNF-alpha.

    PubMed

    Löntz, W; Sirsjö, A; Liu, W; Lindberg, M; Rollman, O; Törmä, H

    1995-02-01

    Because reactive oxygen species have been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of the antioxidant enzyme superoxide dismutase was studied in psoriatic skin tissue. By using reverse transcription-PCR we found similar expression of copper, zinc superoxide dismutase (CuZnSOD) in the involved vs. uninvolved psoriatic skin. In contrast, the level of the manganese superoxide dismutase (MnSOD) mRNA message was consistently higher in lesional psoriatic skin as compared to adjacent uninvolved skin and healthy control skin. Parallel investigation of those cytokines that are thought to be direct or indirect inducers of the MnSOD activity revealed an increased mRNA expression of IL-1 beta, TNF-alpha, and GM-CSF in lesional psoriatic skin. To study if these cytokines exert a direct effect on dismutase expression in epidermal cells, human keratinocytes in culture were challenged with IL-1 beta, TNF-alpha, and GM-CSF. It was found that IL-1 beta and TNF-alpha, but not GM-CSF, induced the mRNA expression of MnSOD, and an additive effect was demonstrated for the two former cytokines. Further, the expression of both CuZnSOD and MnSOD transcripts was similar in cultured keratinocytes maintained at low differentiation (low Ca2+ medium) and cells forced to terminal differentiation (by high Ca2+ medium). Our results indicate that the abnormal expression of MnSOD mRNA in lesional psoriatic skin is not directly linked to the pathologic state of keratinocyte differentiation in the skin. It seems more likely that the cutaneous overexpression of MnSOD in psoriatic epidermis represents a protective cellular response evoked by cytokines released from inflammatory cells invading the diseased skin. PMID:7744320

  7. In vivo cytokine responses to interleukin-2 immunotherapy after autologous stem cell transplantation in children with solid tumors.

    PubMed

    Bönig, H; Laws, H J; Wundes, A; Verheyen, J; Hannen, M; Kim, Y M; Banning, U; Nürnberger, W; Körholz, D

    2000-07-01

    The potent immunostimulatory cytokine interleukin-2 (IL-2) has been extensively investigated for its potential to induce anti-tumor immunity in a number of tumor models. Only recently the complex interplay of mutually suppressive or supportive cytokines of the IL-2-induced network of cytokines has been better characterized. The aim of this study was to assess which of these in vitro findings are reproducible in vivo in recipients of stem cell transplants (SCT), since in these patients long- lasting impairments in cytokine inducibility have been described. We have therefore studied the kinetics of putative modulators and mediators of IL-2-induced immune activation, namely IL-1beta, IL-4, IL-5, IL-10, IL-12, soluble Fas ligand (sFasL), and GM-CSF during IL-2 therapy. All patients were children or adolescents suffering from solid tumors with poor prognosis who received three 5-day courses of high-dose intravenous IL-2 as an adjuvant to their radio-chemotherapy and autologous SCT. While IL-1beta, IL-4 and IL-12 were not, and sFasL was only mildly affected by the IL-2 therapy, we observed a consistent and early rise of IL-10, IL-5, and GM-CSF. These increases were rapidly reversible after discontinuation of IL-2 therapy. The inducibility of IL-10, IL-5 and GM-CSF was more pronounced with increasing time from the SCT, and in the third cycle reached an order of magnitude as in high-dose IL-2 patients without SCT. Together with the abundant in vitro data, these findings may help devise a combination immunotherapy permitting stronger anti-tumor effects, but lesser adverse effects.

  8. Liver myeloid-derived suppressor cells expand in response to liver metastases in mice and inhibit the anti-tumor efficacy of anti-CEA CAR-T

    PubMed Central

    Burga, Rachel A.; Thorn, Mitchell; Point, Gary R.; Guha, Prajna; Nguyen, Cang T.; Licata, Lauren A.; DeMatteo, Ronald P.; Ayala, Alfred; Espat, N. Joseph; Junghans, Richard P.; Katz, Steven C.

    2015-01-01

    Chimeric antigen receptor modified T cell (CAR-T) technology, a promising immunotherapeutic tool, has not been applied specifically to treat liver metastases (LM). While CAR-T delivery to LM can be optimized by regional intrahepatic infusion, we propose that liver CD11b+Gr-1+ myeloid-derived suppressor cells (L-MDSC) will inhibit the efficacy of CAR-T in the intrahepatic space. We studied anti-CEA CAR-T in a murine model of CEA+ LM and identified mechanisms through which L-MDSC expand and inhibit CAR-T function. We established CEA+ LM in mice and studied purified L-MDSC and responses to treatment with intrahepatic anti-CEA CAR-T infusions. L-MDSC expanded three-fold in response to LM and their expansion was dependent on GM-CSF, which was produced by tumor cells. L-MDSC utilized PD-L1 to suppress anti-tumor responses through engagement of PD-1 on CAR-T. GM-CSF, in cooperation with STAT3, promoted L-MDSC PD-L1 expression. CAR-T efficacy was rescued when mice received CAR-T in combination with MDSC depletion, GM-CSF neutralization to prevent MDSC expansion, or PD-L1 blockade. As L-MDSC suppressed anti-CEA CAR-T, infusion of anti-CEA CAR-T in tandem with agents targeting L-MDSC is a rational strategy for future clinical trials. PMID:25850344

  9. The prolyl hydroxylase PHD3 identifies proinflammatory macrophages and its expression is regulated by activin A.

    PubMed

    Escribese, María M; Sierra-Filardi, Elena; Nieto, Concha; Samaniego, Rafael; Sánchez-Torres, Carmen; Matsuyama, Takami; Calderon-Gómez, Elisabeth; Vega, Miguel A; Salas, Azucena; Sánchez-Mateos, Paloma; Corbí, Angel L

    2012-08-15

    Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163(+) lung macrophages under basal homeostatic conditions, whereas PHD3(+) macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn's disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro. PMID:22778395

  10. Dendritic cell maturation requires STAT1 and is under feedback regulation by suppressors of cytokine signaling.

    PubMed

    Jackson, Sharon H; Yu, Cheng-Rong; Mahdi, Rashid M; Ebong, Samuel; Egwuagu, Charles E

    2004-02-15

    In this study we show that activation of STAT pathways is developmentally regulated and plays a role in dendritic cell (DC) differentiation and maturation. The STAT6 signaling pathway is constitutively activated in immature DC (iDC) and declines as iDCs differentiate into mature DCs (mDCs). However, down-regulation of this pathway during DC differentiation is accompanied by dramatic induction of suppressors of cytokine signaling 1 (SOCS1), SOCS2, SOCS3, and cytokine-induced Src homology 2-containing protein expression, suggesting that inhibition of STAT6 signaling may be required for DC maturation. In contrast, STAT1 signaling is most robust in mDCs and is not inhibited by the up-regulated SOCS proteins, indicating that STAT1 and STAT6 pathways are distinctly regulated in maturing DC. Furthermore, optimal activation of STAT1 during DC maturation requires both IL-4 and GM-CSF, suggesting that synergistic effects of both cytokines may in part provide the requisite STAT1 signaling intensity for DC maturation. Analyses of STAT1(-/-) DCs reveal a role for STAT1 in repressing CD86 expression in precursor DCs and up-regulating CD40, CD11c, and SOCS1 expression in mDCs. We further show that SOCS proteins are differentially induced by IL-4 and GM-CSF in DCs. SOCS1 is primarily induced by IL-4 through a STAT1-dependent mechanism, whereas SOCS3 is induced mainly by GM-CSF. Taken together, these results suggest that cytokine-induced maturation of DCs is under feedback regulation by SOCS proteins and that the switch from constitutive activation of the STAT6 pathway in iDCs to predominant use of STAT1 signals in mDC is mediated in part by STAT1-induced SOCS expression.

  11. Neutrophil priming occurs in a sequential manner and can be visualized in living animals by monitoring IL-1β promoter activation

    PubMed Central

    Yao, Yi; Matsushima, Hironori; Ohtola, Jennifer A.; Geng, Shuo; Lu, Ran; Takashima, Akira

    2014-01-01

    Rapid enhancement of phagocyte functionality is a hallmark of neutrophil priming. GeneChip analyses unveiled elevated CD54, dectin-2, and IL-1β mRNA expression by neutrophils isolated from inflammatory sites. In fact, CD54 and dectin-2 protein expression was detected on neutrophils recovered from skin, peritoneal and lung inflammation lesions, but not on those in bone marrow or peripheral blood. Neutrophils elevated CD54 and dectin-2 mRNA during migration in Boyden chambers and acquired CD54 and dectin-2 surface expression after subsequent exposure to GM-CSF. Neutrophils purified from IL-1β promoter-driven DsRed transgenic mice acquired DsRed signals during cell migration or exposure to GM-CSF. CD54 and dectin-2 were expressed by DsRed+ (but not DsRed–) neutrophils in GM-CSF-supplemented culture, and neutrophils recovered from inflammatory sites exhibited strong DsRed signals. The dynamic process of neutrophil priming was then studied in chemically induced inflammatory skin lesions by monitoring DsRed expression under confocal microscopy. A majority (>80%) of Ly6G+ neutrophils expressed DsRed, and those DsRed+/Ly6G+ cells exhibited crawling motion with a higher velocity compared to the DsRed–/Ly6G+ counterpart. This is the first report showing motile behaviors of primed neutrophils in living animals. We propose that neutrophil priming occurs in a sequential manner with rapid enhancement of phagocyte functionality followed by CD54 and dectin-2 mRNA and protein expression, IL-1β promoter activation, and accelerated motility. Not only do these findings provide a new conceptual framework for our understanding of the process of neutrophil priming, they also unveil new insights into the pathophysiology of many inflammatory disorders characterized by neutrophil infiltration. PMID:25527787

  12. Glioma-specific cytotoxic T cells can be effectively induced by subcutaneous vaccination of irradiated wild-type tumor cells without artificial cytokine production.

    PubMed

    Iwadate, Yasuo; Yamaura, Akira; Sakiyama, Shigeru; Sato, Yasuo; Tagawa, Masatoshi

    2003-08-01

    Effective induction of systemic antitumor immunity is a crucial step for success of immune gene therapy for intracerebral gliomas. We examined in this study the ability to induce glioma-specific cytotoxic T lymphocytes (CTL) by subcutaneous (s.c.) immunization of irradiated whole-tumor cell vaccine with or without artificial cytokine production, and also examined in vivo efficacy of the induced CTL against a rat brain tumor model with 9L gliosarcoma cells. Murine neuroblastoma C1300 cells transduced with the interleukin-2 (IL-2), IL-4 or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene (C1300/IL-2, C1300/IL-4 or C1300/GM-CSF) were used as cytokine-producers. Glioma-specific CTL activity was equivalently induced in the rats vaccinated s.c. with irradiated 9L, irradiated IL-2-producing 9L cells or the mixed population of irradiated 9L and C1300/IL-2 cells, while the activity was relatively lower in the rats vaccinated with irradiated 9L cells mixed with either C1300/IL-4 or C1300/GM-CSF cells. In the rats immunized s.c. with irradiated 9L cells, intracerebral (i.c.) 9L tumors implanted together with either C1300/IL-2 or C1300/IL-4 were completely rejected. Pre-established brain tumor also could be eliminated by the s.c. immunization of irradiated 9L cells and i.c. transplantation of IL-2-producers. These results suggest that glioma-specific CTLs could be effectively induced by s.c. immunization of irradiated wild-type tumor cells without artificial cytokine production.

  13. Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-{kappa}B translocation

    SciTech Connect

    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.

    2008-06-15

    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 {mu}M after 48 h incubation. Pretreatment with 100 {mu}M 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)-{alpha}, 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 I{kappa}B{alpha}, as well as the nuclear translocation of NF-{kappa}B primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-{kappa}B 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.

  14. Timing of the loss of Pten protein determines disease severity in a mouse model of myeloid malignancy

    PubMed Central

    Yan, Yan; Webster, Cody; Shao, Lijian; Lensing, Shelly Y.; Ni, Hongyu; Feng, Wei; Colorado, Natalia; Pathak, Rupak; Xiang, Zhifu; Hauer-Jensen, Martin; Li, Shaoguang; Zhou, Daohong; Emanuel, Peter D.

    2016-01-01

    Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN). JMML leukemogenesis is linked to a hyperactivated RAS pathway, with driver mutations in the KRAS, NRAS, NF1, PTPN11, or CBL genes. Previous murine models demonstrated how those genes contributed to the selective hypersensitivity of JMML cells to granulocyte macrophage–colony-stimulating factor (GM-CSF), a unifying characteristic in the disease. However, it is unclear what causes the early death in children with JMML, because transformation to acute leukemia is rare. Here, we demonstrate that loss of Pten (phosphatase and tensin homolog) protein at postnatal day 8 in mice harboring Nf1 haploinsufficiency results in an aggressive MPN with death at a murine prepubertal age of 20 to 35 days (equivalent to an early juvenile age in JMML patients). The death in the mice was due to organ infiltration with monocytes/macrophages. There were elevated activities of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) in cells at physiological concentrations of GM-CSF. These were more pronounced in mice with Nf1 haploinsufficiency than in littermates with wild-type Nf1, but this model is insufficient to cause cells to be GM-CSF hypersensitive. This new model represents a murine MPN model with features of a pediatric unclassifiable mixed MDS/MPN and mimics many clinical manifestations of JMML in terms of age of onset, aggressiveness, and organ infiltration with monocytes/macrophages. Our data suggest that the timing of the loss of PTEN protein plays a critical role in determining the disease severity in myeloid malignancies. This model may be useful for studying the pathogenesis of pediatric diseases with alterations in the Ras pathway. PMID:26764354

  15. Antigen activation of THP-1 human monocytic cells after stimulation with lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

    PubMed

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

    1999-05-01

    A human THP-1 monocyte cell line culture system has been utilized to evaluate the morphological changes in THP-1 cells and to measure expression of activation antigens (CD-11b, CD-11c, CD-14, CD-35, CD-68, CD-71 and HLA-DR) as evidence of maturation of THP-1 cells in response to stimulation by lipopolysaccharide (LPS) from the oral microorganisms, Fusobacterium nucleatum and Porphyromonas gingivalis, and granulocyte-macrophage colony-stimulating factor. THP-1 cells were stimulated with LPS (1 microgram/ml) of P. gingivalis or F. nucleatum for different time periods (1, 2, 4 and 7 d). Detection of different activation antigens on THP-1 cells was performed by indirect immunohistochemical staining followed by light microscopy. Confirmational studies were performed in parallel using indirect immunofluorescence and immunogold electron microscopy for detection of the corresponding activation antigens. Expression of different activation antigens by resting THP-1 cells revealed HLA-DR to be on 3% of the cells; CD-11b, 9%; CD-11c, 8%; CD-14, 22%; CD-35, 9% and CD-68, 7%. The CD-71 activation antigen was not expressed in untreated THP-1 cells. LPS stimulation increased expression of all activation antigens. A significant (p < 0.05) increase in expression of CD-11b, CD-11c, CD-14, CD-35, CD-68 and CD-71 was observed when GM-CSF (50 IU/ml) was supplemented during the treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis. Activation and differentiation of THP-1 cells by LPS from oral microorganisms in the presence of GM-CSF supports a role for human macrophages in acute and chronic periodontal diseases and may explain the clinically observable periodontal exacerbations in some patients after GM-CSF therapy.

  16. Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.

    PubMed

    Labeur, M S; Roters, B; Pers, B; Mehling, A; Luger, T A; Schwarz, T; Grabbe, S

    1999-01-01

    Bone marrow-derived dendritic cells (BmDC) are potent APC and can promote antitumor immunity in mice when pulsed with tumor Ag. This study aimed to define the culture conditions and maturation stages of BmDC that enable them to optimally function as APC in vivo. BmDC cultured under various conditions (granulocyte-macrophage CSF (GM-CSF) or GM-CSF plus IL-4 alone or in combination with Flt3 ligand, TNF-alpha, LPS, or CD40 ligand (CD40L)) were analyzed morphologically, phenotypically, and functionally and were tested for their ability to promote prophylactic and/or therapeutic antitumor immunity. Each of the culture conditions generated typical BmDC. Whereas cells cultured in GM-CSF alone were functionally immature, cells incubated with CD40L or LPS were mature BmDC, as evident by morphology, capacity to internalize Ag, migration into regional lymph nodes, IL-12 secretion, and alloantigen or peptide Ag presentation in vitro. The remaining cultures exhibited intermediate dendritic cell maturation. The in vivo Ag-presenting capacity of BmDC was compared with respect to induction of both protective tumor immunity and immunotherapy of established tumors, using the poorly immunogenic squamous cell carcinoma, KLN205. In correspondence to their maturation stage, BmDC cultured in the presence of CD40L exhibited the most potent immunostimulatory effects. In general, although not entirely, the capacity of BmDC to induce an antitumor immune response in vivo correlated to their degree of maturation. The present data support the clinical use of mature, rather than immature, tumor Ag-pulsed dendritic cells as cancer vaccines and identifies CD40L as a potent stimulus to enhance their in vivo Ag-presenting capacity.

  17. Local transgenic expression of granulocyte macrophage-colony stimulating factor initiates autoimmunity.

    PubMed

    Biondo, M; Nasa, Z; Marshall, A; Toh, B H; Alderuccio, F

    2001-02-01

    Mechanisms leading to breakdown of immunological tolerance and initiation of autoimmunity are poorly understood. Experimental autoimmune gastritis is a paradigm of organ-specific autoimmunity arising from a pathogenic autoimmune response to gastric H/K ATPase. The gastritis is accompanied by autoantibodies to the gastric H/K ATPase. The best characterized model of experimental autoimmune gastritis requires neonatal thymectomy. This procedure disrupts the immune repertoire, limiting its usefulness in understanding how autoimmunity arises in animals with intact immune systems. Here we tested whether local production of GM-CSF, a pro-inflammatory cytokine, is sufficient to break tolerance and initiate autoimmunity. We generated transgenic mice expressing GM-CSF in the stomach. These transgenic mice spontaneously developed gastritis with an incidence of about 80% after six backcrosses to gastritis-susceptible BALBc/CrSlc mice. The gastritis is accompanied by mucosal hypertrophy, enlargement of draining lymph nodes and autoantibodies to gastric H/K ATPase. An infiltrate of dendritic cells and macrophages preceded CD4 T cells into the gastric mucosa. T cells from draining lymph nodes specifically proliferated to the gastric H/K ATPase. CD4 but not CD8 T cells transferred gastritis to nude mouse recipients. CD4(+) CD25(+) T cells from the spleen retained anergic suppressive properties that were reversed by IL-2. We conclude that local expression of GM-CSF is sufficient to break tolerance and initiate autoimmunity mediated by CD4 T cells. This new mouse model should be useful for studies of organ-specific autoimmunity.

  18. Multiplex Analysis of Pro- or Anti-Inflammatory Serum Cytokines and Chemokines in relation to Gender and Age among Tanzanian Tuberculous Lymphadenitis Patients

    PubMed Central

    Mustafa, Tehmina; Brokstad, Karl Albert; Mfinanga, Sayoki G.; Wiker, Harald G.

    2015-01-01

    Objectives. Tuberculous lymphadenitis is the most common form of extrapulmonary tuberculosis (TB) with a female and paediatric preponderance, postulated to be due to differences in the immune response. The aim of this study was to analyze the differences in the serum cytokine levels of tuberculous lymphadenitis patients with respect to age and gender. Methods. A multiplex bead-based enzyme-linked immunosorbent assay was used to measure IFN-γ, TNF-α, GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, and IL-17 levels in sera of patients (n = 86) and healthy controls (n = 23). Results. Levels of IFN-γ, TNF-α, GM-CSF, IL-1β, IL-2, IL-4, and IL-6 were higher in adult patients than in controls, while those of IL-12 were lower (P < 0.05). Children had lower levels of TNF-α, GM-CSF, and IL-5 and higher levels of IL-2 compared with adult patients (P < 0.05). The male adult patients had higher levels of IL-17 and lower levels of IL-12 compared with female adult patients (P < 0.05).  Conclusion. There were significant differences in the levels of circulating cytokines with respect to gender and age. Children had generally lower levels of cytokines as compared to adults, which could make them more susceptible. Findings do not support that female preponderance is due to differences in immune response. PMID:26060581

  19. Increased inflammasome related gene expression profile in PBMC may facilitate T helper 17 cell induction in multiple sclerosis.

    PubMed

    Peelen, E; Damoiseaux, J; Muris, A-H; Knippenberg, S; Smolders, J; Hupperts, R; Thewissen, M

    2015-02-01

    The NLRP3 inflammasome is a macromolecular complex importantly involved in IL-1β processing. A role for this has been described in multiple sclerosis (MS). One mechanism by which IL-1β might be involved in MS is by inducing pathogenic Th17 cells, i.e. GM-CSF+ Th17 cells. In the present study, we show that expression of the inflammasome related genes, NLRP3, caspase-1, IL-1β and the IL-1β/IL-1Ra ratio, was increased in PBMC from MS patients compared to healthy controls (HC). However, in an in vitro inflammasome activity assay with PBMC, IL-1β protein secretion and the IL-1β/IL-1Ra protein ratio were similar in MS patients and HC. Th cells cultured in the presence of supernatant derived from LPS/ATP inflammasome activated PBMC showed increased Th17 and GM-CSF+ Th17 cell frequencies in HC and MS patients and decreased anti-inflammatory IL-10+Th cell frequency in HC compared to Th cells cultured in the presence of control supernatant. Moreover, addition of the immune modulator calcitriol to the former condition resulted in reduced frequencies of Th17 and GM-CSF+Th17 cells, and also of IL-10+ Th cells. Evidently, our data indicate that inflammasome activity can skew the Th cell population toward a more pro-inflammatory composition, an effect that might be inhibited by vitamin D, and that might be importantly involved in inflammation within the central nervous system.

  20. Human macrophage polarization in vitro: maturation and activation methods compared.

    PubMed

    Vogel, Daphne Y S; Glim, Judith E; Stavenuiter, Andrea W D; Breur, Marjolein; Heijnen, Priscilla; Amor, Sandra; Dijkstra, Christine D; Beelen, Robert H J

    2014-09-01

    Macrophages form a heterogeneous cell population displaying multiple functions, and can be polarized into pro- (M1) or anti-inflammatory (M2) macrophages, by environmental factors. Their activation status reflects a beneficial or detrimental role in various diseases. Currently several in vitro maturation and activation protocols are used to induce an M1 or M2 phenotype. Here, the impact of different maturation factors (NHS, M-CSF, or GM-CSF) and activation methods (IFN-γ/LPS, IL-4, dexamethason, IL-10) on the macrophage phenotype was determined. Regarding macrophage morphology, pro-inflammatory (M1) activation stimulated cell elongation, and anti-inflammatory (M2) activation induced a circular appearance. Activation with pro-inflammatory mediators led to increased CD40 and CD64 expression, whereas activation with anti-inflammatory factors resulted in increased levels of MR and CD163. Production of pro-inflammatory cytokines was induced by activation with IFN-γ/LPS, and TGF-β production was enhanced by the maturation factors M-CSF and GM-CSF. Our data demonstrate that macrophage marker expression and cytokine production in vitro is highly dependent on both maturation and activation methods. In vivo macrophage activation is far more complex, since a plethora of stimuli are present. Hence, defining the macrophage activation status ex vivo on a limited number of markers could be indecisive. From this study we conclude that maturation with M-CSF or GM-CSF induces a moderate anti- or pro-inflammatory state respectively, compared to maturation with NHS. CD40 and CD64 are the most distinctive makers for human M1 and CD163 and MR for M2 macrophage activation and therefore can be helpful in determining the activation status of human macrophages ex vivo.

  1. Analysis of splenic Gr-1int immature myeloid cells in tumor-bearing mice.

    PubMed

    Yamamoto, Yoshiko; Ishigaki, Hirohito; Ishida, Hideaki; Itoh, Yasushi; Noda, Yoichi; Ogasawara, Kazumasa

    2008-01-01

    It is known that the number of ImC, expressing myeloid markers, CD11b and Gr-1, increase with tumor growth and ImC play a role in the escape of tumor cells from immunosurveillance in tumor-bearing mice and cancer patients. However, the mechanisms by which ImC suppress immune responses in tumor-bearing mice have not been completely elucidated. In the present study, we investigated the function of splenic ImC freshly isolated from tumor-bearing mice and splenic ImC differentiated in vitro by GM-CSF. Freshly isolated splenic ImC were divided into two groups depending on Gr-1 expression, Gr-1 high (Gr-1hi) and intermediate (Gr-1int). Freshly isolated splenic Gr-1int ImC, but not Gr-1hi ImC, from tumor-bearing mice reduced production of IFN-gamma in CD8+ T cells, but neither splenic Gr-1int ImC nor Gr-1hi ImC isolated from naive mice did. Both Gr-1int and Gr-1hi ImC differentiated in vitro by GM-CSF inhibited production of IFN-gamma in both CD8+ and CD4+ T cells. In addition, the differentiated Gr-1int ImC, one-third of which were CD11c+F4/80+ cells, and their culture supernatants suppressed proliferative responses of T cells stimulated by CD3 ligation, but the differentiated Gr-1hi ImC and their culture supernatants did not. These results suggest that Gr-1int ImC are altered to immune-suppressive cells in tumor circumstances and that they are differentiated by GM-CSF progressively into CD11c+F4/80+ cells with further suppressive activity against T cells.

  2. Genetic variation in the immunosuppression pathway genes and breast cancer susceptibility: a pooled analysis of 42,510 cases and 40,577 controls from the Breast Cancer Association Consortium.

    PubMed

    Lei, Jieping; Rudolph, Anja; Moysich, Kirsten B; Behrens, Sabine; Goode, Ellen L; Bolla, Manjeet K; Dennis, Joe; Dunning, Alison M; Easton, Douglas F; Wang, Qin; Benitez, Javier; Hopper, John L; Southey, Melissa C; Schmidt, Marjanka K; Broeks, Annegien; Fasching, Peter A; Haeberle, Lothar; Peto, Julian; Dos-Santos-Silva, Isabel; Sawyer, Elinor J; Tomlinson, Ian; Burwinkel, Barbara; Marmé, Frederik; Guénel, Pascal; Truong, Thérèse; Bojesen, Stig E; Flyger, Henrik; Nielsen, Sune F; Nordestgaard, Børge G; González-Neira, Anna; Menéndez, Primitiva; Anton-Culver, Hoda; Neuhausen, Susan L; Brenner, Hermann; Arndt, Volker; Meindl, Alfons; Schmutzler, Rita K; Brauch, Hiltrud; Hamann, Ute; Nevanlinna, Heli; Fagerholm, Rainer; Dörk, Thilo; Bogdanova, Natalia V; Mannermaa, Arto; Hartikainen, Jaana M; Van Dijck, Laurien; Smeets, Ann; Flesch-Janys, Dieter; Eilber, Ursula; Radice, Paolo; Peterlongo, Paolo; Couch, Fergus J; Hallberg, Emily; Giles, Graham G; Milne, Roger L; Haiman, Christopher A; Schumacher, Fredrick; Simard, Jacques; Goldberg, Mark S; Kristensen, Vessela; Borresen-Dale, Anne-Lise; Zheng, Wei; Beeghly-Fadiel, Alicia; Winqvist, Robert; Grip, Mervi; Andrulis, Irene L; Glendon, Gord; García-Closas, Montserrat; Figueroa, Jonine; Czene, Kamila; Brand, Judith S; Darabi, Hatef; Eriksson, Mikael; Hall, Per; Li, Jingmei; Cox, Angela; Cross, Simon S; Pharoah, Paul D P; Shah, Mitul; Kabisch, Maria; Torres, Diana; Jakubowska, Anna; Lubinski, Jan; Ademuyiwa, Foluso; Ambrosone, Christine B; Swerdlow, Anthony; Jones, Michael; Chang-Claude, Jenny

    2016-01-01

    Immunosuppression plays a pivotal role in assisting tumors to evade immune destruction and promoting tumor development. We hypothesized that genetic variation in the immunosuppression pathway genes may be implicated in breast cancer tumorigenesis. We included 42,510 female breast cancer cases and 40,577 controls of European ancestry from 37 studies in the Breast Cancer Association Consortium (2015) with available genotype data for 3595 single nucleotide polymorphisms (SNPs) in 133 candidate genes. Associations between genotyped SNPs and overall breast cancer risk, and secondarily according to estrogen receptor (ER) status, were assessed using multiple logistic regression models. Gene-level associations were assessed based on principal component analysis. Gene expression analyses were conducted using RNA sequencing level 3 data from The Cancer Genome Atlas for 989 breast tumor samples and 113 matched normal tissue samples. SNP rs1905339 (A>G) in the STAT3 region was associated with an increased breast cancer risk (per allele odds ratio 1.05, 95 % confidence interval 1.03-1.08; p value = 1.4 × 10(-6)). The association did not differ significantly by ER status. On the gene level, in addition to TGFBR2 and CCND1, IL5 and GM-CSF showed the strongest associations with overall breast cancer risk (p value = 1.0 × 10(-3) and 7.0 × 10(-3), respectively). Furthermore, STAT3 and IL5 but not GM-CSF were differentially expressed between breast tumor tissue and normal tissue (p value = 2.5 × 10(-3), 4.5 × 10(-4) and 0.63, respectively). Our data provide evidence that the immunosuppression pathway genes STAT3, IL5, and GM-CSF may be novel susceptibility loci for breast cancer in women of European ancestry.

  3. MOUSE MODEL FOR PRE-CLINICAL STUDY OF HUMAN CANCER IMMUNOTHERAPY

    PubMed Central

    Ya, Zhiya; Hailemichael, Yared; Overwijk, Willem; Restifo, Nicholas

    2015-01-01

    This unit describes protocols for developing tumors in mice including subcutaneous growth, pulmonary metastases of B16 melanoma, and spontaneous melanoma in B-Raf V600E/PTEN deletion transgenic mouse models. Two immunization methods to prevent B16 tumor growth are described using B16.GM-CSF and recombinant vaccinia virus. A therapeutic approach is also included that uses adoptive transfer of tumor antigen-specific T cells. Methods including CTL induction, isolation, testing, and genetic modification of mouse T cells for adoptive transfer by using retrovirus expressing genes of interest are provided. Additional sections, including growing B16 melanoma, enumerating pulmonary metastases, tumor imaging technique and use of recombinant viruses for vaccination, are discussed together with safety concerns. PMID:25640991

  4. Size-partitioning of an urban aerosol to identify particle determinants involved in the proinflammatory response induced in airway epithelial cells

    PubMed Central

    Ramgolam, Kiran; Favez, Olivier; Cachier, Hélène; Gaudichet, Annie; Marano, Francelyne; Martinon, Laurent; Baeza-Squiban, Armelle

    2009-01-01

    Background The contribution of air particles in human cardio-respiratory diseases has been enlightened by several epidemiological studies. However the respective involvement of coarse, fine and ultrafine particles in health effects is still unclear. The aim of the present study is to determine which size fraction from a chemically characterized background aerosol has the most important short term biological effect and to decipher the determinants of such a behaviour. Results Ambient aerosols were collected at an urban background site in Paris using four 13-stage low pressure cascade impactors running in parallel (winter and summer 2005) in order to separate four size-classes (PM0.03–0.17 (defined here as ultrafine particles), PM0.17–1 (fine), PM1–2.5(intermediate) and PM2.5–10 (coarse)). Accordingly, their chemical composition and their pro-inflammatory potential on human airway epithelial cells were investigated. Considering isomass exposures (same particle concentrations for each size fractions) the pro-inflammatory response characterized by Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) release was found to decrease with aerosol size with no seasonal dependency. When cells were exposed to isovolume of particle suspensions in order to respect the particle proportions observed in ambient air, the GM-CSF release was maximal with the fine fraction. In presence of a recombinant endotoxin neutralizing protein, the GM-CSF release induced by particles is reduced for all size-fractions, with exception of the ultra-fine fraction which response is not modified. The different aerosol size-fractions were found to display important chemical differences related to the various contributing primary and secondary sources and aerosol age. The GM-CSF release was correlated to the organic component of the aerosols and especially its water soluble fraction. Finally, Cytochrome P450 1A1 activity that reflects PAH bioavailability varied as a function of the season

  5. Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

    PubMed Central

    Longhi, Silvia A.; Atienza, Augusto; Perez Prados, Graciela; Buying, Alcinette; Balouz, Virginia; Buscaglia, Carlos A.; Santos, Radleigh; Tasso, Laura M.; Bonato, Ricardo; Chiale, Pablo; Gómez, Karina A.

    2014-01-01

    Background Trypanosoma cruzi ribosomal P proteins, P2β and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with β1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals. Methodology/Principal findings We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2β, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2β or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells. Conclusions/Significance Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection. PMID

  6. Differentiation therapy in poor risk myeloid malignancies: Results of companion phase II studies.

    PubMed

    Norsworthy, Kelly J; Cho, Eunpi; Arora, Jyoti; Kowalski, Jeanne; Tsai, Hua-Ling; Warlick, Erica; Showel, Margaret; Pratz, Keith W; Sutherland, Lesley A; Gore, Steven D; Ferguson, Anna; Sakoian, Sarah; Greer, Jackie; Espinoza-Delgado, Igor; Jones, Richard J; Matsui, William H; Smith, B Douglas

    2016-10-01

    Pre-clinical data in non-M3 AML supports the use of differentiation therapy, but clinical activity has been limited. Myeloid growth factors can enhance anti-leukemic activity of differentiating agents in vitro. We conducted companion phase II trials investigating sargramostim (GM-CSF) 125μg/m(2)/day plus 1) bexarotene (BEX) 300mg/m(2)/day or 2) entinostat (ENT) 4-8mg/m(2)/week in patients with MDS or relapsed/refractory AML. Primary endpoints were response after at least two treatment cycles and toxicity. 26 patients enrolled on the BEX trial had a median of 2 prior treatments and 24 enrolled on the ENT trial had a median of 1. Of 13 response-evaluable patients treated with BEX, the best response noted was hematologic improvement in neutrophils (HI-N) seen in 4 (31%) patients; none achieved complete (CR) or partial remission (PR). Of 10 treated with ENT, there was 1 (10%) partial remission (PR) and 2 (20%) with HI-N. The secondary endpoint responses of HI-N with each combination were accompanied by a numerical increase in ANC (BEX: 524 to 931 cells/mm(3), p=0.096; ENT: 578 to 1 137 cells/mm(3), p=0.15) without increasing marrow blasts. Shared grade 3-4 non-hematologic toxicities included febrile neutropenia, bone pain, fatigue, and dyspnea. GM-CSF plus either BEX or ENT are well tolerated in resistant and refractory MDS and AML and showed modest clinical and biologic activity, most commonly HI-N.

  7. The granulocyte-macrophage colony-stimulating factor receptor: linking its structure to cell signaling and its role in disease.

    PubMed

    Hercus, Timothy R; Thomas, Daniel; Guthridge, Mark A; Ekert, Paul G; King-Scott, Jack; Parker, Michael W; Lopez, Angel F

    2009-08-13

    Already 20 years have passed since the cloning of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, the first member of the GM-CSF/interleukin (IL)-3/IL-5 family of hemopoietic cytokine receptors to be molecularly characterized. The intervening 2 decades have uncovered a plethora of biologic functions transduced by the GM-CSF receptor (pleiotropy) and revealed distinct signaling networks that couple the receptor to biologic outcomes. Unlike other hemopoietin receptors, the GM-CSF receptor has a significant nonredundant role in myeloid hematologic malignancies, macrophage-mediated acute and chronic inflammation, pulmonary homeostasis, and allergic disease. The molecular mechanisms underlying GM-CSF receptor activation have recently been revealed by the crystal structure of the GM-CSF receptor complexed to GM-CSF, which shows an unexpected higher order assembly. Emerging evidence also suggests the existence of intracellular signosomes that are recruited in a concentration-dependent fashion to selectively control cell survival, proliferation, and differentiation by GM-CSF. These findings begin to unravel the mystery of cytokine receptor pleiotropy and are likely to also apply to the related IL-3 and IL-5 receptors as well as other heterodimeric cytokine receptors. The new insights in GM-CSF receptor activation have clinical significance as the structural and signaling nuances can be harnessed for the development of new treatments for malignant and inflammatory diseases.

  8. Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization.

    PubMed

    Heinzelman, Pete; Carlson, Sharon J; Cox, George N

    2015-10-01

    Crohn's Disease (CD) afflicts over half a million Americans with an annual economic impact exceeding $10 billion. Granulocyte macrophage colony-stimulating factor (GM-CSF) can increase patient immune responses against intestinal microbes that promote CD and has been effective for some patients in clinical trials. We have made important progress toward developing GM-CSF variants that could be more effective CD therapeutics by virtue of being less prone to neutralization by the endogenous GM-CSF autoantibodies that are highly expressed in CD patients. Yeast display engineering revealed mutations that increase GM-CSF variant binding affinity by up to ∼3-fold toward both GM-CSF receptor alpha and beta subunits in surface plasmon resonance experiments. Increased binding affinity did not reduce GM-CSF half-maximum effective concentration (EC50) values in conventional in vitro human leukocyte proliferation assays. Affinity-enhancing mutations did, however, promote a 'refacing effect' that imparted all five evaluated GM-CSF variants with increased in vitro bioactivity in the presence of GM-CSF-neutralizing polyclonal antisera. The most improved variant, H15L/R23L, was 6-fold more active than wild-type GM-CSF. Incorporation of additional known affinity-increasing mutations could augment the refacing effect and concomitant bioactivity improvements described here. PMID:25855658

  9. Effect of space flight on cytokine production and other immunologic parameters of rhesus monkeys

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, G.; Davis, S.; Taylor, G. R.; Mandel, A. D.; Konstantinova, I. V.; Lesnyak, A.; Fuchs, B. B.; Peres, C.; Tkackzuk, J.; Schmitt, D. A.

    1996-01-01

    During a recent flight of a Russian satellite (Cosmos #2229), initial experiments examining the effects of space flight on immunologic responses of rhesus monkeys were performed to gain insight into the effect of space flight on resistance to infection. Experiments were performed on tissue samples taken from the monkeys before and immediately after flight. Additional samples were obtained approximately 1 month after flight for a postflight restraint study. Two types of experiments were carried out throughout this study. The first experiment determined the ability of leukocytes to produce interleukin-1 and to express interleukin-2 receptors. The second experiment examined the responsiveness of rhesus bone marrow cells to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Human reagents that cross-reacted with monkey tissue were utilized for the bulk of the studies. Results from both studies indicated that there were changes in immunologic function attributable to space flight. Interleukin-1 production and the expression of interleukin-2 receptors was decreased after space flight. Bone marrow cells from flight monkeys showed a significant decrease in their response to GM-CSF compared with the response of bone marrow cells from nonflight control monkeys. These results suggest that the rhesus monkey may be a useful surrogate for humans in future studies that examine the effect of space flight on immune response, particularly when conditions do not readily permit human study.

  10. Clinical Development of the E75 Vaccine in Breast Cancer.

    PubMed

    Clifton, Guy T; Gall, Victor; Peoples, George E; Mittendorf, Elizabeth A

    2016-04-01

    E75 is an immunogenic peptide derived from the human epidermal growth factor receptor 2 (HER2) protein. A large amount of preclinical work evaluated the immunogenicity of E75, after which phase I trials investigated using E75 mixed with an immunoadjuvant as a vaccine. Those studies showed the vaccine to be safe and capable of stimulating an antigen-specific immune response. Subsequent to that, our group conducted trials evaluating E75 + granulocyte macrophage colony-stimulating factor (GM-CSF) in the adjuvant setting. The studies enrolled node-positive and high-risk node-negative breast cancer patients, with the goal being to determine if vaccination could decrease the recurrence risk. The studies included 187 evaluable patients: 108 vaccinated ones and 79 controls. The 5-year disease-free survival for the vaccinated patients was 89.7% compared to 80.2% for the control patients, a 48% reduction in relative risk of recurrence. Based on these data, E75 + GM-CSF, now known as NeuVax™, is being evaluated in a phase III trial. In this article, we review preclinical data and results of the early-phase trials and provide an update on the ongoing phase III study. We also present additional strategies for employing the vaccine to be included as a component of combination immunotherapy as well as in the setting of ductal carcinoma in situ as an initial step towards primary prevention. PMID:27239173

  11. Characterization of Inflammatory Response in Acute-on-Chronic Liver Failure and Relationship with Prognosis

    PubMed Central

    Solé, Cristina; Solà, Elsa; Morales-Ruiz, Manuel; Fernàndez, Guerau; Huelin, Patricia; Graupera, Isabel; Moreira, Rebeca; de Prada, Gloria; Ariza, Xavier; Pose, Elisa; Fabrellas, Núria; Kalko, Susana G.; Jiménez, Wladimiro; Ginès, Pere

    2016-01-01

    ACLF is characterized by a systemic inflammatory response, but the cytokines involved in this process have not been well studied. The aim of this study was to characterize the systemic inflammatory response in patients with cirrhosis and ACLF and its relationship with prognosis. Fifty-five patients with cirrhosis, 26 with ACLF, were studied prospectively. Systemic inflammatory response was analyzed by measuring a large array of plasma cytokines by using a multiplex kit. A principal component analysis show noticeable differences between ACLF and decompensated cirrhosis without ACLF. Patients with ACLF had significant abnormal levels of 12 cytokines compared to those without ACLF, including: VCAM-1, VEGF-A, Fractalkine, MIP-1α, Eotaxin, IP-10, RANTES, GM-CSF, IL-1β, IL-2, ICAM-1, and MCP-1. Cytokines showing the most marked relationship with ACLF were VCAM-1 and VEGF-A (AUCROC 0.77; p = 0.001). There was a significant relationship between some of inflammatory mediators and 3-month mortality, particularly VCAM-1, ICAM-1, and GM-CSF (AUCROC>0.7; p < 0.05). Functional Enrichment Analysis showed that inflammatory markers differentially expressed in ACLF patients were enriched in leukocyte migration, particularly monocytes and macrophages, and chemotaxis pathways. In conclusion, ACLF is characterized by a marked inflammatory reaction with activation of mediators of adhesion and migration of leukocytes. The intensity of the inflammatory reaction correlates with prognosis. PMID:27578545

  12. Characterization of Inflammatory Response in Acute-on-Chronic Liver Failure and Relationship with Prognosis.

    PubMed

    Solé, Cristina; Solà, Elsa; Morales-Ruiz, Manuel; Fernàndez, Guerau; Huelin, Patricia; Graupera, Isabel; Moreira, Rebeca; de Prada, Gloria; Ariza, Xavier; Pose, Elisa; Fabrellas, Núria; Kalko, Susana G; Jiménez, Wladimiro; Ginès, Pere

    2016-01-01

    ACLF is characterized by a systemic inflammatory response, but the cytokines involved in this process have not been well studied. The aim of this study was to characterize the systemic inflammatory response in patients with cirrhosis and ACLF and its relationship with prognosis. Fifty-five patients with cirrhosis, 26 with ACLF, were studied prospectively. Systemic inflammatory response was analyzed by measuring a large array of plasma cytokines by using a multiplex kit. A principal component analysis show noticeable differences between ACLF and decompensated cirrhosis without ACLF. Patients with ACLF had significant abnormal levels of 12 cytokines compared to those without ACLF, including: VCAM-1, VEGF-A, Fractalkine, MIP-1α, Eotaxin, IP-10, RANTES, GM-CSF, IL-1β, IL-2, ICAM-1, and MCP-1. Cytokines showing the most marked relationship with ACLF were VCAM-1 and VEGF-A (AUCROC 0.77; p = 0.001). There was a significant relationship between some of inflammatory mediators and 3-month mortality, particularly VCAM-1, ICAM-1, and GM-CSF (AUCROC>0.7; p < 0.05). Functional Enrichment Analysis showed that inflammatory markers differentially expressed in ACLF patients were enriched in leukocyte migration, particularly monocytes and macrophages, and chemotaxis pathways. In conclusion, ACLF is characterized by a marked inflammatory reaction with activation of mediators of adhesion and migration of leukocytes. The intensity of the inflammatory reaction correlates with prognosis. PMID:27578545

  13. Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome

    PubMed Central

    Wood, Lauren W.; Cox, Nicole I.; Phelps, Cody A.; Lai, Shao-Chiang; Poddar, Arjun; Talbot, Conover; Mu, David

    2016-01-01

    Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1+ lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1+ lung cancer cells (designated EDM-TTF-1+) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1+ conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1+ lung adenocarcinomas. PMID:26912193

  14. Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome.

    PubMed

    Wood, Lauren W; Cox, Nicole I; Phelps, Cody A; Lai, Shao-Chiang; Poddar, Arjun; Talbot, Conover; Mu, David

    2016-02-25

    Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1(+) lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1(+) lung cancer cells (designated EDM-TTF-1(+)) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1(+) conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1(+) lung adenocarcinomas.

  15. Effects of hypergravity on immunologic function

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, G.; Koebel, D. A.; Davis, S.

    1995-01-01

    The purpose of this study was to compare the effects of hypergravity exposure (2g) with those of exposure to space flight in the Cosmos 2044 flight. To do so, rats were centrifuged continuously for 14 days. Two different experiments were carried out on tissue obtained from the centrifuged rats. In the first experiment, rat bone marrow cells were examined for their response to recombinant murine colony stimulating factor-granulocyte/monocyte (GM-CSF). In the second experiment, rat spleen and bone marrow cells were stained in with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and analyzed on a flow cytometer. The results of the studies indicated that bone marrow cells from centrifuged rats showed no significant change in response to GM-CSF as compared to bone marrow cells from control rats. Spleen cells from flown rats showed some statistically significant changes in leukocytes subset distribution, but no differences that appeared to be of biological significance. These results indicate that hypergravity did not greatly affect the same immunological parameters affected by space flight in the Cosmos 2044 mission.

  16. Effects of hypergravity on immunologic function

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, G.; Koebel, D. A.; Davis, S.

    1994-01-01

    The purpose of this study was to compare the effects of hypergravity exposure (2g) with those of exposure to space flight in the Cosmos 2044 flight. To do so, rats were centrifuged continuously for 14 days. Two different experiments were carried out on tissue obtained from the centrifuged rats. In the first experiment, rat bone marrow cells were examined for their response to recombinant murine colony stimulating factor-granulocyte/monocyte (GM-CSF). In the second experiment, rate spleen and bone marrow cells were stained in with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and analyzed on a flow cytometer. The results of the studies indicated that bone marrow cells from centrifuged rats showed no significant change in response to GM-CSF as compared to bone marrow cells from control rats. Spleen cells from flown rats showed some statistically significant changes in leukocytes subset distribution, but no differences that appeared to be of biological significance. These results indicate that hypergravity did not greatly affect the same immunological parameters affected by space flight in the Cosmos 2044 mission.

  17. Vaccination with Irradiated Tumor Cells Engineered to Secrete Murine Granulocyte-Macrophage Colony-Stimulating Factor Stimulates Potent, Specific, and Long-Lasting Anti-Tumor Immunity

    NASA Astrophysics Data System (ADS)

    Dranoff, Glenn; Jaffee, Elizabeth; Lazenby, Audrey; Golumbek, Paul; Levitsky, Hyam; Brose, Katja; Jackson, Valerie; Hamada, Hirofumi; Pardoll, Drew; Mulligan, Richard C.

    1993-04-01

    To compare the ability of different cytokines and other molecules to enhance the immunogenicity of tumor cells, we generated 10 retroviruses encoding potential immunomodulators and studied the vaccination properties of murine tumor cells transduced by the viruses. Using a B16 melanoma model, in which irradiated tumor cells alone do not stimulate significant anti-tumor immunity, we found that irradiated tumor cells expressing murine granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated potent, long-lasting, and specific anti-tumor immunity, requiring both CD4^+ and CD8^+ cells. Irradiated cells expressing interleukins 4 and 6 also stimulated detectable, but weaker, activity. In contrast to the B16 system, we found that in a number of other tumor models, the levels of anti-tumor immunity reported previously in cytokine gene transfer studies involving live, transduced cells could be achieved through the use of irradiated cells alone. Nevertheless, manipulation of the vaccine or challenge doses made it possible to demonstrate the activity of murine GM-CSF in those systems as well. Overall, our results have important implications for the clinical use of genetically modified tumor cells as therapeutic cancer vaccines.

  18. Injectable, Pore-Forming Hydrogels for In Vivo Enrichment of Immature Dendritic Cells.

    PubMed

    Verbeke, Catia S; Mooney, David J

    2015-12-01

    Biomaterials-based vaccines have emerged as a powerful method to evoke potent immune responses directly in vivo, without the need for ex vivo cell manipulation, and modulating dendritic cell (DC) responses in a noninflammatory context could enable the development of tolerogenic vaccines to treat autoimmunity. This study describes the development of a noninflammatory, injectable hydrogel system to locally enrich DCs in vivo without inducing their maturation or activation, as a first step toward this goal. Alginate hydrogels that form pores in situ are characterized and used as a physical scaffold for cell infiltration. These gels are also adapted to control the release of granulocyte-macrophage colony stimulating factor (GM-CSF), a potent inducer of DC recruitment and proliferation. In vivo, sustained release of GM-CSF from the pore-forming gels leads to the accumulation of millions of cells in the material. These cells are highly enriched in CD11b(+) CD11c(+) DCs, and further analysis of cell surface marker expression indicates these DCs are immature. This study demonstrates that a polymeric delivery system can mediate the accumulation of a high number and percentage of immature DCs, and may provide the basis for further development of materials-based, therapeutic vaccines.

  19. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity.

    PubMed Central

    Dranoff, G; Jaffee, E; Lazenby, A; Golumbek, P; Levitsky, H; Brose, K; Jackson, V; Hamada, H; Pardoll, D; Mulligan, R C

    1993-01-01

    To compare the ability of different cytokines and other molecules to enhance the immunogenicity of tumor cells, we generated 10 retroviruses encoding potential immunomodulators and studied the vaccination properties of murine tumor cells transduced by the viruses. Using a B16 melanoma model, in which irradiated tumor cells alone do not stimulate significant anti-tumor immunity, we found that irradiated tumor cells expressing murine granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated potent, long-lasting, and specific anti-tumor immunity, requiring both CD4+ and CD8+ cells. Irradiated cells expressing interleukins 4 and 6 also stimulated detectable, but weaker, activity. In contrast to the B16 system, we found that in a number of other tumor models, the levels of anti-tumor immunity reported previously in cytokine gene transfer studies involving live, transduced cells could be achieved through the use of irradiated cells alone. Nevertheless, manipulation of the vaccine or challenge doses made it possible to demonstrate the activity of murine GM-CSF in those systems as well. Overall, our results have important implications for the clinical use of genetically modified tumor cells as therapeutic cancer vaccines. PMID:8097319

  20. Differential expression of p38 MAPK α, β, γ, δ isoforms in nucleus pulposus modulates macrophage polarization in intervertebral disc degeneration

    PubMed Central

    Yang, Chen; Cao, Peng; Gao, Yang; Wu, Ming; Lin, Yun; Tian, Ye; Yuan, Wen

    2016-01-01

    P38MAPK mediates cytokine induced inflammation in nucleus pulposus (NP) cells and involves in multiple cellular processes which are related to intervertebral disc degeneration (IDD). The aim of this study was to investigate the expression, activation and function of p38 MAPK isoforms (α,β, γ and δ) in degenerative NP and the effect of p38 activation in NP cells on macrophage polarization. P38 α, β and δ isoforms are preferential expressed, whereas the p38γ isoform is absent in human NP tissue. LV-sh-p38α, sh-p38β transfection in NP cells significantly decreased the ADAMTS-4,-5, MMP-13,CCL3 expression and restored collagen-II and aggrecan expression upon IL-1β stimulation. As compared with p38α and p38β, p38δ exhibited an opposite effect on ADAMTS-4,-5, MMP-13 and aggrecan expression in NP cells. Furthermore, the production of GM-CSF and IFNγ which were trigged by p38α or p38β in NP cells induced macrophage polarization into M1 phenotype. Our finding indicates that p38 MAPK α, β and δ isoform are predominantly expressed and activated in IDD. P38 positive NP cells modulate macrophage polarization through the production of GM-CSF and IFNγ. Hence, Our study suggests that selectively targeting p38 isoforms could ameliorate the inflammation in IDD and regard IDD progression. PMID:26911458

  1. Differential induction of bone marrow macrophage proliferation by mycoplasmas involves granulocyte-macrophage colony-stimulating factor.

    PubMed Central

    Stuart, P M; Cassell, G H; Woodward, J G

    1990-01-01

    We have studied the ability of three different Mycoplasma species to induce proliferation of bone marrow-derived macrophages (BMM). We observed a significant mitogenic effect when BMM cells from BALB/c, DBA/2J, SJL, and C57BL/6 mice were incubated with membranes derived from Mycoplasma arginini or M. arthritidis but not when they were incubated with an equivalent amount of M. pulmonis membrane. We also determined that pretreatment of mycoplasma membrane preparations with papain eliminated the ability of these preparations to induce BMM proliferation. To determine whether these membrane fractions acted indirectly by stimulating the production of soluble factors known to stimulate proliferation of BMM cells, we performed blocking studies with antibodies directed against colony-stimulating factor 1 (CSF-1), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor. Our results indicate that antibodies directed against either CSF-1 or IL-3 failed to block mycoplasma-initiated proliferation of BMM cells. However, when anti-GM-CSF was added to proliferative cultures at the time of initiation, we saw a dose-dependent reduction of mycoplasma-initiated proliferation. We conclude that the ability of mycoplasma membranes to initiate the proliferation of BMM is not shared by all species of mycoplasma and that it involves the production of GM-CSF by an as yet undetermined cell. PMID:2228227

  2. Culture and Identification of Mouse Bone Marrow-Derived Dendritic Cells and Their Capability to Induce T Lymphocyte Proliferation

    PubMed Central

    Wang, Wenguang; Li, Jia; Wu, Kun; Azhati, Baihetiya; Rexiati, Mulati

    2016-01-01

    Background The aim of this study was to establish a culture method for mouse dendritic cells (DCs) in vitro and observe their morphology at different growth stages and their ability to induce the proliferation of T lymphocytes. Material/Methods Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were used in combination to induce differentiation of mouse bone marrow (BM) mononucleocytes into DCs. The derived DCs were then assessed for morphology, phenotype, and function. Results The mouse BM-derived mononucleocytes had altered cell morphology 3 days after induction by GM-CSF and IL-4 and grew into colonies. Typical dendrites appeared 8 days after induction. Many mature DCs were generated, with typical dendritic morphology observed under scanning electron microscopy. Expression levels of CD11c, a specific marker of BM-derived DCs, and of co-stimulatory molecules such as CD40, CD80, CD86, and MHC-II were elevated in the mature DCs. Furthermore, the mature DCs displayed a strong potency in stimulating the proliferation of syngenic or allogenic T lymphocytes. Conclusions Mouse BM-derived mononucleocytes cultured in vitro can produce a large number of DCs, as well as immature DCs, in high purity. The described in vitro culture method lays a foundation for further investigations of anti-tumor vaccines. PMID:26802068

  3. Inorganic arsenite alters macrophage generation from human peripheral blood monocytes.

    PubMed

    Sakurai, Teruaki; Ohta, Takami; Fujiwara, Kitao

    2005-03-01

    Inorganic arsenite has caused severe inflammatory chronic poisoning in humans through the consumption of contaminated well water. In this study, we examined the effects of arsenite at nanomolar concentrations on the in vitro differentiation of human macrophages from peripheral blood monocytes. While arsenite was found to induce cell death in a culture system containing macrophage colony stimulating factor (M-CSF), macrophages induced by granulocyte-macrophage CSF (GM-CSF) survived the treatment, but were morphologically, phenotypically, and functionally altered. In particular, arsenite-induced cells expressed higher levels of a major histocompatibility complex (MHC) class II antigen, HLA-DR, and CD14. They were more effective at inducing allogeneic or autologous T cell responses and responded more strongly to bacterial lipopolysaccharide (LPS) by inflammatory cytokine release as compared to cells induced by GM-CSF alone. On the other hand, arsenite-induced cells expressed lower levels of CD11b and CD54 and phagocytosed latex beads or zymosan particles less efficiently. We also demonstrated that the optimum amount of cellular reactive oxygen species (ROS) induced by nM arsenite might play an important role in this abnormal monocyte differentiation. This work may have implications in chronic arsenic poisoning because the total peripheral blood arsenic concentrations of these patients are at nM levels.

  4. Assessment of lymphocyte subpopulations and cytokine secretion in children exposed to arsenic.

    PubMed

    Soto-Peña, Gerson A; Luna, Ana L; Acosta-Saavedra, Leonor; Conde, Patricia; López-Carrillo, Lizbeth; Cebrián, Mariano E; Bastida, Mariana; Calderón-Aranda, Emma S; Vega, Libia

    2006-04-01

    Exposure of several human populations to arsenic has been associated with a high incidence of detrimental dermatological and carcinogenic effects. To date, studies examining the immunotoxic effects of arsenic in humans, and specifically in children, are lacking. Therefore, we evaluated several parameters of immunological status in a group of children exposed to arsenic through their drinking water. Peripheral blood mononuclear cells (PBMCs) of 90 children (6 to 10 years old) were collected. Proportions of lymphocyte subpopulations, PBMC mitogenic proliferative response, and urinary arsenic levels were evaluated. Increased urine arsenic levels were associated with a reduced proliferative response to phytohemaglutinin (PHA) stimulation (P=0.005), CD4 subpopulation proportion (P=0.092), CD4/CD8 ratio (P=0.056), and IL-2 secretion levels (P=0.003). Increased arsenic exposure was also associated with an increase in GM-CSF secretion by mononucleated cells (P=0.000). We did not observe changes in CD8, B, or NK cell proportions, nor did we observe changes in the secretion of IL-4, IL-10, or IFN-gamma by PHA-activated PBMCs. These data indicate that arsenic exposure could alter the activation processes of T cells, such that an immunosuppression status that favors opportunistic infections and carcinogenesis is produced together with increased GM-CSF secretion that may be associated with chronic inflammation.

  5. A Modular Enhancer Is Differentially Regulated by GATA and NFAT Elements That Direct Different Tissue-Specific Patterns of Nucleosome Positioning and Inducible Chromatin Remodeling▿

    PubMed Central

    Bert, Andrew G.; Johnson, Brett V.; Baxter, Euan W.; Cockerill, Peter N.

    2007-01-01

    We investigated alternate mechanisms employed by enhancers to position and remodel nucleosomes and activate tissue-specific genes in divergent cell types. We demonstrated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene enhancer is modular and recruits different sets of transcription factors in T cells and myeloid cells. The enhancer recruited distinct inducible tissue-specific enhanceosome-like complexes and directed nucleosomes to different positions in these cell types. In undifferentiated T cells, the enhancer was activated by inducible binding of two NFAT/AP-1 complexes which disrupted two specifically positioned nucleosomes (N1 and N2). In myeloid cells, the enhancer was remodeled by GATA factors which constitutively displaced an upstream nucleosome (N0) and cooperated with inducible AP-1 elements to activate transcription. In mast cells, which express both GATA-2 and NFAT, these two pathways combined to activate the enhancer and generate high-level gene expression. At least 5 kb of the GM-CSF locus was organized as an array of nucleosomes with fixed positions, but the enhancer adopted different nucleosome positions in T cells and mast cells. Furthermore, nucleosomes located between the enhancer and promoter were mobilized upon activation in an enhancer-dependent manner. These studies reveal that distinct tissue-specific mechanisms can be used either alternately or in combination to activate the same enhancer. PMID:17283044

  6. Eosinophil-specific deletion of IκBα in mice reveals a critical role of NF-κB–induced Bcl-xL for inhibition of apoptosis

    PubMed Central

    Schwartz, Christian; Willebrand, Ralf; Huber, Silke; Rupec, Rudolf A.; Wu, Davina; Locksley, Richard

    2015-01-01

    Eosinophils are associated with type 2 immune responses to allergens and helminths. They release various proinflammatory mediators and toxic proteins on activation and are therefore considered proinflammatory effector cells. Eosinophilia is promoted by the cytokines interleukin (IL)-3, IL-5, and granulocyte macrophage–colony-stimulating factor (GM-CSF) and can result from enhanced de novo production or reduced apoptosis. In this study, we show that only IL-5 induces differentiation of eosinophils from bone marrow precursors, whereas IL-5, GM-CSF, and to a lesser extent IL-3 promote survival of mature eosinophils. The receptors for these cytokines use the common β chain, which serves as the main signaling unit linked to signal transducer and activator of transcription 5, p38 mitogen-activated protein kinase, and nuclear factor (NF)-κB pathways. Inhibition of NF-κB induced apoptosis of in vitro cultured eosinophils. Selective deletion of IκBα in vivo resulted in enhanced expression of Bcl-xL and reduced apoptosis during helminth infection. Retroviral overexpression of Bcl-xL promoted survival, whereas pharmacologic inhibition of Bcl-xL in murine or human eosinophils induced rapid apoptosis. These results suggest that therapeutic strategies targeting Bcl-xL in eosinophils could improve health conditions in allergic inflammatory diseases. PMID:25862560

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

    PubMed

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

    2012-11-01

    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.

  8. Idiopathic Choroidal Neovascularization: Intraocular Inflammatory Cytokines and the Effect of Intravitreal Ranibizumab Treatment

    PubMed Central

    Yin, Houfa; Fang, Xiaoyun; Ma, Jian; Chen, Min; Yang, Yabo; Guo, Shenchao; Chen, Zhiqing; Su, Zhaoan; Feng, Lei; Ye, Panpan; Wu, Fang; Yin, Jinfu

    2016-01-01

    Idiopathic choroidal neovascularization (ICNV) is a disorder that primarily affecting patients younger than 50 years and can cause severe loss of vision. Choroidal abnormalities, especially choroidal inflammation, have been thought to be involved in the pathophysiology of ICNV. However, the exact pathogenesis of ICNV remains unclear. The aim of our study was investigate the levels of 27 inflammatory cytokines in the aqueous humor of eyes with ICNV, and to determine the effect of intravitreal injection of ranibizumab (IVR) on cytokine levels. Significantly higher levels of IL-2, IL-10, IL-15, IL-17, basic FGF, and GM-CSF were observed in patients with ICNV compared with controls. However, only IL-17 levels were significantly higher in patients with ICNV compared with controls after adjusting for axial length. Furthermore, there were significant correlations between the levels of IL-10, IL-17, GM-CSF, and VEGF and the lesion area. Significant changes in visual acuity and central retinal thickness were observed after IVR. Besides VEGF, IVR also significantly reduced the levels of IL-2, IL-10, basic FGF, and IL-12, however, the IL-6 levels were significantly increased. Our results suggest that there may be an involvement of IL-17-related inflammatory processes in the etiology of ICNV. PMID:27558944

  9. T(H)2 cytokines modulate the IL-9R expression on human neutrophils.

    PubMed

    Dragon, Stéphane; Takhar, Manrit Kaur; Shan, Lianyu; Hayglass, Kent T; Simons, F Estelle; Gounni, Abdelilah S

    2009-06-26

    Interleukin (IL)-9 is associated with key pathological features of asthma such as airway hyperresponsiveness, bronchoconstriction and mucus production. Inflammatory responses mediated by IL-9 rely on the expression of the IL-9R which has been reported on lung epithelial cells, T lymphocytes and recently on airway granulocyte infiltrates. In this study, we assessed the regulatory and constitutive cell surface expression of the IL-9Ralpha in unfractionated and purified human neutrophils from atopic asthmatics, atopic non-asthmatics and healthy normal controls. We demonstrate that T(H)2 cytokines (IL-4 or IL-13) and granulocyte macrophage-colony stimulating factor (GM-CSF) up-regulated mRNA and cell surface expression levels of the IL-9Ralpha in primary human and HL-60 differentiated neutrophils. Pharmacological inhibition of NF-kappaB did not affect T(H)2-mediated IL-9Ralpha expression in human neutrophils although IFN-gamma and IL-10 down-regulated IL-9Ralpha expression when co-incubated with IL-4, IL-13 or GM-CSF. Collectively, our results reveal a regulatory function for IFN-gamma and IL-10 on modulating the inducible IL-9Ralpha expression levels on peripheral blood neutrophils by T(H)2 cytokines. PMID:19401191

  10. Oncolytic and immunotherapeutic vaccinia induces antibody-mediated complement-dependent cancer cell lysis in humans.

    PubMed

    Kim, Mi Kyung; Breitbach, Caroline J; Moon, Anne; Heo, Jeong; Lee, Yu Kyoung; Cho, Mong; Lee, Jun Woo; Kim, Seong-Geun; Kang, Dae Hwan; Bell, John C; Park, Byeong Ho; Kirn, David H; Hwang, Tae-Ho

    2013-05-15

    Oncolytic viruses cause direct cytolysis and cancer-specific immunity in preclinical models. The goal of this study was to demonstrate induction of functional anticancer immunity that can lyse target cancer cells in humans. Pexa-Vec (pexastimogene devacirepvec; JX-594) is a targeted oncolytic and immunotherapeutic vaccinia virus engineered to express human granulocyte-macrophage colony-stimulating factor (GM-CSF). Pexa-Vec demonstrated replication, GM-CSF expression, and tumor responses in previous phase 1 trials. We now evaluated whether Pexa-Vec induced functional anticancer immunity both in the rabbit VX2 tumor model and in patients with diverse solid tumor types in phase 1. Antibody-mediated complement-dependent cancer cell cytotoxicity (CDC) was induced by intravenous Pexa-Vec in rabbits; transfer of serum from Pexa-Vec-treated animals to tumor-bearing animals resulted in tumor necrosis and improved survival. In patients with diverse tumor types treated on a phase 1 trial, CDC developed within 4 to 8 weeks in most patients; normal cells were resistant to the cytotoxic effects. T lymphocyte activation in patients was evidenced by antibody class switching. We determined that patients with the longest survival duration had the highest CDC activity, and identified candidate target tumor cell antigens. Thus, we demonstrated that Pexa-Vec induced polyclonal antibody-mediated CDC against multiple tumor antigens both in rabbits and in patients with diverse solid tumor types.

  11. Effect of Blood Component Coatings of Enosseal Implants on Proliferation and Synthetic Activity of Human Osteoblasts and Cytokine Production of Peripheral Blood Mononuclear Cells

    PubMed Central

    Hulejova, Hana; Bartova, Jirina; Riedel, Tomas; Pesakova, Vlasta

    2016-01-01

    The study monitored in vitro early response of connective tissue cells and immunocompetent cells to enosseal implant materials coated by different blood components (serum, activated plasma, and plasma/platelets) to evaluate human osteoblast proliferation and synthetic activity and inflammatory response presented as a cytokine profile of peripheral blood mononuclear cells (PBMCs) under conditions imitating the situation upon implantation. The cells were cultivated on coated Ti-plasma-sprayed (Ti-PS), Ti-etched (Ti-Etch), Ti-hydroxyapatite (Ti-HA), and ZrO2 surfaces. The plasma/platelets coating supported osteoblast proliferation only on osteoconductive Ti-HA and Ti-Etch whereas activated plasma enhanced proliferation on all surfaces. Differentiation (BAP) and IL-8 production remained unchanged or decreased irrespective of the coating and surface; only the serum and plasma/platelets-coated ZrO2 exhibited higher BAP and IL-8 expression. RANKL production increased on serum and activated plasma coatings. PBMCs produced especially cytokines playing role in inflammatory phase of wound healing, that is, IL-6, GRO-α, GRO, ENA-78, IL-8, GM-CSF, EGF, and MCP-1. Cytokine profiles were comparable for all tested surfaces; only ENA-78, IL-8, GM-CSF, and MCP-1 expression depended on materials and coatings. The activated plasma coating led to uniformed surfaces and represented a favorable treatment especially for bioinert Ti-PS and ZrO2 whereas all coatings had no distinctive effect on bioactive Ti-HA and Ti-Etch.

  12. Lysyl oxidase-like 2 (LOXL2) and E47 EMT factor: novel partners in E-cadherin repression and early metastasis colonization.

    PubMed

    Canesin, G; Cuevas, E P; Santos, V; López-Menéndez, C; Moreno-Bueno, G; Huang, Y; Csiszar, K; Portillo, F; Peinado, H; Lyden, D; Cano, A

    2015-02-19

    Epithelial-mesenchymal transition (EMT) has been associated with increased aggressiveness and acquisition of migratory properties providing tumor cells with the ability to invade into adjacent tissues. Downregulation of E-cadherin, a hallmark of EMT, is mediated by several transcription factors (EMT-TFs) that act also as EMT inducers, among them, Snail1 and the bHLH transcription factor E47. We previously described lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, as a Snail1 regulator and EMT inducer. Here we show that LOXL2 is also an E47-interacting partner and functionally collaborates in the repression of E-cadherin promoter. Loss and gain of function analyses combined with in vivo studies in syngeneic breast cancer models demonstrate the participation of LOXL2 and E47 in tumor growth and their requirement for lung metastasis. Furthermore, LOXL2 and E47 contribute to early steps of metastatic colonization by cell and noncell autonomous functions regulating the recruitment of bone marrow progenitor cells to the lungs and by direct transcriptional regulation of fibronectin and cytokines TNFα, ANG-1 and GM-CSF. Moreover, fibronectin and GM-CSF proved to be necessary for LOXL2/E47-mediated modulation of tumor growth and lung metastasis.

  13. T(H)2 cytokines modulate the IL-9R expression on human neutrophils.

    PubMed

    Dragon, Stéphane; Takhar, Manrit Kaur; Shan, Lianyu; Hayglass, Kent T; Simons, F Estelle; Gounni, Abdelilah S

    2009-06-26

    Interleukin (IL)-9 is associated with key pathological features of asthma such as airway hyperresponsiveness, bronchoconstriction and mucus production. Inflammatory responses mediated by IL-9 rely on the expression of the IL-9R which has been reported on lung epithelial cells, T lymphocytes and recently on airway granulocyte infiltrates. In this study, we assessed the regulatory and constitutive cell surface expression of the IL-9Ralpha in unfractionated and purified human neutrophils from atopic asthmatics, atopic non-asthmatics and healthy normal controls. We demonstrate that T(H)2 cytokines (IL-4 or IL-13) and granulocyte macrophage-colony stimulating factor (GM-CSF) up-regulated mRNA and cell surface expression levels of the IL-9Ralpha in primary human and HL-60 differentiated neutrophils. Pharmacological inhibition of NF-kappaB did not affect T(H)2-mediated IL-9Ralpha expression in human neutrophils although IFN-gamma and IL-10 down-regulated IL-9Ralpha expression when co-incubated with IL-4, IL-13 or GM-CSF. Collectively, our results reveal a regulatory function for IFN-gamma and IL-10 on modulating the inducible IL-9Ralpha expression levels on peripheral blood neutrophils by T(H)2 cytokines.

  14. Recombinant hybrid protein, Shiga toxin and granulocyte macrophage colony stimulating factor effectively induce apoptosis of colon cancer cells

    PubMed Central

    Roudkenar, Mehryar Habibi; Bouzari, Saeid; Kuwahara, Yoshikazu; Roushandeh, Amaneh Mohammadi; Oloomi, Mana; Fukumoto, Manabu

    2006-01-01

    AIM: To investigate the selective cytotoxic effect of constructed hybrid protein on cells expressing granulocyte macrophage colony stimulating factor (GM-CSF) receptor. METHODS: HepG2 (human hepatoma) and LS174T (colon carcinoma) were used in this study. The fused gene was induced with 0.02 % of arabinose for 4 h and the expressed protein was detected by Western blotting. The chimeric protein expressed in E.coli was checked for its cytotoxic activity on these cells and apoptosis was measured by comet assay and nuclear staining. RESULTS: The chimeric protein was found to be cytotoxic to the colon cancer cell line expressing GM-CSFRs, but not to HepG2 lacking these receptors. Maximum activity was observed at the concentration of 40 ng/mL after 24 h incubation. The IC50 was 20  ±  3.5 ng/mL. CONCLUSION: Selective cytotoxic effect of the hybrid protein on the colon cancer cell line expressing GM-CSF receptors (GM-CSFRs) receptor and apoptosis can be observed in this cell line. The hybrid protein can be considered as a therapeutic agent. PMID:16688822

  15. Lysyl oxidase-like 2 (LOXL2) and E47 EMT factor: novel partners in E-cadherin repression and early metastasis colonization.

    PubMed

    Canesin, G; Cuevas, E P; Santos, V; López-Menéndez, C; Moreno-Bueno, G; Huang, Y; Csiszar, K; Portillo, F; Peinado, H; Lyden, D; Cano, A

    2015-02-19

    Epithelial-mesenchymal transition (EMT) has been associated with increased aggressiveness and acquisition of migratory properties providing tumor cells with the ability to invade into adjacent tissues. Downregulation of E-cadherin, a hallmark of EMT, is mediated by several transcription factors (EMT-TFs) that act also as EMT inducers, among them, Snail1 and the bHLH transcription factor E47. We previously described lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, as a Snail1 regulator and EMT inducer. Here we show that LOXL2 is also an E47-interacting partner and functionally collaborates in the repression of E-cadherin promoter. Loss and gain of function analyses combined with in vivo studies in syngeneic breast cancer models demonstrate the participation of LOXL2 and E47 in tumor growth and their requirement for lung metastasis. Furthermore, LOXL2 and E47 contribute to early steps of metastatic colonization by cell and noncell autonomous functions regulating the recruitment of bone marrow progenitor cells to the lungs and by direct transcriptional regulation of fibronectin and cytokines TNFα, ANG-1 and GM-CSF. Moreover, fibronectin and GM-CSF proved to be necessary for LOXL2/E47-mediated modulation of tumor growth and lung metastasis. PMID:24632622

  16. The Drinking Effect of Hydrogen Water on Atopic Dermatitis Induced by Dermatophagoides farinae Allergen in NC/Nga Mice.

    PubMed

    Ignacio, Rosa Mistica C; Kwak, Hyun-Suk; Yun, Young-Uk; Sajo, Ma Easter Joy V; Yoon, Yang-Suk; Kim, Cheol-Su; Kim, Soo-Ki; Lee, Kyu-Jae

    2013-01-01

    Hydrogen water (HW) produced by electrolysis of water has characteristics of extremely low oxidation-reduction potential (ORP) value and high dissolved hydrogen (DH). It has been proved to have various beneficial effects including antioxidant and anti-inflammatory effects; however, HW effect on atopic dermatitis (AD), an inflammatory skin disorder, is poorly documented. In the present study, we examined the immunological effect of drinking HW on Dermatophagoides farinae-induced AD-like skin in NC/Nga mice. Mice were administered with HW and purified water (PW) for 25 days. We evaluated the serum concentration of pro-inflammatory (TNF- α ), Th1 (IFN- γ , IL-2, and IL-12p70), Th2 (IL-4, IL-5, and IL-10), and cytokine expressed by both subsets (GM-CSF) to assess their possible relationship to the severity of AD. The serum levels of cytokines such as IL-10, TNF- α , IL-12p70, and GM-CSF of mice administered with HW was significantly reduced as compared to PW group. The results suggest that HW affects allergic contact dermatitis through modulation of Th1 and Th2 responses in NC/Nga mice. This is the first note on the drinking effect of HW on AD, clinically implying a promising potential remedy for treatment of AD. PMID:24348704

  17. Interferon-γ enhances both the anti-bacterial and the pro-inflammatory response of human mast cells to Staphylococcus aureus.

    PubMed

    Swindle, Emily J; Brown, Jared M; Rådinger, Madeleine; DeLeo, Frank R; Metcalfe, Dean D

    2015-11-01

    Human mast cells (huMCs) are involved in both innate and adaptive immune responses where they release mediators including amines, reactive oxygen species (ROS), eicosanoids and cytokines. We have reported that interferon-γ (IFN-γ) enhances FcγR-dependent ROS production. The aim of this study was to extend these observations by investigating the effect of IFN-γ on the biological responses of huMCs to Staphylococcus aureus. We found that exposure of huMCs to S. aureus generated intracellular and extracellular ROS, which were enhanced in the presence of IFN-γ. IFN-γ also promoted bacteria killing, β-hexosaminidase release and eicosanoid production. Interferon-γ similarly increased expression of mRNAs encoding CCL1 to CCL4, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-α and CXCL8 in S. aureus-stimulated huMCs. The ability of IFN-γ to increase CXCL8 and GM-CSF protein levels was confirmed by ELISA. Fibronectin or a β1 integrin blocking antibody completely abrogated IFN-γ-dependent S. aureus binding and reduced S. aureus-dependent CXCL8 secretion. These data demonstrate that IFN-γ primes huMCs for enhanced anti-bacterial and pro-inflammatory responses to S. aureus, partially mediated by β1 integrin.

  18. A combination hydrogel microparticle-based vaccine prevents type 1 diabetes in non-obese diabetic mice

    PubMed Central

    Yoon, Young Mee; Lewis, Jamal S.; Carstens, Matthew R.; Campbell-Thompson, Martha; Wasserfall, Clive H.; Atkinson, Mark A.; Keselowsky, Benjamin G.

    2015-01-01

    Targeted delivery of self-antigens to the immune system in a mode that stimulates a tolerance-inducing pathway has proven difficult. To address this hurdle, we developed a vaccine based-approach comprised of two synthetic controlled-release biomaterials, poly(lactide-co-glycolide; PLGA) microparticles (MPs) encapsulating denatured insulin (key self-antigen in type 1 diabetes; T1D), and PuraMatrixTM peptide hydrogel containing granulocyte macrophage colony-stimulating factor (GM-CSF) and CpG ODN1826 (CpG), which were included as vaccine adjuvants to recruit and activate immune cells. Although CpG is normally considered pro-inflammatory, it also has anti-inflammatory effects, including enhancing IL-10 production. Three subcutaneous administrations of this hydrogel (GM-CSF/CpG)/insulin-MP vaccine protected 40% of NOD mice from T1D. In contrast, all control mice became diabetic. In vitro studies indicate CpG stimulation increased IL-10 production, as a potential mechanism. Multiple subcutaneous injections of the insulin containing formulation resulted in formation of granulomas, which resolved by 28 weeks. Histological analysis of these granulomas indicated infiltration of a diverse cadre of immune cells, with characteristics reminiscent of a tertiary lymphoid organ, suggesting the creation of a microenvironment to recruit and educate immune cells. These results demonstrate the feasibility of this injectable hydrogel/MP based vaccine system to prevent T1D. PMID:26279095

  19. A crude extract of Artocarpus integrifolia contains two lectins with distinct biological activities.

    PubMed

    de Miranda-Santos, I K; Delgado, M; Bonini, P V; Bunn-Moreno, M M; Campos-Neto, A

    1992-01-01

    The crude extract derived from seeds of Artocarpus integrifolia (jack fruit) contains two fractions with different biological activities for lymphocytes. One fraction is the D-galactose-binding lectin, jacalin, obtained by affinity purification on a D-galactose agarose column. The other, which is a component of the flow-through fraction (FT), is responsible for the mitogenic activity observed with human PBMC and murine spleen cells. In contrast, jacalin inhibits FT- and ConA-induced proliferative activity of human PMBC and murine spleen cells. This inhibition is not due to toxicity, because: (1) jacalin induces significant levels of IL-3/GM-CSF but not of IL-2 and/or IL-4 in murine spleen cells; (2) jacalin does not affect the capacity of these cells to secrete IL-2 or IL-4 as supernatants obtained from spleen cells sequentially stimulated with jacalin and ConA contain IL-2 and/or IL-4 as well as IL-3/GM-CSF. The ligand for the mitogen contained in the FT fraction is D-mannose as determined by sugar inhibition studies.

  20. T helper (Th) 1 and Th2 cytokine expression profile in dengue and malaria infection using magnetic bead-based bio-plex assay.

    PubMed

    Maneekan, Pannamas; Leaungwutiwong, Pornsawan; Misse, Dorothee; Luplertlop, Natthanej

    2013-01-01

    Dengue and malaria infections are two very common vector-borne diseases annually affecting millions of people around the world. Both diseases show a variety of clinical presentations, ranging from mild symptoms of dengue fever (DF) to severe dengue hemorrhagic fever (DHF) in dengue infection, and low and high parasitemia in malaria infection. T helper (Th)1 and Th2 cytokine expressions in mild and severe forms of dengue virus type-2 (DENV-2) and Plasmodium falciparum infection, were compared to normal human sera using high throughput magnetic bead-based Bio-Plex assay. A significant elevation of Th1 and Th2 cytokines expression [interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha] was detected in DENV-2 and P. falciparum malaria infections compared with normal controls (p < 0.05). DENV-2 infection showed a slight higher expression of Th1 and Th2 cytokines in DHF than DF, except for IL-13. In P. falciparum infection, high parasitemia showed a significantly higher expression of IL-4, IL-10, GM-CSF, and TNF-alpha (p < 0.05). Both DENV-2 and P. falciparum malaria infections manifested high IL-10 expression, greatest among the cytokines examined, and in the severe forms of infection. The results of this study should lead to a better understanding of pathogenesis of dengue infection and P. falciparum malaria.

  1. Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists

    PubMed Central

    Ang, Zhiwei; Er, Jun Zhi; Tan, Nguan Soon; Lu, Jinhua; Liou, Yih-Cherng; Grosse, Johannes; Ding, Jeak Ling

    2016-01-01

    Knockout mice studies implicate the mammalian short-chain fatty acid (SCFA) receptors, FFAR2 and FFAR3– in colitis, arthritis and asthma. However, the correlation with human biology is uncertain. Here, we detected FFAR2 and FFAR3 expression in human monocytes via immunohistochemistry. Upon treatment with acetate SCFA or FFAR2- and FFAR3-specific synthetic agonists, human monocytes displayed elevated p38 phosphorylation and attenuated C5, CCL1, CCL2, GM-CSF, IL-1α, IL-1β and ICAM-1 inflammatory cytokine expression. Acetate and FFAR2 agonist treatment also repressed Akt and ERK2 signalling. Surprisingly, mouse monocytes displayed a distinct response to acetate treatment, elevating GM-CSF, IL-1α, and IL-1β cytokine expression. This effect persisted in FFAR2/3-knockout mouse monocytes and was not reproduced by synthetic agonists, suggesting a FFAR2/3 independent mechanism in mice. Collectively, we show that SCFAs act via FFAR2/3 to modulate human monocyte inflammatory responses– a pathway that is absent in mouse monocytes. PMID:27667443

  2. Transcriptional and functional characterization of CD137L-dendritic cells identifies a novel dendritic cell phenotype

    PubMed Central

    Harfuddin, Zulkarnain; Dharmadhikari, Bhushan; Wong, Siew Cheng; Duan, Kaibo; Poidinger, Michael; Kwajah, Shaqireen; Schwarz, Herbert

    2016-01-01

    The importance of monocyte-derived dendritic cells (DCs) is evidenced by the fact that they are essential for the elimination of pathogens. Although in vitro DCs can be generated by treatment of monocytes with GM-CSF and IL-4, it is unknown what stimuli induce differentiation of DCs in vivo. CD137L-DCs are human monocyte-derived DC that are generated by CD137 ligand (CD137L) signaling. We demonstrate that the gene signature of in vitro generated CD137L-DCs is most similar to those of GM-CSF and IL-4-generated immature DCs and of macrophages. This is reminiscent of in vivo inflammatory DC which also have been reported to share gene signatures with monocyte-derived DCs and macrophages. Performing direct comparison of deposited human gene expression data with a CD137L-DC dataset revealed a significant enrichment of CD137L-DC signature genes in inflammatory in vivo DCs. In addition, surface marker expression and cytokine secretion by CD137L-DCs resemble closely those of inflammatory DCs. Further, CD137L-DCs express high levels of adhesion molecules, display strong attachment, and employ the adhesion molecule ALCAM to stimulate T cell proliferation. This study characterizes the gene expression profile of CD137L-DCs, and identifies significant similarities of CD137L-DCs with in vivo inflammatory monocyte-derived DCs and macrophages. PMID:27431276

  3. The Drinking Effect of Hydrogen Water on Atopic Dermatitis Induced by Dermatophagoides farinae Allergen in NC/Nga Mice

    PubMed Central

    Ignacio, Rosa Mistica C.; Kwak, Hyun-Suk; Yun, Young-Uk; Sajo, Ma. Easter Joy V.; Yoon, Yang-Suk; Kim, Cheol-Su; Kim, Soo-Ki

    2013-01-01

    Hydrogen water (HW) produced by electrolysis of water has characteristics of extremely low oxidation-reduction potential (ORP) value and high dissolved hydrogen (DH). It has been proved to have various beneficial effects including antioxidant and anti-inflammatory effects; however, HW effect on atopic dermatitis (AD), an inflammatory skin disorder, is poorly documented. In the present study, we examined the immunological effect of drinking HW on Dermatophagoides farinae-induced AD-like skin in NC/Nga mice. Mice were administered with HW and purified water (PW) for 25 days. We evaluated the serum concentration of pro-inflammatory (TNF-α), Th1 (IFN-γ, IL-2, and IL-12p70), Th2 (IL-4, IL-5, and IL-10), and cytokine expressed by both subsets (GM-CSF) to assess their possible relationship to the severity of AD. The serum levels of cytokines such as IL-10, TNF-α, IL-12p70, and GM-CSF of mice administered with HW was significantly reduced as compared to PW group. The results suggest that HW affects allergic contact dermatitis through modulation of Th1 and Th2 responses in NC/Nga mice. This is the first note on the drinking effect of HW on AD, clinically implying a promising potential remedy for treatment of AD. PMID:24348704

  4. A phase I clinical study of vaccination of melanoma patients with dendritic cells loaded with allogeneic apoptotic/necrotic melanoma cells. Analysis of toxicity and immune response to the vaccine and of IL-10 -1082 promoter genotype as predictor of disease progression

    PubMed Central

    von Euw, Erika M; Barrio, María M; Furman, David; Levy, Estrella M; Bianchini, Michele; Peguillet, Isabelle; Lantz, Olivier; Vellice, Alejandra; Kohan, Abraham; Chacón, Matías; Yee, Cassian; Wainstok, Rosa; Mordoh, José

    2008-01-01

    Background Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression. Methods PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 × 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC. Results Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 ± 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by

  5. Protective effect of recombinant murine granulocyte-macrophage colony-stimulating factor against Pseudomonas aeruginosa infection in leukocytopenic mice.

    PubMed Central

    Tanaka, T.; Okamura, S.; Okada, K.; Suga, A.; Shimono, N.; Ohhara, N.; Hirota, Y.; Sawae, Y.; Niho, Y.

    1989-01-01

    The effects of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) against Pseudomonas aeruginosa infection in ICR mice were investigated. Mice were treated with cyclophosphamide (CPA) and were then injected intraperitoneally with rmGM-CSF three times daily, beginning on the day after CPA treatment, for 7 days. The number of peripheral blood leukocytes in both CPA- and rmGM-CSF-treated mice and control CPA-treated mice reached a nadir on day 4, when P. aeruginosa was injected intraperitoneally. The administration of rmGM-CSF significantly increased the proportion of survivors among mice infected with a lethal dose of P. aeruginosa. This effect was further analyzed by monitoring sequential changes in leukocyte count and bacterial growth in various organs. The number of bacteria in the peritoneal cavities, peripheral blood samples, and livers of GM-CSF-treated mice decreased to an undetectable level after a transient increase, and the number was significantly lower than that in control mice. In GM-CSF-treated mice, the neutrophil levels in peripheral blood started to increase 5 days after CPA administration and were consistently higher than those in controls. Furthermore, the neutrophils in GM-CSF-treated mice were more mature morphologically. Thus, the prophylactic effect of rmGM-CSF against P. aeruginosa infection may result from a rapid recovery of myelopoiesis and a partial enhancement of mature neutrophil function. PMID:2656523

  6. Structure of the chromosomal gene for granulocyte-macrophage colony stimulating factor: comparison of the mouse and human genes.

    PubMed Central

    Miyatake, S; Otsuka, T; Yokota, T; Lee, F; Arai, K

    1985-01-01

    A cDNA clone that expresses granulocyte-macrophage colony stimulating factor (GM-CSF) activity in COS-7 cells has been isolated from a pcD library prepared from mRNA derived from concanavalin A-activated mouse helper T cell clones. Based on homology with the mouse GM-CSF cDNA sequence, the mouse GM-CSF gene was isolated. The human GM-CSF gene was also isolated based on homology with the human GM-CSF cDNA sequence. The nucleotide sequences determined for the genes and their flanking regions revealed that both the mouse and human GM-CSF genes are composed of three introns and four exons. The organization of the mouse and human GM-CSF genes are highly homologous and strong sequence homology between the two genes is found both in the coding and non-coding regions. A 'TATA'-like sequence was found 20-25 bp upstream from the transcription initiation site. In the 5'-flanking region, there is a highly homologous region extending 330 bp upstream of the putative TATA box. This sequence may play a role in regulation of expression of the GM-CSF gene. These structures are compared with those of different lymphokine genes and their regulatory regions. Images Fig. 2. Fig. 6. PMID:3876930

  7. Nonclinical safety of mavrilimumab, an anti-GMCSF receptor alpha monoclonal antibody, in cynomolgus monkeys: Relevance for human safety

    SciTech Connect

    Ryan, Patricia C.; Sleeman, Matthew A.; Rebelatto, Marlon; Wang, Bing; Lu, Hong; Chen, Xiaomin; Wu, Chi-Yuan; Hinrichs, Mary Jane; Roskos, Lorin; Towers, Heidi; McKeever, Kathleen; Dixit, Rakesh

    2014-09-01

    Mavrilimumab (CAM-3001) is an investigational human IgG4 monoclonal antibody (MAb) targeting GM-CSF receptor alpha which is currently being developed for the treatment of RA. GM-CSF plays a central role in the pathogenesis of rheumatoid arthritis (RA) through the activation, differentiation, and survival of macrophages and neutrophils. To support clinical development, the nonclinical safety of mavrilimumab was evaluated in several studies with cynomolgus monkeys as the pharmacologically relevant species. Comprehensive toxicity parameters were assessed in each study, and treatment duration ranged from 4 to 26 weeks. Mavrilimumab has an acceptable safety profile in monkeys with no changes in any parameters other than microscopic findings in lung. In several studies, minimal accumulation of foamy alveolar macrophages was observed. This finding was only seen in studies of at least 11 weeks duration, was reversible following a dose-free recovery period and was considered non-adverse. At higher dose levels (≥ 30 mg/kg/week), in a 26-week repeat-IV dose study, the presence of lung foreign material, cholesterol clefts, and granulomatous inflammation was also observed in a few animals and was considered adverse. The dose- and time-related accumulation of foamy macrophages in lung following exposure to mavrilimumab observed in several NHP studies was expected based upon the known role of GM-CSFRα signaling in the function of alveolar macrophages. Overall, a clean no-observed-adverse-effect-level (NOAEL) without any effects in lung was established and provided adequate clinical safety margins. In clinical studies in RA patients, mavrilimumab has demonstrated good clinical activity with adequate safety to support further clinical development. A Phase 2b study of mavrilimumab in subjects with RA is in progress. - Highlights: • Mavrilimumab is a MAB targeting GM-CSFRα being developed for RA therapy. • Mavrilimumab has an acceptable safety profile in cynomolgus monkeys.

  8. [A study on the activity of nitric oxide in alveolar macrophages from patients with lung cancer].

    PubMed

    Hu, C; Li, G; Wu, E

    1998-01-01

    Nitrite and nitrate (NO2-/NO2-) in the bronchus alveolar lavage fluid (BALF) and the supernatants of incubated alveolar macrophages (AMs) from patients with primary lung cancer were measured by copper-coated cadmium reduction and Griess method. Mrna expression of AM induced nitric oxide synthase (iNOS) were analyzed by RT-PCR. There was NO2-/NO2- in BALF either from lung cancer patients or from control subjects. When compared with control group and the nontumor-bearing lung, the level of NO2-/NO2-was lower in BALF from the tumor-bearing lung [5.18+/-1.1 vs 2.47+/-0.67nmol x mg protein-1 (P< 0.01); 4.65+/- 2.46 vs 2.47+/- 0.67nmol x mg protein-1(P< 0.01)]. We also found a lower level of NO2-/NO2- in the supernatants of incubated AMs from the lung of cancer patients than from control and nontumor-bearing lung [95.03+/- 21.76 vs 63.37+/- 17.58nmol (P< 0.01); 85.61+/- 16.70 vs 63.37+/- 17.58nmol (P< 0.05)]. No significant difference existed between the MRNA expression of AM iNOS in lung cancer patients (69%) and that of control subjects (91%). After the AMs were stimulated with granulocyte-macrophage colony stimulating factor (GM-CSF), the level of NO2-/NO2- in the supernatants was significantly increased (P< 0.01); while the mRNA expression of AM iNOS from patients with lung cancer resulted in an increase of 16.85+/- 7.58% vs 33.38+/- 8.21% of control group (P< 0.05). These observation suggest that some defects of antitumor function occur in the AMs at the tumor region. GM-CSF can stimulate AMs and thus potentiate their NO activity.

  9. Recovery from severe hematopoietic suppression using recombinant human granulocyte-macrophage colony-stimulating factor

    SciTech Connect

    Monroy, R.L.; Skelly, R.R.; Taylor, P.; Dubois, A.; Donahue, R.E.; MacVittie, T.J.

    1988-06-01

    The ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to enhance recovery of a radiation-suppressed hematopoietic system was evaluated in a nonuniform radiation exposure model using the rhesus monkey. Recombinant human GM-CSF treatment for 7 days after a lethal, nonuniform radiation exposure of 800 cGy was sufficient to enhance hematopoietic reconstitution, leading to an earlier recovery. Monkeys were treated with 72,000 U/kg/day of rhGM-CSF delivered continuously through an Alzet miniosmotic pump implanted subcutaneously on day 3. Treated monkeys demonstrated effective granulocyte and platelet levels in the peripheral blood, 4 and 7 days earlier, respectively, than control monkeys. Granulocyte-macrophage colony-forming unit (CFU-GM) activity in the bone marrow was monitored to evaluate the effect of rhGM-CSF on marrow recovery. Treatment with rhGM-CSF led to an early recovery of CFU-GM activity suggesting that rhGM-CSF acted on an earlier stem cell population to generate CFU-GM. Thus, the effect of rhGM-CSF on hematopoietic regeneration, granulocyte recovery, and platelet recovery are evaluated in this paper.

  10. Recovery from severe hematopoietic suppression using recombinant human granulocyte-macrophage colony-stimulating factor

    SciTech Connect

    Monroy, R.L.; Skelly, R.R.; Taylor, P.; Dubois, A.; Donahue, R.E.

    1988-01-01

    The ability of recombinant human granulocytemacrophage colony-stimulating factor (rhGM-CSF) to enhance recovery of a radiation-suppressed hematopoietic system was evaluated in a nonuniform radiation-exposure model using the rhesus monkey. Recombinant human GM-CSF treatment for 7 days after a lethal, nonuniform radiation exposure of 800 cGy was sufficient to enhance hematopoietic reconstitution, leading to an earlier recovery. Monkeys were treated with 72,000 U/kg/day of rhGm-CSF delivered continuously through an Alzet mini-osmotic pump implanted subcutaneously on day 3. Treated monkeys demonstrated effective granulocyte and platelet levels in the peripheral blood, 4 and 7 days earlier, respectively, than control monkeys. Granulocyte-macrophage colony-forming unit (CFU-GM) activity in the bone marrow was monitored to evaluate the effect of rhGM-CSF on marrow recovery. Treatment with rhGM-CSF led to an early recovery of CFU-GM activity suggesting that rhGM-CSF acted on an earlier stem cell population to generate CFU-GM. Thus, the effect of rhGM-CSF on hematopoietic regeneration, granulocyte recovery, and platelet recovery are evaluated.

  11. Synergistic action of the benzene metabolite hydroquinone on myelopoietic stimulating activity of granulocyte/macrophage colony-stimulating factor in vitro.

    PubMed Central

    Irons, R D; Stillman, W S; Colagiovanni, D B; Henry, V A

    1992-01-01

    The effects of in vitro pretreatment with benzene metabolites on colony-forming response of murine bone marrow cells stimulated with recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF) were examined. Pretreatment with hydroquinone (HQ) at concentrations ranging from picomolar to micromolar for 30 min resulted in a 1.5- to 4.6-fold enhancement in colonies formed in response to rGM-CSF that was due to an increase in granulocyte/macrophage colonies. The synergism equaled or exceeded that reported for the effects of interleukin 1, interleukin 3, or interleukin 6 with GM-CSF. Optimal enhancement was obtained with 1 microM HQ and was largely independent of the concentration of rGM-CSF. Pretreatment with other authentic benzene metabolites, phenol and catechol, and the putative metabolite trans, trans-muconaldehyde did not enhance growth factor response. Coadministration of phenol and HQ did not enhance the maximal rGM-CSF response obtained with HQ alone but shifted the optimal concentration to 100 pM. Synergism between HQ and rGM-CSF was observed with nonadherent bone marrow cells and lineage-depleted bone marrow cells, suggesting an intrinsic effect on recruitment of myeloid progenitor cells not normally responsive to rGM-CSF. Alterations in differentiation in a myeloid progenitor cell population may be of relevance in the pathogenesis of acute myelogenous leukemia secondary to drug or chemical exposure. PMID:1570288

  12. Synergistic action of the benzene metabolite hydroquinone on myelopoietic stimulating activity of granulocyte/macrophage colony-stimulating factor in vitro

    NASA Technical Reports Server (NTRS)

    Irons, R. D.; Stillman, W. S.; Colagiovanni, D. B.; Henry, V. A.; Clarkson, T. W. (Principal Investigator)

    1992-01-01

    The effects of in vitro pretreatment with benzene metabolites on colony-forming response of murine bone marrow cells stimulated with recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF) were examined. Pretreatment with hydroquinone (HQ) at concentrations ranging from picomolar to micromolar for 30 min resulted in a 1.5- to 4.6-fold enhancement in colonies formed in response to rGM-CSF that was due to an increase in granulocyte/macrophage colonies. The synergism equaled or exceeded that reported for the effects of interleukin 1, interleukin 3, or interleukin 6 with GM-CSF. Optimal enhancement was obtained with 1 microM HQ and was largely independent of the concentration of rGM-CSF. Pretreatment with other authentic benzene metabolites, phenol and catechol, and the putative metabolite trans, trans-muconaldehyde did not enhance growth factor response. Coadministration of phenol and HQ did not enhance the maximal rGM-CSF response obtained with HQ alone but shifted the optimal concentration to 100 pM. Synergism between HQ and rGM-CSF was observed with nonadherent bone marrow cells and lineage-depleted bone marrow cells, suggesting an intrinsic effect on recruitment of myeloid progenitor cells not normally responsive to rGM-CSF. Alterations in differentiation in a myeloid progenitor cell population may be of relevance in the pathogenesis of acute myelogenous leukemia secondary to drug or chemical exposure.

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

    SciTech Connect

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

    1990-11-15

    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 {sup 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-{sup 125}I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.

  14. Alteration of the functional effects of granulocyte-macrophage colony-stimulating factor on polymorphonuclear leukocytes by membrane-fluidizing agents.

    PubMed Central

    Buescher, E S; McIlheran, S M; Banks, S M; Vadhan-Raj, S

    1990-01-01

    Locomotion and oxidative metabolism of polymorphonuclear leukocytes from 15 patients receiving recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) were examined in vitro. At the end of each GM-CSF treatment course, polymorphonuclear leukocyte (PMN) chemotactic responses were suppressed and no enhancement of formyl-peptide-stimulated superoxide production was observed. The priming of PMN superoxide production normally seen after in vitro GM-CSF exposure was also blunted in these cells. By using control donor PMN, two membrane-fluidizing agents, pentoxifylline and butanol, were shown to normalize suppressed PMN chemotaxis caused by in vitro GM-CSF (1 nM) exposure. Pentoxifylline, but not butanol, also reversed the effects of in vitro GM-CSF on PMN superoxide production. When PMN obtained from six patients at the end of GM-CSF therapy were exposed to pentoxifylline in vitro, the chemotactic suppression typically observed was significantly improved. The data suggest that GM-CSF may affect PMN function via mechanisms involving membrane fluidity or cell deformability or both. PMID:2167293

  15. Mature dendritic cells generated from patient-derived peripheral blood monocytes in one-step culture using streptococcal preparation OK-432 exert an enhanced antigen-presenting capacity.

    PubMed

    Naito, Kei; Ueda, Yuji; Itoh, Tsuyoshi; Fuji, Nobuaki; Shimizu, Keiji; Yano, Yutaro; Yamamoto, Yoshiki; Imura, Kenichiro; Kohara, Junji; Iwamoto, Arihiro; Shiozaki, Atsushi; Tamai, Hidemasa; Shimizu, Takeshi; Mazda, Osam; Yamagishi, Hisakazu

    2006-06-01

    Dendritic cells (DCs) have been shown to be potent in inducing cytotoxic T cell (CTL) response leading to the efficient anti-tumor effect in active immunotherapy. Myeloid DCs are conventionally generated from human peripheral blood monocytes in the presence of interleukin (IL)-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Streptococcal preparation OK-432, which is known to be a multiple cytokine inducer, has been extensively studied as to its maturation effects on immature DCs using an in vitro culture system. The purpose of this study was to examine whether it could be possible to generate mature DCs directly from peripheral monocytes using OK-432. We specifically focused on the possibility that recombinant cytokines, which are considered to be essential for in vitro DC generation, could be substituted by OK-432. Human peripheral monocytes, which were obtained from patients with advanced cancer, were cultured with IL-4 and OK-432 for 7 days. Cultured cells were compared with DCs generated in the presence of IL-4 and GM-CSF with or without OK-432 with regard to the surface phenotype as well as the antigen-presenting capacity. As a result, the culture of monocytes in the presence of IL-4 followed by the addition of OK-432 on day 4 (IL-4/OK-DC) induced cells with a fully mature DC phenotype. Functional assays also demonstrated that IL-4/OK-DCs had a strong antigen-presenting capacity determined by their enhanced antigen-specific CTL response and exerted a Th1-type T cell response which is critical for the induction of anti-tumor response. In conclusion, human peripheral blood monocytes cultured in the presence of IL-4 and OK-432 without exogenous GM-CSF demonstrated a fully mature DC phenotype and strong antigen-presenting capacity. This one-step culture protocol allows us to generate fully mature DCs directly from monocytes in 7 days and thus, this protocol can be applicable for DC-based anti-tumor immunotherapy.

  16. The physiology of fetal membrane weakening and rupture: Insights gained from the determination of physical properties revisited.

    PubMed

    Kumar, Deepak; Moore, Robert M; Mercer, Brian M; Mansour, Joseph M; Redline, Raymond W; Moore, John J

    2016-06-01

    Rupture of the fetal membranes (FM) is precipitated by stretch forces acting upon biochemically mediated, pre-weakened tissue. Term FM develop a para-cervical weak zone, characterized by collagen remodeling and apoptosis, within which FM rupture is thought to initiate. Preterm FM also have a weak region but are stronger overall than term FM. Inflammation/infection and decidual bleeding/abruption are strongly associated with preterm premature FM rupture (pPROM), but the specific mechanisms causing FM weakening-rupture in pPROM are unknown. There are no animal models for study of FM weakening and rupture. Over a decade ago we developed equipment and methodology to test human FM strength and incorporated it into a FM explant system to create an in-vitro human FM weakening model system. Within this model TNF (modeling inflammation) and Thrombin (modeling bleeding) both weaken human FM with concomitant up regulation of MMP9 and cellular apoptosis, mimicking the characteristics of the spontaneous FM rupture site. The model has been enhanced so that test agents can be applied directionally to the choriodecidual side of the FM explant consistent with the in-vivo situation. With this enhanced system we have demonstrated that the pathways involving inflammation/TNF and bleeding/Thrombin induced FM weakening overlap. Furthermore GM-CSF production was demonstrated to be a critical common intermediate step in both the TNF and the Thrombin induced FM weakening pathways. This model system has also been used to test potential inhibitors of FM weakening and therefore pPROM. The dietary supplement α-lipoic acid and progestogens (P4, MPA and 17α-hydroxyprogesterone) have been shown to inhibit both TNF and Thrombin induced FM weakening. The progestogens act at multiple points by inhibiting both GM-CSF production and GM-CSF action. The use of a combined biomechanical/biochemical in-vitro human FM weakening model system has allowed the pathways of fetal membrane weakening to be

  17. Human casein alpha s1 (CSN1S1) skews in vitro differentiation of monocytes towards macrophages

    PubMed Central

    2013-01-01

    Background The milk-derived protein human Casein alpha s1 (CSN1S1) has recently been detected in blood cells and was shown to possess proinflammatory properties. In the present study, we investigated the effect of CSN1S1 on the differentiation of monocytes. Methods Primary human monocytes were stimulated with recombinant CSN1S1 and compared to cells stimulated with GM-CSF/IL-4 or M-CSF/IFNγ. Morphological changes were assessed by microscopy and quantification of surface markers of differentiation by FACS analysis. Phagocytic activity of CSN1S1 stimulated cells was measured by quantification of zymosan labeled particle uptake. The role of mitogen activated protein kinases for CSN1S1-induced differentiation of monocytes and proinflammatory cytokine expression was assessed by supplementation of specific inhibitors. Results CSN1S1 at a concentration of 10 μg/ml resulted in morphological changes (irregular shape, pseudopodia) and aggregation of cells, comparable to changes observed in M-CSF/IFNγ differentiated macrophages. Surface marker expression was altered after 24 h with an upregulation of CD14 (mean 2.5 fold) and CD64 (1.9 fold) in CSN1S1 stimulated cells. CSN1S1 treated cells showed a characteristic surface marker pattern for macrophages after 120 h of incubation (CD14high, CD64high, CD83low, CD1alow) comparable to changes observed in M-CSF/IFNγ treated monocytes. Furthermore, phagocytic activity was increased 1.4 and 1.9 fold following stimulation with 10 μg/ml CSN1S1 after 24 and 48 h, respectively. Early GM-CSF, but not GM-CSF/IL-4 induced differentiation of monocytes towards dendritic cells (DC) was inhibited by addition of CSN1S1. Finally, CSN1S1 induced upregulation of CD14 was impeded by inhibition of ERK1/2, while inhibition of the mitogen activated protein kinases JNK and p38 did not influence cellular differentiation. However, JNK and p38 inhibitors impeded CSN1S1 induced secretion of the proinflammatory cytokines IL-1b or IL-6. Conclusions CSN1S1

  18. CpG oligonucleotides enhance the tumor antigen-specific immune response of a granulocyte macrophage colony-stimulating factor-based vaccine strategy in neuroblastoma.

    PubMed

    Sandler, Anthony D; Chihara, Hiroshi; Kobayashi, Gen; Zhu, Xiaoyan; Miller, Michal A; Scott, David L; Krieg, Arthur M

    2003-01-15

    Granulocyte macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cells form the basis of many immunotherapeutic strategies. We tested whether combining this approach with T-helper 1 (Th-1)-like immunostimulatory CpG oligodeoxynucleotides (CpG ODNs) would improve therapeutic efficacy in an established model of murine neuroblastoma. The weakly immunogenic Neuro-2a cell line was used in syngeneic A/J mice. CpG 1826 was tested for its antitumor effect alone and as an adjuvant to Neuro-2a cells retrovirally transduced to express murine GM-CSF (GM/Neuro-2a). Three days after wild-type (WT) tumor cell inoculation, mice in different groups were s.c. vaccinated in the opposite leg with combinations of WT neuro2a, irradiated (15 Gy) WT or GM/Neuro-2a transfectants with or without CpG 1826 (200 micro g). To test for the induction of memory responses, mice that rejected their tumor were rechallenged with WT Neuro-2a (1 x 10(6)) 7 weeks after vaccination. All of the mice in the control (unvaccinated) group died within 3 weeks after Neuro-2a inoculation. Most of the vaccinated groups had only minimal-to-modest antitumor responses, and the mice succumbed to tumor. Tumor growth was remarkably inhibited in the group of mice that received irradiated GM/Neuro-2a plus CpG and four (50%) of eight mice in this group survived tumor free. Tumor-free mice were resistant to further WT tumor cell challenge, indicating a memory response. Mechanistic studies showed that CpG alone induced a favorable Th-1-like cytokine immune response and vaccine-induced tumor cell killing was dependent on both CD4 and CD8 T cells that killed tumor cell targets by apoptosis. These results demonstrate that CpG ODNs enhanced the antitumor effect of irradiated GM-CSF secreting Neuro-2a cells. This vaccine strategy elicits a potent tumor antigen-specific immune response against established murine neuroblastoma and generates systemic neuroblastoma-specific immunity.

  19. Human adipose tissue-resident monocytes exhibit an endothelial-like phenotype and display angiogenic properties

    PubMed Central

    2014-01-01

    Introduction Adipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth. However, endothelial progenitor cells contribute minimally to neovascularization. Because myeloid cells have proven to be angiogenic, and monocytes accumulate in expanding adipose tissue, they might contribute to vascularization. Methods The stromal vascular fraction (SVF) cells from human adipose tissue were magnetically separated according to CD45 or CD14 expression. Adipose-derived mesenchymal stromal cells (MSCs) were obtained from SVF CD45- cells. CD14+ monocytes were isolated from peripheral blood (PB) mononuclear cells and then cultured with SVF-derived MSCs. Freshly isolated or cultured cells were characterized with flow cytometry; the conditioned media were analyzed for the angiogenic growth factors, angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) with Luminex Technology; their angiogenic capacity was determined in an in vivo gelatinous protein mixture (Matrigel) plug angiogenesis assay. Results CD45+ hematopoietic cells within the SVF contain CD14+ cells that co-express the CD34 progenitor marker and the endothelial cell antigens VEGF receptor 2 (VEGFR2/KDR), VEGFR1/Flt1, and Tie2. Co-culture experiments showed that SVF-derived MSCs promoted the acquisition of KDR and Tie-2 in PB monocytes. MSCs secreted significant amounts of Ang-2 and HGF, but minimal amounts of bFGF, G-CSF, or GM-CSF, whereas the opposite was observed for SVF CD14+ cells. Additionally, SVF CD14+ cells secreted significantly higher levels of VEGF and bFGF than did MSCs. Culture supernatants of PB monocytes cultured with MSCs contained significantly higher concentrations of VEGF, HGF, G-CSF, and GM-CSF than did the supernatants from cultures without MSCs

  20. Interleukin 18 inhibits osteoclast formation via T cell production of granulocyte macrophage colony-stimulating factor.

    PubMed Central

    Horwood, N J; Udagawa, N; Elliott, J; Grail, D; Okamura, H; Kurimoto, M; Dunn, A R; Martin, T; Gillespie, M T

    1998-01-01

    IL-18 inhibits osteoclast (OCL) formation in vitro independent of IFN-gamma production, and this was abolished by the addition of neutralizing antibodies to GM-CSF. We now establish that IL-18 was unable to inhibit OCL formation in cocultures using GM-CSF-deficient mice (GM-CSF -/-). Reciprocal cocultures using either wild-type osteoblasts with GM-CSF -/- spleen cells or GM-CSF -/- osteoblasts with wild-type spleen cells were examined. Wild-type spleen cells were required to elicit a response to IL-18 indicating that cells of splenic origin were the IL-18 target. As T cells comprise a large proportion of the spleen cell population, the role of T cells in osteoclastogenesis was examined. Total T cells were removed and repleted in various combinations. Addition of wild-type T cells to a GM-CSF -/- coculture restored IL-18 inhibition of osteoclastogenesis. Major subsets of T cells, CD4+ and CD8+, were also individually depleted. Addition of either CD4+ or CD8+ wild-type T cells restored IL-18 action in a GM-CSF -/- background, while IL-18 was ineffective when either CD4+ or CD8+ GM-CSF -/- T cells were added to a wild-type coculture. These results highlight the involvement of T cells in IL-18-induced OCL inhibition and provide evidence for a new OCL inhibitory pathway whereby IL-18 inhibits OCL formation due to action upon T cells promoting the release of GM-CSF, which in turn acts upon OCL precursors. PMID:9449693

  1. Activation of silenced cytokine gene promoters by the synergistic effect of TBP-TALE and VP64-TALE activators.

    PubMed

    Anthony, Kim; More, Abhijit; Zhang, Xiaoliu

    2014-01-01

    Recent work has shown that the combinatorial use of multiple TALE activators can selectively activate certain cellular genes in inaccessible chromatin regions. In this study, we aimed to interrogate the activation potential of TALEs upon transcriptionally silenced immune genes in the context of non-immune cells. We designed a unique strategy, in which a single TALE fused to the TATA-box binding protein (TBP-TALE) is coupled with multiple VP64-TALE activators. We found that our strategy is significantly more potent than multiple TALE activators alone in activating expression of IL-2 and GM-CSF in diverse cell origins in which both genes are otherwise completely silenced. Chromatin analysis revealed that the gene activation was due in part to displacement of a distinctly positioned nucleosome. These studies provide a novel epigenetic mechanism for artificial gene induction and have important implications for targeted cancer immunotherapy, DNA vaccine development, as well as rational design of TALE activators.

  2. Prediagnostic serum levels of inflammatory biomarkers are correlated with future development of lung and esophageal cancer.

    PubMed

    Keeley, Brieze R; Islami, Farhad; Pourshams, Akram; Poustchi, Hossein; Pak, Jamie S; Brennan, Paul; Khademi, Hooman; Genden, Eric M; Abnet, Christian C; Dawsey, Sanford M; Boffetta, Paolo; Malekzadeh, Reza; Sikora, Andrew G

    2014-09-01

    This study tests the hypothesis that prediagnostic serum levels of 20 cancer-associated inflammatory biomarkers correlate directly with future development of head and neck, esophageal, and lung cancers in a high-risk prospective cohort. This is a nested case-control pilot study of subjects enrolled in the Golestan Cohort Study, an ongoing epidemiologic project assessing cancer trends in Golestan, Iran. We measured a panel of 20 21 cytokines, chemokines, and inflammatory molecules using Luminex technology in serum samples collected 2 or more years before cancer diagnosis in 78 aerodigestive cancer cases and 81 controls. Data was analyzed using Wilcoxon rank sum test, odds ratios, receiver operating characteristic areas of discrimination, and multivariate analysis. Biomarkers were profoundly and globally elevated in future esophageal and lung cancer patients compared to controls. Odds ratios were significant for association between several biomarkers and future development of esophageal cancer, including interleukin-1Rα (IL-1Ra; 35.9), interferon α2 (IFN-a2; 34.0), fibroblast growth factor-2 (FGF-2; 17.4), and granulocyte/macrophage colony-stimulating factor (GM-CSF; 17.4). The same pattern was observed among future lung cancer cases for G-CSF (27.7), GM-CSF (13.3), and tumor necrosis factor-α (TNF-a; 8.6). By contrast, the majority of biomarkers studied showed no significant correlation with future head and neck cancer development. This study provides the first direct evidence that multiple inflammatory biomarkers are coordinately elevated in future lung and esophageal cancer patients 2 or more years before cancer diagnosis. PMID:25040886

  3. Prediagnostic serum levels of inflammatory biomarkers are correlated with future development of lung and esophageal cancer

    PubMed Central

    Keeley, Brieze R; Islami, Farhad; Pourshams, Akram; Poustchi, Hossein; Pak, Jamie S; Brennan, Paul; Khademi, Hooman; Genden, Eric M; Abnet, Christian C; Dawsey, Sanford M; Boffetta, Paolo; Malekzadeh, Reza; Sikora, Andrew G

    2014-01-01

    This study tests the hypothesis that prediagnostic serum levels of 20 cancer-associated inflammatory biomarkers correlate directly with future development of head and neck, esophageal, and lung cancers in a high-risk prospective cohort. This is a nested case–control pilot study of subjects enrolled in the Golestan Cohort Study, an ongoing epidemiologic project assessing cancer trends in Golestan, Iran. We measured a panel of 20 21cytokines, chemokines, and inflammatory molecules using Luminex technology in serum samples collected 2 or more years before cancer diagnosis in 78 aerodigestive cancer cases and 81 controls. Data was analyzed using Wilcoxon rank sum test, odds ratios, receiver operating characteristic areas of discrimination, and multivariate analysis. Biomarkers were profoundly and globally elevated in future esophageal and lung cancer patients compared to controls. Odds ratios were significant for association between several biomarkers and future development of esophageal cancer, including interleukin-1Rα (IL-1Ra; 35.9), interferon α2 (IFN-a2; 34.0), fibroblast growth factor-2 (FGF-2; 17.4), and granulocyte/macrophage colony-stimulating factor (GM-CSF; 17.4). The same pattern was observed among future lung cancer cases for G-CSF (27.7), GM-CSF (13.3), and tumor necrosis factor-α (TNF-a; 8.6). By contrast, the majority of biomarkers studied showed no significant correlation with future head and neck cancer development. This study provides the first direct evidence that multiple inflammatory biomarkers are coordinately elevated in future lung and esophageal cancer patients 2 or more years before cancer diagnosis. PMID:25040886

  4. New immunotherapeutic strategies for the treatment of neuroblastoma.

    PubMed

    Croce, Michela; Corrias, Maria Valeria; Rigo, Valentina; Ferrini, Silvano

    2015-01-01

    The prognosis of high-risk neuroblastoma (NB) is still poor, in spite of aggressive multimodal treatment. Recently, adjuvant immunotherapy with anti-GD2 antibodies combined with IL-2 or GM-CSF has been shown to improve survival. Several other immunotherapy strategies proved efficacy in preclinical models of NB, including different types of vaccines, adoptive cell therapies and combined approaches. The remarkable differences in the immunobiology of syngeneic models and human NB may, at least in part, limit the translation of preclinical therapies to a clinical setting. Nonetheless, several preliminary evidences suggest that new antibodies, cancer vaccines and adoptive transfer of lymphocytes, genetically engineered to acquire NB specificity, may result in clinical benefit, and clinical studies are currently ongoing.

  5. A Multi Targeting Conditionally Replicating Adenovirus Displays Enhanced Oncolysis while Maintaining Expression of Immunotherapeutic Agents.

    PubMed

    Dobbins, G Clement; Ugai, Hideyo; Curiel, David T; Gillespie, G Yancey

    2015-01-01

    Studies have demonstrated that oncolytic adenoviruses based on a 24 base pair deletion in the viral E1A gene (D24) may be promising therapeutics for treating a number of cancer types. In order to increase the therapeutic potential of these oncolytic viruses, a novel conditionally replicating adenovirus targeting multiple receptors upregulated on tumors was generated by incorporating an Ad5/3 fiber with a carboxyl terminus RGD ligand. The virus displayed full cytopathic effect in all tumor lines assayed at low titers with improved cytotoxicity over Ad5-RGD D24, Ad5/3 D24 and an HSV oncolytic virus. The virus was then engineered to deliver immunotherapeutic agents such as GM-CSF while maintaining enhanced heterogenic oncolysis. PMID:26689910

  6. Hepatocellular carcinoma from an immunological perspective

    PubMed Central

    Greten, Tim F.; Duffy, Austin G.; Korangy, Firouzeh

    2013-01-01

    Hepatocellular carcinoma is the 3rd most common cancer worldwide. It is an inflammation-associated cancer. Multiple investigators have demonstrated that analysis of the tumor microenvironment may be used to predict patient outcome indicating the importance of local immune responses in this disease. In contrast to other types of cancer, in which surgery, radiation and systemic cytotoxic chemotherapies dominate the treatment options, in HCC loco-regional treatments are widely applied. Such treatments induce rapid tumor cell death and anti-tumor immune responses, which may favor or impair patients’ outcome. Recent immunotherapy studies demonstrating promising results include trials evaluating intra-tumoral injection of an oncolytic virus expressing GM-CSF, glypican-3 targeting treatments and anti-CTLA4 treatment. While some of these novel approaches may provide benefit as single agents, there is a clear opportunity in HCC to evaluate these in combination with the standard modalities to more effectively harness the immune response. PMID:24030702

  7. A Multi Targeting Conditionally Replicating Adenovirus Displays Enhanced Oncolysis while Maintaining Expression of Immunotherapeutic Agents

    PubMed Central

    Dobbins, G. Clement; Ugai, Hideyo; Curiel, David T.; Gillespie, G. Yancey

    2015-01-01

    Studies have demonstrated that oncolytic adenoviruses based on a 24 base pair deletion in the viral E1A gene (D24) may be promising therapeutics for treating a number of cancer types. In order to increase the therapeutic potential of these oncolytic viruses, a novel conditionally replicating adenovirus targeting multiple receptors upregulated on tumors was generated by incorporating an Ad5/3 fiber with a carboxyl terminus RGD ligand. The virus displayed full cytopathic effect in all tumor lines assayed at low titers with improved cytotoxicity over Ad5-RGD D24, Ad5/3 D24 and an HSV oncolytic virus. The virus was then engineered to deliver immunotherapeutic agents such as GM-CSF while maintaining enhanced heterogenic oncolysis. PMID:26689910

  8. Signaling profiling at the single-cell level identifies a distinct signaling signature in murine hematopoietic stem cells

    PubMed Central

    Du, Juan; Wang, Jinyong; Kong, Guangyao; Jiang, Jing; Zhang, Jingfang; Liu, Yangang; Tong, Wei; Zhang, Jing

    2012-01-01

    Hematopoietic stem cell (HSC) function is tightly regulated by cytokine signaling. Although phospho-flow cytometry allows us to study signaling in defined populations of cells, there has been tremendous hurdle to carry out this study in rare HSCs due to unrecoverable critical HSC markers, low HSC number, and poor cell recovery rate. Here, we overcame these difficulties and developed a “HSC phospho-flow” method to analyze cytokine signaling in murine HSCs at the single-cell level and compare HSC signaling profile to that of multipotent progenitors (MPPs), a cell type immediately downstream of HSCs, and commonly used Lin− cKit+ cells (LK cells, enriched for myeloid progenitors). We chose to study signaling evoked from three representative cytokines, stem cell factor (SCF) and thrombopoietin (TPO) that are essential for HSC function, and granulocyte macrophage-colony stimulating factor (GM-CSF) that is dispensable for HSCs. HSCs display a distinct TPO and GM-CSF signaling signature from MPPs and LK cells, which highly correlates with receptor surface expression. In contrast, although majority of LK cells express lower levels of cKit than HSCs and MPPs, SCF-evoked ERK1/2 activation in LK cells shows a significantly increased magnitude for a prolonged period. These results suggest that specific cellular context plays a more important role than receptor surface expression in SCF signaling. Our study of HSC signaling at the homeostasis stage paves the way to investigate signaling changes in HSCs under conditions of stress, aging, and hematopoietic diseases. PMID:22628264

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

    PubMed

    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

  10. Anti-CD69 monoclonal antibody treatment inhibits airway inflammation in a mouse model of asthma*

    PubMed Central

    Wang, Hui-ying; Dai, Yu; Wang, Jiao-li; Yang, Xu-yan; Jiang, Xin-guo

    2015-01-01

    Objective: Airway inflammation and airway hyper-responsiveness (AHR) are principle pathological manifestations of asthma. Cluster of differentiation 69 (CD69) is a well-known co-stimulatory factor associated with the activation, proliferation as well as apoptosis of immune cells. This study aims to examine the effect of anti-CD69 monoclonal antibody (mAb) on the pathophysiology of a mouse model of asthma. Methods: A murine model of ovalbumin (OVA)-induced allergic airway inflammation was used in this study. Briefly, mice were injected with 20 μg chicken OVA intraperitoneally on Days 0 and 14, followed by aerosol provocation with 1% (0.01 g/ml) OVA on Days 24, 25, and 26. Anti-CD69 mAb or isotype IgG was injected intraperitoneally after OVA challenge; dexamethasone (DXM) was administrated either before or after OVA challenge. AHR, mucus production, and eosinophil infiltration in the peribronchial area were examined. The levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) in bronchoalveolar lavage fluid (BALF) were also assayed as indices of airway inflammation on Day 28 following OVA injection. Results: Pretreatment with DXM together with anti-CD69 mAb treatment after OVA provocation completely inhibited AHR, eosinophil infiltration and mucus overproduction, and significantly reduced BALF IL-5. However, treatment with DXM alone after OVA challenge only partially inhibited AHR, eosinophil infiltration and mucus overproduction, and did not diminish BALF IL-5. Treatment with either DXM or anti-CD69 mAb did not alter the concentration of BALF GM-CSF. Conclusions: Anti-CD69 mAb treatment inhibits established airway inflammation as effectively as DXM pretreatment. This study provides a potential alternative therapeutic opportunity for the clinical management of asthma and its exacerbation. PMID:26160720

  11. Identification of immune factors regulating anti-tumor immunity using polymeric vaccines with multiple adjuvants

    PubMed Central

    Ali, Omar A.; Verbeke, Catia; Johnson, Chris; Sands, Warren; Lewin, Sarah A.; White, Des; Doherty, Edward; Dranoff, Glenn; Mooney, David J.

    2014-01-01

    The innate cellular and molecular components required to mediate effective vaccination against weak tumor-associated antigens remain unclear. In this study we utilized polymeric cancer vaccines incorporating different classes of adjuvants to induce tumor protection, in order to identify dendritic cell subsets and cytokines critical to this efficacy. Three-dimensional, porous polymer matrices loaded with tumor lysates and presenting distinct combinations of GM-CSF and various TLR agonists effected 70–90% prophylactic tumor protection in B16-F10 melanoma models. In aggressive, therapeutic B16 models, the vaccine systems incorporating GM-CSF in combination with P(I:C) or CpG-ODN induced the complete regression of solid tumors (≤40mm2) resulting in 33% long-term survival. Regression analysis revealed that the numbers of vaccine-resident CD8(+) DCs and plasmacytoid DCs, along with local IL-12, and G-CSF concentrations correlated strongly to vaccine efficacy regardless of adjuvant type. Further, vaccine studies in Batf3−/− mice revealed that CD8(+) DCs are required to effect tumor protection, as vaccines in these mice were deficient in cytotoxic T cell priming, and IL-12 induction in comparison to wild-type. These studies broadly demonstrate that three-dimensional polymeric vaccines provide a potent platform for prophylactic and therapeutic protection, and can be used as a tool to identify critical components of a desired immune response. Specifically, these results suggest that CD8(+) DCs, plasmacytoid DCs, IL-12, and G-CSF play important roles in priming effective anti-tumor responses with these vaccines. PMID:24480625

  12. The composition of cigarette smoke determines inflammatory cell recruitment to the lung in COPD mouse models.

    PubMed

    John, Gerrit; Kohse, Katrin; Orasche, Jürgen; Reda, Ahmed; Schnelle-Kreis, Jürgen; Zimmermann, Ralf; Schmid, Otmar; Eickelberg, Oliver; Yildirim, Ali Önder

    2014-02-01

    COPD (chronic obstructive pulmonary disease) is caused by exposure to toxic gases and particles, most often CS (cigarette smoke), leading to emphysema, chronic bronchitis, mucus production and a subsequent decline in lung function. The disease pathogenesis is related to an abnormal CS-induced inflammatory response of the lungs. Similar to active (mainstream) smoking, second hand (sidestream) smoke exposure severely affects respiratory health. These processes can be studied in vivo in models of CS exposure of mice. We compared the acute inflammatory response of female C57BL/6 mice exposed to two concentrations [250 and 500 mg/m3 TPM (total particulate matter)] of sidestream and mainstream CS for 3 days and interpreted the biological effects based on physico-chemical differences in the gas and particulate phase composition of CS. BAL (bronchoalveolar lavage fluid) was obtained to perform differential cell counts and to measure cytokine release. Lung tissue was used to determine mRNA and protein expression of proinflammatory genes and to assess tissue inflammation. A strong acute inflammatory response characterized by neutrophilic influx, increased cytokine secretion [KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factor α), MIP-2 (macrophage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein-1)], pro-inflammatory gene expression [KC, MIP-2 and MMP12 (matrix metalloproteinase 12)] and up-regulated GM-CSF (granulocyte macrophage colony-stimulating factor) production was observed in the mainstream model. After sidestream exposure there was a dampened inflammatory reaction consisting only of macrophages and diminished GM-CSF levels, most likely caused by elevated CO concentrations. These results demonstrate that the composition of CS determines the dynamics of inflammatory cell recruitment in COPD mouse models. Different initial inflammatory processes might contribute to COPD pathogenesis in significantly varying ways, thereby

  13. Impact of Whole-Blood Processing Conditions on Plasma and Serum Concentrations of Cytokines.

    PubMed

    Lee, Jae-Eun; Kim, Jong-Wan; Han, Bok-Ghee; Shin, So-Youn

    2016-02-01

    Pre-analytical variations in plasma and serum samples can occur because of variability in whole-blood processing procedures. The aim of this study was to determine the impact of delayed separation of whole blood on the plasma and serum concentrations of cytokines. The concentrations of 16 cytokines were measured in plasma and serum samples when the centrifugation of whole blood at room temperature was delayed for 4, 6, 24, or 48 h, and the values were compared with those observed after separation within 2 h of whole-blood collection. Receiver operating characteristic (ROC) curve analysis was also performed for cytokines to determine whether cytokine levels in plasma and serum samples can be used to assess delayed separation of whole blood. Plasma concentrations of interleukin (IL)-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), and soluble CD40 ligand (sCD40L) and serum concentrations of IL-1β, IL-6, IL-8, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β increased significantly (>2-fold) when separation was delayed at room temperature for 24 h. The concentrations of 6 of these cytokines (all except serum IL-1β and IL-6) demonstrated high diagnostic performance (area under the ROC curve >0.8) for delayed separation of whole blood. Furthermore, these cytokine concentrations typically exhibited high sensitivity and specificity at each optimal cutoff point. Conversely, IL-17A was stable in both plasma and serum samples, even when whole-blood centrifugation was delayed at room temperature for 48 h. This study shows that certain cytokines (IL-1β, GM-CSF, sCD40L, IL-8, MIP-1α, and MIP-1β) could be used for assessing the quality of plasma or serum samples. PMID:26808439

  14. The composition of cigarette smoke determines inflammatory cell recruitment to the lung in COPD mouse models.

    PubMed

    John, Gerrit; Kohse, Katrin; Orasche, Jürgen; Reda, Ahmed; Schnelle-Kreis, Jürgen; Zimmermann, Ralf; Schmid, Otmar; Eickelberg, Oliver; Yildirim, Ali Önder

    2014-02-01

    COPD (chronic obstructive pulmonary disease) is caused by exposure to toxic gases and particles, most often CS (cigarette smoke), leading to emphysema, chronic bronchitis, mucus production and a subsequent decline in lung function. The disease pathogenesis is related to an abnormal CS-induced inflammatory response of the lungs. Similar to active (mainstream) smoking, second hand (sidestream) smoke exposure severely affects respiratory health. These processes can be studied in vivo in models of CS exposure of mice. We compared the acute inflammatory response of female C57BL/6 mice exposed to two concentrations [250 and 500 mg/m3 TPM (total particulate matter)] of sidestream and mainstream CS for 3 days and interpreted the biological effects based on physico-chemical differences in the gas and particulate phase composition of CS. BAL (bronchoalveolar lavage fluid) was obtained to perform differential cell counts and to measure cytokine release. Lung tissue was used to determine mRNA and protein expression of proinflammatory genes and to assess tissue inflammation. A strong acute inflammatory response characterized by neutrophilic influx, increased cytokine secretion [KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factor α), MIP-2 (macrophage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein-1)], pro-inflammatory gene expression [KC, MIP-2 and MMP12 (matrix metalloproteinase 12)] and up-regulated GM-CSF (granulocyte macrophage colony-stimulating factor) production was observed in the mainstream model. After sidestream exposure there was a dampened inflammatory reaction consisting only of macrophages and diminished GM-CSF levels, most likely caused by elevated CO concentrations. These results demonstrate that the composition of CS determines the dynamics of inflammatory cell recruitment in COPD mouse models. Different initial inflammatory processes might contribute to COPD pathogenesis in significantly varying ways, thereby

  15. Evaluation of Dicarbonyls Generated in a Simulated Indoor Air Environment Using an In Vitro Exposure System

    PubMed Central

    Anderson, Stacey E.; Jackson, Laurel G.; Franko, Jennifer; Wells, J. R.

    2010-01-01

    Over the last two decades, there has been increasing awareness regarding the potential impact of indoor air pollution on health. Exposure to volatile organic compounds (VOCs) or oxygenated organic compounds formed from indoor chemistry has been suggested to contribute to adverse health effects. These studies use an in vitro monitoring system called VitroCell, to assess chemicals found in the indoor air environment. The structurally similar dicarbonyls diacetyl, 4-oxopentanal (4-OPA), glyoxal, glutaraldehyde, and methyl glyoxal were selected for use in this system. The VitroCell module was used to determine whether these dicarbonyls were capable of inducing inflammatory cytokine expression by exposed pulmonary epithelial cells (A549). Increases in the relative fold change in messenger RNA expression of the inflammatory mediators, interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α) were identified following exposure to diacetyl, 4-OPA, glyoxal, glutaraldehyde, and methyl glyoxal when compared to a clean air control. Consistent results were observed when the protein levels of these cytokines were analyzed. Exposure to 4-OPA significantly elevated IL-8, IL-6, GM-CSF, and TNF-α while glutaraldehyde caused significant elevations in IL-6, IL-8, and TNF-α. IL-6 and IL-8 were also significantly elevated after exposure to diacetyl, glyoxal, and methyl glyoxal. These studies suggest that exposure to structurally similar oxygenated reaction products may be contributing to some of the health effects associated with indoor environments and may provide an in vitro method for identification and characterization of these potential hazards. PMID:20200221

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

    PubMed

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

    2012-07-30

    Microglia are the innate immune cells of the central nervous system. They help maintaining physiological homeostasis and contribute significantly to inflammatory responses in the course of infection, injury and degenerative processes. To date, there is no standardized simple model available to investigate the biology of human microglia. The aim of this study was to establish a new human microglia model. For that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of M-CSF, GM-CSF, NGF and CCL2 to generate monocyte-derived microglia (M-MG). M-MG were clearly different in morphology, phenotype and function from freshly isolated monocytes, cultured monocytes in the absence of the cytokines and monocyte-derived dendritic cells (M-DC) cultured in the presence of GM-CSF and IL-4. M-MG acquired a ramified morphology with primary and secondary processes. M-MG displayed a comparable phenotype to the human microglia cell line HMC3, expressing very low levels of CD45, CD14 and HLA-DR, CD11b and CD11c; and undetectable levels of CD40, CD80 and CD83, and a distinct pattern of chemokine receptors (positive for CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR3, CX3CR1; negative for CCR6 and CCR7). In comparison with M-DC, M-MG displayed lower T-lymphocyte stimulatory capacity, as well as lower phagocytosis activity. The described protocol for the generation of human monocyte-derived microglia is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. This protocol will certainly be very helpful for future studies investigating the biology and pathology of human microglia. PMID:22659341

  17. Excessive Pro-Inflammatory Serum Cytokine Concentrations in Virulent Canine Babesiosis

    PubMed Central

    Goddard, Amelia; Leisewitz, Andrew L.; Kjelgaard-Hansen, Mads; Kristensen, Annemarie T.; Schoeman, Johan P.

    2016-01-01

    Babesia rossi infection causes a severe inflammatory response in the dog, which is the result of the balance between pro- and anti-inflammatory cytokine secretion. The aim of this study was to determine whether changes in cytokine concentrations were present in dogs with babesiosis and whether it was associated with disease outcome. Ninety-seven dogs naturally infected with B. rossi were studied and fifteen healthy dogs were included as controls. Diagnosis of babesiosis was confirmed by polymerase chain reaction and reverse line blot. Blood samples were collected from the jugular vein at admission, prior to any treatment. Cytokine concentrations were assessed using a canine-specific multiplex assay on an automated analyser. Serum concentrations of interleukin (IL)-2, IL-6, IL-8, IL-10, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF) and monocyte chemotactic protein-1 (MCP-1) were measured. Twelve of the Babesia-infected dogs died (12%) and 85 survived (88%). Babesia-infected dogs were also divided into those that presented within 48 hours from displaying clinical signs, and those that presented more than 48 hours after displaying clinical signs. Cytokine concentrations were compared between the different groups using the Mann-Whitney U test. IL-10 and MCP-1 concentrations were significantly elevated for the Babesia-infected dogs compared to the healthy controls. In contrast, the IL-8 concentration was significantly decreased in the Babesia-infected dogs compared to the controls. Concentrations of IL-6 and MCP-1 were significantly increased in the non-survivors compared to the survivors. Concentrations for IL-2, IL-6, IL-18 and GM-CSF were significantly higher in those cases that presented during the more acute stage of the disease. These findings suggest that a mixed cytokine response is present in dogs with babesiosis caused by B. rossi, and that an excessive pro-inflammatory response may result in a poor outcome. PMID:26953797

  18. The composition of cigarette smoke determines inflammatory cell recruitment to the lung in COPD mouse models

    PubMed Central

    John, Gerrit; Kohse, Katrin; Orasche, Jürgen; Reda, Ahmed; Schnelle-Kreis, Jürgen; Zimmermann, Ralf; Schmid, Otmar; Eickelberg, Oliver; Yildirim, Ali Önder

    2013-01-01

    COPD (chronic obstructive pulmonary disease) is caused by exposure to toxic gases and particles, most often CS (cigarette smoke), leading to emphysema, chronic bronchitis, mucus production and a subsequent decline in lung function. The disease pathogenesis is related to an abnormal CS-induced inflammatory response of the lungs. Similar to active (mainstream) smoking, second hand (sidestream) smoke exposure severely affects respiratory health. These processes can be studied in vivo in models of CS exposure of mice. We compared the acute inflammatory response of female C57BL/6 mice exposed to two concentrations [250 and 500 mg/m3 TPM (total particulate matter)] of sidestream and mainstream CS for 3 days and interpreted the biological effects based on physico-chemical differences in the gas and particulate phase composition of CS. BAL (bronchoalveolar lavage fluid) was obtained to perform differential cell counts and to measure cytokine release. Lung tissue was used to determine mRNA and protein expression of proinflammatory genes and to assess tissue inflammation. A strong acute inflammatory response characterized by neutrophilic influx, increased cytokine secretion [KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factor α), MIP-2 (macrophage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein-1)], pro-inflammatory gene expression [KC, MIP-2 and MMP12 (matrix metalloproteinase 12)] and up-regulated GM-CSF (granulocyte macrophage colony-stimulating factor) production was observed in the mainstream model. After sidestream exposure there was a dampened inflammatory reaction consisting only of macrophages and diminished GM-CSF levels, most likely caused by elevated CO concentrations. These results demonstrate that the composition of CS determines the dynamics of inflammatory cell recruitment in COPD mouse models. Different initial inflammatory processes might contribute to COPD pathogenesis in significantly varying ways, thereby

  19. Activation of human monocyte-derived macrophages cultured on Teflon: response to interferon-gamma during terminal maturation in vitro.

    PubMed

    Andreesen, R; Gadd, S; Brugger, W; Löhr, G W; Atkins, R C

    1988-05-01

    Macrophages (M phi) are potential antitumor effector cells derived from circulating blood monocytes (mo). Most studies on human mo/M phi biology and function have been performed using immature mo precursor cells. However, the conclusions drawn may be questionable, as mo have to undergo terminal differentiation before they reach relevant tissue sites of inflammation and immune reaction. We have analyzed the ability of mo-derived, teflon-cultured M phi to respond to activating stimuli with an increased tumor cytotoxic effector cell function using recombinant interferon-gamma (IFN-gamma), IFN-alpha 2, granulocyte/macrophage colony stimulating factor (GM-CSF), interleukin(IL) 2, IL 1 alpha, and bacterial lipopolysaccharides (LPS) as mediator molecules. It could be shown that the response of M phi to the most potent activator molecule, IFN-gamma, depends on the terminal differentiation from the mo stage to the mature M phi. Whereas adherent mo could be activated only moderately, M phi increased their cytotoxicity by a factor of up to 400. IFN-gamma activation positively correlated with the effector cell number, the time of incubation and the dosage used. Activation did not depend on the presence of LPS, and was lost within 24 to 48 h. LPS itself activated cells only in the microgram range. IFN-alpha 2 activated M phi only at a two log higher concentration than IFN-gamma; GM-CSF was only slightly effective, whereas M phi incubation with IL 1 alpha or IL 2 did not result in M phi activation. Thus, the ability of human M phi to become activated appears to be a function of cellular maturation and is acquired during the terminal step of M phi differentiation. Teflon-cultured M phi could facilitate studies of the activation of human M phi and may be more suitable cells for adoptive immunotherapy in cancer patients than blood monocytes. PMID:3136081

  20. Immunological profiling in chronic rhinosinusitis with nasal polyps reveals distinct VEGF and GMCSF signatures during symptomatic exacerbations

    PubMed Central

    Divekar, Rohit D.; Samant, Shefali; Rank, Matthew A.; Hagan, John; Lal, Devyani; O’Brien, Erin K.; Kita, Hirohito

    2015-01-01

    Background The mechanisms and immune pathways associated with chronic rhinosinusitis (CRS) are not fully understood. Immunological changes during acute exacerbation of CRS may provide valuable clues to the pathogenesis and perpetuation of the disease. Objective To characterize local and systemic immune responses associated with acute worsening of sinonasal symptoms during exacerbation in CRS with nasal polyps (CRSwNP) compared to controls. Methods This was a noninterventional prospective study of individuals with CRSwNP and normal controls. Subjects underwent a baseline visit with collection of nasal secretions, nasal washes, and serum specimens. Within 3 days of acute worsening of sinonasal symptoms, subjects underwent a study visit, followed by a post-visit 2 weeks later. The Sinonasal Outcome Test-22 (SNOT-22) scores and immunological parameters in the specimens were analyzed using a novel, unsupervised learning method and by conventional univariate analysis. Results Both CRSwNP patients and control subjects showed a significant increase in SNOT-22 scores during acute exacerbation. Increased nasal levels of IL-6, IL-5, and eosinophil major basic protein were observed in CRSwNP patients. A network analysis of serum specimens revealed changes in a set of immunological parameters, which are distinctly associated with CRSwNP but not with controls. In particular, systemic increases in VEGF and GM-CSF levels were notable and were validated by a conventional analysis. Conclusions CRSwNP patients demonstrate distinct immunological changes locally and systemically during acute exacerbation. Growth factors VEGF and GM-CSF may be involved in the immunopathogenesis of subjects with CRS and nasal polyps experiencing exacerbation. PMID:25429844

  1. Systemic inflammatory responses in patients with type 2 diabetes with chronic periodontitis

    PubMed Central

    Mesia, Ruben; Gholami, Fatemeh; Huang, Hong; Clare-Salzler, Michael; Aukhil, Ikramuddin; Wallet, Shannon M; Shaddox, Luciana M

    2016-01-01

    Objective The objective of this case–control study was to quantify the immune responsiveness in individuals with type 2 diabetes (T2D) as compared with patients without diabetes (NT2D) diagnosed with periodontitis. Research Design and Methods Peripheral blood was collected from 20 patients with moderate-to-severe chronic periodontitis (10 T2D, 10 NT2D). Blood samples were stimulated with ultrapure Porphyromonas gingivalis and Escherichia coli lipopolysaccharide (LPS) for 24 hours. 14 cytokines/chemokines were quantified in culture supernatants using multiplex technology. Results T2D individuals demonstrated higher unstimulated levels of interleukin 6 (IL-6), IL-1β, tumor necrosis factor α, interferon γ, IL-10, IL-8, macrophage inflammatory protein 1α (MIP1α), and 1β (MIP1β), and higher stimulated levels of IL-6, IL-8, IL-10, MIP1α and MIP1β, along with lower unstimulated and stimulated levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) when compared with NT2D (p<0.05). Importantly, the LPS-induced levels of IL-6, IL-8, IL-10 and MIP1α strongly correlated with severity of disease, measured by pocket depths (PD), within the T2D group (r2≥0.7, p<0.05), but not within NT2D. Conclusions Among patients with chronic periodontitis, patients with T2D seem to have an enhanced LPS-induced immune responsiveness than individuals without diabetes, which correlates with periodontal disease severity, concomitant with a less robust GM-CSF response. This data may in part explain the higher predisposition to periodontitis in this population. PMID:27651910

  2. Effect of Blood Component Coatings of Enosseal Implants on Proliferation and Synthetic Activity of Human Osteoblasts and Cytokine Production of Peripheral Blood Mononuclear Cells.

    PubMed

    Himmlova, Lucie; Kubies, Dana; Hulejova, Hana; Bartova, Jirina; Riedel, Tomas; Stikarova, Jana; Suttnar, Jiri; Pesakova, Vlasta

    2016-01-01

    The study monitored in vitro early response of connective tissue cells and immunocompetent cells to enosseal implant materials coated by different blood components (serum, activated plasma, and plasma/platelets) to evaluate human osteoblast proliferation and synthetic activity and inflammatory response presented as a cytokine profile of peripheral blood mononuclear cells (PBMCs) under conditions imitating the situation upon implantation. The cells were cultivated on coated Ti-plasma-sprayed (Ti-PS), Ti-etched (Ti-Etch), Ti-hydroxyapatite (Ti-HA), and ZrO2 surfaces. The plasma/platelets coating supported osteoblast proliferation only on osteoconductive Ti-HA and Ti-Etch whereas activated plasma enhanced proliferation on all surfaces. Differentiation (BAP) and IL-8 production remained unchanged or decreased irrespective of the coating and surface; only the serum and plasma/platelets-coated ZrO2 exhibited higher BAP and IL-8 expression. RANKL production increased on serum and activated plasma coatings. PBMCs produced especially cytokines playing role in inflammatory phase of wound healing, that is, IL-6, GRO-α, GRO, ENA-78, IL-8, GM-CSF, EGF, and MCP-1. Cytokine profiles were comparable for all tested surfaces; only ENA-78, IL-8, GM-CSF, and MCP-1 expression depended on materials and coatings. The activated plasma coating led to uniformed surfaces and represented a favorable treatment especially for bioinert Ti-PS and ZrO2 whereas all coatings had no distinctive effect on bioactive Ti-HA and Ti-Etch. PMID:27651560

  3. Favorable alteration of tumor microenvironment by immunomodulatory cytokines for efficient T-cell therapy in solid tumors.

    PubMed

    Tähtinen, Siri; Kaikkonen, Saija; Merisalo-Soikkeli, Maiju; Grönberg-Vähä-Koskela, Susanna; Kanerva, Anna; Parviainen, Suvi; Vähä-Koskela, Markus; Hemminki, Akseli

    2015-01-01

    Unfavorable ratios between the number and activation status of effector and suppressor immune cells infiltrating the tumor contribute to resistance of solid tumors to T-cell based therapies. Here, we studied the capacity of FDA and EMA approved recombinant cytokines to manipulate this balance in favor of efficient anti-tumor responses in B16.OVA melanoma bearing C57BL/6 mice. Intratumoral administration of IFN-α2, IFN-γ, TNF-α, and IL-2 significantly enhanced the anti-tumor effect of ovalbumin-specific CD8+ T-cell (OT-I) therapy, whereas GM-CSF increased tumor growth in association with an increase in immunosuppressive cell populations. None of the cytokines augmented tumor trafficking of OT-I cells significantly, but injections of IFN-α2, IFN-γ and IL-2 increased intratumoral cytokine secretion and recruitment of endogenous immune cells capable of stimulating T-cells, such as natural killer and maturated CD11c+ antigen-presenting cells. Moreover, IFN-α2 and IL-2 increased the levels of activated tumor-infiltrating CD8+ T-cells concomitant with reduction in the CD8+ T-cell expression of anergy markers CTLA-4 and PD-1. In conclusion, intratumoral administration of IFN-α2, IFN-γ and IL-2 can lead to immune sensitization of the established tumor, whereas GM-CSF may contribute to tumor-associated immunosuppression. The results described here provide rationale for including local administration of immunostimulatory cytokines into T-cell therapy regimens. One appealing embodiment of this would be vectored delivery which could be advantageous over direct injection of recombinant molecules with regard to efficacy, cost, persistence and convenience.

  4. Favorable Alteration of Tumor Microenvironment by Immunomodulatory Cytokines for Efficient T-Cell Therapy in Solid Tumors

    PubMed Central

    Tähtinen, Siri; Kaikkonen, Saija; Merisalo-Soikkeli, Maiju; Grönberg-Vähä-Koskela, Susanna; Kanerva, Anna; Parviainen, Suvi; Vähä-Koskela, Markus; Hemminki, Akseli

    2015-01-01

    Unfavorable ratios between the number and activation status of effector and suppressor immune cells infiltrating the tumor contribute to resistance of solid tumors to T-cell based therapies. Here, we studied the capacity of FDA and EMA approved recombinant cytokines to manipulate this balance in favor of efficient anti-tumor responses in B16.OVA melanoma bearing C57BL/6 mice. Intratumoral administration of IFN-α2, IFN-γ, TNF-α, and IL-2 significantly enhanced the anti-tumor effect of ovalbumin-specific CD8+ T-cell (OT-I) therapy, whereas GM-CSF increased tumor growth in association with an increase in immunosuppressive cell populations. None of the cytokines augmented tumor trafficking of OT-I cells significantly, but injections of IFN-α2, IFN-γ and IL-2 increased intratumoral cytokine secretion and recruitment of endogenous immune cells capable of stimulating T-cells, such as natural killer and maturated CD11c+ antigen-presenting cells. Moreover, IFN-α2 and IL-2 increased the levels of activated tumor-infiltrating CD8+ T-cells concomitant with reduction in the CD8+ T-cell expression of anergy markers CTLA-4 and PD-1. In conclusion, intratumoral administration of IFN-α2, IFN-γ and IL-2 can lead to immune sensitization of the established tumor, whereas GM-CSF may contribute to tumor-associated immunosuppression. The results described here provide rationale for including local administration of immunostimulatory cytokines into T-cell therapy regimens. One appealing embodiment of this would be vectored delivery which could be advantageous over direct injection of recombinant molecules with regard to efficacy, cost, persistence and convenience. PMID:26107883

  5. Potential Role of Fibroblast-Like Synoviocytes in Joint Damage Induced by Brucella abortus Infection through Production and Induction of Matrix Metalloproteinases ▿

    PubMed Central

    Scian, Romina; Barrionuevo, Paula; Giambartolomei, Guillermo H.; De Simone, Emilio A.; Vanzulli, Silvia I.; Fossati, Carlos A.; Baldi, Pablo C.; Delpino, M. Victoria

    2011-01-01

    Arthritis is one of the most common complications of human brucellosis, but its pathogenic mechanisms have not been elucidated. Fibroblast-like synoviocytes (FLS) are known to be central mediators of joint damage in inflammatory arthritides through the production of matrix metalloproteinases (MMPs) that degrade collagen and of cytokines and chemokines that mediate the recruitment and activation of leukocytes. In this study we show that Brucella abortus infects and replicates in human FLS (SW982 cell line) in vitro and that infection results in the production of MMP-2 and proinflammatory mediators (interleukin-6 [IL-6], IL-8, monocyte chemotactic protein 1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Culture supernatants from Brucella-infected FLS induced the migration of monocytes and neutrophils in vitro and also induced these cells to secrete MMP-9 in a GM-CSF- and IL-6-dependent fashion, respectively. Reciprocally, culture supernatants from Brucella-infected monocytes and neutrophils induced FLS to produce MMP-2 in a tumor necrosis factor alpha (TNF-α)-dependent fashion. The secretion of proinflammatory mediators and MMP-2 by FLS did not depend on bacterial viability, since it was also induced by heat-killed B. abortus (HKBA) and by a model Brucella lipoprotein (L-Omp19). These responses were mediated by the recognition of B. abortus antigens through Toll-like receptor 2. The intra-articular injection of HKBA or L-Omp19 into the knee joint of mice resulted in the local induction of the proinflammatory mediators MMP-2 and MMP-9 and in the generation of a mixed inflammatory infiltrate. These results suggest that FLS, and phagocytes recruited by them to the infection focus, may be involved in joint damage during brucellar arthritis through the production of MMPs and proinflammatory mediators. PMID:21730088

  6. The Inhibitory Effects of Anti-Oxidants on Ultraviolet-Induced Up-Regulation of the Wrinkling-Inducing Enzyme Neutral Endopeptidase in Human Fibroblasts

    PubMed Central

    Nakajima, Hiroaki; Terazawa, Shuko; Niwano, Takao; Yamamoto, Yorihiro; Imokawa, Genji

    2016-01-01

    We recently reported that the over-expression of skin fibroblast-derived neutral endopeptidase (NEP) plays a pivotal role in impairing the three-dimensional architecture of dermal elastic fibers during the biological mechanism of ultraviolet (UV)-induced skin wrinkling. In that process, a UVB-associated epithelial-mesenchymal cytokine interaction as well as a direct UVA-induced cellular stimulation are associated with the up-regulation of NEP in human fibroblasts. In this study, we characterized the mode of action of ubiquinol10 which may abrogate the up-regulation of NEP by dermal fibroblasts, resulting in a reported in vivo anti-wrinkling action, and compared that with 3 other anti-oxidants, astaxanthin (AX), riboflavin (RF) and flavin mononucleotide (FMN). Post-irradiation treatment with all 4 of those anti-oxidants elicited an interrupting effect on the UVB-associated epithelial-mesenchymal cytokine interaction leading to the up-regulation of NEP in human fibroblasts but with different modes of action. While AX mainly served as an inhibitor of the secretion of wrinkle-inducing cytokines, such as interleukin-1α (IL-1α) and granulocyte macrophage colony stimulatory factor (GM-CSF) in UVB-exposed epidermal keratinocytes, ubiquinol10, RF and FMN predominantly interrupted the IL-1α and GM-CSF-stimulated expression of NEP in dermal fibroblasts. On the other hand, as for the UVA-associated mechanism, similar to the abrogating effects reported for AX and FMN, ubiquinol10 but not RF had the potential to abrogate the increased expression of NEP and matrix-metalloproteinase-1 in UVA-exposed human fibroblasts. Our findings strongly support the in vivo anti-wrinkling effects of ubiquinol10 and AX on human and animal skin and provide convincing proof of the UV-induced wrinkling mechanism that essentially focuses on the over-expression of NEP by dermal fibroblasts as an intrinsic causative factor. PMID:27648570

  7. Multiplex cytokine profile from dengue patients: MIP-1beta and IFN-gamma as predictive factors for severity

    PubMed Central

    Bozza, Fernando A; Cruz, Oswaldo G; Zagne, Sonia MO; Azeredo, Elzinandes L; Nogueira, Rita MR; Assis, Edson F; Bozza, Patricia T; Kubelka, Claire F

    2008-01-01

    Background Dengue virus pathogenesis is not yet fully understood and the identification of patients at high risk for developing severe disease forms is still a great challenge in dengue patient care. During the present study, we evaluated prospectively the potential of cytokines present in plasma from patients with dengue in stratifying disease severity. Methods Seventeen-cytokine multiplex fluorescent microbead immunoassay was used for the simultaneous detection in 59 dengue patients. GLM models using bimodal or Gaussian family were determined in order to associate cytokines with clinical manifestations and laboratory diagnosis. Results IL-1β, IFN-γ, IL-4, IL-6, IL-13, IL-7 and GM-CSF were significantly increased in patients with severe clinical manifestations (severe dengue) when compared to mild disease forms (mild dengue). In contrast, increased MIP-1β levels were observed in patients with mild dengue. MIP-1β was also associated with CD56+NK cell circulating rates. IL-1β, IL-8, TNF-α and MCP-1 were associated with marked thrombocytopenia. Increased MCP-1 and GM-CSF levels correlated with hypotension. Moreover, MIP-1β and IFN-γ were independently associated with both dengue severity and disease outcome. Conclusion Our data demonstrated that the use of a multiple cytokine assay platform was suitable for identifying distinct cytokine profiles associated with the dengue clinical manifestations and severity. MIP-β is indicated for the first time as a good prognostic marker in contrast to IFN-γ that was associated with disease severity. PMID:18578883

  8. Selective and efficient generation of functional Batf3-dependent CD103+ dendritic cells from mouse bone marrow

    PubMed Central

    Mayer, Christian Thomas; Ghorbani, Peyman; Nandan, Amrita; Dudek, Markus; Arnold-Schrauf, Catharina; Hesse, Christina; Berod, Luciana; Stüve, Philipp; Puttur, Franz; Merad, Miriam

    2014-01-01

    Multiple subsets of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent dendritic cells (DCs) control T-cell tolerance and immunity. In mice, Batf3-dependent CD103+ DCs efficiently enter lymph nodes and cross-present antigens, rendering this conserved DC subset a promising target for tolerance induction or vaccination. However, only limited numbers of CD103+ DCs can be isolated with current methods. Established bone marrow culture protocols efficiently generate monocyte-derived DCs or produce a mixture of FLT3L-dependent DC subsets. We show that CD103+ DC development requires prolonged culture time and continuous action of both FLT3L and granulocyte macrophage colony-stimulating factor (GM-CSF), explained by a dual effect of GM-CSF on DC precursors and differentiating CD103+ DCs. Accordingly, we established a novel method to generate large numbers of CD103+ DCs (iCD103-DCs) with limited presence of other DC subsets. iCD103-DCs develop in a Batf3- and Irf8-dependent fashion, express a CD8α/CD103 DC gene signature, cross-present cell-associated antigens, and respond to TLR3 stimulation. Thus, iCD103-DCs reflect key features of tissue CD103+ DCs. Importantly, iCD103-DCs express high levels of CCR7 upon maturation and migrate to lymph nodes more efficiently than classical monocyte-derived DCs. Finally, iCD103-DCs induce T cell–mediated protective immunity in vivo. Our study provides insights into CD103+ DC development and function. PMID:25100743

  9. Megakaryocyte growth and development factor is a potent growth factor for primitive hematopoietic progenitors in the human fetus.

    PubMed

    Muench, Marcus O; Bárcena, Alicia

    2004-06-01

    Megakaryocyte growth and development factor (MGDF), or thrombopoietin, has received considerable attention as a therapeutic agent for treating thrombocytopenia or for its use in the ex vivo culture of hematopoietic stem cells. MGDF is known to support the growth of a broad spectrum of hematopoietic precursors obtained from adult or neonatal tissues, but its effects on the growth of fetal progenitors and stem cells has not been studied. Human CD38(+)CD34(2+) progenitors and CD38(-)CD34(2+) cells, a population that contains stem cells, were isolated from midgestation liver and grown under defined conditions with MGDF and various cytokines known to support the growth of primitive hematopoietic precursors. In clonal assays of colony-forming cells (CFCs), MGDF supported the growth of 15-25% of candidate stem cells when combined with granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), flk-2/flt3 ligand, or stem cell factor. MGDF was observed to strongly support the early stages of hematopoiesis and expansion of high proliferative potential CFCs. More mature progenitors were expanded nearly 78-fold in 1 wk of culture with MGDF+SCF+GM-CSF. MGDF alone was also found to support the short-term (2 d) survival of CD38(-)CD34(2+) high proliferative potential CFCs. The effects of MGDF were more modest on CD38(+)CD34(2+) progenitors with only additive increases in colony formation being observed. These findings suggest that MGDF administration in fetuses and neonates may strongly affect the growth and mobilization of primitive hematopoietic progenitors and that MGDF may find use in the ex vivo growth and expansion of fetal stem cells.

  10. Effect of Blood Component Coatings of Enosseal Implants on Proliferation and Synthetic Activity of Human Osteoblasts and Cytokine Production of Peripheral Blood Mononuclear Cells

    PubMed Central

    Hulejova, Hana; Bartova, Jirina; Riedel, Tomas; Pesakova, Vlasta

    2016-01-01

    The study monitored in vitro early response of connective tissue cells and immunocompetent cells to enosseal implant materials coated by different blood components (serum, activated plasma, and plasma/platelets) to evaluate human osteoblast proliferation and synthetic activity and inflammatory response presented as a cytokine profile of peripheral blood mononuclear cells (PBMCs) under conditions imitating the situation upon implantation. The cells were cultivated on coated Ti-plasma-sprayed (Ti-PS), Ti-etched (Ti-Etch), Ti-hydroxyapatite (Ti-HA), and ZrO2 surfaces. The plasma/platelets coating supported osteoblast proliferation only on osteoconductive Ti-HA and Ti-Etch whereas activated plasma enhanced proliferation on all surfaces. Differentiation (BAP) and IL-8 production remained unchanged or decreased irrespective of the coating and surface; only the serum and plasma/platelets-coated ZrO2 exhibited higher BAP and IL-8 expression. RANKL production increased on serum and activated plasma coatings. PBMCs produced especially cytokines playing role in inflammatory phase of wound healing, that is, IL-6, GRO-α, GRO, ENA-78, IL-8, GM-CSF, EGF, and MCP-1. Cytokine profiles were comparable for all tested surfaces; only ENA-78, IL-8, GM-CSF, and MCP-1 expression depended on materials and coatings. The activated plasma coating led to uniformed surfaces and represented a favorable treatment especially for bioinert Ti-PS and ZrO2 whereas all coatings had no distinctive effect on bioactive Ti-HA and Ti-Etch. PMID:27651560

  11. Dendritic Cell (DC) Vaccine in Mouse Lung Cancer Minimal Residual Model; Comparison of Monocyte-derived DC vs. Hematopoietic Stem Cell Derived-DC.

    PubMed

    Baek, Soyoung; Lee, Seog Jae; Kim, Myoung Joo; Lee, Hyunah

    2012-12-01

    The anti-tumor effect of monocyte-derived DC (MoDC) vaccine was studied in lung cancer model with feasible but weak Ag-specific immune response and incomplete blocking of tumor growth. To overcome this limitation, the hematopoietic stem cell-derived DC (SDC) was cultured and the anti-tumor effect of MoDC & SDC was compared in mouse lung cancer minimal residual model (MRD). Therapeutic DCs were cultured from either CD34(+) hematopoietic stem cells with GM-CSF, SCF and IL-4 for 14 days (SDC) or monocytes with GM-CSF and IL-4 for 7 days (MoDC). DCs were injected twice by one week interval into the peritoneum of mice that are inoculated with Lewis Lung Carcinoma cells (LLC) one day before the DC injection. Anti-tumor responses and the immune modulation were observed 3 weeks after the final DC injection. CD11c expression, IL-12 and TGF-β secretion were higher in SDC but CCR7 expression, IFN-γ and IL-10 secretion were higher in MoDC. The proportion of CD11c(+)CD8a(+) cells was similar in both DC cultures. Although both DC reduced the tumor burden, histological anti-tumor effect and the frequencies of IFN-γ secreting CD8(+) T cells were higher in SDC treated group than in MoDC. Conclusively, although both MoDC and SDC can induce the anti-tumor immunity, SDC may be better module as anti-tumor vaccine than MoDC in mouse lung cancer. PMID:23396889

  12. Engineering HSV-1 vectors for gene therapy.

    PubMed

    Goins, William F; Huang, Shaohua; Cohen, Justus B; Glorioso, Joseph C

    2014-01-01

    Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications, and with the approval of Glybera (alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20: 1831-1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme, a fatal form of brain cancer, and in malignant melanoma. In fact, T-VEC (talimogene laherparepvec, formerly known as OncoVex GM-CSF) displayed efficacy in a recent Phase III trial when compared to standard GM-CSF treatment alone (Andtbacka et al. J Clin Oncol 31: sLBA9008, 2013) and may soon become the second FDA-approved gene therapy product used in standard patient care. In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy, and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.

  13. Development of dextran nanoparticles for stabilizing delicate proteins

    NASA Astrophysics Data System (ADS)

    Wu, Fei; Zhou, Zhihua; Su, Jing; Wei, Liangming; Yuan, Weien; Jin, Tuo

    2013-04-01

    One of the most challenging problems in the development of protein pharmaceuticals is to deal with stabilities of proteins due to its complicated structures. This study aims to develop a novel approach to stabilize and encapsulate proteins into dextran nanoparticles without contacting the interface between the aqueous phase and the organic phase. The bovine serum albumin, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), β-galactosidase, and myoglobin were selected as model proteins. The proteins were added into an aqueous solution containing the dextran and polyethylene glycol, and then encapsulated into dextran nanoparticles by aqueous-aqueous freezing-induced phase separation. The encapsulation efficiency and recovery of dextran nanoparticles were determined. The dextran nanoparticles loaded with proteins were characterized by scanning electron microscopy and particle size analysis. The protein aggregation was determined by size-exclusion chromatography-high-performance chromatography, and the bioactivity of proteins recovered during formulation steps was determined. The bioactivity of GM-CSF, G-CSF, and β-galactosidase were examined by the proliferation of TF-1 cell, NSF-60 cell, and ortho-nitrophenyl- β-galactoside assay, respectively. The results of bioactivity recovered show that this novel dextran nanoparticle can preserve the protein's bioactivity during the preparation process. LysoSensor™ Yellow/Blue dextran, a pH-sensitive indicator with fluorescence excited at two channels, was encapsulated into dextran nanoparticles to investigate the ability of dextran nanoparticles to resist the acidic microenvironment (pH < 2.5). The result shows that the dextran nanoparticles attenuate the acidic microenvironment in the poly (lactic-co-glycolic acid) microsphere by means of the dilution effect. These novel dextran nanoparticles provided an appealing approach to stabilize the delicate proteins for

  14. Autocrine enhancement of leukotriene synthesis by endogenous leukotriene B4 and platelet-activating factor in human neutrophils.

    PubMed Central

    McDonald, P. P.; McColl, S. R.; Braquet, P.; Borgeat, P.

    1994-01-01

    1. Platelet-activating factor (PAF) and leukotriene B4 (LTB4), two potent lipid mediators synthesized by activated neutrophils, are known to stimulate several neutrophil functional responses. In this study, we have determined that endogenous LTB4 and PAF exert autocrine effects on LT synthesis, as well as the underlying mechanism involved. 2. Pretreatment of neutrophils with either pertussis toxin (PT), or with receptor antagonists for LTB4 and PAF, resulted in an inhibition of LT synthesis induced by calcium ionophore, A23187. This inhibition was most marked at submaximal (100-300 nM) A23187 concentrations, whilst it was least at ionophore concentrations which induce maximal LT synthesis (1-3 microM). Thus newly-synthesized PAF and LTB4 can enhance LT synthesis induced by A23187 under conditions where the LT-generating system is not fully activated. 3. In recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, LT synthesis in response to chemoattractants (fMet-Leu-Phe or rhC5a) was also significantly inhibited by the LTB4 receptor antagonist, and to a lesser extent by PAF receptor antagonists. 4. Further investigation revealed that LTB4 and/or PAF exert their effects on LT synthesis via an effect on arachidonic acid (AA) availability, as opposed to 5-lipoxygenase (5-LO) activation. Indeed, the receptor antagonists, as well as PT, inhibited LT synthesis and AA release to a similar extent, whereas 5-LO activation (assessed with an exogenous 5-LO substrate) was virtually unaffected under the same conditions. Accordingly, we showed that addition of exogenous LTB4 could enhance AA availability in response to chemoattractant challenge in rhGM-CSF-primed cells, without significantly affecting the 5-LO activation status.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8019762

  15. Constitutive production of multiple cytokines and a human chorionic gonadotrophin beta-subunit by a human bladder cancer cell line (KU-19-19): possible demonstration of totipotential differentiation.

    PubMed Central

    Tachibana, M.; Miyakawa, A.; Nakashima, J.; Murai, M.; Nakamura, K.; Kubo, A.; Hata, J. I.

    1997-01-01

    Bladder cancer cells have been shown to secrete a variety of factors that are not related to cells of urothelial origin. The histogenesis of these tumour developments is uncertain, and a variety of theories have been previously reported. In the present manuscript, we identify the factors constitutively produced by a human bladder cancer cell line (KU-19-19) that was found to produce beta human chorionic gonadotrophin (beta-hCG), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1alpha (IL-1alpha), interleukin 6 (IL-6) and interleukin 8 (IL-8). The cells were obtained from a case of metastatic carcinoma that was originally diagnosed to be a grade 3 (WHO classification), invasive transitional cell carcinoma of the bladder. On microscopic observation, the cultured cells exhibited an epithelial appearance with vacuole formation in their cytoplasm. Ultrastructural observations revealed relatively marked microvilli and a tight junction. Significant amounts of beta-hCG, G-CSF, GM-CSF, IL-1alpha, IL-6 and IL-8 concentrations in the supernatant from cultured cells were demonstrated by enzyme-linked immunosorbent assays, while the expression of mRNA of these marker proteins in cancer cells was also significantly exhibited by reverse transcription polymerase chain reaction (RT-PCR). In addition, the expression of G-CSF receptor and IL-6 receptor mRNA was also shown by RT-PCR. Xenograft transplantability using nude mice was observed in association with the presence of severe neutrophilia in the peripheral blood. These results indicate that this cell line appears to be an effective model for the study of transitional cell carcinoma of the bladder with multipotent differentiation potentials. Images Figure 1 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:9231915

  16. The Inhibitory Effects of Anti-Oxidants on Ultraviolet-Induced Up-Regulation of the Wrinkling-Inducing Enzyme Neutral Endopeptidase in Human Fibroblasts.

    PubMed

    Nakajima, Hiroaki; Terazawa, Shuko; Niwano, Takao; Yamamoto, Yorihiro; Imokawa, Genji

    2016-01-01

    We recently reported that the over-expression of skin fibroblast-derived neutral endopeptidase (NEP) plays a pivotal role in impairing the three-dimensional architecture of dermal elastic fibers during the biological mechanism of ultraviolet (UV)-induced skin wrinkling. In that process, a UVB-associated epithelial-mesenchymal cytokine interaction as well as a direct UVA-induced cellular stimulation are associated with the up-regulation of NEP in human fibroblasts. In this study, we characterized the mode of action of ubiquinol10 which may abrogate the up-regulation of NEP by dermal fibroblasts, resulting in a reported in vivo anti-wrinkling action, and compared that with 3 other anti-oxidants, astaxanthin (AX), riboflavin (RF) and flavin mononucleotide (FMN). Post-irradiation treatment with all 4 of those anti-oxidants elicited an interrupting effect on the UVB-associated epithelial-mesenchymal cytokine interaction leading to the up-regulation of NEP in human fibroblasts but with different modes of action. While AX mainly served as an inhibitor of the secretion of wrinkle-inducing cytokines, such as interleukin-1α (IL-1α) and granulocyte macrophage colony stimulatory factor (GM-CSF) in UVB-exposed epidermal keratinocytes, ubiquinol10, RF and FMN predominantly interrupted the IL-1α and GM-CSF-stimulated expression of NEP in dermal fibroblasts. On the other hand, as for the UVA-associated mechanism, similar to the abrogating effects reported for AX and FMN, ubiquinol10 but not RF had the potential to abrogate the increased expression of NEP and matrix-metalloproteinase-1 in UVA-exposed human fibroblasts. Our findings strongly support the in vivo anti-wrinkling effects of ubiquinol10 and AX on human and animal skin and provide convincing proof of the UV-induced wrinkling mechanism that essentially focuses on the over-expression of NEP by dermal fibroblasts as an intrinsic causative factor. PMID:27648570

  17. Systemic inflammatory responses in patients with type 2 diabetes with chronic periodontitis

    PubMed Central

    Mesia, Ruben; Gholami, Fatemeh; Huang, Hong; Clare-Salzler, Michael; Aukhil, Ikramuddin; Wallet, Shannon M; Shaddox, Luciana M

    2016-01-01

    Objective The objective of this case–control study was to quantify the immune responsiveness in individuals with type 2 diabetes (T2D) as compared with patients without diabetes (NT2D) diagnosed with periodontitis. Research Design and Methods Peripheral blood was collected from 20 patients with moderate-to-severe chronic periodontitis (10 T2D, 10 NT2D). Blood samples were stimulated with ultrapure Porphyromonas gingivalis and Escherichia coli lipopolysaccharide (LPS) for 24 hours. 14 cytokines/chemokines were quantified in culture supernatants using multiplex technology. Results T2D individuals demonstrated higher unstimulated levels of interleukin 6 (IL-6), IL-1β, tumor necrosis factor α, interferon γ, IL-10, IL-8, macrophage inflammatory protein 1α (MIP1α), and 1β (MIP1β), and higher stimulated levels of IL-6, IL-8, IL-10, MIP1α and MIP1β, along with lower unstimulated and stimulated levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) when compared with NT2D (p<0.05). Importantly, the LPS-induced levels of IL-6, IL-8, IL-10 and MIP1α strongly correlated with severity of disease, measured by pocket depths (PD), within the T2D group (r2≥0.7, p<0.05), but not within NT2D. Conclusions Among patients with chronic periodontitis, patients with T2D seem to have an enhanced LPS-induced immune responsiveness than individuals without diabetes, which correlates with periodontal disease severity, concomitant with a less robust GM-CSF response. This data may in part explain the higher predisposition to periodontitis in this population.

  18. Recombinant human interleukin-3: pharmacokinetics after intravenous and subcutaneous bolus injection and effects on granulocyte kinetics.

    PubMed

    Hovgaard, D J; Folke, M; Mortensen, B T; Nissen, N I

    1994-08-01

    The pharmacokinetics of E. coli derived recombinant human interleukin-3 (rhIL-3) was studied following intravenous (i.v.) and subcutaneous (s.c.) bolus injection of rhIL-3. After i.v. bolus injection in eight patients, serum peak levels of 34.5-135.0 ng/ml were reached, followed by a rapid decline with a t1/2 alpha of 17 +/- 2 min and a t1/2 beta of 59 +/- 7 min. After s.c. bolus injection in five patients, the absorption was more prolonged with peak serum levels reached at 2.8 +/- 0.4 h. Elimination was also more protracted, and serum base-line levels were reached at 14-24 h. The immediate effect of rhIL-3 on peripheral white blood cells was less pronounced and more variable than previously found for G- or GM-CSF. Following i.v. administration, neutrophils showed a moderate drop to median 64% of initial values (range 42-85%) at median 30 min after injection (range 15-60 min) followed by an increase at 24 h to 69-288% of initial values. Eosinophils dropped to a median nadir of 34% and then gradually increased to maximum values in the range 135-720% at 18-24 h. The effect of rhIL-3 was further examined following i.v. injection of autologous 111Indium-labelled granulocytes in six patients. In steady state, i.v. injection of rhIL-3 caused a moderate drop in 111Indium activity of peripheral blood within 20 min without tendency to subsequent recovery. No change occurred in the activity recorded over the lungs and liver. The activity over the spleen decreased moderately in two patients. These results are strikingly different from those previously obtained after i.v. injection of rhGM-CSF.

  19. Dendritic cell appearance and differentiation during early and late stages of rat stomach carcinogenesis.

    PubMed

    Takeuchi, Motoi; Yamamoto, Masami; Tatematsu, Masae; Miki, Kazumasa; Sakaki, Yoshiyuki; Furihata, Chie

    2002-08-01

    Dendritic cell appearance and differentiation during early and late stages of rat stomach carcinogenesis were studied in the pyloric mucosa. Young male rats were given drinking water with or without N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 100 mg/liter) for 14 days. Use of competitive RT-PCR and northern blotting showed that MNNG exposure induced 3- to 4-fold greater expression of the genes for integrin beta7 and integrin alphaE2 (identical with antigen OX-62, a dendritic cell marker), as well as three cytokines, IL-4, GM-CSF and TNFalpha, in the stomach pyloric mucosa of resistant Buffalo rats compared to sensitive ACI rats. These genes were minimally expressed in control animals. The results confirm the appearance of dendritic cells in the target pyloric mucosa and suggest the possibility that dendritic cell differentiation and maturation are induced by various cytokines, at least in Buffalo rats. Competitive RT-PCR showed expression of integrin alphaE2 and beta7, MHC class II-associated invariant chain (Ii), MHC class II, B7-1, CD28, GM-CSF and TNFalpha genes in all 12 examined stomach adenocarcinomas and adenomas induced in male Lewis and WKY rats with 30 weeks' MNNG exposure, suggesting the presence of dendritic cells in tumors. OX-62 staining and western blotting for OX-62 also confirmed the presence of dendritic cells in tumors. However, the population of dendritic cells in tumors was less than that in the pyloric mucosa after 14 days' MNNG exposure. The present results suggest that immune defense involving dendritic cells is marshaled from the very early initiation stage during rat stomach cancer development, but is downgraded in developed tumors.

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

    SciTech Connect

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

    1991-03-15

    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.

  1. A role for granulocyte-macrophage colony-stimulating factor in the regulation of CD8{sup +} T cell responses to rabies virus

    SciTech Connect

    Wanjalla, Celestine N.; Goldstein, Elizabeth F.; Wirblich, Christoph; Schnell, Matthias J.

    2012-05-10

    Inflammatory cytokines have a significant role in altering the innate and adaptive arms of immune responses. Here, we analyzed the effect of GM-CSF on a RABV-vaccine vector co-expressing HIV-1 Gag. To this end, we immunized mice with RABV expressing HIV-1 Gag and GM-CSF and analyzed the primary and recall CD8{sup +} T cell responses. We observed a statistically significant increase in antigen presenting cells (APCs) in the spleen and draining lymph nodes in response to GM-CSF. Despite the increase in APCs, the primary and memory anti HIV-1 CD8{sup +} T cell response was significantly lower. This was partly likely due to lower levels of proliferation in the spleen. Animals treated with GM-CSF neutralizing antibodies restored the CD8{sup +} T cell response. These data define a role of GM-CSF expression, in the regulation of the CD8{sup +} T cell immune responses against RABV and has implications in the use of GM-CSF as a molecular adjuvant in vaccine development.

  2. Cytokine-mediated cPLA(2) phosphorylation is regulated by multiple MAPK family members.

    PubMed

    Geijsen, N; Dijkers, P F; Lammers, J J; Koenderman, L; Coffer, P J

    2000-04-01

    Cytosolic phospholipase A(2) (cPLA(2)) plays a critical role in various neutrophil functions including the generation of leukotrienes and platelet-activating factor release. Enzyme activity is regulated both by translocation to the membrane in a Ca(2+)-dependent manner and serine phosphorylation by members of the mitogen-activated protein kinase (MAPK) family. In this report, we have investigated the role of granulocyte/macrophage colony-stimulating factor (GM-CSF)-mediated signalling pathways in the regulation of cPLA(2). GM-CSF-induced cPLA(2) phosphorylation was not affected by pharmacological inhibition of p38 MAPK, phosphatidylinositol 3-kinase or Src. However, inhibition of extracellular signal-regulated kinase (ERK) MAPK activation resulted in a partial inhibition of cPLA(2) phosphorylation, revealed in a slower onset of phosphorylation. A cell line stably transfected with the GM-CSF receptor was used to further analyze GM-CSF-mediated cPLA(2) phosphorylation. Mutation of tyrosine residues 577 and 612 resulted in a delayed cPLA(2) phosphorylation similar to the pharmacological ERK inhibition. Furthermore, inhibition of p38 MAPK in cells bearing the double mutant betac577/612 completely abrogated GM-CSF-induced cPLA(2) phosphorylation. We conclude that GM-CSF can mediate cPLA(2) phosphorylation through the redundant activation of both p38 and ERK MAP kinases.

  3. Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor

    SciTech Connect

    Messner, H.A.; Yamasaki, K.; Jamal, N.; Minden, M.M.; Yang, Y.C.; Wong, G.G.; Clark, S.C.

    1987-10-01

    Supernatants of COS-1 cells transfected with gibbon cDNA encoding interleukin 3 (IL-3) with homology to sequences for human IL-3 were tested for ability to promote growth of various human hemopoietic progenitors. The effect of these supernatants as a source of recombinant IL-3 was compared to that of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) as well as to that of medium conditioned by phytohemagglutinin-stimulated leukocytes. The frequency of multilineage colonies, erythroid bursts, and megakaryocyte colonies in cultures containing the COS-1 cell supernatant was equivalent to the frequency observed in the controls and significantly higher than found in cultures plated with recombinant GM-CSF. G-CSF did not support the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. In contrast, growth of granulocyte-macrophage colonies was best supported with GM-CSF, while recombinant IL-3 yielded colonies at lower or at best equivalent frequency. The simultaneous addition of higher concentrations of GM-CSF to cultures containing IL-3 in optimal amounts did not enhance the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. However, the frequency of such colonies and bursts increased with GM-CSF when cultures were plated with suboptimal concentrations of IL-3. Growth of colonies within the granulocyte-macrophage lineage is optimally supported by GM-CSF and does not increase with further addition of IL-3.

  4. Study on the anti-H1N1 virus effects of quercetin and oseltamivir and their mechanism related to TLR7 pathway.

    PubMed

    Chen, Chen; Jiang, Zhen-You; Yu, Bin; Wu, Xian-Lin; Dai, Cong-Qi; Zhao, Chang-Lin; Ju, Da-Hong; Chen, Xiao-Yin

    2012-01-01

    The antivirus effect of quercetin and oseltamivir on the Toll-like receptor 7 (TLR7) signaling pathway was observed when dendritic cells and macrophages were infected with H1N1. Leukomonocytes were obtained from umbilical cord blood and harvested after stimulation by recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (rhGM-CSF) and recombinant human Interleukin 4 (rhIL-4). Virus-infected cell model was established by human bronchial epithelial cells (16HBE) infected with H1N1. After immunological cells and virus-infected cells were co-cultured, quercetin and oseltamivir were also added into the medium as a treatment intervention. Then the immunological cells were collected for Real Time PCR (RT-PCR) and Western blot to determine the expression levels of genes related to TLR7 pathway. Viral infection led to cell death and increased the gene expression levels of TLR7 signal pathway. Quercetin and oseltamivir increased cell viability and reduced the expression levels of TLR7 signal pathway.

  5. Effects of histamine and its antagonists on murine T-cells and bone marrow-derived dendritic cells.

    PubMed

    Hu, Xiufen; Zafar, Mohammad Ishraq; Gao, Feng

    2015-01-01

    We determined the effects of histamine and its antagonists on the surface marker expression of dendritic cells (DCs) and the influence of lipopolysaccharide (LPS), histamine, and histamine receptor antagonists on DCs and T-cells. The bone marrow was extracted from the femurs and tibiae of 6- to 8-week-old female Balb/c mice and cultured in medium containing penicillin, streptomycin, L-glutamine, fetal calf serum, or granulocyte macrophage colony-stimulating factor (GM-CSF) alone or with interleukin (IL)-4. The cells received three different doses of LPS and histamine, plus three different doses of descarboethoxyloratadine (DCL). We assayed the supernatant for various cytokines. The spleen cells of DO11.10 mice were examined by flow cytometry, which included labeling and sorting CD4+ T-cells, as well as coculture of DCs and T-cells with ovalbumin (OVA)323-339 peptide. Histamine or histamine plus DCL did not affect the expression of major histocompatibility complex class II, CD11c, CD11b, CD86, and CD80. However, GM-CSF increased the expression of all markers except CD80. Histamine increased interferon-γ production in GM-CSF + IL-4-cultured cells; it also enhanced IL-10 production, but suppressed IL-12 production in LPS-stimulated DCs with no DCL. Cimetidine inhibited IL-10 production and restored IL-12 secretion in LPS-treated DCs. LPS increased IL-10 and decreased IL-12 levels. GM-CSF + IL-4-generated DCs had a stronger stimulatory effect on DO11.10 T-cell proliferation than GM-CSF-generated DCs. Inducible costimulator ligand expression was higher in GM-CSF + IL-4- than in GM-CSF-generated DC groups after 2 days of coculture, but decreased 4 days later. IL-13 production was higher in bone marrow DCs generated with GM-CSF than in those generated with GM-CSF + IL-4. OVA-pulsed DCs and OVA-plus-DCL DCs showed increased IL-12 levels. OVA plus LPS increased both IL-10 and interferon-α. Although histamine or histamine receptor-1 antagonists did not influence DC LPS

  6. Inactivated genotype 1 Japanese encephalitis vaccine for swine

    PubMed Central

    2014-01-01

    Purpose Japanese encephalitis is a reproductive disorder caused by Japanese encephalitis virus (JEV) in swine. Recent genotype (G) shift phenomenon (G3 to G1) in the Asia-wide has posed a challenge for proper prevention by the current vaccine strain. Thus, new kinds of JEV G1 vaccines with enhanced immunogenicity have been required for pigs. Materials and Methods Recombinant porcine granulocyte monocyte-colony stimulating factor (reporGM-CSF) protein was expressed in Spodoptera frugiperda (Sf-9) cells using baculovirus expression system. Two kinds of trials with inactivated JEV vaccines containing IMS1313 adjuvant (Seppic, France) were prepared with or without reporGM-CSF protein. Safety and immunogenicity of the pigs inoculated with the JEV vaccines via intramuscular route was evaluated for 28 days after inoculation. Results Mice, guinea pigs, and fattening pigs inoculated with the inactivated vaccine showed no signs for 14 and 21 days. Both hemagglutination inhibition and plaque reduction neutralizing antibody titers were significantly higher in pigs immunized with the vaccine containing reporGM-CSF protein after boosting. However, on the side of vaccine efficacy, most mice (87%) immunized with the inactivated JEV vaccine survived after virulent JEV challenge. Whereas the group with the vaccine containing reporGM-CSF protein showed lower protective effects than the vaccine alone for the biological activity of the GM-CSF depending on species specific. Conclusion Our data indicate that animals inoculated with the JEV vaccines was safe and pigs inoculated with inactivated JEV vaccine containing reporGM-CSF protein showed higher humoral immune responses than that of inactivated JEV vaccine without reporGM-CSF protein. PMID:25003095

  7. Differential augmentation of in vivo natural killer cytotoxicity in normal primates with recombinant human interleukin-1 and granulocyte-macrophage colony-stimulating factor.

    PubMed Central

    Davis, T A; Monroy, R L; Skelly, R R; Donahue, R E; MacVittie, T J

    1990-01-01

    The effect of recombinant human interleukin-1 (IL-1) alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and combined factor therapy (CFT) on Rhesus monkey peripheral blood natural killer (NK) activity in vivo was compared. During a 14-day treatment period, IL-1-treated animals demonstrated a 170% increase in NK activity against K562 target cells by day 4, reaching maximal levels (300%) at day 16, and returning to baseline by day 30. NK activity of GM-CSF-treated monkeys increased slightly (60-100%) during days 4-12, as did saline-treated monkeys, but returned to baseline values by day 16. A delayed increase in NK activity resulted after GM-CSF treatment, reaching a peak (260%) on day 23 and remaining elevated through day 39. CFT resulted in a bimodal response pattern, with two peaks of NK activity: one at day 16 and a second at day 39. The first peak of activity (223%) was significantly less than the activity attained with IL-1 alone; the second peak (300%) was of greater duration and occurred later than the peak observed in GM-CSF-treated monkeys. Unlike IL-1, GM-CSF treatment did not lead to a immediate stimulation of NK activity; augmentation was delayed by more than 7 days post treatment. CFT results suggest that GM-CSF reduced the direct NK response to IL-1; while IL-1 led to an enhanced delayed NK response. Therefore, IL-1 and GM-CSF augment NK activity through different but interrelated pathways. PMID:2180599

  8. Therapeutic effect of autologous dendritic cell vaccine on patients with chronic hepatitis B: A clinical study

    PubMed Central

    Chen, Min; Li, Yong-Guo; Zhang, Da-Zhi; Wang, Zhi-Yi; Zeng, Wei-Qun; Shi, Xiao-Feng; Guo, Yuan; Guo, Shu-Hua; Ren, Hong

    2005-01-01

    AIM: To investigate the therapeutic effect of autologous HBsAg-loaded dendritic cells (DCs) on patients with chronic hepatitis B. METHODS: Monocytes were isolated from fresh peripheral blood of 19 chronic HBV-infected patients by Ficoll-Hypaque density gradient centrifugation and cultured by plastic-adherence methods. DCs were induced and proliferated in the culture medium with recombinant human granulocyte-macrophage-colony- stimulating factor (rhGM-CSF) and human interleukin-4 (rhIL-4). DCs pulsed with HBsAg for twelve hours were injected into patients subcutaneously twice at intervals of two weeks. Two patients received 100 mg oral lamivudine daily for 12 mo at the same time. HBV-DNA and viral markers in sera of patients were tested every two months. RESULTS: By the end of 2003, 11 of 19 (57.9%) patients had a clinical response to DC-treatment. HBeAg of 10 (52.6%) patients became negative, and the copies of HBV-DNA decreased 101.77±2.39 averagely (t = 3.13, P<0.01).Two cases co-treated with DCs and lamivudine had a complete clinical response. There were no significant differences in the efficient rate between the cases with ALT level lower than 2×ULN and those with ALT level higher than 2×ULN before treatment (χ2 = 0.0026). CONCLUSION: Autologous DC-vaccine induced in vitro can effectively suppress HBV replication, reduce the virus load in sera, eliminate HBeAg and promote HBeAg/anti-HBe transformation. Not only the patients with high serum ALT levels but also those with normal ALT levels can respond to DC vaccine treatment, and the treatment combining DCs with lamivudine can eliminate viruses more effectively. PMID:15793869

  9. Endothelin receptor B protects granulocyte macrophage colony-stimulating factor mRNA from degradation.

    PubMed

    Jungck, David; Knobloch, Jürgen; Körber, Sandra; Lin, Yingfeng; Konradi, Jürgen; Yanik, Sarah; Stoelben, Erich; Koch, Andrea

    2015-06-01

    Evidence is lacking on the differential effects of the two therapeutic concepts of endothelin receptor antagonists (ERAs): the blockade of only the endothelin receptor A (ETAR; selective antagonism) versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker, are both available for therapy. We hypothesized that there are differences in the potential of ERAs to ameliorate inflammatory processes in human airway smooth muscle cells (HASMCs) and aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reverse-transcription polymerase chain reaction. Tumor necrosis factor α (TNFα) induced transcription and expression of chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), granulocyte macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 12 (MMP12) in HASMCs. In concentration-response experiments, bosentan led to a significantly greater reduction of GM-CSF and MMP12 protein release than ambrisentan, whereas there was no significant difference in their effect on GM-CSF and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA equally but had no effect on CXCL2. Blocking mitogen-activated protein kinases revealed that both ETAR and ETBR signal through p38 mitogen-activated protein kinase, but ETBR also signals through extracellular signal-regulated kinase (ERK) 1/2 to induce GM-CSF expression. In the presence of the transcription inhibitor actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF but not MMP12 or CXCL3 mRNA. In conclusion, blockade of each endothelin receptor subtype reduces GM-CSF transcription, but blocking ETBR additionally protects GM-CSF mRNA from degradation via ERK-1/2. Accordingly, blocking both ETAR and ETBR leads to a stronger reduction of TNFα-induced GM-CSF protein expression. This mechanism might be specific to GM-CSF. Our data stress the anti-inflammatory potential

  10. Granulocyte Macrophage-Colony Stimulating Factor-induced Zn Sequestration Enhances Macrophage Superoxide and Limits Intracellular Pathogen Survival

    PubMed Central

    Vignesh, Kavitha Subramanian; Landero Figueroa, Julio A.; Porollo, Aleksey; Caruso, Joseph A.; Deepe, George S.

    2013-01-01

    SUMMARY Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like Zn. The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7 and the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H+ channel function and triggered reactive oxygen species (ROS) generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function. PMID:24138881

  11. The hematopoietic cytokine granulocyte-macrophage colony stimulating factor is important for cognitive functions

    PubMed Central

    Krieger, Markus; Both, Martin; Kranig, Simon A.; Pitzer, Claudia; Klugmann, Matthias; Vogt, Gerhard; Draguhn, Andreas; Schneider, Armin

    2012-01-01

    We recently reported expression of hematopoietic growth factor GM-CSF and its receptor (GM-CSFR) in CNS neurons. Here we evaluated this system in learning and memory formation using GM-CSF deficient mice. In complementation, GM-CSF signalling was manipulated specifically in adult murine hippocampus by adeno-associated virus (AAV)-mediated GM-CSFR alpha overexpression or knock-down. GM-CSF ablation caused various hippocampus and amygdala-dependent deficits in spatial and fear memory while rendering intact basic parameters like motor function, inherent anxiety, and pain threshold levels. Corroborating these data, spatial memory of AAV-injected mice was positively correlated with GM-CSFRα expression levels. Hippocampal neurons of knock-out mice showed markedly pruned dendritic trees, reduced spine densities, and lower percentages of mature spines. Despite such morphological alterations, long-term potentiation (LTP) was unimpaired in the knock-out hippocampus. Collectively, these results suggest that GM-CSF signalling plays a major role in structural plasticity relevant to learning and memory. PMID:23019518

  12. Restoring cigarette smoke-induced impairment of efferocytosis in alveolar macrophages.

    PubMed

    Subramaniam, R; Mukherjee, S; Chen, H; Keshava, S; Neuenschwander, P; Shams, H

    2016-07-01

    Cigarette smoke has been associated with susceptibility to different pulmonary and airway diseases. Impaired alveolar macrophages (AMs) that are major phagocytes in the lung have been associated with patients with airway diseases and active smokers. In the current report, we show that exposure to second-hand cigarette smoke (SHS) significantly reduced efferocytosis in vivo. More importantly, delivery of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) to the alveolar space restored and refurbished the efferocytosis capability of AMs. Exposure to SHS significantly reduced expression of CD16/32 on AMs, and treatment with GM-CSF not only restored but also significantly increased the expression of CD16/32 on AMs. GM-CSF treatment increased uptake and digestion/removal of apoptotic cells by AMs. The latter was attributed to increased expression of Rab5 and Rab7. Increased efferocytosis of AMs was also tested in a disease condition. AMs from GM-CSF-treated, influenza-infected, SHS-exposed mice showed significantly better efferocytosis activity, and mice had significantly less morbidity compared with phosphate-buffered saline-treated group. GM-CSF-treated mice had increased amphiregulin levels in the lungs, which in addition to efferocytosis of AMs may have attributed to their protection against influenza. These results will have great implications for developing therapeutic approaches by harnessing mucosal innate immunity to treat lung and airway diseases and protect against pneumonia. PMID:26577570

  13. Treatment of methimazole-induced severe aplastic anemia with recombinant human granulocyte-monocyte colony-stimulating factor and glucocorticosteroids.

    PubMed

    López-Karpovitch, X; Ulloa-Aguirre, A; von Eiff, C; Hurtado-Monroy, R; Alanis, A

    1992-01-01

    The in vivo response to recombinant human granulocyte-monocyte colony-stimulating factor (rHu GM-CSF) in facilitating the reconstitution of granulomonopoiesis was evaluated in a patient with Graves' disease who developed severe aplastic anemia during methimazole therapy. After 10 days of treatment with rHu GM-CSF, the neutrophil and monocyte counts rose to 1.65 x 10(9)/l and 0.41 x 10(9)/l, respectively. However, the patient was still dependent on erythrocyte and platelet transfusions. Two days after rHu GM-CSF withdrawal, the neutrophil count dropped off to 0.41 x 10(9)/l.rHu GM-CSF was reinitiated for 2 days along with glucocorticosteroids. With this combined therapeutic approach, the neutrophil count returned to normal and remained stable, and both Hb and platelet values began to improve. It is concluded that the combination of rHu GM-CSF and glucocorticosteroids can be used as a therapeutic option that may lead to beneficial results in drug-induced aplastic anemia.

  14. Activation-dependent cell death of human monocytes is a novel mechanism of fine-tuning inflammation and autoimmunity.

    PubMed

    Däbritz, Jan; Weinhage, Toni; Varga, Georg; Wirth, Timo; Ehrchen, Jan M; Barczyk-Kahlert, Katarzyna; Roth, Johannes; Schwarz, Tobias; Foell, Dirk

    2016-08-01

    In patients with juvenile idiopathic arthritis (JIA), increased release of IFN-γ and GM-CSF in cells infiltrating synovial tissue can be a potent driver of monocyte activation. Given the fundamental role of monocyte activation in remodeling the early phases of inflammatory responses, here we analyze the GM-CSF/IFN-γ induced activity of human monocytes in such a situation in vitro and in vivo. Monocytes from healthy donors were isolated and stimulated with GM-CSF ± IFN-γ. Monocyte activation and death were analyzed by flow cytometry, immunofluorescence microscopy, ELISA, and qPCR. T-cell GM-CSF/IFN-γ expression and monocyte function were determined in synovial fluid and peripheral blood from 15 patients with active JIA and 21 healthy controls. Simultaneous treatment with GM-CSF and IFN-γ induces cell death of monocytes. This cell death is partly cathepsin B-associated and has morphological characteristics of necrosis. Monocytes responding to costimulation with strong proinflammatory activities are consequently eliminated. Monocytes surviving this form of hyperactivation retain normal cytokine production. Cathepsin B activity is increased in monocytes isolated from synovial fluid from patients with active arthritis. Our data suggest GM-CSF/IFN-γ induced cell death of monocytes as a novel mechanism to eliminate overactivated monocytes, thereby potentially balancing inflammation and autoimmunity in JIA. PMID:27159026

  15. Human conjunctival epithelial cell responses to platelet-activating factor (PAF): signal transduction and release of proinflammatory cytokines

    PubMed Central

    Xu, Shouxi; Hellberg, Peggy E.; Pang, Iok-Hou; Gamache, Daniel A.; Yanni, John M.

    2009-01-01

    Purpose The aims of the study were to characterize the signal transduction responses to platelet-activating factor (PAF) and to monitor the downstream effects of PAF on the production of proinflammatory cytokines in human conjunctival epithelial cells (HCECs). Methods The generation of inositol phosphates ([3H]IPs) from [3H]phosphoinositide (PI) hydrolysis and the mobilization of intracellular calcium ([Ca2+]i) were evaluated using ion exchange chromatography and Fura-2 fluorescence techniques, respectively. The production of the cytokines (interleukin-6 [IL-6], interleukin-8 [IL-8], and granulocyte macrophage colony-stimulating factor [GM-CSF]) from PAF-stimulated HCECs was quantified using specific ELISA assays. Specific PAF antagonists were used to study the pharmacological aspects of PAF actions in HCECs. Results PAF (100 nM) maximally stimulated PI turnover in HCECs by 2.3±0.02 fold (n=21) above basal levels and with a potency (EC50) of 5.9±1.7 nM (n=4). PAF or its stabilized analog, methyl carbamyl (mc)PAF (EC50=0.8 nM), rapidly mobilized [Ca2+]i, which peaked within 30–60 s and remained elevated for 3 min. PAF (10 nM–1 µM) stimulated the release of the proinflammatory cytokines, IL-6, IL-8, and GM-CSF, 1.4–3.5 fold above basal levels. The effects of PAF (100 nM) on PI turnover and [Ca2+]i were potently antagonized by the PAF antagonists, 1-o-hexadecyl-2-o-acetyl–sn-glycero-3-phospho (N,N,N-trimethyl) hexanolamine (IC50=0.69 µM; Ki=38 nM), methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethyl-pyridine-3,5-dicsrboxylate (PCA-42481; IC50=0.89 µM; Ki=50 nM), rac-3-(N-octadecylcarbomoyl)-2-methoxy) propyl-(2-thiazolioethyl) phosphate (CV-3988; IC50=13 µM; Ki=771 nM), and (+/−)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl (SM-10661; IC50=14 µM; Ki=789 nM [n=3 for each antagonist]). PAF-induced production of IL-6, IL-8, and GM-CSF from HCECs was also blocked by these PAF antagonists (IC50=4.6– 8.6 µM). Conclusions HCECs respond to PAF by

  16. The in vivo effects of interleukin-3 on histamine levels in non-Hodgkin's lymphoma patients.

    PubMed

    Hovgaard, D J; Stahl Skov, P; Nissen, N I

    1997-06-01

    Recombinant human Interleukin-3 (RhIL-3) is a haemopoietic growth factor with effect both on early and differentiated cells, such as eosinophils and basophils, and it also acts as a histamine-releasing agent. The purpose of the present study was to examine whether in vivo rhIL-3 administration after chemotherapy affected basophil histamine levels and whether a concordance between rhIL-3 induced histamine release and side effects during the treatment could be demonstrated. Thirty patients with non-Hodgkin's lymphoma entered the study. All patients received 6 courses of chemotherapy, rhIL-3 was administered subcutaneously once daily after the second and the fourth course of chemotherapy from cycle day 2-15 at the dose levels 0.5, 1.0, 5.0, 7.5 and 10 micrograms/kg with 6 patients at each dose level. In cycle 6 recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (rhGM-CSF) (3.0 micrograms/kg) was administered sequential/concurrent day 9-15 to rhIL-3 (day 2-15) at all dose levels except 7.5 micrograms/kg, where rhIL-3 was given day 2-8 and rhGM-CSF sequential day 9-15. Cycles 1, 3 and 5 served as control cycles with no cytokine therapy. During rhIL-3 treatment, and after CHOP chemotherapy, the basophil counts increased moderately especially during the recovery period day 15-22, and mainly at the two highest dose levels 7.5 and 10 micrograms/kg, but never exceeded the normal upper limit. Histamine levels in basophils were the same in patients before chemotherapy and healthy volunteers, and except from a trend to increased histamine level at 10 micrograms/kg on day 15, no difference was noted between rhIL-3 cycles and control cycles. Within 3-4 hr after rhIL-3 administration, a drop in histamine level in basophils was noted, which could be due to histamine-releasing properties of rhIL-3 as previously demonstrated by in vitro studies. No serious side effects were noted during the cytokine treatment, and despite that most patients had mild flushing of the

  17. The impact of weight loss on circulating cytokines in Beagle dogs.

    PubMed

    Bastien, Berenice C; Patil, Avinash; Satyaraj, Ebenezer

    2015-02-15

    Chronic low-grade inflammation in obesity is characterized by an increased production of pro-inflammatory and chemotactic cytokines that are contributing to insulin resistance and related co-morbidities. Cytokines act in networks and exhibit pleiotropic effects so we investigated the circulating levels of a wide array of cytokines (pro and anti-inflammatory, chemotactic and growth factors) in a canine model of weight loss. The dogs served as their own control in order to study the impact of weight loss independent of potential confounding factors, such as history of excess weight or gender. While low-grade inflammation had been previously investigated in obese dogs by measuring changes in adipokines, acute phase proteins and key pro-inflammatory cytokines, to the best of our knowledge this is the first study to evaluate how weight loss impacts a wide array of circulating cytokines. Eighteen overweight Beagle dogs were recruited (six spayed females and 12 neutered males), and none of them were grossly obese according to the body condition score (BCS). All the dogs reached an ideal weight by the end of the program. Parameters were assessed before (baseline), at mid-point (month 3) and at end-point (month 6). Plasma GM-CSF, IL-2, Il-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IFNγ, IP-10, TNFα, monocyte chemotactic protein 1 (MCP-1), keratinocyte chemokine (KC) were measured with canine multiplex immunoassays. Fat mass was assessed by dual energy X-ray absorption (DEXA). Several cytokines decreased throughout the weight loss program (p<0.01) and were correlated with the percentage of fat measured by DEXA (p<0.05): chemotactic (MCP-1), growth factors (GM-CSF, IL-7 and IL-2), and pro-inflammatory (KC and IL-18). We could not show trends for several cytokines, possibly because their level may be lower than the assay sensitivity: anti-inflammatory (IL-4 and IL-10), and pro-inflammatory (IL-6 and TNFα). In conclusion, while our findings for several pro-inflammatory and

  18. Structural and Topographic Dynamics of Pulmonary Histopathology and Local Cytokine Profiles in Paracoccidioides brasiliensis Conidia-Infected Mice

    PubMed Central

    Cruz, Oswaldo G.; Restrepo, Angela; Cano, Luz Elena; Lenzi, Henrique Leonel

    2011-01-01

    Background Paracoccidioidomycosis (PCM), an endemic systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), usually results in severe lung damage in patients. Methods and Findings Considering the difficulties to sequentially study the infection in humans, this work was done in mice inoculated intranasally with infective Pb-conidia. Lungs of control and Pb-infected mice were studied after 2-hours, 4, 8, 12 and 16-weeks post-infection (p.i) in order to define histopathologic patterns of pulmonary lesions, multiplex-cytokine profiles and their dynamics during the course of this mycosis. Besides the nodular/granulomatous lesions previously informed, results revealed additional non-formerly described lung abnormalities, such as periarterial sheath inflammation and pseudotumoral masses. The following chronologic stages occurring during the course of the experimental infection were defined: Stage one (2-hours p.i): mild septal infiltration composed by neutrophils and macrophages accompanied by an intense “cytokine burst” represented by significant increases in IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL12p70, IL-13, IL-17, Eotaxin, G-CSF, MCP1, MIP1α, GM-CSF, IFN-γ, MIP1β and TNFα levels. Stage two (4-weeks p.i): presence of nodules, evidence of incipient periarterial- and intense but disperse parenchymal- inflammation, abnormalities that continued to be accompanied by hyper-secretion of those cytokines and chemokines mentioned in the first stage of infection. Stages three and four (8 and 12-weeks p.i.): fungal proliferation, inflammation and collagenesis reached their highest intensity with particular involvement of the periarterial space. Paradoxically, lung cytokines and chemokines were down-regulated with significant decreases in IL-2,IL-3,IL-5,IL-9,IL-13,IL-15,GM-CSF,IFN-γ,MIP1β and TNFα. Stage five (16-weeks p.i.): inflammation decreased becoming limited to the pseudotumoral masses and was accompanied by a “silent” cytokine response

  19. Lead effects on development and function of bone marrow-derived dendritic cells promote Th2 immune responses

    SciTech Connect

    Gao Donghong; Mondal, Tapan K.; Lawrence, David A. . E-mail: lawrencd@wadsworth.org

    2007-07-01

    Although lead (Pb) has significant effects on the development and function of macrophages, B cells, and T cells and has been suggested to promote allergic asthma in mice and humans, Pb modulation of bone marrow (BM)-derived dendritic cells (DCs) and the resultant DC effects on Th1 and Th2 development have not been examined. Accordingly, we cultured BM cells with murine granulocyte macrophage-colony stimulating factor (mGM-CSF) {+-} PbCl{sub 2}. At day 10, culture supernatant (SN) and non-adherent cells were harvested for analysis. Additionally, day 10 non-adherent BM-DCs were harvested and recultured with mGM-CSF + LPS {+-} Pb for 2 days. The day 10 Pb exposure significantly inhibited BM-DC generation, based on CD11c expression. Although fewer DCs were generated with Pb, the existing Pb-exposed DCs had significantly greater MHC-II expression than did the non-Pb-exposed DCs. However, these differences diminished upon LPS stimulation. After LPS stimulation, CD80, CD86, CD40, CD54, and MHC-II were all up-regulated on both Pb-DCs and DCs, but Pb-DCs expressed significantly less CD80 than did DCs. The CD86:CD80 ratio suggests a Pb-DC potential for Th2 cell development. After LPS stimulation, IL-6, IL-10, IL-12 (p70), and TNF-{alpha} levels significantly increased with both Pb-DCs and DCs, but Pb-DCs produced significantly less cytokines than did DCs, except for IL-10, which further supports Pb-DC preferential skewing toward type-2 immunity. In vitro studies confirm that Pb-DCs have the ability to polarize antigen-specific T cells to Th2 cells. Pb-DCs also enhanced allogeneic and autologous T cell proliferation in vitro, and in vivo studies suggested that Pb-DCs inhibited Th1 effects on humoral and cell-mediated immunity. The Pb effect was mainly on DCs, rather than on T cells, and Pb's modification of DC function appears to be the main cause of Pb's promotion of type-2-related immunity, which may relate to Pb's enhanced activation of the Erk/MAP kinase pathway.

  20. Evaluation of cytotoxic, genotoxic and inflammatory responses of nanoparticles from photocopiers in three human cell lines

    PubMed Central

    2013-01-01

    Background Photocopiers emit nanoparticles with complex chemical composition. Short-term exposures to modest nanoparticle concentrations triggered upper airway inflammation and oxidative stress in healthy human volunteers in a recent study. To further understand the toxicological properties of copier-emitted nanoparticles, we studied in-vitro their ability to induce cytotoxicity, pro-inflammatory cytokine release, DNA damage, and apoptosis in relevant human cell lines. Methods Three cell types were used: THP-1, primary human nasal- and small airway epithelial cells. Following collection in a large volume photocopy center, nanoparticles were extracted, dispersed and characterized in the cell culture medium. Cells were doped at 30, 100 and 300 μg/mL administered doses for up to 24 hrs. Estimated dose delivered to cells, was ~10% and 22% of the administered dose at 6 and 24 hrs, respectively. Gene expression analysis of key biomarkers was performed using real time quantitative PCR (RT-qPCR) in THP-1 cells at 5 μg nanoparticles/mL for 6-hr exposure for confirmation purposes. Results Multiple cytokines, GM-CSF, IL-1β, IL-6, IL-8, IFNγ, MCP-1, TNF-α and VEGF, were significantly elevated in THP-1 cells in a dose-dependent manner. Gene expression analysis confirmed up-regulation of the TNF-α gene in THP-1 cells, consistent with cytokine findings. In both primary epithelial cells, cytokines IL-8, VEGF, EGF, IL-1α, TNF-α, IL-6 and GM-CSF were significantly elevated. Apoptosis was induced in all cell lines in a dose-dependent manner, consistent with the significant up-regulation of key apoptosis-regulating genes P53 and Casp8 in THP-1 cells. No significant DNA damage was found at any concentration with the comet assay. Up-regulation of key DNA damage and repair genes, Ku70 and Rad51, were also observed in THP-1 cells, albeit not statistically significant. Significant up-regulation of the key gene HO1 for oxidative stress, implicates oxidative stress induced by

  1. Exposure to Palladium Nanoparticles Affects Serum Levels of Cytokines in Female Wistar Rats

    PubMed Central

    Iavicoli, Ivo; Fontana, Luca; Corbi, Maddalena; Leso, Veruscka; Marinaccio, Alessandro; Leopold, Kerstin; Schindl, Roland; Sgambato, Alessandro

    2015-01-01

    Background Information currently available on the impact of palladium on the immune system mainly derives from studies assessing the biological effects of palladium salts. However, in the last years, there has been a notable increase in occupational and environmental levels of fine and ultrafine palladium particles released from automobile catalytic converters, which may play a role in palladium sensitization. In this context, the evaluation of the possible effects exerted by palladium nanoparticles (Pd-NPs) on the immune system is essential to comprehensively assess palladium immunotoxic potential. Aim Therefore, the aim of this study was to investigate the effects of Pd-NPs on the immune system of female Wistar rats exposed to this xenobiotic for 14 days, by assessing possible quantitative changes in a number of cytokines: IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, INF-γ and TNF-α. Methods Twenty rats were randomly divided into four exposure groups and one of control. Animals were given a single tail vein injection of vehicle (control group) and different concentrations of Pd-NPs (0.012, 0.12, 1.2 and 12 μg/kg). A multiplex biometric enzyme linked immunosorbent assay was used to evaluate cytokine serum levels. Results The mean serum concentrations of all cytokines decreased after the administration of 0.012 μg/kg of Pd-NPs, whereas exceeded the control levels at higher exposure doses. The highest concentration of Pd-NPs (12 μg/kg) induced a significant increase of IL-1α, IL-4, IL-6, IL-10, IL-12, GM-CSF and INF-γ compared to controls. Discussion and Conclusions These results demonstrated that Pd-NP exposure can affect the immune response of rats inducing a stimulatory action that becomes significant at the highest administered dose. Our findings did not show an imbalance between cytokines produced by CD4+ T helper (Th) cells 1 and 2, thus suggesting a generalized stimulation of the immune system with a simultaneous activation and polarization of the

  2. Anti-inflammatory effects of myrtol standardized and other essential oils on alveolar macrophages from patients with chronic obstructive pulmonary disease

    PubMed Central

    2009-01-01

    Introduction Myrtol standardized is established in the treatment of acute and chronic bronchitis and sinusitis. It increases mucociliar clearance and has muco-secretolytic effects. Additional anti-inflammatory and antioxidative properties have been confirmed for Myrtol standardized, eucalyptus oil, and orange oil in several in vitro studies. Objective The aim of this study was to prove the ability of essential oils to reduce cytokines release and reactive oxygen species (ROS) production derived from ex vivo cultured alveolar macrophages. Material and methods Alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD, n = 26, GOLD III-IV) were pre-cultured with essential oils (10-3-10-8%) for 1 h and then stimulated with LPS (1 μg/ml). After 4 h and 20 h respectively a) cellular reactive oxygen species (ROS) using 2',7'-dichlorofluorescein (DCF), and b) TNF-α, IL-8, and GM-CSF secretion were quantified. Results In comparison with negative controls, pre-cultured Myrtol, eucalyptus oil and orange oil (10-4%) reduced in the LPS-activated alveolar macrophages ROS release significantly after 1+20 h as follows: Myrtol - 17.7% (P = 0.05), eucalyptus oil -21.8% (P < 0.01) and orange oil -23.6% (P < 0.01). Anti-oxidative efficacy was comparable to NAC (1 mmol/l). Essential oils also induced a TNF-α reduction: Myrtol (-37.3%, P < 0.001), eucalyptus oil (-26.8%, P < 0.01) and orange oil (-26.6%, P < 0.01). TNF-α reduction at 1+4 h and 1+20 h did not vary (Myrtol: -31.9% and -37.3% respectively, P = 0.372) indicating that this effect occurs early and cannot be further stimulated. Myrtol reduced the release of GMCSF by -35.7% and that of IL-8 only inconsiderably. Conclusions All essential oils tested have effective antioxidative properties in ex vivo cultured and LPS-stimulated alveolar macrophages. Additionally, Myrtol inhibited TNF-α and GM-CSF release best indicating additional potent anti-inflammator y activity. PMID:20156758

  3. [The 72nd Annual Meeting Education Lecture. Cord factor].

    PubMed

    Yano, I

    1998-01-01

    IL-1, IFN-gamma, TNF-alpha, GM-CSF and chemotactic factor were observed and in vitro, TNF-alpha, GM-CSF, chemotactic factor, complement, NO, PGE2 inductions and protein kinase C activation were demonstrated. Furthermore, recently, we have demonstrated that cord factor induced a marked thymic atrophy due to the cortical lymphocyte apoptosis before granuloma formation in mice. It was also established that cord factor showed antigenicity in mice and rabbits and human tuberculous patient sera contained specific antibody (IgG) reactive against cord factor. From above results, cord factor seems to be one of the most potent immunomodulators in the mycobacterial cell wall components pathologically and beneficially.

  4. Influence of malignant cell clonogenic capacities and position along the maturation pathway on their susceptibility to lymphokine-activated killer cell cytotoxicity.

    PubMed

    Thomas, X; Anglaret, B; Adeleine, P; Maritaz, O; Bailly, M; Fiere, D; Archimbaud, E

    1998-01-01

    In order to investigate the sensitivity of malignant target cells to lysis by LAK cells according to their clonogenic capacities and their position along the maturation pathway, we compared clonogenic and chromium release cytotoxicity assays performed on human hematopoietic cell lines using Effector: Target ratios of 1:1, 3:1, 6:1, 12:1, 24:1, 48:1 and 96:1, and studied the sensitivity of HL-60 and U937 human cell lines after exposure to different factors including GM-CSF, SCF, IFN, Retinoic acid (RA), DMSO, and TPA which are able to recruit cells into the cell cycle or to induce cell differentiation. There was a good correlation between the lysis of the target cells using 51Cr release and the growth inhibition in semisolid medium. The degree of inhibition was significantly higher using the colony growth assay (p = 0.006). Regarding the effects of culturing cell lines with proliferating and differentiating agents on the sensitivity of these cell lines to LAK cytolysis, a correlation was noted between the proliferative response of the U937 cell line and susceptibility to LAK cell lysis (p = 0.01), while results appeared close to significance with HL-60. The most significant effects were a decreased sensitivity of HL-60 to LAK lysis with RA (p < 0.001) and TPA (p < 0.001), and an increased susceptibility of U937 to LAK lysis with GM-CSF (p < 0.0001). In studies planned to investigate whether susceptibility of treated cells to LAK activity was a consequence of a downregulation of adhesion molecules expressed on target cell surface, the proportion of cells expressing adhesion molecules was not significantly changed, except for CD54 expression on HL-60 cells which showed a higher expression, after cells were treated with RA or DMSO. We conclude that clonogenic cells are more sensitive to LAK cell lysis and that cell line sensitivity to LAK cytolysis can be modulated by a variety of agents of potential therapeutic use. The poor correlation between adhesion molecules

  5. Synergistic effects of p38 mitogen-activated protein kinase inhibition with a corticosteroid in alveolar macrophages from patients with chronic obstructive pulmonary disease.

    PubMed

    Armstrong, J; Harbron, C; Lea, S; Booth, G; Cadden, P; Wreggett, K A; Singh, D

    2011-09-01

    Corticosteroids partially suppress cytokine production by chronic obstructive pulmonary disease (COPD) alveolar macrophages. p38 mitogen-activated protein kinase (MAPK) inhibitors are a novel class of anti-inflammatory drug. We have studied the effects of combined treatment with a corticosteroid and a p38 MAPK inhibitor on cytokine production by COPD alveolar macrophages, with the aim of investigating dose-sparing and efficacy-enhancing effects. Alveolar macrophages from 10 patients with COPD, six smokers, and six nonsmokers were stimulated with lipopolysaccharide (LPS) after preincubation with five concentrations of dexamethasone alone, five concentrations of the p38 MAPK inhibitor 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB-796) alone, and all combinations of these concentrations. After 24 h, the supernatants were analyzed for interleukin (IL)-8, IL-6, tumor necrosis factor α (TNFα), granulocyte macrophage-colony-stimulating factor (GM-CSF), IL-1α, IL-1β, IL-1ra, IL-10, monocyte chemoattractant protein 3, macrophage-derived chemokine (MDC), and regulated on activation normal T cell expressed and secreted (RANTES). The effect of dexamethasone on p38 MAPK activation was analyzed by Western blotting. Dexamethasone and BIRB-796 both reduced LPS-induced cytokine production in a dose-dependent manner in all subject groups, with no differences between groups. Increasing the concentration of BIRB-796 in combination with dexamethasone produced progressively greater inhibition of cytokine production than dexamethasone alone. There were significant efficacy-enhancing benefits and synergistic dose-sparing effects (p < 0.05) for the combination treatment for IL-8, IL-6, TNFα, GM-CSF, IL-1ra, IL-10, MDC, and RANTES in one or more subject groups. Dexamethasone had no effect on LPS-induced p38 MAPK activation. We conclude that p38 MAPK activation in alveolar macrophages is corticosteroid-insensitive. Combining a p38

  6. Dendritic cell-based vaccines in the setting of peripheral blood stem cell transplantation: CD34+ cell-depleted mobilized peripheral blood can serve as a source of potent dendritic cells.

    PubMed

    Choi, D; Perrin, M; Hoffmann, S; Chang, A E; Ratanatharathorn, V; Uberti, J; McDonagh, K T; Mulé, J J

    1998-11-01

    We are investigating the use of tumor-pulsed dendritic cell (DC)-based vaccines in the treatment of patients with advanced cancer. In the current study, we evaluated the feasibility of obtaining both CD34+ hematopoietic stem/ progenitor cells (HSCs) and functional DCs from the same leukapheresis collection in adequate numbers for both peripheral blood stem cell transplantation (PBSCT) and immunization purposes, respectively. Leukapheresis collections of mobilized peripheral blood mononuclear cells (PBMCs) were obtained from normal donors receiving granulocyte colony-stimulating factor (G-CSF) (for allogeneic PBSCT) and from intermediate grade non-Hodgkin's lymphoma or multiple myeloma patients receiving cyclophosphamide plus G-CSF (for autologous PBSCT). High enrichment of CD34+ HSCs was obtained using an immunomagnetic bead cell separation device. After separation, the negative fraction of mobilized PBMCs from normal donors and cancer patients contained undetectable levels of CD34+ HSCs by flow cytometry. This fraction of cells was then subjected to plastic adherence, and the adherent cells were cultured for 7 days in GM-CSF (100 ng/ml) and interleukin 4 (50 ng/ml) followed by an additional 7 days in GM-CSF, interleukin 4, and tumor necrosis factor alpha (10 ng/ml) to generate DCs. Harvested DCs represented yields of 4.1+/-1.4 and 5.8+/-5.4% of the initial cells plated from the CD34+ cell-depleted mobilized PBMCs of normal donors and cancer patients, respectively, and displayed a high level expression of CD80, CD86, HLA-DR, and CD11c but not CD14. This phenotypic profile was similar to that of DCs derived from non-CD34+ cell-depleted mobilized PBMCs. DCs generated from CD34+ cell-depleted mobilized PBMCs elicited potent antitetanus as well as primary allogeneic T-cell proliferative responses in vitro, which were equivalent to DCs derived from non-CD34+ cell-depleted mobilized PBMCs. Collectively, these results demonstrate the feasibility of obtaining both DCs and

  7. Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice

    SciTech Connect

    Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.; Pinheiro-Torres, A.S.; Roncalho-Buck, I.A.; Squebola-Cola, D.M.; Mello, G.C.; Anhê, G.F.; Antunes, E.; DeSouza, I.A.

    2015-09-15

    Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. - Highlights: • Airway exposure to SEA causes acute lung inflammation. • SEA induces accumulation of bone marrow (BM) in immature and mature neutrophils. • SEA increases BM granulocyte or BM PMN adhesion to ICAM-1 and VCAM-1, and MAC-1 expression. • SEA induces BM elevations of CXCL-1, INF-γ, TNF-α, GM-CSF, G-CSF and

  8. Role of double-stranded RNA pattern recognition receptors in rhinovirus-induced airway epithelial cell responses

    PubMed Central

    Wang, Qiong; Nagarkar, Deepti R.; Bowman, Emily R.; Schneider, Dina; Gosangi, Babina; Lei, Jing; Zhao, Ying; McHenry, Christina L.; Burgens, Richai V.; Miller, David J.; Sajjan, Umadevi; Hershenson, Marc B.

    2010-01-01

    Rhinovirus (RV), a single-stranded RNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral double-stranded RNA produced during replication may be recognized by the host pattern recognition receptors Toll-like receptor (TLR)-3, retinoic acid inducible gene (RIG)-I and melanoma-differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV double-stranded (ds)-RNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and interferon (IFN) and IFN-stimulated gene expression determined by quantitative PCR. The separate requirements of RIG-I, MDA5 and IFN response factor (IRF)-3 were determined using their respective siRNAs. The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule TRIF. Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-β̤, IFN-λ̣1, IFN-λ2/3, IRF7, RIG-I, MDA5, IP-10/CXCL10, IL-8/CXCL8 and GM-CSF. siRNA against IRF3, MDA5 and TRIF, but not RIG-I, decreased RV1B-induced expression of IFN-β̤ IFN-λ̣1, IFN-λ2/3, IRF7, RIG-I, MDA5 and IP-10/CXCL10, but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA, and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3. PMID:19890046

  9. Ex Vivo Innate Immune Cytokine Signature of Enhanced Risk of Relapsing Brucellosis

    PubMed Central

    Feldman, Kristyn E.; Loriaux, Paul M.; Saito, Mayuko; Tuero, Iskra; Villaverde, Homarh; Siva, Tenaya; Gotuzzo, Eduardo; Gilman, Robert H.; Hoffmann, Alexander; Vinetz, Joseph M.

    2013-01-01

    Background Brucellosis, a zoonotic infection caused by one of the Gram-negative intracellular bacteria of the Brucella genus, is an ongoing public health problem in Perú. While most patients who receive standard antibiotic treatment recover, 5–40% suffer a brucellosis relapse. In this study, we examined the ex vivo immune cytokine profiles of recovered patients with a history of acute and relapsing brucellosis. Methodology/Principal Findings Blood was taken from healthy control donors, patients with a history of acute brucellosis, or patients with a history of relapsing brucellosis. Peripheral blood mononuclear cells were isolated and remained in culture without stimulation or were stimulated with a panel of toll-like receptor agonists or heat-killed Brucella melitensis (HKBM) isolates. Innate immune cytokine gene expression and protein secretion were measured by quantitative real-time polymerase chain reaction and a multiplex bead-based immunoassay, respectively. Acute and relapse patients demonstrated consistently elevated cytokine gene expression and secretion levels compared to controls. Notably, these include: basal and stimulus-induced expression of GM-CSF, TNF-α, and IFN-γ in response to LPS and HKBM; basal secretion of IL-6, IL-8, and TNF-α; and HKBM or Rev1-induced secretion of IL-1β, IL-2, GM-CSF, IFN-Υ, and TNF-α. Although acute and relapse patients were largely indistinguishable by their cytokine gene expression profiles, we identified a robust cytokine secretion signature that accurately discriminates acute from relapse patients. This signature consists of basal IL-6 secretion, IL-1β, IL-2, and TNF-α secretion in response to LPS and HKBM, and IFN-γ secretion in response to HKBM. Conclusions/Significance This work demonstrates that informative cytokine variations in brucellosis patients can be detected using an ex vivo assay system and used to identify patients with differing infection histories. Targeted diagnosis of this signature may

  10. Characterization of the growth-inhibitory and apoptosis-inducing activities of a triterpene saponin, securioside B against BAC1.2F5 macrophages.

    PubMed Central

    Yui, Satoru; Kudo, Tomoya; Hodono, Kazumi; Mimaki, Yoshihiro; Kuroda, Minpei; Sashida, Yutaka; Yamazaki, Masatoshi

    2003-01-01

    BACKGROUND: Since the growth state of macrophages in local pathological sites is considered a factor that regulates the processes of many disease, such as tumors, inflammation, and atherosclerosis, the substances that regulate macrophage growth or survival may be useful for disease control. We previously reported that securiosides A and B, novel triterpene saponins, exerted macrophage-oriented cytotoxicity in the presence of a L-cell-conditioned medium containing macrophage colony-stimulating factor (M-CSF), while the compounds did not exhibit an effect on macrophages in the absence of the growth-stimulating factors. AIM: This study was undertaken to characterize the growth-inhibitory and the apoptosis-inducing activities of securioside B, focusing on the effects of the macrophage-growth factor(s), and to examine the implication of a mitochondria pathway in apoptosis induction. METHODS: The effect of securioside B on a murine macrophage cell line (BAC1.2F5) was examined by MTT assay and lactose dehydrogenase release assay in the presence of L-cell-conditioned medium, M-CSF, or granulocyte-macrophage CSF (GM-CSF). RESULT: Securioside B inhibited the growth of the cells stimulated by recombinant M-CSF or GM-CSF, but it scarcely induced cytolysis of the cells under the same conditions. Moreover, securioside B did not induce cell death when the compound only was added to the cells. On the other hand, the compound extensively induced apoptotic cell death in the presence of L-cell-conditioned medium, suggesting that apoptosis induction by securioside B requires the additional factor(s) present in L-cell-conditioned medium. Securioside B plus L-cell-conditioned medium induced the activation of caspase-3 and caspase-9, but not caspase-8. In addition, cytochrome c release from the mitochondria into the cytosol, and disrupted mitochondria membrane potential, was also observed in the apoptotic BAC1.2F5 cells. CONCLUSION: These data suggest that securioside B has growth

  11. Reconstituted Human Upper Airway Epithelium as 3-D In Vitro Model for Nasal Polyposis

    PubMed Central

    de Borja Callejas, Francisco; Martínez-Antón, Asunción; Alobid, Isam; Fuentes, Mireya; Cortijo, Julio; Picado, César

    2014-01-01

    Background Primary human airway epithelial cells cultured in an air-liquid interface (ALI) develop a well-differentiated epithelium. However, neither characterization of mucociliar differentiation overtime nor the inflammatory function of reconstituted nasal polyp (NP) epithelia have been described. Objectives 1st) To develop and characterize the mucociliar differentiation overtime of human epithelial cells of chronic rhinosinusitis with nasal polyps (CRSwNP) in ALI culture system; 2nd) To corroborate that 3D in vitro model of NP reconstituted epithelium maintains, compared to control nasal mucosa (NM), an inflammatory function. Methods Epithelial cells were obtained from 9 NP and 7 control NM, and differentiated in ALI culture for 28 days. Mucociliary differentiation was characterized at different times (0, 7, 14, 21, and 28 days) using ultrastructure analysis by electron microscopy; ΔNp63 (basal stem/progenitor cell), β-tubulin IV (cilia), and MUC5AC (goblet cell) expression by immunocytochemistry; and mucous (MUC5AC, MUC5B) and serous (Lactoferrin) secretion by ELISA. Inflammatory function of ALI cultures (at days 0, 14, and 28) through cytokine (IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70) and chemokine (RANTES, MIG, MCP-1, IP-10, eotaxin-1, and GM-CSF) production was analysed by CBA (Cytometric Bead Array). Results In both NP and control NM ALI cultures, pseudostratified epithelium with ciliated, mucus-secreting, and basal cells were observed by electron microscopy at days 14 and 28. Displaying epithelial cell re-differentation, β-tubulin IV and MUC5AC positive cells increased, while ΔNp63 positive cells decreased overtime. No significant differences were found overtime in MUC5AC, MUC5B, and lactoferrin secretions between both ALI cultures. IL-8 and GM-CSF were significantly increased in NP compared to control NM regenerated epithelia. Conclusion Reconstituted epithelia from human NP epithelial cells cultured in ALI system provides a 3D in vitro model

  12. Vaccination with a mixed vaccine of autogenous and allogeneic breast cancer cells and tumor associated antigens CA15-3, CEA and CA125--results in immune and clinical responses in breast cancer patients.

    PubMed

    Jiang, X P; Yang, D C; Elliott, R L; Head, J F

    2000-10-01

    In breast cancer there is often overexpression of the breast cancer antigen CA15-3, the carcinoembryonic antigen (CEA) and the ovarian cancer antigen CA125, which makes them potential target antigens for immunotherapy. In this study, we used a multi-antigen vaccine, which included the following antigens: autologous breast cancer cells (AUTOC), allogeneic breast cancer MCF-7 cells (ALLOC), and the tumor associated antigens CA15-3, CEA and CA125, plus low doses of granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interleukin 2 (IL-2). Forty-two breast cancer patients received weekly subcutaneous vaccination at the 1st, 2nd, 3rd, 7th, 11th and 15th weeks. Their lymphocyte proliferative responses to AUTOC, ALLOC, CA15-3, CEA and CA125 were tested in lymphocyte blastogenesis assays (LBA) before and after vaccination. The disease stage and serum CA15-3, CEA and CA125 concentrations were also determined pre- and post-vaccination. We found that the vaccine was safe, and the only major side effects were swelling at the site of injection, muscle pain, and weakness or fatigue. The vaccine induced a significant increase in post-vaccination lymphocyte proliferative responses to AUTOC, CA15-3, CEA and CA125 but not ALLOC, compared to pre-vaccination (p < 0.05, p < 0.01, p < 0.05, p < 0.01 and p > 0.05, respectively, a paired t Test). Computed tomography (CT), ultrasound or bone scan showed evidence of disease improvement in 2 (12%) patients after vaccination. Hepatic metastases were reduced in size and number and some actually disappeared one patient. Metastatic disease in the L5 vertebra and the skull decreased in size and some osteolytic sites completely healed in a second patient. In addition, 7 patients (44%) had stable disease and 7 patients (44%) had disease progression. We did not find vaccination significantly reduced serum tumor markers CA15-3, CEA and CA125 of these breast cancer patients. These results suggest that the vaccine mixture of autologous and

  13. Th1, Th2 and Treg/T17 cytokines in two types of proliferative glomerulonephritis

    PubMed Central

    Stangou, M.; Bantis, C.; Skoularopoulou, M.; Korelidou, L.; Kouloukouriotou, D.; Scina, M.; Labropoulou, I. T.; Kouri, N. M.; Papagianni, A.; Efstratiadis, G.

    2016-01-01

    IgA nephropathy (IgAN) and focal segmental necrotizing glomerulonephritis (FSNGN) are characterized by proliferation of native glomerular cells and infiltration by inflammatory cells. Several cytokines act as mediators of kidney damage in both diseases. The aim of the present study was to investigate the role of Th1, Th2 and Treg/T17 cytokines in these types of proliferative glomerulonephritis. Simultaneous measurement of Th1 interleukin (IL-2, IL-12, tumor necrosis factor-alpha [TNF-α], interferon-gamma [INF-γ]), Th2 (IL-4, IL-5, IL-6, IL-10, IL-13), Treg/T17 transforming growth factor-beta 1 (TGF-β1, granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-17) cytokines and C-C chemokines Monocyte chemoattractant protein-1 (MCP-1, macrophage inflammatory protein-1 [MIP-1] β) was performed in first-morning urine samples, at the day of renal biopsy, using a multiplex cytokine assay. Cytokine concentrations were correlated with histological findings and renal function outcome. Urinary excretion of Th1, Th2 and Treg/Th17 cytokines were significantly higher in FSNGN compared to IgAN patients. In IgAN patients (n = 50, M/F: 36/14, M age: 40.7 [17–67] years), Th1, Th2 and T17 cytokines correlated significantly with the presence of endocapillary proliferation, while in FSNGN patients (n = 40, M/F: 24/16, M age: 56.5 [25–80] years), MCP-1 and TGF-β1 had a positive correlation with severe extracapillary proliferation (P = 0.001 and P = 0.002, respectively). Urinary IL-17 was the only independent parameter associated with endocapillary proliferation in IgAN and with MCP-1 urinary excretion in FSNGN. Response to treatment was mainly predicted by IL-6 in IgAN, and by Th2 (IL-4, IL-6), Treg (GM-CSF) cytokines and MIP-1 β in FSNGN. Th1, Th2 and T17 cytokines were directly implicated in renal pathology in IgAN and possibly through MCP-1 production in FSNGN. IL-17 and IL-6 seem to have a central role in inflammation and progression of kidney injury. PMID:27194829

  14. Role of PU.1 in MHC Class II Expression via CIITA Transcription in Plasmacytoid Dendritic Cells.

    PubMed

    Miura, Ryosuke; Kasakura, Kazumi; Nakano, Nobuhiro; Hara, Mutsuko; Maeda, Keiko; Okumura, Ko; Ogawa, Hideoki; Yashiro, Takuya; Nishiyama, Chiharu

    2016-01-01

    The cofactor CIITA is a master regulator of MHC class II expression and several transcription factors regulating the cell type-specific expression of CIITA have been identified. Although the MHC class II expression in plasmacytoid dendritic cells (pDCs) is also mediated by CIITA, the transcription factors involved in the CIITA expression in pDCs are largely unknown. In the present study, we analyzed the role of a hematopoietic lineage-specific transcription factor, PU.1, in CIITA transcription in pDCs. The introduction of PU.1 siRNA into mouse pDCs and a human pDC cell line, CAL-1, reduced the mRNA levels of MHC class II and CIITA. When the binding of PU.1 to the 3rd promoter of CIITA (pIII) in CAL-1 and mouse pDCs was analyzed by a chromatin immunoprecipitation assay, a significant amount of PU.1 binding to the pIII was detected, which was definitely decreased in PU.1 siRNA-transfected cells. Reporter assays showed that PU.1 knockdown reduced the pIII promoter activity and that three Ets-motifs in the human pIII promoter were candidates of cis-enhancing elements. By electrophoretic mobility shift assays, it was confirmed that two Ets-motifs, GGAA (-181/-178) and AGAA (-114/-111), among three candidates, were directly bound with PU.1. When mouse pDCs and CAL-1 cells were stimulated by GM-CSF, mRNA levels of PU.1, pIII-driven CIITA, total CIITA, MHC class II, and the amount of PU.1 binding to pIII were significantly increased. The GM-CSF-mediated up-regulation of these mRNAs was canceled in PU.1 siRNA-introduced cells. Taking these results together, we conclude that PU.1 transactivates the pIII through direct binding to Ets-motifs in the promoter in pDCs.

  15. THE IMPACT OF CONCURRENT GRANULOCYTE MACROPHAGE-COLONY STIMULATING FACTOR ON QUALITY OF LIFE IN HEAD AND NECK CANCER PATIENTS: RESULTS OF THE RANDOMIZED, PLACEBO-CONTROLED RADIATION THERAPY ONCOLOGY GROUP 9901 TRIAL

    PubMed Central

    Hoffman, Karen E.; Pugh, Stephanie; James, Jennifer L.; Scarantino, Charles; Movsas, Benjamin; Valicenti, Richard K.; Fortin, Andre; Pollock, JonDavid; Kim, Harold; Brachman, David G.; Berk, Lawrence B.; Bruner, Deborah Watkins; Kachnic, Lisa A.

    2015-01-01

    Purpose The Radiation Therapy Oncology Group (RTOG) conducted a randomized, placebo-controlled, trial evaluating the efficacy of GM-CSF in reducing mucosal injury and symptom burden from curative radiotherapy for head-and-neck (H&N) cancer. Methods Eligible patients with H&N cancer receiving radiation encompassing ≥ 50% of the oral cavity or oropharynx received subcutaneous GM-CSF or placebo. Quality of life (QoL) was assessed using the RTOG modified University of Washington H&N symptom questionnaire at baseline, 4, 13, 26 and 48 weeks from radiation initiation. Results Of 125 eligible patients, 114 were evaluable for QoL (58 GM-CSF, 56 placebo). Patient demographics, clinical characteristics, and baseline symptom scores were well balanced between the treatment arms. At the end of the acute period (13 weeks) patients in both arms reported negative change in total symptom score indicating increase in symptom burden relative to baseline (mean −18.4 GM-CSF, −20.8 placebo). There was no difference in change in total symptom score (p>0.05) or change in mucous, pain, eating, or activity domain scores (p>0.01) between patients in the GM-CSF and placebo arms. Analysis limited to patients treated per protocol or with an acceptable protocol deviation also found no difference in change in total symptom score (p>0.05) or change in domain scores (p>0.01) between treatment arms. Provider assessment of acute mucositis during treatment did not correlate with patient-reported mucous domain and total symptom scores (p>0.05) Conclusion GM-CSF administered concurrently during head-and-neck radiation does not appear to significantly improve patient-reported QoL symptom burden. PMID:24492945

  16. Secretion of cytokines by natural killer cells primed with interleukin-2 and stimulated with different lipoproteins.

    PubMed Central

    De Sanctis, J B; Blanca, I; Bianco, N E

    1997-01-01

    Natural killer (NK) cells were shown to secrete differentially interleukins (IL), IL-1 alpha, IL-1 beta, IL-2, IL-8, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukaemia inhibitory factor (LIF) upon stimulation with optimal concentrations of chylomicrons (CM), very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL) or acetyl-modified low-density lipoprotein (AcLDL). CM, VLDL, LDL and AcLDL induced LIF secretion which was absent in nonstimulated cells. CM, VLDL, and LDL did not affect IL-1 alpha secretion. CM stimulated IL-8 > TNF-alpha > IL-1 alpha > IL-2 = IFN-gamma, and decreased seventeen-fold GM-CSF secretion. VLDL stimulated IL-8 secretion > IL-1 alpha = IL-2 > IFN-gamma > TNF-alpha and decreased fivefold GM-CSF secretion. LDL stimulated IL-8 secretion > IL-1 alpha > IL-2 = IFN-gamma, it did not modify TNF-alpha and inhibited five hundred-fold GM-CSF secretion. HDL stimulated IL-2 secretion = IFN-gamma > IL-8, it decreased GM-CSF secretion > IL-1 alpha > IL-1 beta > TNF-alpha without affecting LIF. AcLDL stimulated IL-8 secretion > TNF-alpha > IL-1 alpha > IL-2 = IFN-gamma = IL-1 beta, and decreased GM-CSF secretion eightfold. When NK cells were primed with 10, 100 or 500 IU/ml of IL-2 before the addition of lipoproteins, a decrease in the secretion of cytokines was observed as compared with cells primed with IL-2 only. Differences in cytokine secretion were observed among the diverse type of lipoproteins used for cell stimulus. Thus, lipoproteins may condition NK cytokine secretion and cell activation. Images Figure 1 PMID:9176105

  17. Ca2+ ionophore A23187-dependent stabilization of granulocyte-macrophage colony-stimulating factor messenger RNA in murine thymoma EL-4 cells is mediated through two distinct regions in the 3'-untranslated region.

    PubMed

    Iwai, Y; Akahane, K; Pluznik, D H; Cohen, R B

    1993-05-15

    We analyze the role of the Ca2+ ionophore A23187 in the induction of GM-CSF mRNA expression in EL-4 thymoma cells. Northern analysis shows that A23187 increases the half-life of GM-CSF mRNA. To identify potential Ca2+ response elements in the GM-CSF mRNA, we produced stable transfectants containing pRSV-CAT (EL-4cat) or hybrid constructs in which most of the GM-CSF 3'-untranslated region (EL-4gm) or the adenosine-uridine boxes alone (EL-4au) were placed in a downstream position from the CAT coding region. A23187 induces a 4.4-fold increase in CAT activity in EL-4cat cells and a 210-fold and 48-fold increase in CAT activity in EL-4gm and EL-4au cells, respectively. Actinomycin D chase experiments in transfected cells demonstrate that A23187 increases the half-life of CAT mRNA from 15 min to 3 h in EL-4au cells and more than 3 h in EL-4gm cells, suggesting that the effect of Ca2+ is mediated predominantly by the adenosine-uridine boxes with a smaller contribution from upstream regions. To map these upstream regions, we transfected cells with constructs containing mutations of the 3'-untranslated region. With two of these mutations, corresponding to a region located about 160 bases upstream of the adenosine-uridine boxes, CAT activity was induced only 50-fold compared to 200-fold in EL-4gm cells. These data indicate that two regions within the GM-CSF 3'-untranslated region interact to modulate Ca2+ effects on GM-CSF mRNA half-life. PMID:8482841

  18. Kinetics of human hemopoietic cells after in vivo administration of granulocyte-macrophage colony-stimulating factor.

    PubMed Central

    Aglietta, M; Piacibello, W; Sanavio, F; Stacchini, A; Aprá, F; Schena, M; Mossetti, C; Carnino, F; Caligaris-Cappio, F; Gavosto, F

    1989-01-01

    The kinetic changes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) on hemopoietic cells were assessed in physiological conditions by administering GM-CSF (8 micrograms/kg per d) for 3 d to nine patients with solid tumors and normal bone marrow (BM), before chemotherapy. GM-CSF increased the number of circulating granulocytes and monocytes; platelets, erythrocytes, lymphocyte number, and subsets were unmodified. GM-CSF increased the percentage of BM S phase BFU-E (from 32 +/- 7 to 79 +/- 16%), day 14 colony-forming unit granulocyte-macrophage (CFU-GM) (from 43 +/- 20 to 82 +/- 11%) and day 7 CFU-GM (from 41 +/- 14 to 56 +/- 20%). The percentage of BM myeloblasts, promyelocytes, and myelocytes in S phase increased from 26 +/- 14 to 41 +/- 6%, and that of erythroblasts increased from 25 +/- 12 to 30 +/- 12%. This suggests that GM-CSF activates both erythroid and granulomonopoietic progenitors but that, among the morphologically recognizable BM precursors, only the granulomonopoietic lineage is a direct target of the molecule. GM-CSF increased the birth rate of cycling cells from 1.3 to 3.4 cells %/h and decreased the duration of the S phase from 14.3 to 9.1 h and the cell cycle time from 86 to 26 h. After treatment discontinuation, the number of circulating granulocytes and monocytes rapidly fell. The proportion of S phase BM cells dropped to values lower than pretreatment levels, suggesting a period of relative refractoriness to cell cycle-active antineoplastic agents. PMID:2643633

  19. Combined suicide and granulocyte-macrophage colony-stimulating factor gene therapy induces complete tumor regression and generates antitumor immunity.

    PubMed

    Jones, R K; Pope, I M; Kinsella, A R; Watson, A J; Christmas, S E

    2000-12-01

    The use of prodrug-activated ("suicide") gene therapy has been shown to be effective in inducing tumor regression when only a small proportion of tumor cells contains the suicide gene. These experiments were designed to test whether additional therapeutic benefit may be obtained by stimulating the immune response. Murine MC26 colon carcinoma cells, either untransduced or transduced with genes for herpes simplex virus-1 thymidine kinase (HSV1-TK) or human GM-CSF, were injected subcutaneously into syngeneic BALB/c mice in various combinations. Inoculation of equal numbers of untransduced and HSV1-TK-containing cells followed by ganciclovir (GCV) treatment resulted in almost complete tumor regression, but by 7 weeks, tumors had recurred in all mice. A similar initial regression was obtained using equal numbers of cells containing HSV1-TK and GM-CSF genes, but >80% of these mice remained tumor-free after 3 months. Groups of tumor-free mice that had received GM-CSF-containing cells were left for different periods of time and rechallenged with unmodified MC26 cells on the opposite flank. Of the mice rechallenged 14, 28, and 108 days later, 100%, 88%, and 57%, respectively, showed complete resistance to unmodified tumor cells. In mice that showed tumor regrowth, tumor volume was much less than in control mice. Adoptive transfer of spleen cells from resistant mice to naïve syngeneic mice resulted in partial resistance to challenge with unmodified tumor cells. Specific cytotoxicity against MC26 cells was only demonstrable in mice receiving GM-CSF- and HSV1-TK-containing tumor cells. These experiments show that the presence of cells secreting GM-CSF in HSV1-TK-containing, regressing tumor is able to induce complete or partial resistance to tumor rechallenge. This indicates the potential usefulness of GM-CSF in enhancing other antitumor therapies.

  20. Interleukin-10 inhibits burst-forming unit-erythroid growth by suppression of endogenous granulocyte-macrophage colony-stimulating factor production from T cells.

    PubMed

    Oehler, L; Kollars, M; Bohle, B; Berer, A; Reiter, E; Lechner, K; Geissler, K

    1999-02-01

    Numerous cytokines released from accessory cells have been shown to exert either stimulatory or inhibitory growth signals on burst-forming unit-erythroid (BFU-E) growth. Because of its cytokine synthesis-inhibiting effects on T cells and monocytes, interleukin-10 (IL-10) may be a potential candidate for indirectly affecting erythropoiesis. We investigated the effects of IL-10 on BFU-E growth from normal human peripheral blood mononuclear cells (PBMC) using a clonogenic progenitor cell assay. The addition of recombinant human IL-10 to cultures containing recombinant human erythropoietin suppressed BFU-E growth in a dose-dependent manner (by 55.2%, range 47.3-63.3%, p < 0.01, at 10 ng/mL). In contrast, no inhibitory effect of IL-10 was seen when cultivating highly enriched CD34+ cells. BFU-E growth from PBMC also was markedly suppressed in the presence of a neutralizing anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (by 48.7%, range 32.9-61.2% inhibition,p < 0.01), but not by neutralizing antibodies against granulocyte colony-stimulating factor and interleukin-3. This suggests a stimulatory role of endogenously released GM-CSF on BFU-E formation. Also, the addition of exogenous GM-CSF completely restored IL-10-induced suppression of BFU-E growth. To determine the cellular source of GM-CSF production, we analyzed GM-CSF levels in suspension cultures containing PBMC that were either depleted of monocytes or T cells. Monocyte-depleted PBMC showed spontaneous production of increasing amounts of GM-CSF on days 3, 5, and 7, respectively, which could be suppressed by IL-10, whereas GM-CSF levels did not increase in cultures containing T-cell-depleted PBMC. Our data indicate that IL-10 inhibits the growth of erythroid progenitor cells in vitro, most likely by suppression of endogenous GM-CSF production from T cells.

  1. Safety evaluation of intravenously administered mono-thioated aptamer against E-selectin in mice

    SciTech Connect

    Kang, Shin-Ae; Tsolmon, Bilegtsaikhan; Mann, Aman P.; Zheng, Wei; Zhao, Lichao; Zhao, Yan Daniel; Volk, David E.; Lokesh, Ganesh L.-R.; Morris, Lynsie; Gupta, Vineet; Razaq, Wajeeha; Rui, Hallgeir; Suh, K. Stephen; Gorenstein, David G.; Tanaka, Takemi

    2015-08-15

    The medical applications of aptamers have recently emerged. We developed an antagonistic thioaptamer (ESTA) against E-selectin. Previously, we showed that a single injection of ESTA at a dose of 100 μg inhibits breast cancer metastasis in mice through the functional blockade of E-selectin. In the present study, we evaluated the safety of different doses of intravenously administered ESTA in single-dose acute and repeat-dose subacute studies in ICR mice. Our data indicated that intravenous administration of up to 500 μg ESTA did not result in hematologic abnormality in either study. Additionally, intravenous injection of ESTA did not affect the levels of plasma cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF, IFN-γ, and TNF-α) or complement split products (C3a and C5a) in either study. However, repeated injections of ESTA slightly increased plasma ALT and AST activities, in accordance with the appearance of small necrotic areas in the liver. In conclusion, our data demonstrated that intravenous administration of ESTA does not cause overt hematologic, organs, and immunologic responses under the experimental conditions. - Highlights: • Intravenous administration of ESTA was well tolerated. • ESTA up to 500 μg does not cause hematologic, organs, and immunologic responses. • ESTA-mediated hepatic abnormality was considered minor.

  2. A locus regulating bronchial hyperresponsiveness maps to chromosome 5q

    SciTech Connect

    Levitt, R.C.; Meyers, D.A.; Bleecker, E.R.

    1994-09-01

    Bronchial hyperresponsiveness (BHR) is one of the hallmarks of asthma. BHR correlates well with asthmatic symptoms and the response to treatment. Moreover, BHR appears to be closely related to airways inflammation. Numerous studies have demonstrated a familial aggregation; however, this phenotype is not likely inherited as a simple Mendelian trait. BHR is also closely associated with total serum IgE levels, as are allergy and asthma. We studied 92 families from Northern Holland ascertained through a parent with asthma who were originally studied between 1962-1970. Since there are a number of candidate genes on chromosome 5q potentially important in producing BHR, families were genotyped for markers in this region. These genes regulate IgE production and the cellular elements that are likely involved in inflammation associated with BHR, allergy and asthma. They include IL-4, IL-3, IL-5, IL-9, IL-12, IL-13 and GM-CSF. Linkage of BHR with markers on 5q was tested using a model free sib-pair method. The data suggest a locus for BHR maps near the cytokine gene cluster on 5q. This region appears critical in producing susceptibility to BHR and possibly to asthma.

  3. A locus regulating total serum IgE maps to chromosome 5q

    SciTech Connect

    Amelung, P.J.; Panhuysen, C.I.M.; Postma, D.S. |

    1994-09-01

    Familial aggregation of allergy has been demonstrated in numerous past studies. However, allergy is a complex disorder which is not inherited as a simple Mendelian trait. Total serum IgE levels correlate with the clinical expression of allergy and asthma and can be utilized as a quantitative measure of the allergic phenotype. We studied 92 families from Northern Holland ascertained through a parent with asthma who were originally studied between 1962-1970. Since there is a large number of candidate genes on chromosome 5q, families were genotyped for markers in this region. These genes either directly or indirectly regulate IgE production and the activation and proliferation of cellular elements that are involved in inflammation associated with allergy and asthma. They include IL-4, IL-3, IL-5, IL-9, IL-12, IL-13 and GM-CSF. Segregation analyses revealed recessive inheritance of `high` levels with a mean for the `low` phenotype of 1.51 (32 IU) and 2.52 (331 IU) for the `high` phenotype. Linkage of log IgE with markers on 5q was tested using the sib-pair and the LOD score methods with the genetic model obtained from the segregation analyses. These results provide evidence for a locus controlling IgE levels near the cytokine gene cluster on 5q. This region appears critical in susceptibility to allergy and asthma.

  4. The vaccine-site microenvironment induced by injection of incomplete Freund's adjuvant, with or without melanoma peptides

    PubMed Central

    Harris, Rebecca C.; Chianese-Bullock, Kimberly A.; Petroni, Gina R.; Schaefer, Jochen T.; Brill, Louis B.; Molhoek, Kerrington R.; Deacon, Donna H.; Patterson, James W.; Slingluff, Craig L.

    2011-01-01

    Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in two clinical trials of melanoma patients. In one study (Mel38), patients received one injection with an adjuvant mixture alone, comprised of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature dendritic cells (DC) and vessels resembling high endothelial venules (HEV). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DC, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4+ and CD8+ T lymphocytes, as well as FoxP3+CD4+ lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination. PMID:22130163

  5. The vaccine-site microenvironment induced by injection of incomplete Freund's adjuvant, with or without melanoma peptides.

    PubMed

    Harris, Rebecca C; Chianese-Bullock, Kimberly A; Petroni, Gina R; Schaefer, Jochen T; Brill, Louis B; Molhoek, Kerrington R; Deacon, Donna H; Patterson, James W; Slingluff, Craig L

    2012-01-01

    Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell (DC) activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in 2 clinical trials of melanoma patients. In 1 study (Mel38), patients received 1 injection with an adjuvant mixture alone, composed of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature DCs, and vessels resembling high endothelial venules (HEVs). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DCs, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4and CD8 T lymphocytes, as well as FoxP3CD4 lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination. PMID:22130163

  6. Protein-based profiling of the immune response to uropathogenic Escherichia coli in adult patients immediately following hospital admission for acute cystitis.

    PubMed

    Sundac, Lana; Dando, Samantha J; Sullivan, Matthew J; Derrington, Petra; Gerrard, John; Ulett, Glen C

    2016-08-01

    Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are common infections in humans. Despite the substantial healthcare cost represented by these infections, the human immune response associated with the infection immediately following the onset of symptoms in patients remains largely undefined. We performed a prospective study aimed at defining the milieu of urinary cytokines in adult inpatients in the 24-48 h period immediately following hospital admission for acute cystitis due to UPEC. Urine samples, analyzed using 27-target multiplex protein assays, were used to generate immune profiles for patients and compared to age- and gender-matched healthy controls. The levels of multiple pro-inflammatory cytokines were significantly elevated in urine as a result of infection, an observation consistent with prior findings in murine models and clinical literature. We also identified significant responses for several novel factors not previously associated with the human response to UTI, including Interleukin (IL)-4, IL-7, IL-9, IL-17A, eotaxin, Granulocyte-macrophage colony-stimulating factor (GM-CSF) and several growth factors. These data establish crucial parallels between the human immune response to UPEC and murine model UTI studies, and emphasize the complex but poorly defined nature of the human immune response to UPEC, particularly in the immediate period following the onset of symptoms for acute cystitis. PMID:27354295

  7. Inflammatory and Oxidative Stress Responses of an Alveolar Epithelial Cell Line to Airborne Zinc Oxide Nanoparticles at the Air-Liquid Interface: A Comparison with Conventional, Submerged Cell-Culture Conditions

    PubMed Central

    Lenz, Anke-Gabriele; Karg, Erwin; Brendel, Ellen; Hinze-Heyn, Helga; Maier, Konrad L.; Eickelberg, Oliver; Stoeger, Tobias; Schmid, Otmar

    2013-01-01

    The biological effects of inhalable nanoparticles have been widely studied in vitro with pulmonary cells cultured under submerged and air-liquid interface (ALI) conditions. Submerged exposures are experimentally simpler, but ALI exposures are physiologically more realistic and hence potentially biologically more meaningful. In this study, we investigated the cellular response of human alveolar epithelial-like cells (A549) to airborne agglomerates of zinc oxide (ZnO) nanoparticles at the ALI, compared it to the response under submerged culture conditions, and provided a quantitative comparison with the literature data on different types of particles and cells. For ZnO nanoparticle doses of 0.7 and 2.5 μg ZnO/cm2 (or 0.09 and 0.33 cm2 ZnO/cm2), cell viability was not mitigated and no significant effects on the transcript levels of oxidative stress markers (HMOX1, SOD-2 and GCS) were observed. However, the transcript levels of proinflammatory markers (IL-8, IL-6, and GM-CSF) were induced to higher levels under ALI conditions. This is consistent with the literature data and it suggests that in vitro toxicity screening of nanoparticles with ALI cell culture systems may produce less false negative results than screening with submerged cell cultures. However, the database is currently too scarce to draw a definite conclusion on this issue. PMID:23484138

  8. Entamoeba histolytica trophozoites induce an inflammatory cytokine response by cultured human cells through the paracrine action of cytolytically released interleukin-1 alpha.

    PubMed Central

    Eckmann, L; Reed, S L; Smith, J R; Kagnoff, M F

    1995-01-01

    Infection with the protozoan parasite Entamoeba histolytica results in a high mortality worldwide. To initiate infection, E. histolytica trophozoites in the bowel lumen penetrate the epithelium, and cause extensive lysis of host cells. The acute amebic lesions in animal models are characterized by infiltration with inflammatory cells, particularly neutrophils. The acute host response is likely important for determining whether the infection will spread systemically, but little is known regarding the signals which initiate an acute inflammatory response to E. histolytica. In the studies reported herein, we used an in vitro model system to define the proinflammatory signals produced by epithelial and other host cells in response to infection with E. histolytica trophozoites. Coculture of human epithelial and stromal cells and cell lines with trophozoites is shown to increase expression and secretion of an array of chemoattractant and proinflammatory cytokines, including IL-8, GRO alpha, GM-CSF, IL-1 alpha, and IL-6. Moreover, high-level secretion of those cytokines is regulated by the paracrine action of cytolytically released IL-1 alpha. A second mechanism for trophozoite-induced IL-8 production involves trophozoite-target cell contact via a galactose-inhibitable amebic adherence protein, and appears to be mediated through increased intracellular calcium levels. These studies define novel mechanisms through which acute inflammation can be initiated in the host in response to a cytolytic pathogen, such as E. histolytica. PMID:7657801

  9. Peptide vaccines in breast cancer: The immunological basis for clinical response.

    PubMed

    Peres, Lívia de Paula; da Luz, Felipe Andrés Cordero; Pultz, Brunna dos Anjos; Brígido, Paula Cristina; de Araújo, Rogério Agenor; Goulart, Luiz Ricardo; Silva, Marcelo José Barbosa

    2015-12-01

    This review discusses peptide-based vaccines in breast cancer, immune responses and clinical outcomes, which include studies on animal models and phase I, phase I/II, phase II and phase III clinical trials. Peptide-based vaccines are powerful neoadjuvant immunotherapies that can directly target proteins expressed in tumor cells, mainly tumor-associated antigens (TAAs). The most common breast cancer TAA epitopes are derived from MUC1, HER2/neu and CEA proteins. Peptides derived from TAAs could be successfully used to elicit CD8 and CD4 T cell-specific responses. Thus, choosing peptides that adapt to natural variations of human leukocyte antigen (HLA) genes is critical. The most attractive advantage is that the target response is more specific and less toxic than for other therapies and vaccines. Prominent studies on NeuVax - E75 (epitope for HER2/neu and GM-CSF) in breast cancer and DPX-0907 (HLA-A2-TAAs) expressed in breast cancer, ovarian and prostate cancer have shown the efficacy of peptide-based vaccines as neoadjuvant immunotherapy against cancer. Future peptide vaccine strategies, although a challenge to be applied in a broad range of breast cancers, point to the development of degenerate multi-epitope immunogens against multiple targets.

  10. Multiple Costimulatory Modalities Enhance CTL Avidity

    PubMed Central

    Hodge, James W.; Chakraborty, Mala; Kudo-Saito, Chie; Garnett, Charlie T.; Schlom, Jeffrey

    2007-01-01

    Recent studies in both animal models and clinical trials have demonstrated that the avidity of T cells is a major determinant of anti-tumor and anti-viral immunity. Here, we evaluated several different vaccine strategies for their ability to enhance both the quantity and avidity of CTL responses. CD8+ T-cell quantity was measured by tetramer binding precursor frequency, and avidity was measured by both tetramer dissociation and quantitative cytolytic function. We have evaluated a peptide, a viral vector expressing the antigen transgene alone, with one costimulatory molecule (B7-1), and with three costimulatory molecules (B7-1, ICAM-1 and LFA-3), with anti-CTLA-4 mAb, with GM-CSF, and combinations of the above. We have evaluated these strategies in both a foreign antigen model employing β-gal as immunogen, and in a “self” antigen model, employing CEA as immunogen in CEA transgenic (Tg) mice. The combined use of several of these strategies was shown to enhance not only the quantity, but, to a greater magnitude, the avidity of T cells generated; a combination strategy is also shown to enhance anti-tumor effects. The studies reported here thus demonstrate multiple strategies that can be employed in both anti-tumor and anti-viral vaccine settings to generate higher avidity host T-cell responses. PMID:15879092

  11. Antitumor cell-complex vaccines employing genetically modified tumor cells and fibroblasts.

    PubMed

    Miguel, Antonio; Herrero, María José; Sendra, Luis; Botella, Rafael; Diaz, Ana; Algás, Rosa; Aliño, Salvador F

    2014-02-19

    The present study evaluates the immune response mediated by vaccination with cell complexes composed of irradiated B16 tumor cells and mouse fibroblasts genetically modified to produce GM-CSF. The animals were vaccinated with free B16 cells or cell complexes. We employed two gene plasmid constructions: one high producer (pMok) and a low producer (p2F). Tumor transplant was performed by injection of B16 tumor cells. Plasma levels of total IgG and its subtypes were measured by ELISA. Tumor volumes were measured and survival curves were obtained. The study resulted in a cell complex vaccine able to stimulate the immune system to produce specific anti-tumor membrane proteins (TMP) IgG. In the groups vaccinated with cells transfected with the low producer plasmid, IgG production was higher when we used free B16 cell rather than cell complexes. Nonspecific autoimmune response caused by cell complex was not greater than that induced by the tumor cells alone. Groups vaccinated with B16 transfected with low producer plasmid reached a tumor growth delay of 92% (p ≤ 0.01). When vaccinated with cell complex, the best group was that transfected with high producer plasmid, reaching a tumor growth inhibition of 56% (p ≤ 0.05). Significant survival (40%) was only observed in the groups vaccinated with free transfected B16 cells.

  12. Antitumor Cell-Complex Vaccines Employing Genetically Modified Tumor Cells and Fibroblasts

    PubMed Central

    Miguel, Antonio; Herrero, María José; Sendra, Luis; Botella, Rafael; Diaz, Ana; Algás, Rosa; Aliño, Salvador F.

    2014-01-01

    The present study evaluates the immune response mediated by vaccination with cell complexes composed of irradiated B16 tumor cells and mouse fibroblasts genetically modified to produce GM-CSF. The animals were vaccinated with free B16 cells or cell complexes. We employed two gene plasmid constructions: one high producer (pMok) and a low producer (p2F). Tumor transplant was performed by injection of B16 tumor cells. Plasma levels of total IgG and its subtypes were measured by ELISA. Tumor volumes were measured and survival curves were obtained. The study resulted in a cell complex vaccine able to stimulate the immune system to produce specific anti-tumor membrane proteins (TMP) IgG. In the groups vaccinated with cells transfected with the low producer plasmid, IgG production was higher when we used free B16 cell rather than cell complexes. Nonspecific autoimmune response caused by cell complex was not greater than that induced by the tumor cells alone. Groups vaccinated with B16 transfected with low producer plasmid reached a tumor growth delay of 92% (p ≤ 0.01). When vaccinated with cell complex, the best group was that transfected with high producer plasmid, reaching a tumor growth inhibition of 56% (p ≤ 0.05). Significant survival (40%) was only observed in the groups vaccinated with free transfected B16 cells. PMID:24556729

  13. The long polar fimbriae of STEC O157:H7 induce expression of pro-inflammatory markers by intestinal epithelial cells.

    PubMed

    Farfan, Mauricio J; Cantero, Lidia; Vergara, Alejandra; Vidal, Roberto; Torres, Alfredo G

    2013-03-15

    Infection with Shiga toxin-producing Escherichia coli (STEC) O157:H7 is characterized by acute inflammation of the colonic mucosa. STEC O157:H7 contains two non-identical loci encoding long polar fimbriae (Lpf), which play a role in the STEC colonization of the intestinal epithelial cells. However, no information is available regarding the involvement of Lpf in the STEC-induced host inflammatory response. Hence, in this study we assess the role of Lpf as an inducer of inflammation on intestinal epithelial cells. Secretion of pro-inflammatory cytokines in response to STEC wild type and lpf isogenic mutants was evaluated on intestinal T84 cells. Of the 27 cytokines assayed, IL-6, IL-8, IL-15, FGF, GM-CSF and IP-10 were significantly reduced, when compared to the wild-type strain, in the lpfA1 lpfA2 double mutant. Further, the host intracellular signaling pathways activated in response to Lpf were determined by using an array containing genes representative of 18 different signal transduction pathways. The analysis indicated that the NF-κB pathway is activated in response to Lpf-expressing STEC. Therefore, our study supports the role of Lpf as a STEC factor mediating intestinal inflammation.

  14. Therapeutic efficacy of PD-L1 blockade in a breast cancer model is enhanced by cellular vaccines expressing B7-1 and glycolipid-anchored IL-12

    PubMed Central

    Bozeman, Erica N; He, Sara; Shafizadeh, Yalda; Selvaraj, Periasamy

    2016-01-01

    Immunotherapeutic approaches have emerged as promising strategies to treat various cancers, including breast cancer. A single approach, however, is unlikely to effectively combat the complex, immune evasive strategies found within the tumor microenvironment, thus novel, effective combination treatments must be explored. In this study, we investigated the efficacy of a combination therapy consisting of PD-L1 immune checkpoint blockade and whole cell vaccination in a HER-2 positive mouse model of breast cancer. We demonstrate that tumorigenicity is completely abrogated when adjuvanted with immune stimulatory molecules (ISMs) B7-1 and a cell-surface anchored (GPI) form of IL-12 or GM-CSF. Irradiated cellular vaccines expressing the combination of adjuvants B7-1 and GPI-IL-12 completely inhibited tumor formation which was correlative with robust HER-2 specific CTL activity. However, in a therapeutic setting, both cellular vaccination and PD-L1 blockade induced only 10–20% tumor regression when administered alone but resulted in 50% tumor regression as a combination therapy. This protection was significantly hindered following CD4 or CD8 depletion indicating the essential role played by cellular immunity. Collectively, these pre-clinical studies provide a strong rationale for further investigation into the efficacy of combination therapy with tumor cell vaccines adjuvanted with membrane-anchored ISMs along with PD-L1 blockade for the treatment of breast cancer. PMID:26308597

  15. Cardiovascular risk in pulmonary alveolar proteinosis.

    PubMed

    Manali, Effrosyni D; Papadaki, Georgia; Konstantonis, Dimitrios; Tsangaris, Iraklis; Papaioannou, Andriana I; Kolilekas, Likurgos; Schams, Andrea; Kagouridis, Konstantinos; Karakatsani, Anna; Orfanos, Stylianos; Griese, Matthias; Papiris, Spyros A

    2016-02-01

    We hypothesized that cardiovascular events and/or indices of cardiac dysfunction may be increased in pulmonary alveolar proteinosis (PAP). Systemic and pulmonary arterial hypertension, arrhythmias, pulmonary embolism, stroke and ischemic heart attack were reported. Patients underwent serum anti-GM-CSF antibodies, disease severity score (DSS), Doppler transthoracic echocardiograph, glucose, thyroid hormones, lipids, troponin and pro-Brain natriuretic peptide (BNP) examination. Thirteen patients (8 female) were studied, median age of 47. Pro-BNP inversely related to DLCO% and TLC%; troponin directly related to DSS, age, P(A-a)O2, left atrium-, left ventricle-end-diastole diameter and BMI. On multiple regression analysis DSS was the only parameter significantly and strongly related with troponin (R(2) = 0.776, p = 0.007). No cardiovascular event was reported during follow-up. In PAP cardiovascular risk indices relate to lung disease severity. Therefore, PAP patients could be at increased risk for cardiovascular events. Quantitation of its magnitude and potential links to lungs' physiologic derangement will be addressed in future studies. PMID:26558331

  16. BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7

    SciTech Connect

    Goupille, Olivier; Penglong, Tipparat; Lefevre, Carine; Granger, Marine; Kadri, Zahra; Fucharoen, Suthat; Maouche-Chretien, Leila; Leboulch, Philippe; Chretien, Stany

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer UT7 erythroleukemia cells are known to be refractory to differentiate. Black-Right-Pointing-Pointer Brief JQ1 treatment initiates the first steps of erythroid differentiation program. Black-Right-Pointing-Pointer Engaged UT7 cells then maturate in the presence of erythropoietin. Black-Right-Pointing-Pointer Sustained JQ1 treatment inhibits both proliferation and erythroid differentiation. -- Abstract: Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

  17. IL-10 derived from CD1dhiCD5⁺ B cells regulates experimental autoimmune myasthenia gravis.

    PubMed

    Sheng, Jian Rong; Quan, Songhua; Soliven, Betty

    2015-12-15

    IL-10-competent subset within CD1d(hi)CD5(+) B cells, also known as B10 cells, has been shown to regulate autoimmune diseases. In our previous study, adoptive transfer of CD1d(hi)CD5(+) B cells expanded in vivo by GM-CSF prevented and suppressed experimental autoimmune myasthenia gravis (EAMG). The goal of this study was to further examine the role and mechanism of IL-10 in the regulatory function of B10 cells in EAMG. We found that only IL-10 competent CD1d(hi)CD5(+) B cells sorted from WT mice, but not IL-10 deficient CD1d(hi)CD5(+) B cells exhibited regulatory function in vitro and in vivo. Adoptive transfer of IL-10 competent CD1d(hi)CD5(+) B cells led to higher frequency of Tregs and B10 cells, and low levels of proinflammatory cytokines and autoantibody production. We conclude that IL-10 production within CD1d(hi)CD5(+) B cells plays an important role in immune regulation of EAMG. PMID:26616882

  18. Ex vivo generation of myeloid-derived suppressor cells that model the tumor immunosuppressive environment in colorectal cancer

    PubMed Central

    Dufait, Inès; Schwarze, Julia Katharina; Liechtenstein, Therese; Leonard, Wim; Jiang, Heng; Escors, David

    2015-01-01

    Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate in tumor-bearing subjects and which strongly inhibit anti-cancer immune responses. To study the biology of MDSC in colorectal cancer (CRC), we cultured bone marrow cells in conditioned medium from CT26 cells, which are genetically modified to secrete high levels of granulocyte-macrophage colony-stimulating factor. This resulted in the generation of high numbers of CD11b+ Ly6G+ granulocytic and CD11b+ Ly6C+ monocytic MDSC, which closely resemble those found within the tumor but not the spleen of CT26 tumor-bearing mice. Such MDSC potently inhibited T-cell responses in vitro, a process that could be reversed upon blocking of arginase-1 or inducible nitric oxide synthase (iNOS). We confirmed that inhibition of arginase-1 or iNOS in vivo resulted in the stimulation of cytotoxic T-cell responses. A delay in tumor growth was observed upon functional repression of both enzymes. These data confirm the role of MDSC as inhibitors of T-cell-mediated immune responses in CRC. Moreover, MDSC differentiated in vitro from bone marrow cells using conditioned medium of GM-CSF-secreting CT26 cells, represent a valuable platform to study/identify drugs that counteract MDSC activities. PMID:25869209

  19. Protein-based profiling of the immune response to uropathogenic Escherichia coli in adult patients immediately following hospital admission for acute cystitis.

    PubMed

    Sundac, Lana; Dando, Samantha J; Sullivan, Matthew J; Derrington, Petra; Gerrard, John; Ulett, Glen C

    2016-08-01

    Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are common infections in humans. Despite the substantial healthcare cost represented by these infections, the human immune response associated with the infection immediately following the onset of symptoms in patients remains largely undefined. We performed a prospective study aimed at defining the milieu of urinary cytokines in adult inpatients in the 24-48 h period immediately following hospital admission for acute cystitis due to UPEC. Urine samples, analyzed using 27-target multiplex protein assays, were used to generate immune profiles for patients and compared to age- and gender-matched healthy controls. The levels of multiple pro-inflammatory cytokines were significantly elevated in urine as a result of infection, an observation consistent with prior findings in murine models and clinical literature. We also identified significant responses for several novel factors not previously associated with the human response to UTI, including Interleukin (IL)-4, IL-7, IL-9, IL-17A, eotaxin, Granulocyte-macrophage colony-stimulating factor (GM-CSF) and several growth factors. These data establish crucial parallels between the human immune response to UPEC and murine model UTI studies, and emphasize the complex but poorly defined nature of the human immune response to UPEC, particularly in the immediate period following the onset of symptoms for acute cystitis.

  20. Acrolein induced both pulmonary inflammation and the death of lung epithelial cells.

    PubMed

    Sun, Yang; Ito, Sachiko; Nishio, Naomi; Tanaka, Yuriko; Chen, Nana; Isobe, Ken-Ichi

    2014-09-01

    Acrolein, a compound found in cigarette smoke, is a major risk factor for respiratory diseases. Previous research determined that both acrolein and cigarette smoke produced reactive oxygen species (ROS). As many types of pulmonary injuries are associated with inflammation, this study sought to ascertain the extent to which exposure to acrolein advanced inflammatory state in the lungs. Our results showed that intranasal exposure of mice to acrolein increased CD11c(+)F4/80(high) macrophages in the lungs and increased ROS formation via induction of NF-κB signaling. Treatment with acrolein activated macrophages and led to their increased production of ROS and expression of several key pro-inflammatory cytokines. In in vitro studies, acrolein treatment of bone marrow-derived GM-CSF-dependent immature macrophages (GM-IMs), activated the cells and led to their increased production of ROS and expression of several key pro-inflammatory cytokines. Acrolein treatment of macrophages induced apoptosis of lung epithelial cells. Inclusion of an inhibitor of ROS formation markedly decreased acrolein-mediated macrophage activation and reduced the extent of epithelial cell death. These results indicate that acrolein can cause lung damage, in great part by mediating the increased release of pro-inflammatory cytokines/factors by macrophages.

  1. Immunoprotective mechanisms in swine within the "grey zone" in antibody response after immunization with foot-and-mouth disease vaccine.

    PubMed

    Zhang, Liu; Feng, Xia; Jin, Ye; Ma, Junwu; Cai, Hu; Zhang, Xiaoxia

    2016-07-15

    Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals caused by the FMD virus (FMDV). Vaccination represents one approach for limiting the effects of FMD. The level of protection in vaccinated animals after challenge with foot and mouth disease virus (FMDV) is closely related to the antibody titer, which can be classified into three zones: a "white zone", a "grey zone", and a "black zone". The aim of the present study was to clarify the immunoprotective mechanisms operating in the grey zone, in which vaccinated animals have intermediate antibody titers, making it difficult to predict the level of protection. Thirty-three pigs were used to analyze the distribution of lymphocyte subpopulations in whole blood and the expression levels of 40 cytokines before vaccination and challenge. The antibody titer in pigs in the grey zone ranged from 1:6-1:45. Cytotoxic T lymphocyte subpopulations, expression levels of Th1 cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-12, IL-15, IL-18, and monocyte interferon gamma inducing factor (MIG), and of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1α, transforming growth factor-α (TGF-α), and TWEAK R varied between protected and unprotected animals. The results of this study suggest that the cellular immune response is the key factor responsible for immunoprotection in vaccinated animals with antibody titers within the grey zone. PMID:27067203

  2. Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events.

    PubMed

    Mihalache, Cristina C; Yousefi, Shida; Conus, Sébastien; Villiger, Peter M; Schneider, E Marion; Simon, Hans-Uwe

    2011-06-01

    The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

  3. Insulin-like growth factor-1 delays Fas-mediated apoptosis in human neutrophils through the phosphatidylinositol-3 kinase pathway.

    PubMed

    Himpe, Eddy; Degaillier, Céline; Coppens, Astrid; Kooijman, Ron

    2008-10-01

    Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We previously showed that insulin-like growth factor-1 (IGF1) delays spontaneous neutrophil apoptosis without influencing the secretion of cytokines by these cells. In the present study, we further addressed the role of IGF1 in regulating neutrophil survival in the presence of other factors present during inflammation, and the mechanism involved in delaying apoptosis. We show that IGF1 delays neutrophil apoptosis triggered by the agonistic anti-Fas antibody CH11 and that the effect of IGF1 is comparable in magnitude to that of the acknowledged anti-apoptotic cytokines interferon-gamma (IFNG) and granulocyte-macrophage colony-stimulating factor (GM-CSF; now known as CSF2). Furthermore, IGF1 exerted additional effects on cell survival in the presence of these cytokines. IGF1 did not affect Fas expression or activation by anti-Fas of caspase-8, but inhibited the depolarization of the mitochondrial membrane. Inhibitor studies indicate that the phosphatidylinositol-3 kinase (PI3K) pathway, but not the MEK-ERK pathway, mediates the effects of IGF1. However, in contrast to CSF2, IGF1 did not induce phosphorylation and translocation to the membrane of AKT, the canonical downstream target of PI3K. We therefore speculate that other downstream targets of PI3K are involved in the delay of neutrophil apoptosis by IGF1, possibly through stabilization of the mitochondrial membrane.

  4. A vaccine combining two Leishmania braziliensis proteins offers heterologous protection against Leishmania infantum infection.

    PubMed

    Duarte, Mariana C; Lage, Daniela P; Martins, Vívian T; Costa, Lourena E; Lage, Letícia M R; Carvalho, Ana Maria R S; Ludolf, Fernanda; Santos, Thaís T O; Roatt, Bruno M; Menezes-Souza, Daniel; Fernandes, Ana Paula; Tavares, Carlos A P; Coelho, Eduardo A F

    2016-08-01

    In the present study, two Leishmania braziliensis proteins, one hypothetical and the eukaryotic initiation factor 5a (EiF5a), were cloned and used as a polyproteins vaccine for the heterologous protection of BALB/c mice against infantum infection. Animals were immunized with the antigens separately or in association, and in both cases saponin was used as an adjuvant. In the results, spleen cells from mice inoculated with the individual or polyproteins vaccine and lately challenged produced significantly higher levels of protein- and parasite-specific IFN-γ, IL-12, and GM-CSF, when both a capture ELISA and flow cytometry assays were performed. Evaluating the parasite load by a limiting dilution as well as by RT-PCR, these animals presented significant reductions in the parasite number in all evaluated organs, when compared to the control (saline and saponin) groups. The best protection was reached when the polyproteins vaccine was employed. Protection was associated with the IFN-γ production against parasite extracts, which was mediated by both CD4(+) and CD8(+) T cells and correlated with the antileishmanial nitrite production. In this context, this vaccine combining two L. braziliensis proteins was able to induce a heterologous protection against VL, and could be considered in future studies to be tested against other Leishmania species or in other mammalian hosts.

  5. Host factors associated with serologic inflammatory markers assessed using multiplex assays.

    PubMed

    McKay, Heather S; Bream, Jay H; Margolick, Joseph B; Martínez-Maza, Otoniel; Phair, John P; Rinaldo, Charles R; Abraham, Alison G; Jacobson, Lisa P

    2016-09-01

    Chronic systemic inflammation contributes to the development of adverse health conditions, yet the influence of fixed and modifiable risk factors on many serologic biomarkers of inflammation remains largely unknown. Serum concentrations of twenty-three biomarkers, including C-reactive protein (CRP), cytokines (CXCL11, CXCL8, CXCL10, CCL2, CCL13, CCL4, CCL17, CXCL13, IL-10, IL-12p70, IL-6, TNF-α, IL-2, IFN-γ, IL-1β, GM-CSF, BAFF), and soluble immune receptors (sCD14, sIL-2Rα, sCD27, sgp130, sTNF-R2) were measured longitudinally using multiplexed immunometric assays in 250 HIV-uninfected men followed in the Multicenter AIDS Cohort Study (1984-2009). Generalized gamma regression was used to determine the statistical significance of factors associated with each biomarker. After accounting for age, race, and education, and for analysis of multiple biomarkers, higher concentrations of specific individual biomarkers were significantly (P<0.002) associated with hypertension, obesity, hepatitis C infection, stimulant use, and diabetes and lower concentrations with hypercholesterolemia. These associations should be taken into account in epidemiological studies of these biomarkers, and may provide potential targets for disease prevention and treatment. PMID:27295613

  6. A vaccine combining two Leishmania braziliensis proteins offers heterologous protection against Leishmania infantum infection.

    PubMed

    Duarte, Mariana C; Lage, Daniela P; Martins, Vívian T; Costa, Lourena E; Lage, Letícia M R; Carvalho, Ana Maria R S; Ludolf, Fernanda; Santos, Thaís T O; Roatt, Bruno M; Menezes-Souza, Daniel; Fernandes, Ana Paula; Tavares, Carlos A P; Coelho, Eduardo A F

    2016-08-01

    In the present study, two Leishmania braziliensis proteins, one hypothetical and the eukaryotic initiation factor 5a (EiF5a), were cloned and used as a polyproteins vaccine for the heterologous protection of BALB/c mice against infantum infection. Animals were immunized with the antigens separately or in association, and in both cases saponin was used as an adjuvant. In the results, spleen cells from mice inoculated with the individual or polyproteins vaccine and lately challenged produced significantly higher levels of protein- and parasite-specific IFN-γ, IL-12, and GM-CSF, when both a capture ELISA and flow cytometry assays were performed. Evaluating the parasite load by a limiting dilution as well as by RT-PCR, these animals presented significant reductions in the parasite number in all evaluated organs, when compared to the control (saline and saponin) groups. The best protection was reached when the polyproteins vaccine was employed. Protection was associated with the IFN-γ production against parasite extracts, which was mediated by both CD4(+) and CD8(+) T cells and correlated with the antileishmanial nitrite production. In this context, this vaccine combining two L. braziliensis proteins was able to induce a heterologous protection against VL, and could be considered in future studies to be tested against other Leishmania species or in other mammalian hosts. PMID:27387277

  7. The long polar fimbriae of STEC O157:H7 induce expression of pro-inflammatory markers by intestinal epithelial cells.

    PubMed

    Farfan, Mauricio J; Cantero, Lidia; Vergara, Alejandra; Vidal, Roberto; Torres, Alfredo G

    2013-03-15

    Infection with Shiga toxin-producing Escherichia coli (STEC) O157:H7 is characterized by acute inflammation of the colonic mucosa. STEC O157:H7 contains two non-identical loci encoding long polar fimbriae (Lpf), which play a role in the STEC colonization of the intestinal epithelial cells. However, no information is available regarding the involvement of Lpf in the STEC-induced host inflammatory response. Hence, in this study we assess the role of Lpf as an inducer of inflammation on intestinal epithelial cells. Secretion of pro-inflammatory cytokines in response to STEC wild type and lpf isogenic mutants was evaluated on intestinal T84 cells. Of the 27 cytokines assayed, IL-6, IL-8, IL-15, FGF, GM-CSF and IP-10 were significantly reduced, when compared to the wild-type strain, in the lpfA1 lpfA2 double mutant. Further, the host intracellular signaling pathways activated in response to Lpf were determined by using an array containing genes representative of 18 different signal transduction pathways. The analysis indicated that the NF-κB pathway is activated in response to Lpf-expressing STEC. Therefore, our study supports the role of Lpf as a STEC factor mediating intestinal inflammation. PMID:23078900

  8. Use of RDA analysis of knockout mice to identify myeloid genes regulated in vivo by PU.1 and C/EBPalpha.

    PubMed Central

    Iwama, A; Zhang, P; Darlington, G J; McKercher, S R; Maki, R; Tenen, D G

    1998-01-01

    PU.1 and C/EBPalpha are transcription factors essential for normal myeloid development. Loss-of-function mutation of PU.1 leads to an absolute block in monocyte/macrophage development and abnormal granulocytic development while that of C/EBPalpha causes a selective block in neutrophilic differentiation. In order to understand these phenotypes, we studied the role of PU.1 and C/EBPalpha in the regulation of myeloid target genes in vivo . Northern blot analysis revealed that mRNAs encoding receptors for M-CSF, G-CSF and GM-CSF, were expressed at low levels in PU.1(-/-) fetal liver compared with wild type. To identify additional myeloid genes regulated by PU.1 and C/EBPalpha, we performed representational difference analysis (RDA), a PCR-based subtractive hybridization using fetal livers from wild type and PU.1 or C/EBPalpha knockout mice. By introducing a new modification of RDA, that of tissue-specific gene suppression, we could selectively identify a set of differentially expressed genes specific to myeloid cells. Differentially expressed genes included both primary and secondary granule protein genes. In addition, eight novel genes were identified that were upregulated in expression during myeloid differentiation. These methods provide a general strategy for elucidating the genes affected in murine knockout models. PMID:9611252

  9. Artificial cytokine storm combined with hyperthermia induces significant anti-tumor effect in mice inoculated with lewis lung carcinoma and B16 melanoma cells.

    PubMed

    Kushida, Shigeki; Ohmae, Hiroshi; Kamma, Hiroshi; Totsuka, Rumiko; Matsumura, Masayuki; Takeuchi, Akira; Saiki, Ikuo; Yanagawa, Toru; Onizawa, Kojiro; Ishii, Tetsuro; Ohn, Tadao

    2006-12-01

    In cancer immunotherapies combined with hyperthermia, one or two cytokines have been tested to augment the anti-tumor effect. However, the therapies have not shown sufficient improvement. The aim of this study is to find a new potent tumor immunotherapy in order to augment antitumor effect of hyperthermia by the cytokine cocktails in vivo. We used a combination therapy of local hyperthermia (LH) and various cytokine cocktails composed of IFNs (IFN-alpha, -beta, and -gamma), Thl cytokines (IL-2, -12, -15, and -18), a Th2 cytokine (IL-4), inflammatory cytokines (IL-lalpha and TNF-alpha), and dendritic cell-inducible cytokines (IL-3 and GM-CSF). These cytokines in a proper combination augmented the anti-tumor effect of LH and prolonged survival time in Lewis lung carcinoma or B16 melanoma significantly. Moreover, the 12-cytokine cocktail suppressed B 16 metastasis to the lung and lymph nodes, and complete regression of the tumors without regrowth occurred in 3 of 5 mice. In the cured three B16 mice, there was hyperplasia of lymphatic organs with many CD3-positive T lymphocytes. The most effective cytokine combination should be able to augment the anti-tumor effect of other therapies besides hyperthermia that induce the necrosis of tumor cells.

  10. Induction of pro-inflammatory cytokine gene expression and apoptosis in human chorion cells of fetal membranes by influenza virus infection: possible implications for maintenance and interruption of pregnancy during infection.

    PubMed

    Uchide, Noboru; Ohyama, Kunio; Bessho, Toshio; Toyoda, Hiroo

    2005-01-01

    Human fetal membranes are composed of amnion, chorion and decidua tissues, which play a critical role in defense barriers as well as maintenance of pregnancy and parturition. Pro-inflammatory cytokines, such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha, produced by the tissues are postulated to facilitate parturition. Influenza virus infection is one of causes of pregnancy-associated complications, such as premature delivery, abortion and stillbirth. Recent studies have demonstrated that influenza virus infection induced the gene expression of a set of pro-inflammatory cytokines, such as IL-1beta, IL-6, TNF-alpha, interferon (IFN)-beta, IFN-gamma and granulocyte macrophage colony-stimulating factor (GM-CSF), and the secretion of unidentified monocyte differentiation-inducing factor(s) from primary cultured chorion cells undergoing apoptosis. These phenomena were not observed in primary cultured amnion cells infected with the virus. This article reviews, (1) the production of cytokines in fetal membrane tissues and their functions; (2) the differential induction of pro-inflammatory cytokine gene expression and apoptosis in fetal membrane chorion and amnion cells by influenza virus infection. An accumulating number of evidence suggests that interactive reactions between fetal membrane chorion cells and maternal monocytes/macrophages may play a critical role in defense barriers against the virus infection. Understanding the interactions would make important contributions to the elucidation of the pathogenesis of influenza virus infection during pregnancy. PMID:15614205

  11. Store-Operated Ca2+ Release-Activated Ca2+ Channels Regulate PAR2-Activated Ca2+ Signaling and Cytokine Production in Airway Epithelial Cells.

    PubMed

    Jairaman, Amit; Yamashita, Megumi; Schleimer, Robert P; Prakriya, Murali

    2015-09-01

    The G-protein-coupled protease-activated receptor 2 (PAR2) plays an important role in the pathogenesis of various inflammatory and auto-immune disorders. In airway epithelial cells (AECs), stimulation of PAR2 by allergens and proteases triggers the release of a host of inflammatory mediators to regulate bronchomotor tone and immune cell recruitment. Activation of PAR2 turns on several cell signaling pathways of which the mobilization of cytosolic Ca(2+) is likely a critical but poorly understood event. In this study, we show that Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by stromal interaction molecule 1 and Orai1 are a major route of Ca(2+) entry in primary human AECs and drive the Ca(2+) elevations seen in response to PAR2 activation. Activation of CRAC channels induces the production of several key inflammatory mediators from AECs including thymic stromal lymphopoietin, IL-6, and PGE2, in part through stimulation of gene expression via nuclear factor of activated T cells (NFAT). Furthermore, PAR2 stimulation induces the production of many key inflammatory mediators including PGE2, IL-6, IL-8, and GM-CSF in a CRAC channel-dependent manner. These findings indicate that CRAC channels are the primary mechanism for Ca(2+) influx in AECs and a vital checkpoint for the induction of PAR2-induced proinflammatory cytokines.

  12. Anti-Inflammatory Effect of Quercetin on RAW 264.7 Mouse Macrophages Induced with Polyinosinic-Polycytidylic Acid.

    PubMed

    Kim, Young-Jin; Park, Wansu

    2016-04-04

    Quercetin (3,3',4',5,6-pentahydroxyflavone) is a well-known antioxidant and a flavonol found in many fruits, leaves, and vegetables. Quercetin also has known anti-inflammatory effects on lipopolysaccharide-induced macrophages. However, the effects of quercetin on virus-induced macrophages have not been fully reported. In this study, the anti-inflammatory effect of quercetin on double-stranded RNA (dsRNA)-induced macrophages was examined. Quercetin at concentrations up to 50 μM significantly inhibited the production of NO, IL-6, MCP-1, IP-10, RANTES, GM-CSF, G-CSF, TNF-α, LIF, LIX, and VEGF as well as calcium release in dsRNA (50 μg/mL of polyinosinic-polycytidylic acid)-induced RAW 264.7 mouse macrophages (p < 0.05). Quercetin at concentrations up to 50 μM also significantly inhibited mRNA expression of signal transducer and activated transcription 1 (STAT1) and STAT3 in dsRNA-induced RAW 264.7 cells (p < 0.05). In conclusion, quercetin had alleviating effects on viral inflammation based on inhibition of NO, cytokines, chemokines, and growth factors in dsRNA-induced macrophages via the calcium-STAT pathway.

  13. Radioprotection with interleukin-1 comparison with other cytokines

    SciTech Connect

    Neta, R.; Oppenheim, J.J.; Douches, S.D.; Imbra, R.J.

    1986-01-01

    Murine interleukin IL-1alpha and human IL-1 alpha and IL-1beta protect mice from lethal effects of radiation-induced hematopoietic syndrome. Studies of possible mechanisms of radioprotection by IL -1 showed that radioprotection of LD 100/30 irradiated mice could not be induced with IL-2, IFN-gamma, or GM-CSF. Radioprotection with IL-1 did not depend on the release of prostaglandins, since indomethacin did not diminish survival of IL-1 treated mice. Unlike C57Bl/6 and DBA/1 mice, C3H/HeJ mice were not protected from lethal irradiation by IL-1. Nevertheless, both high-responder C57BL/6 and low-responder C3H/HeJ strains, treated with IL-1, develop similarly enhanced levels of acute-phase proteins: metallothionein and ceruloplasmin, with known radioprotective abilities. Therefore, the observed differences in radioprotection with IL-1 in murine strains probably cannot be attributed to differences in levels of these scavenging metalloproteins. Striking differences were observed in the recovery of bone-marrow cells after lethal and midlethal irradiations, the radioprotective effect of IL-1 was similar following sublethal irradiation of the two strains when recovery of endogenous hematopoietic splenic colonies was compared. IL-1 treatment, as is the case for many radioprotective agents, induces recovery of erytropoiesis.

  14. Dendritic cell profile induced by Schistosoma mansoni antigen in cutaneous leishmaniasis patients.

    PubMed

    Lopes, Diego Mota; Fernandes, Jamille Souza; Cardoso, Thiago Marconi de Souza; Bafica, Aline Michele Barbosa; Oliveira, Sérgio Costa; Carvalho, Edgar M; Araujo, Maria Ilma; Cardoso, Luciana Santos

    2014-01-01

    The inflammatory response in cutaneous leishmaniasis (CL), although responsible for controlling the infection, is associated with the pathogenesis of disease. Conversely, the immune response induced by S. mansoni antigens is able to prevent immune-mediated diseases. The aim of this study was to evaluate the potential of the S. mansoni Sm29 antigen to change the profile of monocyte-derived dendritic cells (MoDCs) from subjects with cutaneous leishmaniasis (CL) in vitro. Monocytes derived from the peripheral blood mononuclear cells of twelve patients were cultured with GM-CSF and IL-4 for differentiation into dendritic cells and then stimulated with soluble Leishmania antigen (SLA) in the presence or absence of Sm29 antigen. The expression of surface molecules associated with maturation and activation (HLA-DR, CD40, CD83, CD80, and CD86), inflammation (IL-12, TNF), and downregulation (IL-10, IL-10R) was evaluated using flow cytometry. We observed that the frequencies of HLA-DR, CD83, CD80, and CD86 as well as of IL-10 and IL-10R on MoDCs were higher in cultures stimulated with Sm29, compared to the unstimulated cell cultures. Our results indicate that the Sm29 antigen is able to activate regulatory MoDCs in patients with cutaneous leishmaniasis. It might be useful to control the inflammatory process associated with this disease.

  15. Microbe-specific unconventional T-cells induce human neutrophil differentiation into antigen cross-presenting cells

    PubMed Central

    Liuzzi, Anna Rita; Tyler, Christopher J.; Khan, Mohd Wajid A.; Szakmany, Tamas; Hall, Judith E.; Moser, Bernhard; Eberl, Matthias

    2014-01-01

    The early immune response to microbes is dominated by the recruitment of neutrophils whose primary function is to clear invading pathogens. However, there is emerging evidence that neutrophils play additional effector and regulatory roles. The present study demonstrates that human neutrophils assume antigen cross-presenting functions, and suggests a plausible scenario for the local generation of APC-like neutrophils through the mobilization of unconventional T-cells in response to microbial metabolites. Vγ9/Vδ2 T-cells and MAIT cells are abundant in blood, inflamed tissues and mucosal barriers. Here, both human cell types responded rapidly to neutrophils after phagocytosis of Gram-positive and Gram-negative bacteria producing the corresponding ligands, and in turn mediated the differentiation of neutrophils into APCs for both CD4+ and CD8+ T-cells through secretion of GM-CSF, IFN-γ and TNF-α. In patients with acute sepsis, circulating neutrophils displayed a similar APC-like phenotype and readily processed soluble proteins for cross-presentation of antigenic peptides to CD8+ T-cells, at a time when peripheral Vγ9/Vδ2 T-cells were highly activated. Our findings indicate that unconventional T-cells represent key controllers of neutrophil-driven innate and adaptive responses to a broad range of pathogens. PMID:25165152

  16. Polyfunctionality of a DKK1 self-antigen-specific CD8(+) T lymphocyte clone in lung cancer.

    PubMed

    Forget, Marie-Andrée; Reuben, Alexandre; Turcotte, Simon; Martin, Jocelyne; Lapointe, Réjean

    2011-08-01

    Polyfunctionality is the capacity of a T-cell to execute a variety of effector functions mainly mediated by production of cytokines, chemokines, and cytolytic enzymes. Studies in anti-viral immunity have acknowledged the importance of polyfunctionality in the clearance of infections and maintenance of protection. Although accepted in the field, this concept has not been as well characterized in cancer immunology. Here, we report the polyfunctionality profile analysis of a CD8(+) T-cell clone isolated from a lung cancer patient and directed against Dickkopf-1, a potentially new tumor-associated antigen (TAA). The clone showed Tc1/Th1 effector tendencies confirmed by secretion of cytokines such as IFN-γ, IP-10, MIP-1β, MIP-1α, IL-2, GM-CSF, and expression of cytolytic enzyme granzyme B. This secretion profile is of particular interest in the context of an anti-tumor response. Although secretion of IL-5 and IL-13 was also detected, absence of IL-4 and IL-10 opposes the idea of cytokine-dependent Th1 inhibition. Establishing a comprehensive cytokine secretion profile may help predict T cells' specific response against a novel TAA in a peptide vaccination context. It may further help in selecting clones with an optimal functional profile from the peripheral blood of cancer patients for expansion and adoptive cell transfer therapy.

  17. SPI-1-encoded type III secretion system of Salmonella enterica is required for the suppression of porcine alveolar macrophage cytokine expression.

    PubMed

    Pavlova, Barbora; Volf, Jiri; Ondrackova, Petra; Matiasovic, Jan; Stepanova, Hana; Crhanova, Magdalena; Karasova, Daniela; Faldyna, Martin; Rychlik, Ivan

    2011-01-24

    Genes localized at Salmonella pathogenicity island-1 (SPI-1) are involved in Salmonella enterica invasion of host non-professional phagocytes. Interestingly, in macrophages, SPI-1-encoded proteins, in addition to invasion, induce cell death via activation of caspase-1 which also cleaves proIL-1β and proIL-18, precursors of 2 proinflammatory cytokines. In this study we were therefore interested in whether SPI-1-encoded type III secretion system (T3SS) may influence proinflammatory response of macrophages. To test this hypothesis, we infected primary porcine alveolar macrophages with wild-type S. Typhimurium and S. Enteritidis and their isogenic SPI-1 deletion mutants. ΔSPI1 mutants of both serovars invaded approx. 5 times less efficiently than the wild-type strains and despite this, macrophages responded to the infection with ΔSPI1 mutants by increased expression of proinflammatory cytokines IL-1β, IL-8, TNFα, IL-23α and GM-CSF. Identical macrophage responses to that induced by the ΔSPI1 mutants were also observed to the infection with sipB but not the sipA mutant. The hilA mutant exhibited an intermediate phenotype between the ΔSPI1 mutant and the wild-type S. Enteritidis. Our results showed that the SPI-1-encoded T3SS is required not only for cell invasion but in macrophages also for the suppression of early proinflammatory cytokine expression.

  18. Heterogeneity of cytokine and growth factor gene expression in human melanoma cells with different metastatic potentials.

    PubMed

    Singh, R K; Gutman, M; Radinsky, R

    1995-01-01

    The purpose of this study was to determine the mRNA expression level of multiple cytokine and growth factor genes in human malignant melanoma. Melanoma cells were isolated from several surgical specimens, adapted to growth in culture, characterized for their ability to produce experimental metastases in nude mice, and assessed for cytokine and growth factor steady-state gene expression. Highly metastatic in vivo- and in vitro-derived variants isolated from a single melanoma, A375, were also analyzed. Northern blot analyses revealed that all melanomas analyzed constitutively expressed steady-state mRNA transcripts for the growth and angiogenic factors, basic fibroblast growth factor (bFGF), and transforming growth factor alpha (TGF-alpha), which correlated with metastatic propensity. Only one highly metastatic melanoma, TXM-1, originally isolated from a lymph node metastasis, expressed mRNA transcripts specific for monocyte chemotactic and activating factor (MCAF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Similarly, of the nine melanomas examined, only TXM-1 expressed interleukin (IL)-1 alpha, IL-1 beta, and IL-6, important immunomodulatory cytokines. These data demonstrate the differential and heterogeneous expression of cytokine and growth factor genes in human malignant melanoma. PMID:7648437

  19. Paracoccidioides brasiliensis Interferes on Dendritic Cells Maturation by Inhibiting PGE2 Production

    PubMed Central

    2015-01-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE2, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE2 inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-α. Assays using exogenous PGE2 confirmed an association between PGE2 inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus. PMID:25793979

  20. Structure-Based Design and Synthesis of 3-Amino-1,5-dihydro-4H-pyrazolopyridin-4-one Derivatives as Tyrosine Kinase 2 Inhibitors.

    PubMed

    Yogo, Takatoshi; Nagamiya, Hiroyuki; Seto, Masaki; Sasaki, Satoshi; Shih-Chung, Huang; Ohba, Yusuke; Tokunaga, Norihito; Lee, Gil Nam; Rhim, Chul Yun; Yoon, Cheol Hwan; Cho, Suk Young; Skene, Robert; Yamamoto, Syunsuke; Satou, Yousuke; Kuno, Masako; Miyazaki, Takahiro; Nakagawa, Hideyuki; Okabe, Atsutoshi; Marui, Shogo; Aso, Kazuyoshi; Yoshida, Masato

    2016-01-28

    We report herein the discovery and optimization of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one TYK2 inhibitors. High-throughput screening against TYK2 and JAK1-3 provided aminoindazole derivative 1 as a hit compound. Scaffold hopping of the aminoindazole core led to the discovery of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one derivative 3 as a novel chemotype of TYK2 inhibitors. Interestingly, initial SAR study suggested that this scaffold could have a vertically flipped binding mode, which prompted us to introduce a substituent at the 7-position as a moiety directed toward the solvent-exposed region. Introduction of a 1-methyl-3-pyrazolyl moiety at the 7-position resulted in a dramatic increase in TYK2 inhibitory activity, and further optimization led to the discovery of 20. Compound 20 inhibited IL-23-induced IL-22 production in a rat PD assay, as well as inhibited IL-23 signaling in human PBMC. Furthermore, 20 showed selectivity for IL-23 signaling inhibition against GM-CSF, demonstrating the unique cytokine selectivity of the novel TYK2 inhibitor. PMID:26701356

  1. Serum IL-10 from systemic lupus erythematosus patients suppresses the differentiation and function of monocyte-derived dendritic cells

    PubMed Central

    Sun, Zhida; Zhang, Rong; Wang, Huijuan; Jiang, Pengtao; Zhang, Jiangquan; Zhang, Mingshun; Gu, Lei; Yang, Xiaofan; Zhang, Miaojia; Ji, Xiaohui

    2012-01-01

    The role played by cytokines, other than interferon (IFN)-α, in the differentiation and function of dendritic cells (DCs) in systemic lupus erythematosus (SLE), remains unclear. Serum interleukin-10 (IL-10) levels are generally elevated in SLE patients, which might modulate the differentiation of DCs. In this study, DCs were induced from monocytes either by transendothelial trafficking or by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) + IL-4 + tumor necrosis factor (TNF)-α. Both systems were used to investigate the effects of elevated serum IL-10 level on DC differentiation in SLE patients. The results showed that monocyte-derived DCs induced by either SLE serum or exogenous IL-10 reduced the expression of human leukocyte antigen (HLA)-DR and CD80, decreased IL-12p40 level, and increased IL-10 level, and exhibited an impaired capacity to stimulate allogenic T-cell proliferation. These results indicate that serum IL-10 may be involved in the pathogenesis of SLE by modulating the differentiation and function of DCs. PMID:23554785

  2. The Influence of Ouabain on Human Dendritic Cells Maturation

    PubMed Central

    Nascimento, C. R.; Valente, R. C.; Echevarria-Lima, J.; Fontes, C. F. L.; de Araujo-Martins, L.; Araujo, E. G.; Rumjanek, V. M.

    2014-01-01

    Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance. PMID:25609892

  3. Dendritic Cell Profile Induced by Schistosoma mansoni Antigen in Cutaneous Leishmaniasis Patients

    PubMed Central

    Lopes, Diego Mota; Fernandes, Jamille Souza; Cardoso, Thiago Marconi de Souza; Bafica, Aline Michele Barbosa; Oliveira, Sérgio Costa; Carvalho, Edgar M.; Araujo, Maria Ilma; Cardoso, Luciana Santos

    2014-01-01

    The inflammatory response in cutaneous leishmaniasis (CL), although responsible for controlling the infection, is associated with the pathogenesis of disease. Conversely, the immune response induced by S. mansoni antigens is able to prevent immune-mediated diseases. The aim of this study was to evaluate the potential of the S. mansoni Sm29 antigen to change the profile of monocyte-derived dendritic cells (MoDCs) from subjects with cutaneous leishmaniasis (CL) in vitro. Monocytes derived from the peripheral blood mononuclear cells of twelve patients were cultured with GM-CSF and IL-4 for differentiation into dendritic cells and then stimulated with soluble Leishmania antigen (SLA) in the presence or absence of Sm29 antigen. The expression of surface molecules associated with maturation and activation (HLA-DR, CD40, CD83, CD80, and CD86), inflammation (IL-12, TNF), and downregulation (IL-10, IL-10R) was evaluated using flow cytometry. We observed that the frequencies of HLA-DR, CD83, CD80, and CD86 as well as of IL-10 and IL-10R on MoDCs were higher in cultures stimulated with Sm29, compared to the unstimulated cell cultures. Our results indicate that the Sm29 antigen is able to activate regulatory MoDCs in patients with cutaneous leishmaniasis. It might be useful to control the inflammatory process associated with this disease. PMID:25309922

  4. Managing Macrophages in Rheumatoid Arthritis by Reform or Removal

    PubMed Central

    Li, Jun; Hsu, Hui-Chen

    2013-01-01

    Macrophages play a central role in the pathogenesis of rheumatoid arthritis (RA). There is an imbalance of inflammatory and antiinflammatory macrophages in RA synovium. Although the polarization and heterogeneity of macrophages in RA have not been fully uncovered, the identity of macrophages in RA can potentially be defined by their products, including the co-stimulatory molecules, scavenger receptors, different cytokines/chemokines and receptors, and transcription factors. In the last decade, efforts to understand the polarization, apoptosis regulation, and novel signaling pathways in macrophages, as well as how distinct activated macrophages influence disease progression, have led to strategies that target macrophages with varied specificity and selectivity. Major targets that are related to macrophage development and apoptosis include TNF-α, IL-1, IL-6, GM-CSF, M-CSF, death receptor 5 (DR5), Fas, and others, as listed in Table 1. Combined data from clinical, preclinical, and animal studies of inhibitors of these targets have provided valuable insights into their roles in the disease progression and, subsequently, have led to the evolving therapeutic paradigms in RA. In this review, we propose that reestablishment of macrophage equilibrium by inhibiting the development of, and/or eliminating, the proinflammatory macrophages will be an effective therapeutic approach for RA and other autoimmune diseases. PMID:22855296

  5. Establishment and characterization of a new erythropoietin-dependent acute myeloid leukemia cell line, AS-E2.

    PubMed

    Miyazaki, Y; Kuriyama, K; Higuchi, M; Tsushima, H; Sohda, H; Imai, N; Saito, M; Kondo, T; Tomonaga, M

    1997-11-01

    We have established an erythropoietin-dependent human leukemia cell line, AS-E2, from a patient with acute myeloid leukemia. These cells have many characteristics of late erythroid progenitor cells, they are positive for CD36, Glycophorin A, and CD71 but negative for CD41, and positive for benzidine and PAS staining. These cells express GATA-1 and have low affinity erythropoietin (EPO) receptor on their surface. Interestingly, AS-E2 cells are strictly dependent on EPO for their growth and survival; other cytokines including GM-CSF, stem cell factor, or IL-3 cannot support the growth of this cell line. These features are similar to late erythroid lineage cells, like normal BFU-E or CFU-E, and we have demonstrated that EPO stimulation induces the tyrosine phosphorylation of several proteins in AS-E2 cells including the EPO receptor and JAK2 kinase. This new cell line is a useful reagent to study biological and molecular events during the late stages of erythropoiesis, and to understand transforming events in human erythroid cells.

  6. RXRα ablation in epidermal keratinocytes enhances UVR-induced DNA damage, apoptosis, and proliferation of keratinocytes and melanocytes.

    PubMed

    Wang, Zhixing; Coleman, Daniel J; Bajaj, Gaurav; Liang, Xiaobo; Ganguli-Indra, Gitali; Indra, Arup K

    2011-01-01

    We show here that keratinocytic nuclear receptor retinoid X receptor-α (RXRα) regulates mouse keratinocyte and melanocyte homeostasis following acute UVR. Keratinocytic RXRα has a protective role in UVR-induced keratinocyte and melanocyte proliferation/differentiation, oxidative stress-mediated DNA damage, and cellular apoptosis. We discovered that keratinocytic RXRα, in a cell-autonomous manner, regulates mitogenic growth responses in skin epidermis through secretion of heparin-binding EGF-like growth factor, GM-CSF, IL-1α, and cyclooxygenase-2 and activation of mitogen-activated protein kinase pathways. We identified altered expression of several keratinocyte-derived mitogenic paracrine growth factors such as endothelin 1, hepatocyte growth factor, α-melanocyte stimulating hormone, stem cell factor, and fibroblast growth factor-2 in skin of mice lacking RXRα in epidermal keratinocytes (RXRα(ep-/-) mice), which in a non-cell-autonomous manner modulated melanocyte proliferation and activation after UVR. RXRα(ep-/-) mice represent a unique animal model in which UVR induces melanocyte proliferation/activation in both epidermis and dermis. Considered together, the results of our study suggest that RXR antagonists, together with inhibitors of cell proliferation, can be effective in preventing solar UVR-induced photocarcinogenesis.

  7. Induction and identification of rabbit peripheral blood derived dendritic cells

    NASA Astrophysics Data System (ADS)

    Zhou, Jing; Yang, FuYuan; Chen, WenLi

    2012-03-01

    Purpose: To study a method of the induction of dendritic cells (DCs) from rabbit peripheral blood. Methods: Peripheral blood cells were removed from rabbit, filtered through nylon mesh. Peripheral blood mononuclear cells (PBMC) were isolated from the blood cells by Ficoll-Hypaque centrifugation (density of 1.077g/cm3).To obtain DCs, PBMC were cultured in RPMI1640 medium containing 10% fetal calf serum, 50U/mL penicillin and streptomycin, referred to subsequently as complete medium, at 37°C in 5% CO2 atmosphere for 4 hours. Nonadherent cells were aspirated, adherent cells were continued incubated in complete medium, supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF, 50ng/ml),and interleukin 4 (IL-4, 50ng/ml) for 9 days. Fluorescein labeled antibodies(anti-CD14, anti-HLA-DR, anti-CD86) were used to sign cells cultured for 3,6,9 days respectively, Then flow cytometry was performed. Results: Ratio of anti-HLA-DR and anti-CD86 labeled cells increased with induction time extension, in contrast with anti-CD14. Conclusion: Dendritic cells can be effectively induced by the method of this experiment, cell maturation status increased with induction time extension.

  8. High IFN-γ Release and Impaired Capacity of Multi-Cytokine Secretion in IGRA Supernatants Are Associated with Active Tuberculosis

    PubMed Central

    Pisoni, Amandine; Bendriss, Sophie; Marin, Grégory; Peries, Marianne; Bolloré, Karine; Terru, Dominique; Godreuil, Sylvain; Bourdin, Arnaud; Van de Perre, Philippe; Tuaillon, Edouard

    2016-01-01

    Interferon gamma (IFN-γ) release assays (IGRAs) detect Mycobacterium tuberculosis (Mtb) infection regardless of the active (ATB) or latent (LTBI) forms of tuberculosis (TB). In this study, Mtb-specific T cell response against region of deletion 1 (RD1) antigens were explored by a microbead multiplex assay performed in T-SPOT TB assay (T-SPOT) supernatants from 35 patients with ATB and 115 patients with LTBI. T-SPOT is positive when over 7 IFN-γ secreting cells (SC)/250 000 peripheral blood mononuclear cells (PBMC) are enumerated. However, over 100 IFN-γ SC /250 000 PBMC were more frequently observed in the ATB group compared to the LTBI group. By contrast, lower cytokine concentrations and lower cytokine productions relative to IFN-γ secretion were observed for IL 4, IL-12, TNF-α, GM-CSF, Eotaxin and IFN-α when compared to LTBI. Thus, high IFN-γ release and low cytokine secretions in relation with IFN-γ production appeared as signatures of ATB, corroborating that multicytokine Mtb-specific response against RD1 antigens reflects host capacity to contain TB reactivation. In this way, testing cytokine profile in IGRA supernatants would be helpful to improve ATB screening strategy including immunologic tests. PMID:27603919

  9. "Accelerating aging" chemotherapy on aged animals: protective effect from nutraceutical modulation.

    PubMed

    Marotta, Francesco; Harada, Masatoshi; Minelli, Emilio; Ono-Nita, Suzanne K; Marandola, Paulo

    2008-04-01

    The aim of this study was to test a novel phytocompound in an experimental model of antitumor-induced immunosuppression. Five groups of mice were considered: young (Y) and aged (A) that were given intraperitoneally 10 doses of cyclophosphamide (CPX, 25mg/kg/bw) or CPX plus (150 mg/kg/bw) of the nutraceutical DTS (Denshichi-Tochiu-Sen), and control. After sacrifice, macrophage chemotaxis and serum levels of IFN-gamma, IL-2, and GM-CSF were determined. Liver and urinary bladder were examined histologically, as were the liver and kidney for redox enzymes. CPX significantly decreased macrophage chemotaxis and all cytokines (p < 0.05, A > Y). DTS restored macrophage function and cytokine concentration (p < 0.001) and partly improved the necro-inflammatory score and substance P receptor expression in the bladder and the redox status in liver and kidney (p < 0.05). Such data suggest that DTS effectively prevents CPX-induced immune suppression and oxidative-inflammatory damage, which are particularly enhanced in aged organisms.

  10. A Natural Variant of the T Cell Receptor-Signaling Molecule Vav1 Reduces Both Effector T Cell Functions and Susceptibility to Neuroinflammation

    PubMed Central

    Kassem, Sahar; Bernard, Isabelle; Dejean, Anne S.; Liblau, Roland; Fournié, Gilbert J.; Colacios, Céline

    2016-01-01

    The guanine nucleotide exchange factor Vav1 is essential for transducing T cell antigen receptor signals and therefore plays an important role in T cell development and activation. Our previous genetic studies identified a locus on rat chromosome 9 that controls the susceptibility to neuroinflammation and contains a non-synonymous polymorphism in the major candidate gene Vav1. To formally demonstrate the causal implication of this polymorphism, we generated a knock-in mouse bearing this polymorphism (Vav1R63W). Using this model, we show that Vav1R63W mice display reduced susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide immunization. This is associated with a lower production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Despite increased proportion of Foxp3+ regulatory T cells in Vav1R63W mice, we show that this lowered cytokine production is intrinsic to effector CD4 T cells and that Treg depletion has no impact on EAE development. Finally, we provide a mechanism for the above phenotype by showing that the Vav1R63W variant has normal enzymatic activity but reduced adaptor functions. Together, these data highlight the importance of Vav1 adaptor functions in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation. PMID:27438086

  11. Structure-Based Design and Synthesis of 3-Amino-1,5-dihydro-4H-pyrazolopyridin-4-one Derivatives as Tyrosine Kinase 2 Inhibitors.

    PubMed

    Yogo, Takatoshi; Nagamiya, Hiroyuki; Seto, Masaki; Sasaki, Satoshi; Shih-Chung, Huang; Ohba, Yusuke; Tokunaga, Norihito; Lee, Gil Nam; Rhim, Chul Yun; Yoon, Cheol Hwan; Cho, Suk Young; Skene, Robert; Yamamoto, Syunsuke; Satou, Yousuke; Kuno, Masako; Miyazaki, Takahiro; Nakagawa, Hideyuki; Okabe, Atsutoshi; Marui, Shogo; Aso, Kazuyoshi; Yoshida, Masato

    2016-01-28

    We report herein the discovery and optimization of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one TYK2 inhibitors. High-throughput screening against TYK2 and JAK1-3 provided aminoindazole derivative 1 as a hit compound. Scaffold hopping of the aminoindazole core led to the discovery of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one derivative 3 as a novel chemotype of TYK2 inhibitors. Interestingly, initial SAR study suggested that this scaffold could have a vertically flipped binding mode, which prompted us to introduce a substituent at the 7-position as a moiety directed toward the solvent-exposed region. Introduction of a 1-methyl-3-pyrazolyl moiety at the 7-position resulted in a dramatic increase in TYK2 inhibitory activity, and further optimization led to the discovery of 20. Compound 20 inhibited IL-23-induced IL-22 production in a rat PD assay, as well as inhibited IL-23 signaling in human PBMC. Furthermore, 20 showed selectivity for IL-23 signaling inhibition against GM-CSF, demonstrating the unique cytokine selectivity of the novel TYK2 inhibitor.

  12. Differences between Mycobacterium-Host Cell Relationships in Latent Tuberculous Infection of Mice Ex Vivo and Mycobacterial Infection of Mouse Cells In Vitro

    PubMed Central

    Ufimtseva, Elena

    2016-01-01

    The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells. PMID:27066505

  13. TGF-β blockade depletes T regulatory cells from metastatic pancreatic tumors in a vaccine dependent manner

    PubMed Central

    Soares, Kevin C.; Rucki, Agnieszka A.; Kim, Victoria; Foley, Kelly; Solt, Sara; Wolfgang, Christopher L.; Jaffee, Elizabeth M.; Zheng, Lei

    2015-01-01

    Our neoadjuvant clinical trial of a GM-CSF secreting allogeneic pancreas tumor vaccine (GVAX) revealed the development of tertiary lymphoid aggregates (TLAs) within the pancreatic ductal adenocarcinoma (PDA) tumor microenvironment 2 weeks after GVAX treatment. Microarray studies revealed that multiple components of the TGF-β pathway were suppressed in TLAs from patients who survived greater than 3 years and who demonstrated vaccine-enhanced mesothelin-specific T cell responses. We tested the hypothesis that combining GVAX with TGF-β inhibitors will improve the anti-tumor immune response of vaccine therapy. In a metastatic murine model of pancreatic cancer, combination therapy with GVAX vaccine and a TGF-β blocking antibody improved the cure rate of PDA-bearing mice. TGF-β blockade in combination with GVAX significantly increased the infiltration of effector CD8+ T lymphocytes, specifically anti-tumor-specific IFN-γ producing CD8+ T cells, when compared to monotherapy controls (all p < 0.05). TGF-β blockade alone did not deplete T regulatory cells (Tregs), but when give in combination with GVAX, GVAX induced intratumoral Tregs were depleted. Therefore, our PDA preclinical model demonstrates a survival advantage in mice treated with an anti-TGF-β antibody combined with GVAX therapy and provides strong rational for testing this combinational therapy in clinical trials. PMID:26515728

  14. IκBNS regulates murine Th17 differentiation during gut inflammation and infection.

    PubMed

    Annemann, Michaela; Wang, Zuobai; Plaza-Sirvent, Carlos; Glauben, Rainer; Schuster, Marc; Ewald Sander, Frida; Mamareli, Panagiota; Kühl, Anja A; Siegmund, Britta; Lochner, Matthias; Schmitz, Ingo

    2015-03-15

    IL-17-producing Th17 cells mediate immune responses against a variety of fungal and bacterial infections. Signaling via NF-κB has been linked to the development and maintenance of Th17 cells. We analyzed the role of the unusual inhibitor of NF-κB, IκBNS, in the proliferation and effector cytokine production of murine Th17 cells. Our study demonstrates that nuclear IκBNS is crucial for murine Th17 cell generation. IκBNS is highly expressed in Th17 cells; in the absence of IκBNS, the frequencies of IL-17A-producing cells are drastically reduced. This was measured in vitro under Th17-polarizing conditions and confirmed in two colitis models. Mechanistically, murine IκBNS (-/-) Th17 cells were less proliferative and expressed markedly reduced levels of IL-2, IL-10, MIP-1α, and GM-CSF. Citrobacter rodentium was used as a Th17-inducing infection model, in which IκBNS (-/-) mice displayed an increased bacterial burden and diminished tissue damage. These results demonstrate the important function of Th17 cells in pathogen clearance, as well as in inflammation-associated pathology. We identified IκBNS to be crucial for the generation and function of murine Th17 cells upon inflammation and infection. Our findings may have implications for the therapy of autoimmune conditions, such as inflammatory bowel disease, and for the treatment of gut-tropic infections. PMID:25694610

  15. Woodchuck dendritic cells generated from peripheral blood mononuclear cells and transduced with recombinant human adenovirus serotype 5 induce antigen-specific cellular immune responses.

    PubMed

    Ochoa-Callejero, Laura; Berraondo, Pedro; Crettaz, Julien; Olagüe, Cristina; Vales, Africa; Ruiz, Juan; Prieto, Jesús; Tennant, Bud C; Menne, Stephan; González-Aseguinolaza, Gloria

    2007-05-01

    Woodchucks infected with the woodchuck hepatitis virus (WHV) is the best available animal model for testing the immunotherapeutic effects of dendritic cells (DCs) in the setting of a chronic infection, as woodchucks develop a persistent infection resembling that seen in humans infected with the hepatitis B virus. In the present study, DCs were generated from woodchuck peripheral blood mononuclear cells (wDCs) in the presence of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and human interleukin 4 (hIL-4). After 7 days of culture, cells with morphology similar to DCs were stained positively with a cross-reactive anti-human CD86 antibody. Functional analysis showed that uptake of FITC-dextran by wDCs was very efficient and was partially inhibited after LPS-induced maturation. Furthermore, wDCs stimulated allogenic lymphocytes and induced proliferation. Moreover, wDCs were transduced efficiently with a human adenovirus serotype 5 for the expression of beta-galactosidase. Following transduction and in vivo administration of such DCs into woodchucks, an antigen-specific cellular immune response was induced. These results demonstrate that wDCs can be generated from the peripheral blood. Following transfection with a recombinant adenovirus wDCs can be used as a feasible and effective tool for eliciting WHV-specific T-cell responses indicating their potential to serve as prophylactic and therapeutic vaccines. PMID:17385694

  16. Chronic and Invasive Fungal Infections in a Family with CARD9 Deficiency.

    PubMed

    Alves de Medeiros, Ana Karina; Lodewick, Evelyn; Bogaert, Delfien J A; Haerynck, Filomeen; Van Daele, Sabine; Lambrecht, Bart; Bosma, Sara; Vanderdonckt, Laure; Lortholary, Olivier; Migaud, Mélanie; Casanova, Jean-Laurent; Puel, Anne; Lanternier, Fanny; Lambert, Jo; Brochez, Lieve; Dullaers, Melissa

    2016-04-01

    Chronic mucocutaneous or invasive fungal infections are generally the result of primary or secondary immune dysfunction. Patients with autosomal recessive CARD9 mutations are also predisposed to recurrent mucocutaneous and invasive fungal infections with Candida spp., dermatophytes (e.g., Trichophyton spp.) and phaeohyphomycetes (Exophiala spp., Phialophora verrucosa). We study a consanguineous family of Turkish origin in which three members present with distinct clinical phenotypes of chronic mucocutaneous and invasive fungal infections, ranging from chronic mucocutaneous candidiasis (CMC) in one patient, treatment-resistant cutaneous dermatophytosis and deep dermatophytosis in a second patient, to CMC with Candida encephalitis and endocrinopathy in a third patient. Two patients consented to genetic testing and were found to have a previously reported homozygous R70W CARD9 mutation. Circulating IL-17 and IL-22 producing T cells were decreased as was IL-6 and granulocyte/macrophage colony-stimulating factor (GM-CSF) secretion upon stimulation with Candida albicans. Patients with recurrent fungal infections in the absence of known immunodeficiencies should be analyzed for CARD9 gene mutations as the cause of fungal infection predisposition. PMID:26961233

  17. Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells

    PubMed Central

    Kalbe, Benjamin; Knobloch, Jürgen; Schulz, Viola M.; Wecker, Christine; Schlimm, Marian; Scholz, Paul; Jansen, Fabian; Stoelben, Erich; Philippou, Stathis; Hecker, Erich; Lübbert, Hermann; Koch, Andrea; Hatt, Hanns; Osterloh, Sabrina

    2016-01-01

    Pathophysiological mechanisms in human airway smooth muscle cells (HASMCs) significantly contribute to the progression of chronic inflammatory airway diseases with limited therapeutic options, such as severe asthma and COPD. These abnormalities include the contractility and hyperproduction of inflammatory proteins. To develop therapeutic strategies, key pathological mechanisms, and putative clinical targets need to be identified. In the present study, we demonstrated that the human olfactory receptors (ORs) OR1D2 and OR2AG1 are expressed at the RNA and protein levels in HASMCs. Using fluorometric calcium imaging, specific agonists for OR2AG1 and OR1D2 were identified to trigger transient Ca2+ increases in HASMCs via a cAMP-dependent signal transduction cascade. Furthermore, the activation of OR2AG1 via amyl butyrate inhibited the histamine-induced contraction of HASMCs, whereas the stimulation of OR1D2 with bourgeonal led to an increase in cell contractility. In addition, OR1D2 activation induced the secretion of IL-8 and GM-CSF. Both effects were inhibited by the specific OR1D2 antagonist undecanal. We herein provide the first evidence to show that ORs are functionally expressed in HASMCs and regulate pathophysiological processes. Therefore, ORs might be new therapeutic targets for these diseases, and blocking ORs could be an auspicious strategy for the treatment of early-stage chronic inflammatory lung diseases. PMID:27540365

  18. The Effects of Low Dose Irradiation on Inflammatory Response Proteins in a 3D Reconstituted Human Skin Tissue Model

    SciTech Connect

    Varnum, Susan M.; Springer, David L.; Chaffee, Mary E.; Lien, Katie A.; Webb-Robertson, Bobbie-Jo M.; Waters, Katrina M.; Sacksteder, Colette A.

    2012-12-01

    Skin responses to moderate and high doses of ionizing radiation include the induction of DNA repair, apoptosis, and stress response pathways. Additionally, numerous studies indicate that radiation exposure leads to inflammatory responses in skin cells and tissue. However, the inflammatory response of skin tissue to low dose radiation (<10 cGy) is poorly understood. In order to address this, we have utilized a reconstituted human skin tissue model (MatTek EpiDerm FT) and assessed changes in 23 cytokines twenty-four and forty eight hours following treatment of skin with either 3 or 10 cGy low-dose of radiation. Three cytokines, IFN-γ, IL-2, MIP-1α, were significantly altered in response to low dose radiation. In contrast, seven cytokines were significantly altered in response to a high radiation dose of 200 cGy (IL-2, IL-10, IL-13, IFN-γ, MIP-1α, TNF α, and VEGF) or the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (G-CSF, GM-CSF, IL-1α, IL-8, MIP-1α, MIP-1β, RANTES). Additionally, radiation induced inflammation appears to have a distinct cytokine response relative to the non-radiation induced stressor, TPA. Overall, these results indicate that there are subtle changes in the inflammatory protein levels following exposure to low dose radiation and this response is a sub-set of what is seen following a high dose in a human skin tissue model.

  19. Acute Generalized Exanthematous Pustulosis: Pathogenesis, Genetic Background, Clinical Variants and Therapy

    PubMed Central

    Feldmeyer, Laurence; Heidemeyer, Kristine; Yawalkar, Nikhil

    2016-01-01

    Acute generalized exanthematous pustulosis (AGEP) is a severe, usually drug-related reaction, characterized by an acute onset of mainly small non-follicular pustules on an erythematous base and spontaneous resolution usually within two weeks. Systemic involvement occurs in about 20% of cases. The course is mostly benign, and only in rare cases complications lead to life-threatening situations. Recent studies highlight the importance of genetic variations in interleukin-36 receptor antagonist gene (IL-36RN) in the pathogenesis of this disease. The physiopathology of AGEP remains unclear, but an involvement of innate and acquired immune cells together with resident cells (keratinocytes), which recruit and activate neutrophils via production of cytokines/chemokines such as IL-17, IL-36, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα) and chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, has been postulated. Treatment is based on the removal of the causative drug, supportive care, infection prevention and use of potent topical or systemic steroids. PMID:27472323

  20. Dendritic cells generated from the blood of patients with multiple myeloma are phenotypically and functionally identical to those similarly produced from healthy donors.

    PubMed

    Pfeiffer, S; Gooding, R P; Apperley, J F; Goldschmidt, H; Samson, D

    1997-09-01

    Using a combination of GM-CSF, SCF, flk-2/flt-3 ligand, and IL-4, dendritic cells (DC) have been generated in vitro from the adherent fraction of mononuclear cells isolated from the blood of patients with MM. Analysis of cell yield showed no significant difference in DC yield (numbers or percentage of leucocytes) or total number of leucocytes generated in myeloma cultures compared to similar cultures prepared using mononuclear cells from the blood of healthy donors. The mean number of DC produced after 10d of culture were 8.19 x 10(5) and 9.87 x 10(5) cells (41% and 51% of all leucocytes) for the myeloma and normal cultures respectively. Flow cytometry investigation of phenotypic markers including CD1a, HLA-DR, CD80 (BB1/B7.1) and CD86 (B70/B7.2), and functional status (stimulatory potential in allogeneic mixed leucocyte reactions (MLR)) confirmed the generation of cells phenotypically identified as cultured DC. In addition, these cells were more effective than PBMC at stimulating allogeneic PBMC proliferation. These data demonstrate no difference between DC generated from patients with MM and healthy donors. This study was consider